NL2037265B1

NL2037265B1 - Means and methods for assessing cytotoxicity of effector cells

Info

Publication number: NL2037265B1
Application number: NL2037265A
Authority: NL
Inventors: Dalla Vecchia Simone; Suñer Niño Luis; Muffatto Federico
Original assignee: Digi Bio B V
Priority date: 2024-03-15
Filing date: 2024-03-15
Publication date: 2025-09-26
Also published as: WO2025193104A1

Abstract

A B S T R A C T Here are provided advantageous means and methods for assessing target cell killing, i.e. cytotoxicity, e.g. as induced by (engineered) effector cells, such as CAR-T cells, or as induced by effector cells when combined e.g. with an agent, such as an (effector) cell engager, e.g. a bispecific antibody having a binding domain that binds with an effector cell and another binding domain that binds with a target cell. Highly advantageously, an EWOD microfluidic device was found to be useful for accommodating such improved means and methods.

Description

Title: Means and methods for assessing cytotoxicity of effector cells

Introduction

Despite tremendous clinical success of T cell therapy, including CAR-T cell therapy, for the treatment of hematological cancers, many obstacles still remain for the therapy in treatment for a wider range of (cancer) indications. Predicting success of T-cell therapy using in vitro data alone is still a challenge. A standard cytotoxicity assay used for assessing e.g. CAR-T cell function towards a target antigen expressed on a target cell is applied during the in vitro testing, which involves assessment of target cell death 24 hours after initiating contact between CAR-T effector cell and target cells. However, this does not does not fully define

CAR-T cell activity. With the increasing complexity of constructing novel methodologies to further improve the clinical outcome of immunotherapies, screening methods to quickly identify the best lead candidates are becoming even more essential. To date, it has become evident that merely measuring cytotoxicity, utilizing standard assays will not accurately predict in vitro and in vivo outcomes, during development of CAR-T cell therapies, and the like, as well as in application of CAR-T cell therapies in the clinic.

One of the main obstacles in the development is the lack of fast, specific accurate tools to assess cytotoxicity, and the like.

Summary of the invention

The current inventors, in aiming to improve cytotoxicity assessment, advantageously now provide for highly improved methods that allow for fast, specific and accurate assessment of cytotoxicity in great detail. The current inventors now provide advantageous methods for assessing target cell killing, i.e. cytotoxicity, e.g. as induced by (engineered) effector cells, such as CAR-T cells, or as induced by effector cells when combined e.g. with an agent, such as an (effector) cell engager, e.g. a bispecific antibody having a binding domain that binds with an effector cell and another binding domain that binds with a target cell. Highly advantageously, the inventors have utilized an EWOD microfluidic device (such as described e.g. in detail in the EWOD microfluidic device section herein and as shown in the examples) for such assays, and now provide for highly advantageous methods, to which may also be referred to as workflows herein, which can be performed fast, specific and accurately, and simultaneously in parallel, on such EWOD microfluidic devices providing detailed information with regard to functionality of effector cells against target cells, including e.g. target cell killing, frequency of cell killing, motility, and also detection of molecules of interest, such as induced cytokines representing anti-tumor effector, stimulatory, regulatory and/or inflammatory functions.

EWOD is a microfluidic technique used to control and manipulate tiny droplets of liquids on a solid surface. It involves applying an electrical voltage to a droplet of liquid that is placed on a dielectric material, which is an insulating material that does not conduct electricity. When an electric field is applied, it interacts with the liquid's dipoles, aligning them with the electric field. This alignment changes the surface tension of the liquid, causing the droplet to spread on an attractive electrode (increasing wettability) and shrink on a repulsive electrode (decreasing wettability). By controlling the voltage applied to the liquid at different electrode positions in a matrix of electrodes, EWOD techniques allow for precise control over the movement and mixing of tiny droplets of liquids inside the EWOD microfluidic device. Using EWOD actuation, droplets are manipulated within the microfluidic device by dispensing microliter droplets from the input reservoir, splitting, merging and mixing operations and transporting them across the grid of electrodes towards electrodes where imaging sensors, magnetic forces or fluidic inputs and output might intervene.

Hence, EWOD microfluidic devices are, as described herein, provided with means that allows for manipulation of droplets of fluids, i.e. generating, moving, merging, splitting and mixing droplets (such as shown in the examples herein), and with means that allows for microscopy imaging, i.e. the EWOD microfluidic device allows for taking images utilizing a microscope, for which the EWOD microfluidic device can be, and highly preferably is, advantageously provided with an area therefor which has an optical black background.

More specifically, different microfluidic droplet operations like dispensing and splitting from the reservoirs with the original analytes (e.g. liquids with cells and/or culture medium) and subsequent merging, mixing and splitting to perform dilutions to make the desired concentrations, may be required as sample preparation steps prior to, or part of, the workflow assay procedure to be performed on the droplets. After such automating sample preparation steps, as part of the workflow assay, the various droplets containing the effector and target cells to be analysed, are imaged. These analytes with effector cells and target cells in co-culture may also secret proteins which can subsequently be purified /analysed from the sample through magnetic bead capture steps as described herein. After performing the workflow assay procedure, with or without magnetic bead capture steps, the various droplets can leave the microfluidic device through an outlet opening and may be collected.

Highly advantageously, this way, multiple droplets with effector cells and target cells can be prepared separately, and can be simultaneously merged, with or without mixing of the droplets, and subsequently effector cells and target cells can be closely monitored, this way performing a cytotoxicity evaluation from microscopy images, e.g. time-lapse images and/or in real-time, and assessed e.g. with regard to cell morphology, or motility and including at least target cell killing. Hence, in a preferred embodiment, effector cells and target cells are merged simultaneously. By merging effector cells and target cells without mixing, the effector cells and target cells are separated at opposing sides of the droplet at the start of the assay, which is advantageous as it allows measurement of the motility towards each other when tracking them from the onset of the experiment. This is advantageous because effector cells can act fast, targeting and moving towards the target cells, and this way can be monitored from the start of the incubation. In another embodiment after merging of effector and target cells prior to imaging both populations are controllably mixed using EWOD forces to produce a more homogeneous mixing of the cells, which is also advantageously well controlled.

In addition, the EWOD microfluidic device may allow for detection, e.g. quantification, of target molecules generated by the cells through magnetic bead capture from the mixed droplets. This can be performed at the end of the effector cells and target cell incubation step. The EWOD microfluidic device is then highly advantageously provided with a magnet positioned such that a magnetic force can be exerted which is orthogonal to the forces exerted by EWOD for manipulating the droplets. By allowing the mixed droplets to interact with captured beads, which capture beads are provided e.g. with an antibody for a target of interest, such as IFNy, and subsequently separate or isolating the capture beads from the droplet using said magnet, a target molecule of interest, such as IFNy, can be quantified, after which the effector and target cells can be extracted through the outlet of the microfluidic device. This way, highly efficiently, utilizing relatively low amounts of effector cells and target cells, in a short timeframe, a large amount of useful data regarding a large set of biological parameters can be collected through imaging from a plurality of effector cell and target cell incubations, which because these can be performed simultaneously under the same conditions, allows for accurate assessment of e.g. differences between varied conditions tested.

Figures

Figure 1. Generating, merging and mixing droplets on an EWOD microfluidic device. Shown are from A to | snapshots of generating droplets. In each snapshot, in the upper left and to the right thereof, reservoirs filled with different mediums are shown, such as comprising effector cells and target cells respectively (A), each different medium in a different reservoir having a different color, i.e. shown as different shades of gray. From both containers, droplets exit the reservoir and droplets are generated covering 1 tile surface (B-D). The droplets are subsequently brought into each other's proximity and merged (E-H), generating a merged droplet covering two tiles (H). The boundary between the two merged droplets due to the different colors of the droplets is clearly visible in F, G and H. The merged droplet is subsequently mixed, by circulating the droplet, obtaining a homogenous mixed droplet, without boundary and a mixed color.

Figure 2. Splitting of droplets.

Figure 3. Droplets with magnetic beads. Shown are images of capturing magnetic beads from droplets, and washing and elution of magnetic beads. In Figure 3A-C, the first scenario illustrates the magnet separation of a liquid from a solid phase by the coalescence of beads.

Here, magnetic beads within a droplet are attracted to an external magnet shown in Figure 3D, causing them to accumulate at the droplet's surface. However, as the droplet moves away from the magnetic field generated by the magnet, the force exerted by its movement can overcome the weak magnetic force holding the beads, causing them to remain in the surrounding oil or air (Figure 3D). In contrast, the second scenario depicted in Figure 3E-G demonstrates the use of a train of droplets to remove non-bound agents without disrupting the magnetic beads. As the droplet train, consisting of washing buffer droplets, moves sequentially across the surface of the EWOD microfluidic device, it generates a fluid flow that effectively sweeps away non-bound agents while leaving the magnetic bead-bound analytes intact. This process, known as magnetophoresis, ensures that the magnetic beads remain within the fluid and are not forced out into the surrounding environment (Figure 3H).

By employing this approach, the risk of displacing the magnetic beads can be minimized, advantageous for preserving the integrity of the assay.

Figure 4. Examples of part of microscope images taken of effector cells and target cells. A) shows a dark field microscope image. B} shows a red fluorescent image, showing cells stained with cell death fluorescence marker C) shows a green fluorescent image, showing target cells which were labeled with a green fluorescent label. In D), an integrated image is shown, with each individual cell labeled with an identifier and characterized morphology, integrating dark field and fluorescent image. In E we observe the tracking of individual cells over time. 5

Figures 5-8. Shown are real-time death rates of target cells by effector cells at different effector to target cell ratios (Fig. 5) shows tumor cell death rates by effector cell line FMC63-

BBz shown by a ratio of the number of dead tumors by the total number of cells over time (against control) death rates (Fig. 6), normalized death rates from Fig. 5 using a control without effector cells (Fig. 7) tumor cell death rates by a standard Mock effector cell line shown by a ratio between the number of dead tumors by the total number of cells over time normalized using a control without effector cells (Fig. 8). Figure 8 also shows absolute tumor cell count and dead tumor cell count, and the death rate curve obtained when calculating the ratio between them.

Figure 9: Plot comparing the output from the device (B) and the one obtained from an external laboratory for benchmarking (A). For this, in both the Specific lysis (%) was calculated at T-final using the samples containing either of the two different CAR-T constructs (FMC63-28z or FMC63-BBz) and Mock-transduced T cells (Mock). Additionally, multiple E:T ratios were used, the external laboratory included 3:1, 1:1, 1:3, 1:10, and 1:30, while in the device 1:1, 1:3, and 1:10 was used. Therefore, the comparison was just made in the overlapping E:T ratios. Each data point represents the average between two replicates.

Figure 10. Frequency of contact events made by a single effector cell to target cells performed with an EWOD microfluidic device over an experiment.

Figure 11. Cell contact events denoted in grey as O in legenda and cell killing events denoted in black as 1 in legenda in cell killing assays with effector cells and target cells performed with an EWOD microfluidic device over time where each track represents a single cell.

Figure 12. Comparison of frequency of attacks performed by a single effector to other target cells in an experiment using effector population A vs the frequency of attacks of population

B.

Figure 13: Calibration curve for protein quantification on an EWOD device, a chip. Scatter plot showing the strong logarithmic correlation (R squared = 0.945) between the mean particle fluorescence intensity arbitrary units (A.U.) and the antigen (Ag) concentration. In this case Interferon gamma (IFN-y) was used as Ag. All data points are an average of six replicates.

Figure 14: Linear range of strong linear correlation between fluorescence intensity and protein concentration. Scatter plot showing the range in which the mean particle fluorescence intensity arbitrary units (A.U.) and the antigen (Ag) concentration are strongly correlated (R squared = 0.856). In this case Interferon gamma (IFN-y) was used as Ag. All data points are an average of six replicates.

Figure 15: Distribution of mean particle fluorescence values across different protein concentrations. Box plot showing the range of mean particle fluorescence values obtained at each of the antigen (Ag) concentrations tested. In this case Interferon gamma (IFN-y) was used as Ag.

Figure 16. Flow diagram of workflow / method for cell killing assay with effector cells and target cells on an EWOD device in accordance with the invention.

Figure 17. Shows the flow chart for the proof of principle for an immunoassay, i.e. capture bead assay, using an EWOD microfluidic device.

Figure 18. Shown are two droplets of two different fluorescence particles merging. A shows droplets being one electrode away from each other and hence not being in contact. Figure 18 B shows the droplets are brought to contact and merged. As visible in Figure 18 C the particles do not mix after the merging of the two droplets.

In figure 19 is shown two droplets merged but not yet mixed (Figure 19 A). By EWOD actuation (as depicted in Figure 20) particles are actively mixed (see Figures 19 B and 19

C).

Figure 20. Shows EWOD actuation resulting in the back and forth movement of the merged droplets above electrodes, making it possible to actively mix droplets and cells.

Figure 21. Droplet operations tree showing exemplary droplet microfluidic steps performed in an EWOD device in accordance with the means and methods as described herein, including unit operations for input volume, dispensing, mixing, splitting and imaging to perform an effector cell - killing target cells assay.

Detailed description

Accordingly, the current invention provides for means and methods for determining target cell killing of target cells by effector cells in an EWOD microfluidic device, wherein the

EWOD microfluidic device is equipped with a microscope for imaging of the cells. Such a method, also referred to as workflow, is schematically depicted e.g. in Figures 16 and 21.

Hence, in one embodiment, a method is provided for determining target cell killing by effector cells with an electrowetting on dielectric (EWOD) microfluidic device, comprising the steps of: a) providing an EWOD microfluidic device comprising means for holding liquid comprising cells and/or agents in a plurality of areas, configured for generating, moving, resuspending, merging, splitting and mixing droplets with a series of alternating electrodes by generating an electrical field to individually addressable electrodes, and comprising means for imaging of droplets and cells and/or agents therein using microscopy imaging; b) providing effector cells in culture medium;

Cc) providing target cells in culture medium; qd) providing culture medium; e) transferring the effector cells and target cells, suspended in a culture medium, and culture medium, each to a separate reservoir area, respectively, on the EWOD microfluidic device;

f) generating droplets of the effector and target cells in culture medium, and culture medium, of defined (microliter) volumes, and optionally, determining the number of effector or target cells in each generated droplet using imaging, and subsequently; q) preparing droplets of the effector and the target cells in culture medium, respectively, with a defined concentration of cells, and a defined volume, each in a separate sample preparation area, thereby obtaining prepared droplets of effector cells and of target cells ; ) wherein the prepared droplets are obtained by splitting, merging, and/or mixing of generated droplets to obtain a desired amount or concentration of cells in the prepared droplets;

Ih wherein the prepared droplets have a volume preferably of at least 0.1 microliter, preferably up to 10 microliter, preferably between 0.5 to 5 microliter, more preferably between 1 to 2 microliter; and

In wherein the effector cells and target cells are homogeneously distributed inside of their respective droplets;

IV) wherein the number of effector or target cells in each prepared droplet preferably is between 10 and 1x10*4, more preferably between 1x10*2 and 1x103,

V) optionally, determining the number, of effector and/or target cells in each prepared droplet using imaging; h) merging the prepared droplets of effector cells and prepared droplets of target cells in a culturing area wherein effector cells and target cells are in different ratios of effector cells to target cells, thereby obtaining merged droplets; ) wherein preferably the different ratios of effector cells to target cells in the merged droplets range from 1:10 to 10:1,

In wherein optionally, for each ratio the number of technical repeats is at least 2

Il) determining the number, of effector and target cells, and, preferably, individual location and cell morphology effector and target cells;

IV) wherein after merging, the merged droplet comprising effector and target cells is mixed, or, is not mixed; i) incubating the merged droplets on the EWOD microfluidic device, preferably in an area with an optical black background for imaging;

j) imaging the merged droplets using preferably fluorescent and/or optical automated microscopy, at least at the end of incubation,;

Kk) determining from the captured images for each merged droplet the number of at least viable target cells at least at the end of incubation.

Accordingly, in step a) of the method, an EWOD microfluidic device is provided comprising means for holding liquid comprising cells and/or agents in a plurality of areas, configured for generating, moving, resuspending, merging, splitting and mixing droplets with a series of alternating electrodes by generating an electrical field, and comprising means for imaging of droplets and cells and/or agents therein using microscopy imaging. As shown in the examples herein, with the EWOD microfluidic device, droplets can be very well manipulated, and unit operations like generated droplets, moving, resuspending, merging, splitting and mixing can be performed. The EWOD microfluidic device does not need to have channels or wells, i.e. the electrostatic forces generated by the electrodes guide the fluids, allowing fluid to be contained in an area, and droplets generated and manipulated around. The configuration of the electrodes determines the tile size and combined with the height of the chamber, thus droplet size, or droplet volume. The droplets may be used in a droplet-in-oil microfluidic approach, which means that the droplets may be used in an oil environment, permeable for gasses to allow for gas exchange. Herewith, evaporation of liquid is reduced to a non-observable rate. This also provides an additional benefit: oils reduce the friction experienced by a droplet moving on a surface, and the hydrophobic oil phase will allow for improved droplet splitting behaviour.

In steps b) and c), respectively, effector cells are provided in culture medium and target cells in culture medium. Furthermore, culture medium is provided. The medium that can be used is a standard medium, but without any fluorophores in the medium, such as phenol red. For T cells, a standard RPMI medium is e.g. suitable, supplemented with 10% v/v serum, e.g. FCS. As a EWOD microfluidic device is filled with oil, to these liquids, if necessary, a surfactant may be added in case medium or the like used exhibits specific viscosities and electrostatic properties, surfactant may be added to the medium. For example, synperonic, tetronics, pluronics, PEG2000 or the like may be added in appropriate amounts. Such surfactants can improve EWOD performance, as well as may serve to reduce biofouling of electrodes (accumulation of proteins and/or lipids on surfaces with sequential reduced hydrophobicity of said tile/surface may reduce EWOD). It may also be opted to include gels or extracellular matrix components or the like to modify viscosity of the medium to optimize experimental conditions, e.g. providing for a more biological-like environment. In any case, the person skilled in the art is well capable of optimizing medium conditions to allow for unit operations to be performed in the EWOD microfluidic device, i.e. generating, moving, resuspending, merging, splitting and mixing droplets.

Effector cells include effector cells of the immune system that can exert an effect, e.g. via a receptor. For example, a T cell carrying a T cell receptor can bind an antigen on a cancer cell, upon which it can e.g. exert a cytotoxic effect and kill the target cell. Effector cells can be derived from nature, e.g. obtained from a host, e.g. a human, and can also include genetically modified cells wherein e.g. a receptor in particular useful is provided to an effector cell. It is also understood that effector cells may also interact with an antigen on a target cells via a cell engager, e.g. a bispecific antibody that can bind with the effector cell and with the target cell, with respective binding domains.

Target cells in accordance with the invention are the cells on which the effector cells are to exert an effect, e.g. bind therewith and trigger an immune reaction thereto. Target cells include cells presenting an antigen, such as cancer cells. An antigen may be presented by MHC, i.e. HLA in humans, which are specialized receptors that present peptides e.g. derived from digested proteins expressed by the cell (e.g. usually 8-11 amino acids in length for MHCI). An antigen may also be a protein or other biomolecule that is presented on the surface of a cell, e.g. epidermal growth factor receptors or checkpoint proteins, which in the case of cancer cells are overexpressed therewith providing a differentiating feature when compared with non-diseased cells. Target cells may also include cells expressing auto- antigens, e.g. known to be involved in autoimmune diseases or cells infected with a pathogen, e.g. a virus.

Next, in step e), the effector cells and target cells, suspended in a culture medium, and culture medium, are transferred, each to a separate reservoir area, respectively, on the

EWOD microfluidic device. Hence, the EWOD microfluidic device is provided with an inlet such that these fluids can be transferred to the chamber. It is understood that a reservoir area may not necessarily be a physically restrained area, but may be advantageously defined by a configuration of electrodes, such as depicted in the examples herein (see e.g.

Figure 1).

Once the EWOD microfluidic device, to which may also be referred to as EWOD microfluidic chip instead, has been provided with the effector cells and target cells, suspended in a culture medium, and culture medium, the next step f) involves generating droplets of the effector and target cells in culture medium (as shown e.g. in Fig. 1A-1D), and culture medium, of defined volumes, and optionally, determining the number of effector or target cells in each generated droplet using imaging, and subsequently; in step g) preparing droplets of the effector and the target cells in culture medium, respectively, with a defined concentration of cells, and a defined volume, each in a separate sample preparation area, thereby obtaining prepared droplets of effector cells and of target cells;

I) wherein the prepared droplets are obtained by splitting, merging, and/or mixing of generated droplets to obtain a desired amount or concentration of cells in the prepared droplets;

II) wherein the prepared droplets have a volume preferably of at least 0.1 microliter, preferably up to 10 microliter, preferably between 0.5 to 5 microliter, more preferably between 1 to 2 microliter; and

II) wherein the effector cells and target cells are homogeneously distributed inside of their respective droplets;

IV) wherein the number of effector or target cells in each prepared droplet preferably is between 10 and 1x104, more preferably between 1x10%2 and 1x1043,

V) optionally, determining the number of effector and/or target cells in each prepared droplet using imaging;

It is understood that with regard to these steps in the EWOD microfluidic device, advantageously, by manipulating generating droplets (as shown e.g. Fig. 1A-1D), of defined volume, and by splitting (as a shown e.g. in Fig.2A-2C), merging (shown e.g. in Fig.1E-1G) and/or mixing (shown e.g. in Fig.1 H-11 and in Fig. 20) of droplets, droplets can be prepared with a defined concentration of cells and a defined volume. For example, in a preparation area, the tile volume is 1 pl, by having one or more consecutive steps of merging, droplets of cells in medium with medium, and, subsequently mixing, and splitting, one can obtain different droplets with different concentrations of cells. Of course, in these steps, effector cells and target cells are not mixed, as that would mean the experiment would start. By repeating the cycle of merge, mix and split, with medium, cells can be diluted to obtain suitable concentrations of cells. Hence, it is not required in accordance with the invention when utilizing an EWOD microfluidic device to provide for multiple reservoirs with different concentrations of cells, one can simply generate the desired concentration of cells in a droplet in the device by different operating steps. It is highly preferred to have consecutive steps of merging, mixing and splitting, but of course, it can always be contemplated to have multiple reservoir areas for different concentrations of cells.

The volume of the prepared droplets are to have a volume, preferably of at least 0.1 microliter. Preferably, the prepared droplets have a volume up to 10 microliter. As said, the volume of droplets is determined by tile size, i.e. defined by the electrode configuration and height of the chamber of the device. Of course, as part of process control it may be opted to determine volume of prepared droplets, or of any droplets in the device. In any case, a suitable volume is selected and/or determined, to allow for a substantial amount of cells with which effector cell and target cell interaction can be well studied. As said, a range of between 0.1 microliter and up to 10 microliter suffices, as such range of volume was found to be well suitable for the EWOD microfluidic device for the methods in accordance with the invention, though larger and smaller volumes may be contemplated. More preferably, the volume of the prepared droplets is between 0.5 to 5 microliter, more preferably between 1 to 2 microliter.

In the droplets prepared, the effector cells and target cells are homogeneously distributed inside of their respective droplets. Having the effector cells and target cells homogeneously distributed is advantageous for the next step, merging of the prepared droplets, as this contributes to the reproducibility of the method. The number of effector or target cells in each prepared droplet preferably is between 10 and 1x1044, more preferably between 1x10%2 and 1x1013.

Once prepared droplets are provided, optionally, next is determining the number of effector and/or target cells in each prepared droplet using imaging. Of course, as one may know already the concentration of the cells this may not be necessary. Nevertheless, prior to having the effector cells and target cells initiating contact, it can be advantageous to have the number of cells checked, as part of the quality control of the assay, as an in process monitoring of the generated droplets, which can include monitoring of merged, mixed, and split droplets in the process as well. Anyhow, as said, this is not required as the concentration of cells in the reservoir is known a priori, and the process steps for preparing droplets is well controlled.

The next step involves step h), i.e. merging the prepared droplets of effector cells and prepared droplets of target cells in a culturing area wherein effector cells and target cells are in different ratios of effector cells to target cells, thereby obtaining merged droplets; ) wherein preferably the different ratios of effector cells to target cells in the merged droplets range from 1:10 to 10:1,

Il) and, determining the number, of effector and target cells, and, preferably, individual location and cell morphology effector and target cells;

IV) wherein after merging, the merged droplet comprising effector and target cells is mixed, or, is not mixed.

In this step, the prepared droplets of effector cells and prepared droplets of target cells are merged in a culturing area, having a single culturing area for each effector to target ratio and repetition. In a cytotoxicity assay for effector cells, i.e. a workflow/method for determining target cell killing by effector cells, typically various ranges are tested in order to assess dose response effect. For example, ranges can be selected to include one or more of 1:10, 1:3, 1:1, 3:1 and 10:1. In addition, technical repeats may be performed. With regard to the ranges selected, it may be contemplated to have the number of target cells substantially constant, while varying with the number of effector cells. Conversely, it may be contemplated to have the number of effector cells substantially constant, while varying the number of target cells. Preferably, it may be contemplated to have the total number of effector cells and target cells combined substantially constant in order to have a similar concentration of nutrients for each cell across all droplets.

Highly advantageously after merging, the two prepared droplets, one having the effector cells and the other having the target cells, form an interphase within the merged droplet formed, i.e. the effector cells and target cells are still physically separated but are comprised in a single merged droplet. Immediately when merged, the effector cells, as these can be highly mobile cells, can start moving towards the target cells to interact therewith, and cross the interphase. Hence, it is advantageous to not have the merged cells mixed, as when imaging is initiated at the start of merging the prepared droplets, highly relevant information regarding effector cell function and effect can be obtained. Of course, it can also be contemplated to have the merged prepared droplets immediately mixed after merging.

In any case, once merged, or merged and immediately mixed thereafter, or just prior to merging, the number of effector and target cells is determined. Thus, step Ill) and step

IV) relating to determining the number and, respectively, mixing of effector cells and target cells or not, are not necessarily in chronological order. As long as the number of effector and target cells is determined and thus known at the start of the incubation, such a determination of the number of cells can be contemplated. It is understood that this determination can include identification or classification of cells as effector or target cells,

and by this determination, the number of effector and target cells can be effectuated. As said, it is preferred to know the number of effector and target cells, and, advantageously individual location and cell morphology of effector and target cells as well, at the start of incubation, such that the effector cells and target cells can be imaged and monitored throughout the entire incubation and information on the interaction between cells collected.

Hence, highly preferably, individual location and cell morphology of effector and target cells is determined, the latter information being of course preferably of use after merging (when not mixing), or after merging and mixing, as when cells are mixed, prior location information on individual cells is no longer or of less relevance. Nevertheless, the information gathered on individual cells may be sufficient to identify individual cells after mixing from images gathered prior to mixing, because of individual cells sufficient information may be gathered from images collected to allow for such identification.

Hence, next, merged droplets, i.e. the merged droplets of effector cells and target cells as obtained in step h) are subsequently incubated in step i), for imaging. Such imaging is preferably performed with microscopy. Highly suitable and advantageous microscopy that can be applied includes darkfield microscopy and/or fluorescent microscopy, such as described herein i.a. in the examples. Highly advantageously and preferably, the area in which the merged droplets are incubated has a light absorbing layer, e.g. an optical black background, which is useful for imaging. Preferably, this incubation is in one area, which allows for tracking of the effector cells and/or target cells, in which the merged droplet is preferably not manipulated and remains in position. This area for incubation and imaging, preferably is the area to which the prepared droplets were merged, in particular when the merging step is performed without mixing. This way, manipulation steps with the droplets can be minimized, while being imaged. Hence, preferably imaging, step j) and incubation, step i) are performed in the same area with minimal manipulation of the merged droplets.

The step of imaging the merged droplets, step j), highly preferably involves fluorescent and/or optical automated microscopy, which is performed at least at the end of the incubation. Having cells provided with a fluorescent label is helpful in identifying cells as well as assessing cell viability. Preferably, target cells are provided with a fluorescent label.

Hence, lastly, in step kK), from these captured images, for each merged droplet the number of at least the viable target cells that remain at least at the end of incubation are determined.

By determining at least the number of viable target cells that remain at the end of the incubation, the target cell killing induced by the effector cells can be quantified, which is the minimal information of interest to retrieve from a cytotoxicity assay, or the like.

By combining the images obtained/information available at the start of the incubation with regard to the effector cells and target cells, i.e. information of the number of (viable) target cells, and, optionally, of effector cells with the images obtained at the end of the incubation, in step k) above, for each incubated merged droplet the number of killed target cells can be determined and/or the ratio of viable target cells to total target cells. Of course, in case the number of target cells at the start of the incubation is substantially the same for all conditions tested, simply the number of viable cells at the end of incubation may suffice to provide information with regard to cytotoxicity induced. Of course, having both the initial number and the number at the end, allows for the calculation of a ratio of viable cells remaining (or conversely, determine the ratio of viable cells that were killed). Whichever way of presenting the information is selected, this number, ratio, percentage, or the like, represents a functional property of the effector cells, i.e. their capacity to induce target cell killing. Of course, the present invention is not limited to this particular information and the present invention allows for a diverse and large amount of data to be collected from the images of the target cells and/or the effector cells. It is understood that any analysis of collected data may be performed at the site where the EWOD microfluidic device is used in a method of the invention, e.g. by capturing the images and processing and assessing the images with analysis software. It is understood that such an analysis of the data may also be performed at a site remote from where the device is subjected to the method of the invention. Hence, in the methods in accordance with the invention, at least the data that is generated may be sent to a remote location for storage of the information, and analysis as well, or retrieved later on and send back for analysis. Hence, it is understood that the methods in accordance with the invention may simply entail the collection of data and not necessarily yet comprise the analysis thereof, such as determining at least the number of viable target cells that remain at the end of an incubation.

With regard to the incubation step, it is understood that any suitable time may be selected for this incubation. As shown in the example section, imaging for relatively short time periods, i.e. about 1 hour, or more, may already provide for valuable information with regard to cell killing, as it can be witnessed e.g. in Figure 5-7, that the slopes of plots of death rate (i.e. killed tumor cells/ total tumor cells) vs. time can already show differentiation when comparing different effector to target cell ratios. This is highly advantageous, as standard assays usually are conducted for about 24 hours. Hence, the method in accordance with the invention may allow for shortening of the cytotoxicity assays, while gathering more accuracy than an endpoint. As can also be shown in the plots is that the plots show plateauing. Plateaus may be reached at different moments and may depend on the ratios of effector cells and target cells in merged droplets, as well as the concentration of cells. Moreover, the curve may have an initial steep inclination, which gradually decreases until the plateau is reached. Without being bound by theory, measuring the slope of a plot (of death rate vs. time) for each ratio in the early phase of the incubation may provide for more accurate information as opposed to measuring only the end-result. Hence, it may not even be necessary for conducting incubation steps for 24 hours or more, as when interested in cell killing alone, imaging for shorter time periods may already provide for sufficient and highly accurate information when performing the methods in accordance with the invention. Of course, incubation steps may be performed longer, as information on a plateau, if reached, may be of interest as well, and more information in addition to cell killing may be collected. Hence, it is understood that the incubation step may be performed e.g. for up to 96 hours. Preferably, the incubation step is performed for at least 1 hour, at least 2 hours. The incubation step can be performed for up to 96 hours, such as for 24 hours.

More preferably, the incubation step is 1 hour - 986 hours. In one embodiment, the incubation is about 4 hours. Of course, suitable incubation times can easily be determined for any type of assays by first performing an assay for up to 96 hours, and based on the data collected, a suitable incubation time can be selected that provides for the information required.

In view of the above, it is understood that in the methods in accordance with the invention, the cytotoxicity is assessed of an effector cell towards a target cell. In the method, target cells are provided at a defined concentration, e.g. a cell suspension having substantially single cells in suspension. The target cells are optionally stained, such as shown e.g. in examples herein. It is understood that staining the target cells with a fluorescent marker may be highly advantageous in the methods of the invention as it allows to, using fluorescent microscopy, conveniently detect the target cells. It is understood that any suitable fluorescent staining may suffice, such as e.g. described in the example section.

It is understood that a target cell may also e.g. constitutively express a fluorescent marker instead, e.g. GFP or the like. Of course, the same may apply to effector cells, and if (fluorescent) labels would be used for effector cells, these preferably would be different from labels used for target cells, if both target cells and effector cells are stained. In any case, providing the target cells or effector cells with a fluorescent marker can be contemplated and may be preferred in the methods in accordance with the invention. Any suitable dye may be contemplated in staining target cells and/or effector cells. It may also be contemplated to use in addition a viability staining, which may be helpful to distinguish live from dead cells.

The methods as described above describe therein advantageous optional steps that can be envisioned in accordance with the invention. These methods also describe highly advantageous aspects in accordance with the invention that may be applied independently.

Thus, the methods as described above, may be rephrased without optional steps and focused on the highly advantageous aspects of generating, moving, resuspending, merging, splitting and mixing of droplets, and on the aspect of the highly advantageous merging of prepared droplets of effector cells and target cells, which are both highly well controllable, or focused solely on the aspect of the highly advantageous merging of prepared droplets of effector cells and target cells and automated sample preparation.

These aspects in particular can be highly advantageous with an electrowetting on dielectric (EWOD) microfluidic device.

Hence, in another embodiment, a method is provided for determining target cell killing by effector cells with an electrowetting on dielectric (EWOD) microfluidic device, comprising the steps of: a) providing an EWOD microfluidic device comprising means for holding liquid comprising cells and/or agents in a plurality of areas, configured for generating, moving, resuspending, merging, splitting and mixing droplets with a series of alternating electrodes by applying a voltage to individually addressable electrodes, and comprising means for imaging of droplets and cells and/or agents therein using microscopy imaging; b) providing effector cells in culture medium;

Cc) providing target cells in culture medium; d) providing culture medium; ©) transferring the effector cells and target cells, suspended in a culture medium, and culture medium, each to a separate reservoir area, respectively, on the EWOD microfluidic device; f) generating droplets of the effector and target cells in culture medium, and culture medium, of defined volumes, and subsequently; 9) preparing droplets of the effector and the target cells in culture medium, respectively, with a defined concentration of cells, and a defined volume, each in a separate sample preparation area, thereby obtaining prepared droplets of effector cells and of target cells ;

i) wherein the prepared droplets are obtained by splitting, merging, and/or mixing of generated droplets to obtain a desired amount or concentration of cells in the prepared droplets; ii) wherein the effector cells and target cells are homogeneously distributed inside of their respective droplets; h) merging the prepared droplets of effector cells and prepared droplets of target cells in a culturing area wherein effector cells and target cells are in different ratios of effector cells to target cells, thereby obtaining merged droplets; i) comprising determining the number, of at least the target cells; and ii) wherein after merging, the merged droplet comprising effector and target cells is mixed, or, is not mixed; i) incubating the merged droplets on the EWOD microfluidic device, j) imaging the merged droplets, at least at the end of incubation; k) determining from the captured images for each merged droplet the number of at least viable target cells at least at the end of incubation.

In another embodiment, a method is provided for determining target cell killing by effector cells with an electrowetting on dielectric (EWOD) microfluidic device, comprising the steps of: a) providing an EWOD microfluidic device comprising means for holding liquid comprising cells and/or agents in a plurality of areas, configured for moving, preparing, merging, and mixing droplets with a series of alternating electrodes by applying a voltage to individually addressable electrodes, and comprising means for imaging of droplets and cells and/or agents therein using microscopy imaging; b) providing effector cells in culture medium;

Cc) providing target cells in culture medium; d) optionally, providing culture medium; e) transferring the effector cells and target cells, suspended in a culture medium, and culture medium, each to a separate reservoir area, respectively, on the EWOD microfluidic device; f) preparing droplets of the effector and the target cells in culture medium, respectively, with a defined concentration of cells, and a defined volume, each in a separate sample preparation area, thereby obtaining prepared droplets of effector cells and of target cells,

wherein the effector cells and target cells are homogeneously distributed inside of their respective droplets; a) merging the prepared droplets of effector cells and prepared droplets of target cells in a culturing area wherein effector cells and target cells are in different ratios of effector cells to target cells, thereby obtaining merged droplets; i) comprising determining the number, of at least the target cells; and ii) wherein after merging, the merged droplet comprising effector and target cells is mixed, or, is not mixed; h) incubating the merged droplets on the EWOD microfluidic device, 0) imaging the merged droplets, at least at the end of incubation; i) determining from the captured images for each merged droplet the number of at least viable target cells at least at the end of incubation.

In one embodiment, and highly preferably, in the imaging step (e.g. step i) or j) above) multiple images are captured over time, such as about every 30 seconds or about every minute. As said, in the methods in accordance with the invention, the analysis of the images captured may be performed remotely from the site where the EWOD microfluidic device is located. It is understood that as the EWOD microfluidic device is to allow imaging, the material from which the device is made is to allow for microscopy. Such materials are well known in the art and the skilled person knows how to select appropriate materials to allow for microscopy (as described in the EWOD microfluidics device section herein), in particular darkfield reflective optical and fluorescence microscopy. In addition, the present invention preferably provides for means that allows to reduce optical and fluorescent background.

Such a means may comprise an optical black background. Such backgrounds are highly advantageous in the means and methods of the invention, when utilizing e.g. optical and fluorescence microscopy. In one embodiment, imaging involves microscopy z-stacking. In another embodiment, imaging is in real-time. Hence, in any case, imaging preferably involves collecting images of the droplet to allow for detailed assessment of effector cells and target cells in the droplets.

In yet a further embodiment, preferably location and/or morphology of individual cells is determined in the last determining step (e.g. step j) or k above) to allow tracking of individual cells. In another embodiment, a method in accordance with the invention is provided, wherein individual cells are tracked, and wherein in the last determining step (e.g. step j) or k above)

- target cell killing is plotted against time; - of individual effector cells contact time with target cells and is determined and/or frequency of target cells contacted; - motility of individual effector cells and/or - individual cells are classified as serial killer, mono-killer or non-killer. As shown in the example section, imaging the merged droplets allows to collect for further data in addition to target cell killing. This can be performed by tracking target cells. Cells can be well tracked in accordance with the invention because imaging can be performed continuously, i.e. images are taken at time intervals that allows for cells to be identified because of cell morphology and position. For example, as shown in the example section herein, imaging was performed for merged droplets about every minute. Preferably, imaging is performed every minute, every 50 seconds, every 50 seconds, every 40 seconds, every 30 seconds, every 20 seconds or every 10 seconds. It may also be contemplated to perform imaging in real-time. This way, effector cell motility of individual effector cells can be determined with precision. The more frequent imaging is performed, the more precise effector cell motility and interactions can be monitored. Contact time of individual effector cells with target cells can be determined, and/or frequency of target cells contacted by effector cells. Moreover, killing of target cells can be determined, e.g. based on morphological changes, and effector cells can be classified as non-killer, mono-killer and serial killer. This way, a variety of features can be determined for each individual effector cell, and, the combined information of all individual effector cells provides for quality information representative for the effector cells. The combined information provides phenotypic information on each individual effector cell as provided in the prepared droplets of effector cells, which can furthermore be integrated into a score representing e.g. the quality of the population of effector cells prepared droplets. An analysis framework can further be applied to evaluate the weight of all possible variables measured, e.g. when comparing different merged droplets each subjected to different conditions, in assessing the effect on the effector cells. Such a mathematical framework allows to evaluate if a variable impacts effector cell function and how much it matters in the model. Such a model can then be used to predict how to improve effector cells function through the variables identified.

It is understood that of course, while the advantage of the invention is that merged droplets can be imaged in its entirety, it may also be contemplated to image a defined part of merged droplets, which is representative of the entire merged droplets. As long as a representative dataset can be collected, this can be contemplated. Of course, it is preferred to image the entire merged droplet, as this provides a more comprehensive dataset.

It is understood that as an EWOD microfluidic device can be designed to carry out a large number of different effector cell and target cell incubations of merged droplets, an

EWOD microfluidic device may be provided with different effector cell types, or different effector cell clones. For example, with regard to different effector cell types, these may be effector cells obtained from different donors, e.g. when a defined CAR is provided to effector cells isolated from the PBMCs obtained from different donors. Furthermore, from the same donor, different effector cell types, or cell populations, may be provided, e.g. by providing

T-cells, and NK cells from the same donor, wherein T-cells of said donor may be further differentiated as T-helper cells (CD4+/CD3+) and cytotoxic T cells (CD8+/CD3+).

Furthermore, effector cells may also be provided with different receptors, such as different

CAR receptors. Effector cells carrying the same CAR receptor may be defined as representing or being representative of an effector cell clone, although it is understood and accepted that the effector cells in which the same CAR receptor has been introduced may not necessarily be identical with regard to phenotype or even genotype. Accordingly, in another embodiment, multiple effector cell types or multiple effector cell clones are provided, wherein each of the effector cell types or clones can be subjected to the steps of the methods in accordance with the invention.

However, as the current invention allows for fluorescence multiplexing, it may not be required to provide each effector cell clone or each effector cell type a separate and individual merging step and subsequent incubation and imaging. Highly advantageously, in a further embodiment, wherein multiple effector cell types and/or multiple effector cell clones are provided, each of the effector cell types or each of the effector cell clones is provided with a different staining, and wherein multiple effector cell types or clones are combined and provided to individual areas, i.e. in one merged prepared droplet. For example, in case one wishes to perform experiments in triplicate, and using 4 different fluorescent stainings, one can combine 4 different effector cell clones and/or types per merged droplet. In a EWOD microfluidic device, with 10 incubation areas for merged droplets, this would mean that 40 different effector cell clones and/or types can be analyzed with one device. Because multiple different cell clones and/or types are used in a single area and across an EWOD microfluidic device, cytotoxicity/cell killing assessment can also be better validated across the device because each area is controlled by having multiple samples, and because each unique sample can be provided across the device.

Also, in case images are taken at different time points, having cells with different labels also allows for improved tracking of individual cells in an image. It is understood that in case tracking of cells is of importance, this is likewise advantageous for effector cells that are identical or highly similar, e.g. as having the same phenotype and carrying the same receptor.

Further complexity may be added to the analysis allowing even more different effector cells per cell when cells are allowed to have for example double staining. With double staining with red, blue, and green (r,b,g) 7 unique combinations can be made (no staining, g, b, r, g/b, g/r, b/r). Hence, already with three different fluorescent channels in microscopy, a large variety of experimental conditions involving i.a. different effector genotype and phenotypes, donor types, and/or different effector cell clones may be highly efficiently analysed.

Furthermore, control effector cell clones may be included in each generated droplet with effector cells, or a selection of generated droplets with effector cells, to provide for control across the entire device. Control effector cell clones may include positive effector cell controls, i.e. having a high cellular avidity with the target cells, or negative effector cell controls, i.e. having a low or no detectable cellular avidity with the target cells. One may even contemplate combining both positive and negative effector cell controls. An EWOD microfluidic device may include such controls and when the information obtained complies with a pre-defined reference range, such an EWOD microfluidic device run and analysis thereof may be validated.

Likewise, as it is understood that different target cells may be provided that are differentially stained as well, e.g. with fluorescent markers or the like, e.g. providing target cells originating from different donors, with each donor presenting the same antigens. Such different target cells may include control target cells, for example target cells that do not express a selected antigen and/or do highly efficiently express/present a selected antigen.

Furthermore, with regard to the image analysis highly advantageously, in a further embodiment, convolutional neural networks (CNNs) based cell analysis, as used in cell biology, can be applied for cell type classification and cell death detection based on images collected. CNNs can be trained to classify distinct cellular morphologies and patterns associated with different cell types, allowing for automated categorization of cells based on their structural features. Moreover, CNNs can also be employed to classify cells undergoing apoptosis or necrosis by learning characteristic changes in cell morphology, such as membrane blebbing, nuclear condensation, and cytoplasmic fragmentation. This way, the images collected can be highly advantageously more efficiently analysed and further parameters of interest for effector cells and/or target cells classified. Hence, it is understood that image analysis and tracking and classification of cells, including assessment of contact time, motility, and target cell killing, involves data processing, which data processing may include CNN based cell analysis. It is understood that machine learning techniques or dimensionality reduction based techniques and analysis can be advantageously used to classify cells for viability, and cell type identity, and the like, based on cell morphology collected from the images. It is understood that it may not be necessary to have cells stained, such as described herein, but it maybe advantageous to do so, as it may aid classification of cells.

In a further embodiment, a method is provided as describe above and herein, wherein after incubation and imaging, an assay, preferably an immunoassay is performed with the merged and incubated droplets on the EWOD microfluidic device, wherein the EWOD microfluidic device (for example as described in the EWOD microfluidics device section herein) further comprises an integrated magnet for magnetophoretic elution of magnetic beads, wherein magnetic beads with a capture agent in buffer medium are provided, said capture agent preferably being an antibody, said capture agent capable of binding a target of interest, wherein the magnetic bead is paramagnetic or superparamagnetic, and wherein a detection agent is provided, preferably an antibody with a detectable label, preferably with a fluorescent label, and, comprising means for fluorescence imaging in an analytics area, preferably comprising an optical black background, wherein the merged and incubated droplets are allowed to interact with droplets of the magnetic beads with the capture agent and the detection agent, and subsequently, magnetic beads with capture agent comprising target bound thereto and detection agent, are magnetophoretically eluted and imaged, and quantifying the target.

Following the positioning of the magnetic beads droplet, electric potentials are applied to meticulously control droplet movement, thereby facilitating contact between the magnetic bead droplet and a droplet containing the target analyte, e.g. a liguid droplet containing effector cells and target cells that were incubated. Adequate time is allotted for the binding process to occur, thereby facilitating the desired analyte-magnetic bead interaction.

It is understood that such an assay utilizing magnetic beads can be highly advantageously performed utilizing an EWOD microfluidic device such as described herein, and may not necessarily require a cell killing workflow to be performed prior to such an analysis, though it is highly advantageous therefore. Hence, it is understood that assays utilizing magnetic beads may be advantageously be performed with any type of cell that is to secrete a protein of interest, e.g. when subjected to a defined stimulus, or in screening assays aimed to identify suitable stimuli (e.g. candidate compounds), of which it is of interest to quantify. Accordingly, in an alternative embodiment, a method is provided as describe above and herein, wherein an immunoassay is performed with incubated droplets comprising cells capable of secreting a target of interest, on an EWOD microfluidic device, wherein the EWOD microfluidic device (for example as described in the EWOD microfluidics device section herein) further comprises an integrated magnet for magnetophoretic elution of magnetic beads, wherein magnetic beads with a capture agent in buffer medium are provided, said capture agent preferably being an antibody, said capture agent capable of binding a target of interest, wherein the magnetic bead is paramagnetic or superparamagnetic, and wherein a detection agent is provided, preferably an antibody with a detectable label, preferably with a fluorescent label, and, comprising means for fluorescence imaging in an analytics area, preferably comprising an optical black background, wherein the merged and incubated droplets are allowed to interact with droplets of the magnetic beads with the capture agent and the detection agent, and subsequently, magnetic beads with capture agent comprising target bound thereto and detection agent, are magnetophoretically eluted and imaged, and quantifying the target. It is understood that embodiments herein that apply such magnetophoretical elution in the context of a cell killing assay, do not necessarily require such a cell killing assay, and these embodiments can also be simply be applied in the context of only performing an immunoassay without a cell killing assay.

Though, as said, such an assay that utilizes magnetic beads can be advantageously performed after the cell killing workflow, it is understood that this can also be optionally performed during an incubation step (e.g. of a cell kiling assay), and is not necessarily limited to being performed only thereafter. Of course, performing such a step during incubation means that the incubated effector cells and target cells will have to be mixed, which may be less desirable. Nevertheless, it can be contemplated to perform such an assay, as measuring target molecules of interest during incubation can be of interest.

Hence, in yet another embodiment, an assay is performed with droplets of interest, said droplets comprising, or suspected to comprise, a target of interest, on the EWOD microfluidic device, wherein the EWOD microfluidic device further comprises an integrated magnet for magnetophoretic elution of magnetic beads, wherein magnetic beads with a capture agent in buffer medium are provided, said capture agent capable of binding a target of interest, wherein the magnetic bead is paramagnetic or superparamagnetic, and wherein a detection agent is provided, preferably with a fluorescent label, and, comprising means for (fluorescence) imaging in an analytics area, wherein the droplets of interest are allowed to interact with droplets comprising the magnetic beads with the capture agent and the detection agent, and subsequently, magnetic beads with capture agent comprising target bound thereto and detection agent, are magnetophoretically eluted and imaged, and, quantifying the target of interest.

Hence, utilizing an EWOD microfluidic device, provided with a magnet, allows for the isolation and quantification of targets of interests, utilizing magnetic beads, which is in particular useful when combined with cell killing assays as described herein, but not necessarily limited thereto.

It is understood that with regard to the magnet used, any type of magnet may be used. The EWOD microfluidic device may have an integrated electromagnet, which can be well controlled like the electrodes of the EWOD, such that the magnet can be turned on to capture beads, and turned off when to release beads. A permanent magnet may also be used, which can be brought into the vicinity of a the EWOD microfluidic device by an actuator, to capture beads, and moved away for releasing of beads. Such a magnet may be referred to as a togglable magnet. It is understood that the magnet may be a combination of magnets, i.e. one or more magnets. As long as the magnetic field can be controlled, with regard to location on the EWOD device and allows for capture and release of magnetic beads, such a magnet configuration may be contemplated in accordance with the invention.

As shown in the examples herein (See e.g. Fig. 3 and 17), the EWOD microfluidic device in accordance with the invention well accommodates for isolation of biomolecules, utilizing magnetic beads, from droplets in the EWOD microfluidic device, such as merged and incubated droplets such as generated in a method for determining cell Killing in accordance with the invention. The EWOD microfluidic device allows for unit operations, such as washing of magnetic beads, and mixing of magnetic beads with a droplet. Upon completion of the binding reaction, washing buffer droplets are maneuvered using electric fields to the location of the magnetic bead-bound analyte droplet. This step can be repeated if necessary to eliminate further unbound materials and mitigate nonspecific binding, ensuring the specificity of the assay.

To enable detection, detection reagents such as fluorescently labeled secondary antibodies can be introduced onto the EWOD microfluidics device. Droplets are mixed to promote the reaction between detection reagents and the target of interest bound with the magnetic bead, facilitating the generation of detectable signals. Magnetic separation is effectuated by positioning an external small magnet in proximity to the device to induce a magnetic field. This field attracts the magnetic beads bound with the target analyte, facilitating their separation from the remaining solution. Subsequently, droplets containing the magnetic beads are manipulated to move them away from the bulk of the solution for downstream analysis or disposal.

Any type of biomolecule may be contemplated to be isolated and analysed with the magnetic beads. Preferably, magnetic beads are provided with a capture antibody capable of binding a target, i.e. biomolecule, of interest, and subsequently, allowed to interact with another antibody capable of binding the target of interest, to which may be referred to as detection antibody, which is provided with a detectable label, preferably a fluorescent label.

By washing away unbound detection antibody, quantifying the bound antibody provides for quantification of the biomolecule. It is understood that many iterations of magnetic bead separations utilizing a capture agent that is bound with the magnetic beads may be contemplated in accordance with the invention. For example, the magnetic beads and the detection antibody may be provided in a droplet and mixed with a droplet comprising a biomolecule of interest, such as merged droplet that has been incubated. Following magnetic separation, the EWOD microfluidic device can be imaged to capture e.g. fluorescent signals indicative of analyte binding. The acquired data can then be subjected to analysis to quantitatively determine the concentration or presence of the target analyte based on the generated signal.

In one embodiment, a Fluorescent Immuno Assay is applied, which is useful because of its capacity of multiplexing. Fluorescently tagged beads of varied sizes can be employed, each bound to distinct (capture) antibodies. This enables the identification of individual beads and their specific activity towards targets of interest. Utilizing matching, or mismatching, fluorophores for detection antibodies may facilitate bead recognition. The beads with capture antibodies and detection antibodies can be mixed, which allows for simplifying the process to manage a single bead suspension droplet per analysis, encompassing any necessary controls. In any case, any type of antibody based detection may be contemplated, which utilizes antibody based detection of targets of interests and quantification, such as ELISA, FIA, or the like.

One may of course also contemplate providing magnetic beads separately (e.g. not in a droplet), and subsequently mix the magnetic beads with the droplet comprising a biomolecule of interest first, and subsequently isolate the magnetic beads with biomolecule bound therefrom. This way, the droplet may not be substantially diluted and the composition of the droplet may not be substantially different (with of course bound biomolecules removed therefrom). This may be advantageous in case one wishes to perform further methods on the droplet that comprises the biomolecule of interest, or in case one wishes to perform further incubation. Next, the isolated magnetic beads with the biomolecules bound thereto may be mixed with a droplet comprising detection antibody, and subsequently detection antibody that binds with the captured biomolecules can be quantified. Of course, one may also prepare a droplet with magnetic beads with capture agent and detection agent, and mix that directly with a droplet comprising the biomolecule, i.e. target of interest for the capture agent and detection agent. The end result is always the same, i.e. magnetic beads are obtained which have captured a target of interest, with also detection agent bound thereto. The EWOD device with the magnet thus highly advantageously allows for magnetophoretically elution of the magnetic beads to remove any unbound detection agent, and allow for quantification of bound detection agent. In one embodiment, magnetophoretic elution comprises: - capturing magnetic beads with capture agent with target bound therewith and detection agent, - washing away unbound detection agent, and, optionally target cells and effector cells, if present, from the captured magnetic beads with capture agent with target bound therewith and detection agent, washing away comprising the steps of: 0 moving away from the purification area the droplet with unbound detection agent, and, if present, target cells and effector cells; 0 moving into the purification area a washing droplet, wherein the steps of moving away and moving into the purification area are performed consecutively or simultaneously, and optionally, repeated one or more times; to thereby obtain magnetophoretically eluted magnetic beads with capture agent comprising target bound therewith and detection agent.

In the first scenario, i.e. the steps of moving away and moving into the purification area washing droplets, are performed consecutively, magnet separation of a liquid from a solid phase by coalescence of beads occurs, where the magnetic beads leave the droplet when coalesced, the movement of the droplet away from the magnetic field generated by the external magnet can cause the magnetic beads to separate from the droplet and remain in the surrounding oil or air (Figure 3). This occurs because the magnetic force holding the beads in place within the droplet is not strong enough to overcome the force exerted by the movement of the droplet. Consequently, as the droplet containing the magnetic beads is manipulated away from the magnetic field, the beads are no longer constrained and can remain in the surrounding environment. This scenario poses a risk of losing the magnetic beads and compromising the effectiveness of the assay.

In the second scenario, i.e. the steps of moving away and moving into the purification area washing droplets, are performed simultaneously, a train of droplets is used to remove non-bound agents without exerting force on the magnetic beads themselves. In this approach, the sequential movement of washing buffer droplets forms a train that sweeps across the surface of the EWOD microfluidic device. As the train of droplets advances, it generates a fluid flow that carries away non-bound agents while leaving the magnetic bead- bound analytes intact by magnetophoresis. The washing buffer droplets effectively push non-bound agents towards the edge of the droplet train, clearing them from the vicinity of the magnetic bead-bound analytes (Figure 3). This process ensures that the magnetic beads remain within the fluid and are not forced out into the surrounding oil or air. By employing a train of droplets for sample manipulation, this scenario has a reduced the risk of displacing the magnetic beads and ensures the integrity of the assay.

By subsequently releasing the magnetic beads in both scenarios, subsequently, the droplet comprising the magnetic beads with capture agent comprising target bound therewith and detection agent, can be imaged to quantify the detection agent, thereby quantifying the target.

As imaging is preferably used to quantify the detection agent, the EWOD microfluidic device, like above, for imaging of merged droplets, is to allow for imaging of the magnetic beads comprising target bound therewith and detection agent. Hence, likewise, the material from which the device is made is to allow for microscopy. As said, such materials are well known in the art and the skilled person knows how to select appropriate materials to allow for microscopy, in particular for darkfield microscopy and/or fluorescent microscopy. In addition, in the present invention preferably provides for means that allows to reduce fluorescent background when imaging magnetic beads comprising target bound therewith and detection agent. Such a means may comprise a light absorbing layer, such as an optical black background. Such backgrounds are highly advantageous in the means and methods of the invention, when utilizing e.g. fluorescent microscopy.

As said, the capture agent and detection agent, may be highly preferably antibodies, such as used e.g. in a typical FIA, ELISA or the like. Hence, in one embodiment, a method is provided in accordance with the invention, for determining target cell killing by effector cells, wherein the method after the incubation and imaging step, further comprises

D providing magnetic beads with a capture antibody in buffer medium ; wherein the capture antibody is capable of binding a target of interest; wherein the magnetic bead is paramagnetic or superparamagnetic; m) providing a detection antibody in buffer medium with a fluorescent label; wherein the detection antibody has the same target as the capture antibody, preferably wherein the detection antibody and capture antibody target different epitopes of the same target; 0 optionally wherein the magnetic bead with capture antibody and the detection antibody are provided combined in a single suspension; n) providing buffer media; 0) transferring the magnetic beads with the capture antibody and the detection antibody, suspended in buffer medium, each to a separate reservoir, or combined in a reservoir, on the EWOD microfluidic device; p) resuspending the incubated target and effector cells of step j) in the droplet and merging and mixing the droplet(s) comprising the magnetic beads with capture antibody and the detection antibody therewith thereby allowing the capture and detection antibodies to interact with their respective targets; 9) moving the resuspended droplet to a purification area; wherein the purification area comprises an integrated magnet for magnetophoretic elution of magnetic beads, and, capturing the magnetic beads; - eluting the magnetic beads with capture antibody comprising target bound therewith and detection antibody with a washing droplet, - releasing and resuspending the magnetophoretically eluted magnetic beads with capture antibody comprising target bound thereto and detection antibody; and moving the droplet with magnetophoretically eluted magnetic beads to an analytic area, |) wherein the analytics area comprises an optical black background;

Ih wherein the analytics area comprises means for a reduced fluorescent background; r) quantifying the amount of detection antibody using fluorescent imaging, therewith quantifying the amount of target in the droplet.

Of this embodiment, in a further embodiment, the step of eluting comprises: - washing away unbound detection antibody, and target cells and effector cells from the captured magnetic beads with capture antibody with target bound therewith and detection antibody, washing away comprising the steps of: 0 moving away from the purification area the droplet with unbound detection antibody, and target cells and effector cells; 0 moving into the purification area a washing droplet, wherein the steps of moving away and moving into the purification area performed consecutively or simultaneously, and optionally, repeated one or more times; to thereby obtain magnetophoretically eluted magnetic beads with capture antibody comprising target bound therewith and detection antibody.

In the methods as described above utilizing magnetic beads with capture agents, such as capture antibodies, it is understood that it can be contemplated to perform multiple (immune)assays simultaneously or consecutively. One can perform simultaneous assays by combining e.g. a mixture of beads for a variety of biomolecules of interest. Biomolecules of interest may be cytokines, i.e. interferons, interleukins, chemokines, tumor necrosis factors and adipokines. Such biomolecules can be used to evaluate the safety of effector cells or agents tested. Such biomolecules can be used to evaluate efficacy of effector cells or agents tested. Any biomolecule of interest may be contemplated, and for the means and methods in accordance with the invention there are no restrictions on what type of biomolecule one is interested in. It may even be contemplated to utilize magnetic beads to isolate cells of interest from a droplet. Hence, it is understood that the application of magnetic beads with a capture agent is highly compatible with the EWOD microfluidic device, and any useful application therewith may be contemplated.

With regard to the above embodiments comprising the use of magnetic beads, in one embodiment, the magnetic force for capturing the magnetic beads with capture antibody with target bound therewith and detection antibody, is orthogonal to the moving forces exerted by EWOD on the droplet with unbound detection agent/antibody and washing droplet(s). Having the force orthogonal may be preferred considering that one may wish to move droplets in all directions over captured beads, and having the magnetic force orthogonal allows for conditions to be the same for all moving directions of the droplets. The magnetic purification module can be activated via a linear actuator, displacing the magnet or magnets towards or away from the first component substrate of the EWOD microfluidic device to induce the magnetic force in the droplet, as indicated in Figure 3. It may also be contemplated to use one or more magnets in parallel to perform purification in parallel. It is understood that the magnetic force at droplet locations can be turned on and off.

As said, the means and methods in accordance with the invention are highly useful for assessing cell killing by effector cells. Suitable effectors that may be contemplated in one embodiment, may be selected from (engineered) immune effector cells, such as CAR-

T cells, CAR-NK cells, CAR-macrophages, or non-cytotoxic immune cells (including regulatory T-cells or T-helper cells) cytotoxic T cells, and tumor infiltrating lymphocytes.

The means and methods in accordance with the invention can be carried with any suitable target cells. Target cells that can be contemplated may include, optionally, adherent cells. Organoids and, as said, cancer cells may be contemplated as well.

It may be contemplated in accordance with the invention to include in addition to effector cells and target cells, further cells. Such further cells may e.g. mimic the natural environment that may be encountered in an animal body or human body. For example, effector cells may encounter both target cells and healthy cells, and specificity of the effector cell may be well studied in such a (cell) cytotoxicity assay. Furthermore, in the natural environment, in addition to e.g. cytotoxic T-cells, further immune cells may be present, e.g. helper T cells, macrophages, fibroblasts or regulatory T cells. This way, the interaction of immune effector cells with target cells can be well studied. Hence, it is understood that although the focus is on interaction between effector cells and target cells, further cells may be included in the means and methods in accordance with the invention.

As said, in the means and methods in accordance with the invention for assessing cell killing, in addition to effector cells and target cells, further agents may be included in the methods of the invention. This way, the effects of agents on effector cells and target cells interaction may be well studied. Hence, in another embodiment, the EWOD microfluidic device comprises at least one further reservoir comprising an agent in a medium, wherein droplets of the agent are generated and a defined amount of agent is further merged with a droplet of the effector cells and/or of the target cells and mixed, ) wherein the agent is mixed with the droplet of effector or target cells prior to the merging step of the droplets of effector cells with the target cells in step h),

II) wherein the agent is mixed with the merged droplet of target cells and effector cells in step h).

As described above, the methods of the invention are highly useful to determine the cytotoxicity, i.e. cell killing activity of an effector cell towards a target cell. As it is understood, agents may also be included in the methods of the invention, as these may also affect the cell killing of an effector cell towards a target cell. In particular, cell engagers may be of interest. Cell engagers include antibodies, or the like, which are capable of binding to a target cell and an effector cell. Such antibodies may include single chain antibodies comprising two binding domains such as scFv domain, and include BIiTEs (i.e. bispecific T cell engagers) or the like. A conventional antibody design may includes heavy and light chains, with one half of the antibody (one heavy chain and one light chain) engaging with a target cell, and the other half of the antibody (another heavy chain and another light chain) engaging with an effector cell, wherein preferably, the Fc domain is made inert. In any case, suitable cell engager formats are widely known in the art and the current invention allows to study and/or determine cell killing activity and further features, as induced by a cell engager between a target cell and an effector cell. Hence, accordingly, in the means and methods in accordance with the invention, a cell engager may be provided in addition, and e.g. one or more cell killing parameters, or parameters related thereto, are determined, induced by said cell engager between a cell of interest (e.g. an effector cell) and a target cell. It is thus understood that the incubation step in the methods of the invention can thus be performed in the presence of the cell engager to allow the cells to interact, i.e. effector cells and target cells, and form a bond, via the cell engager, inducing subsequently target cell killing.

Accordingly, in one embodiment, in methods in accordance with the invention, a cell engager is provided capable of binding an effector cell and the target cell and inducing target cell killing by the effector cell, and the cell engager is included in the incubation step.

Furthermore, such methods may be highly useful for screening cell engagers, e.g. by identifying cell engagers that are particularly capable of inducing e.g. target cell killing.

Of course, the methods of the invention are not limited to the use of cell engagers, and other molecules, or combinations thereof, that may affect the interaction between effector cell and target cell may be used therein. Hence, drugs, such as candidate drugs may also be agents that can be selected in accordance with the invention.

Hence, in a further embodiment, the agent is a cell engager, such as a bispecific antibody or a drug, such as a candidate drug.

In yet another embodiment, in the methods in accordance with the invention, the droplets in the EWOD microfluidic chip are surrounded by an oil, preferably a polysiloxane oil, perfluoroalkyl oil or a liquid hydrocarbon. Suitable oils are known in the art, and as long as the selected oil allows for gas exchange and allows for manipulation of droplets as envisioned herein, such oil may be regarded as suitable in accordance with the invention (see Li et al., Advanced Science; Volume 9, Issue 10, 2104510). A preferred oil can be

Silicone Oils 5 csT or Flurinert FC-40. Mineral oil or silicone oil with lower viscosity (between 1 and 5 cst) may be used as well.

As said, defined areas of the EWOD microfluidic device are preferably defined by the electrode configuration and or operations (e.g. mixing, merging, splitting). Hence, in one embodiment, the culturing area comprises at least two further electrodes at different sides of the electrode for hosting the required volume for merging and subsequent mixing the droplets or merging without mixing. In another embodiment, the reservoir area, sample prep area, culturing area, purification area and/or waste area comprise one or more electrodes or more for holding a defined volume, i.e. fluids are advantageously contained by electrostatic forces and not a physical shape like a well or container.

EWOD Microfluidic Device

According to a first example of the disclosure an electrowetting on dielectric, EWOD, microfluidic device structured to subject at least one analyte contained in a sample droplet of fluid to a sequence of process steps defining a workflow assay procedure is proposed.

The EWOD microfluidic device at least comprises a first component substrate and a second component substrate facing and extending next to the first component substrate at a mutual distance from each other, thereby forming a measurement chamber in between, wherein a first surface layer of the first component substrate facing the second component substrate is provided with a plurality of array elements arranged in a conductive electrode matrix array, the array elements being configured to manipulate one or more sample droplets of fluid within the measurement chamber along the electrode matrix array, wherein the plurality of array elements are coated by a visible light absorbing layer, and wherein the second component layer is manufactured from a material at least partly transparent to electromagnetic radiation in the visible region of the electromagnetic spectrum.

By implementing a light absorbing layer, e.g. a black coating to provide for a black background, a substantial portion of incident light for image measurements can be advantageously efficiently absorbed, thus mitigating reflection and scattering. As such a light absorbing layer has a low reflectance, by eliminating reflections, the light absorbing layer minimizes disruptive glare and background noise during the darkfield imaging measurements, enhancing the visibility of scattered light from cells.

Additionally, such a light absorbing layer suppresses light scattering from the first component substrate, curbing artifacts and preserving the quality of (darkfield) images which will otherwise be present in traditional opaque metal electrodes in EWOD. By preventing any light interaction with the material of the first component substrate material, the light absorbing layer redirects scattered light away from the imaging device, thus ensuring high-quality cellular imaging.

In a further advantageous example, the light absorbing layer also exhibits electric properties. Alternatively, the light absorbing layer is coated by a dielectric layer, which also may exhibit hydrophobic properties. In yet another example, the first component substrate is stack layered as on top of the dielectric layer a hydrophobic layer is applied.

In this configuration, the dielectric, hydrophobic layer comprises, but is not limited to, fluorinated ethylene propylene as a dielectric and hydrophobic layer. In other configurations, any form of fluoropolymer can also be used.

Implementing a dielectric coating layer and a hydrophobic coating layer, either in a stacked configuration or in a combined layer, effectively insulates the electrode matrix array against chemical reactions and unwanted electric field interference. Dielectric and hydrophobic functionality also ensures a controlled droplet manipulation, preserving the precision of their movements across the electrode matrix array and within the measurement chamber.

In an embodiment, the first component substrate is made from a non-conductive, opaque material and can be made from a material used for forming a printed circuit board (PCB).

In a further advantageous example, a first surface layer of the second component substrate facing the first component substrate is coated by an optoelectronic layer. The optoelectronic layer serves as the opposing electrode to which the electric field travels to form the PCB's electrode. This layer also exhibits a high light transmittance as well as electric conductivity, ensuring the generation of precise electric fields within the measurement chamber for effective droplet manipulation. Preferably the optoelectronic layer is made from Indium tin oxide, ITO. For example, various FTO/AZO/CNT/pyrroles and in general a variety of organic and inorganic optically conductive inks and coatings can be used as well.

In addition, the optoelectronic layer may be coated by a hydrophobic layer, wherein the insulation properties are as minimal as possible ensuring a controlled droplet manipulation, preserving the precision of their movements across the electrode matrix array and within the measurement chamber.

Preferably, the second component substrate is made from glass thereby ensuring high transmittance into the measurement chamber for performing imaging measurements onthe sample droplets within the measurement chamber. Materials such as quartz may be used for special applications requiring high transmittance of UV light.

In order to manipulate the sample droplet of liquid in a most optimal manner, the

EWOD microfluidic device may further comprise one or multiple inlet and outlet openings being in fluid communication with the measurement chamber. In addition, the measurement chamber comprises a plurality of chamber compartments, and the chamber is delimited by a droplet enclosed by the transparent substrate, the PCB substrate and the perimeter of one electrode, for accommodating different types of reagents or calibrators for performing the workflow assay procedure. Herewith it is possible to implement the microfluidic component for a variety of workflow assay procedures.

The droplets may be used in a droplet-in-oil microfluidic approach, which means that the droplets may be used in an oil environment. Herewith, evaporation of liquid is reduced to a non-observable rate. This also provides an additional benefit: oils reduce the friction experienced by a droplet moving on a surface, and the hydrophobic oil phase will allow for improved droplet splitting behavior.

Accordingly, the EWOD microfluidic device can be constructed in a compact manner and allows a more precise control of the movement and mixing of tiny droplets of liquids in a microfluidic system. Thus a compact design is achieved with limited dimensions, further improving its handling as well as the workflow assay procedures to be performed in a repetitive manner under standardized conditions.

In a further example, a second surface layer of the first component substrate opposite to the first surface layer is provided with an array element circuitry interface electrically connecting the electrode matrix array. Accordingly, the EWOD microfluidic device can be operated (accessed, energized and controlled through suitable control means) from one side of the measurement chamber, which is the side at which the electrode matrix array is positioned, whereas the other side of the measurement chamber, which is the side of the transparent second component layer is not obscured but freely accessible for image measurements.

The present disclosure also pertains to a microfluidic apparatus structured to subject at least one analyte, such as effector cells, target cells, and/or magnetic beads as described herein, contained in a sample droplet of fluid to a sequence of process steps defining a workflow assay procedure such as described herein using an EWOD microfluidic device.

The microfluidic apparatus comprises a housing having a top support face structured to accommodate the second component substrate of the EWOD microfluidic device in a replaceable manner, as well as an imaging device accommodated within the housing and optically facing the top support face of the housing. In particular, the EWOD microfluidic device, when accommodated in the housing, can be positioned above the imaging device.

Herewith an unobscured configuration can be achieved, wherein an imaging device has a free view into the measurement chamber whose view is not obscured by e.g. the electrode matrix array, as the latter is positioned on the opposite side of the whole construction.

In particular, as the EWOD microfluidic device may be positioned upside down with the second component layer being manufactured from a material at least partly transparent to electromagnetic radiation in the visible region of the electromagnetic spectrum facing the imaging device an optimal visibility of the measurement chamber can be guaranteed. This imaging effect is improved with the first surface layer of the second component substrate facing the first component substrate being coated by an optoelectronic layer.

Additionally, as the first surface layer of the second component substrate facing the first component substrate may also be coated by a hydrophobic layer, the droplets present in the measurement chamber and being manipulated by the EWOD conductive electrode matrix array experience less friction. Additionally, the second component layer preferably has a flat surface which allows for easy focus by the imaging device.

Preferably, the imaging device is structured as a darkfield reflective inverted microscopy device.

In particular the microfluidic apparatus according to the disclosure may further comprise an apparatus data interface module structured for mounting on the top face of the housing, the apparatus data interface module comprising array element control circuitry for electrically connecting and controlling the electrode matrix array of the EWOD microfluidic device. Herewith a standardized mounting of the EWOD microfluidic device in the microfluidic apparatus is obtained, allowing for the control of the electrode matrix array, and thus fast and accurate manipulation of the sample droplets of fluid through the measurement chamber for effecting the workflow assay procedures in a repetitive manner and under standardized conditions.

Moreover the apparatus data interface module may further comprise a magnetic module structured to exert a magnetic field on the sample droplet of fluid within the reactor chamber. Herewith, additional process steps can be effected on the sample droplets, such as magnetic purification for solid-state magnetic particle purification or magneto- electrowetting affinity purification.

Examples

Example 1 — Generating droplets from reservoirs, merging and mixing of droplets.

As the electric field reduces the contact angle between the liquid and the substrate surface, decreasing the droplet's surface tension. Dispensing single droplets from a reservoir is done by precisely controlling the voltage, the droplet can be pinched off from the bulk liquid, forming a discrete entity through a voltage applied in adjacent electrodes and a volume of liquid being displaced away from the bulk until separating entirely. Subsequently, by adjusting the electric field, the detached droplet can be moved away from the reservoir, allowing it to separate completely from the bulk liquid. This process facilitates the precise segmentation of the liquid into droplets, which can then be manipulated for various microfluidic applications with high efficiency and control.

Initially, the system begins with the generation of droplets by splitting (shown from

A-D in Figure 1) from a reservoir within the microfluidic chip. In the example, two microlitre droplets are advanced from a reservoir to the adjacent three electrodes in series. Upon deactivating the electrode in the middle, the droplet experiences splitting (further described in Example 2} into two single one microliter droplets. When the voltage is activated in the adjacent droplet, and turned off in the present one, a force propels the droplet across the surface, guided by the gradient of the electric field. Thus, the controlled modulation of electric potentials enables precise movement of the droplet from one electrode to another within the example of the EWOD device in Figure 1. Such reproducible droplet generation is important for the reliability and scalability of the microfluidic system.

Subsequently, the generated droplets are transported (Figure 1 D-E) through predefined electrodes tracks within the chip, facilitated by the manipulation of electric potentials to induce directional movement. This transport mechanism exploits the phenomenon of electrowetting, where the applied electric field alters the wettability of the liquid on the dielectric surface, thereby guiding the droplet along the desired path. For transporting droplets, electric fields are strategically applied to guide droplets along predefined paths on the substrate surface. By modulating the voltage across the different electrodes through the droplet and to the optoelectric material, the direction and speed of droplet movement can be finely controlled, allowing for efficient transport between different regions of the microfluidic device. The automation and reproducibility of this transport process ensures consistent and predictable movement of droplets within the microfluidic system.

Upon reaching a designated location, the droplets are brought into close proximity for subsequent manipulation (Figure 1F). Here, the controlled activation of electric potentials in proximal electrodes facilitates the merging of the droplets without mixing (illustrated by the distinct grayscale difference between the two droplets shown between Figure 1 G and H).

When it comes to merging droplets, electric fields are utilized to bring two or more droplets into proximity, facilitating their coalescence into a single larger droplet. By adjusting the applied voltage and the positioning of the droplets, merging can be achieved with high precision, enabling the mixing of different reagents or samples within the microfluidic system. This precise merging process, reproducible across multiple experiments, is achieved by fine-tuning the contact angles between the droplets and the dielectric surface, thereby preventing unintended coalescence or diffusion at the interface.

Following the initial merging stage, the merged droplets proceed to undergo homogeneous mixing using a mixing procedure like in Figure 20 and resulting in an homogeneous sample shown in Figure 11, driven by controlled electric fields that induce convective and diffusive motion within the droplet volume when activating them in a sequence similar to that of Figure 20. This reproducible mixing process ensures thorough blending of the droplet contents, promoting uniform distribution of reagents, nanoparticles, microparticles, or biomolecules contained within the droplets. The ability to consistently achieve homogeneous mixing can be highly important for the reproducibility and accuracy of biochemical assays, drug screening assays, and other microfluidic applications.

In conclusion, EWOD microfluidics offers a robust and reproducible platform for precise droplet manipulation and mixing, leveraging the controlled modulation of electric fields and wettability properties to achieve reliable results across multiple experimental runs.

Example 2 — Splitting of droplets in electrodes.

As depicted in snapshots A and B of Figure 2, the process of droplet splitting involves the application of voltage potentials to the electrodes. The initial volume in Figure 2A is two microlitres. This creates an electric field across the dielectric layer, which induces changes in the wettability of the droplet's interface with the electrode surface. Through precise activation of the applied electric potentials at the electrodes in the sides of the three electrodes, the forces result in the controlled splitting of the droplet into two equal volumes of one microlitre, as shown in snapshot C. By carefully modulating the voltage and the geometry of the electrodes, droplets can be accurately divided into desired sizes, allowing for precise sample partitioning or parallel processing within the microfluidic device. This splitting mechanism is reproducible and enables the generation of droplets with uniform and controlled volumes, crucial for the reliability and consistency of microfluidic assays and experiments where different concentrations are to be prepared.

Example 3 - Sample preparation of droplets with different effector to target ratios.

Herewith is provided a non-limiting example of the droplet sample preparation of a killing potency assay with effector cells and tumor cells, in accordance with the invention. A killing test was executed in the EWOD microfluidic platform using the unit droplet operations in

Figure 21. The killing test involves five different effector cells (e.g. CAR-T) to target cells (e.g. tumours) or E:T ratios: 2:1, 1:1, 1:2, 1:5 and a control of only target cells 0:1.

After adding the oil to the chip (mostly used were Silicone Oils 5 csT and Flurinert FC-40), a volume (Figure 21) of effectors and of targets was input into separate reservoirs. These ratios were achieved by splitting or dispensing from the reservoir, like shown in Example 1, 1ul droplets from the initial concentrations of cells and mixing them in volume ratios (Evol(ul): Tvol(ul)) equal to the E:T ratio. The first part of the method therefore requires sample preparation. From the CAR-T reservoir we split 5 sets of 5, 4, 3, 2 and 1 ul droplets as in Figure X1. The same is done for the tumour cells. Then we merge and mix them to obtain the five mentioned E:T ratios of 6ul each.

Next, the five 6ul samples were split into 15 2ul droplets, generating three biological replicates for each E:T ratio. Using green fluorescence for the tumour cells and red fluorescence for the CAR-T cells, or vice versa, samples were imaged in co-culture to examine the potency of the CAR-T wrt the tumours for 24 hours. Of course, different incubation times may be selected.

The first operation in the full assay required to isolate 15x 1ul droplets of both CAR-T and tumour cells. To avoid cross-contamination, the two reservoirs were not located adjacent to one another, we first split the 1ul droplet to be merged with the Sul droplet of the opposite sample of cells. Then we split 2x 1ul droplets from each reservoir, then 3x, 4x and 5x.

Meanwhile we transport the droplets to their respective mixing region. Chronologically this looks like this: 1. Dispense n CART and n tumour droplets 2. Displace toward opposite reservoir (i.e. move CAR-T towards tumour reservoir and vice versa) 3. Dispense n+1 CART and n+1 tumour droplets 4. Displace toward opposite reservoir prior to previous dispensed droplets 5. Repeat untiln=>5. 6. Merge 2:1, 1:1, 1:2, 1:5 and 0:1 E:T ratios

Then the five 6ul samples were each split simultaneously into three 2ul biological replicates and incubated. To perform the killing test, the 15 droplets were imaged every minute over 24 hours.

Example 4 - Heterogeneous mixing of Effector and Targets droplets

During a cytotoxicity test executed in an EWOD fluidic device, two ways of mixing effector and target cells can be selected by the user, heterogeneous or homogeneous mixing.

The heterogeneous mixing allows the merging of an effector droplet and a target droplet without the cells to initially mix; the cells form a front where on one side of the boundary between the two original droplets are the target and on the other side of the boundary the effector cells, allowing to study the initial migration at the beginning of the experiment.

The homogenous mixing allows instead to mix the effector and target cells from the beginning of the experiment preventing any effect of the migration to be collected in the experiment start data.

In this example, fluorescent microparticles (PMMA fluorescent microparticles, PolyAnn) were suspended in two separate droplets inside of a microfluidics chip and were used to show the principle of heterogeneous mixing.

The particles are about 3 micrometers in size and are representative of the cells for what concerns this example, and are similar to cells in terms of look, size and concentration. The particles in the two droplets have different fluorescent spectra allowing recognition of the particles during the mixing, and the two particle types will be referred to as red and green for simplicity. In Figure 18 these two different particles are represented by two different intensities of grays.

In Figure 18 it's shown the merging of two droplets of the two different fluorescence particles. In Figure 18 A the two droplets containing the two different red and green particles are shown being one electrode away from each other and hence not being in contact. In

Figure 18 B the droplets are brought to contact and merged. As visible in Figure 18 C the particles do not mix after the merging of the two droplets.

Example 5 - Homogeneous mixing of Effector and Targets droplets

During a cytotoxicity test executed in an EWOD microfluidic device two ways of mixing effector and target cells can be selected by the user, heterogeneous or homogeneous mixing.

In this example, fluorescent microparticles (PMMA fluorescent microparticles, PolyAnn) were suspended in two separate droplets inside of a microfluidics chip and were used to show the principle of heterogeneous mixing. This experiment is effectively a continuation of the experiment shown in example 4 and it temporally follows the steps already shown in example 4 minus the mixing steps, shown here in this example.

The particles are 3 micrometers in size and are representative of the cells for what concerns this example, and are similar to cells in terms of look, size and concentration. The particles in the two droplets have different fluorescent spectra allowing recognition of the particles during the mixing, and the two particle types will be referred to as red and green for simplicity. In Figure 19 these two different particles are represented by two different intensities of grays.

In figure 19 is shown two droplets already merged but not yet mixed (Figure 19 A). By

EWOD actuation (Figure 20) resulting in the back and forth movement of the newly merged droplets above a set of electrodes it is possible to actively mix the cells (or particles in this example, see Figures 19 B and 19 C).

Example 6 — Cell killing assay in EWOD microfluidic device

A cell killing assay was performed in an EWOD microfluidic device. Two chip reservoirs were loaded with effector and target cells respectively and droplets of effector and target cells were generated from each. The newly generated droplets were merged between effector and target cells to generate the desired E:T ratios.

The effector cells were prepared from a donation of T-cells of a patient affected with lymphoma. As shown later in the results, the cytotoxic effect was weak towards the target cells; this data is supported by the benchmark of the partners (see example 7).

The target cells were labeled with DMFDA fluorescent dye prior to the experiment via incubation for 30’ while the effector were not, allowing the recognition and classification of the cell types (target stained in the green fluorescence channel, effector unstained). The cells are suspended in RPMI medium phenol red free with 10% FCS (v:v). The presence of a viability dye in solution (in this case PI) allows to measure cell viability via fluorescence imaging of both cell types.

The E:T ratios for this specific experiment were prepared, similarly to the methods shown in Example 3. In this case however we performed the sample preparation using medium to dilute the concentration needed as shown in the workflow outline of Figure 16. Medium is used when unit droplet operations, such as those in Figure 21, do not sum up to Sul (e..g 1:10 E:T). After preparing the droplets for merging we end up with the volume as shown in the table below.

ET11 ET13 ET CTRL

Vol 53u 15u 05u 00

Effector

Vol 3u 45u 55u 6uL

Table 1.

The control consists of untreated target cells.

The amount of cells present in the final volume of 6 uL is shown hereafter in the table and like in Figure 21. Numbers may vary for different cytotoxicity assays according to the specifications of the test.

E:T ratio | Effector CAR T-cells | Target tumor cells used | Total cells used used ot 300 | 300 | 600 13 150 | 450 | 600

IEE 50 | 550 | 600

Control 600 | 0 | 600

Table 2.

Images of a single E:T ratio are shown in Figure 4. The droplets were merged in the chip via EWOD and imaged over time for 24 hours with fluorescence (Figure 4 B-C) and optical (Figure 4 A) imaging. As a result it is possible to count the viable cells and the cell types over time, allowing the calculation of the death rate of targets.

Figure 5 shows the real-time death rates of tumor cells induced by effector cell line FMC63-

BBz at various effector to target ratios and controls for a cell therapy. It illustrates the tumor cell death rates over time, providing insight into the efficacy of the treatment. This is showing the raw data output of a death curve.

Figure 6 presents the normalized death rates of tumor cells from Figure 5, normalized against the control, to account for variations in experimental conditions. This normalization enables a clearer comparison of the treatment's effectiveness across different ratios. This is the standard data output of the death curve.

In Figure 7 is shown the death rate of the different E:T ratios normalized over the control mean for a mock non-functional cell therapy. This is an alternative use of the death rate curve that can be used to normalize the drug effect over the mack to highlight the effect of the real cell therapy compared to a mock or a cell therapy of interest as comparison. This comparison helps discern the specific impact of the engineered effector cells on tumor cell death, distinguishing the treatment's effect from non-engineered effector cell effects,

As visible in figure 5, 6 and 7 the cells have reached a plateau after 24 hours and differences in the death were not observable anymore. This is due to the fact that the effector cells apparently show a relatively weak cytotoxic effect, and after an initial killing operated by the effectors, the killing speed decreases to the point that it is not distinguishable from the control. This could be due to the effector cells becoming exhausted and stopping to kill over time. The fact that it is possible to observe effector cells reaching a plateau and killing as much as the control after an initial killing shows additional value in this analysis since these behaviors are not observable with standard assessments.

In figure 8 is shown the count of the different cell types; this plot can be used for troubleshooting or to verify possible issues with the cells in the case odd data is obtained for the death curve. It represents the number of viable cells for each class, effectors and targets, and counting of the effector, target or total number of cells over time.

Example 7 — Cell killing assay in EWOD microfluidic device vs external benchmark (BLI assay).

In order to evaluate the cytotoxicity of two different CAR-T cell lines (FMC63-28z and

FMC63-BBz) and Mock transduced T cells (used like a placebo comparison) from the same donor, two tests were conducted in parallel using the Digi.Bio device and an external laboratory for benchmarking. The external laboratory used a conventional bioluminescence assay to measure cell survival after a 24-hour test with no intermediate measurements.

Only the output obtained from the Digi.Bio device at Tfinal (24 hours) was used for comparison. The specific lysis percentage was then calculated using the same standard formula: [(Spontaneous tumor cell death - Experimental tumor cell death) / Spontaneous tumor cell death] * 100.

Figure 9 provides a comparison of the calculated specific lysis (%) values obtained from both the Digi. Bio device and the benchmark assay. This plot visually demonstrates that data points for the same construct and E:T ratio have similar values, particularly for positive values from the CAR-T cell lines, like FMC83-28z A and B at 1:1, with values of 15.38% and 15.52%, respectively. Some negative values were obtained from the benchmark assay, but this was likely due to background noise. In this case, no cytotoxicity is assumed, which also coincided with values close to 0% obtained from the device.

Therefore, the EWOD microfluidic device provided for output with similar results as the conventional benchmark assay, particularly at positive specific lysis values (%), while advantageously not only providing results at T-final but also allows to provide for much further detailed information over time.

Example 8 — Frequency of contact events of effector cells and target cells

In the context of studying interactions between effector cells and tumors, cell tracking and detection methods enable researchers to identify, locate, and track individual cells over time. This capability allows for the computation of contact events between cells and the subsequent killing of tumor cells by effector cells. By knowing the identity of the cells, such as their cell types, researchers can further analyze the specific types of interactions occurring within the experimental system.

To visualize these interactions, various plots can be generated to highlight different aspects of the cell-to-cell interactions. For example, in Figure 10, contact events are represented as a bar plot, providing a summary of how many cells executed attacks during the entire experiment. This type of visualization helps to quantify the overall activity of the effector cells in targeting the tumor cells.

Different plots can be generated to visualize the contact events in different ways and highlight different properties of the cell to cell interactions.

Figure 11 allows the evaluation for each track corresponding to a single cell when exactly it was in contact and for how long during the experiment. These tracks correspond to targets being contacted by effector cells. The same plot can be generated where the tracks are the effector cells and the dark segments are contacts with the targets, allowing for a detailed examination of the timing and duration of interactions between specific cell pairs.

Similarly, Figure 12 provides a summary of attack events similar to Figure 10 but allows for the comparison of different experimental conditions, such as different runs, drugs, or effector-to-target ratios. This enables researchers to assess how various factors influence the efficacy of effector cell killing aver the course of multiple experiments.

Example 9 — Droplets with magnetic beads — magnetophoretic elution.

Here we explain an example of magnetic bead purification in our chip.

In the first scenario, (Figure 3 A-D) magnet separation of a liquid from a solid phase by coalescence of beads occurs, where the magnetic beads leave the droplet when coalesced, the movement of the droplet away from the magnetic field generated by the external magnet can cause the magnetic beads to separate from the droplet and remain in the surrounding oil or air (Figure 3D). This occurs because the magnetic force holding the beads in place within the droplet is not strong enough to overcome the force exerted by the movement of the droplet. Consequently, as the droplet containing the magnetic beads is manipulated away from the magnetic field, the beads are no longer constrained and can remain in the surrounding environment. This scenario poses a risk of losing the magnetic beads and compromising the effectiveness of the assay.

In the second scenario, (Figure 3 E-H) a train of droplets is used to remove non-bound agents without exerting force on the magnetic beads themselves. In this approach, the sequential movement of washing buffer droplets forms a train that sweeps across the surface of the EWOD microfluidic device. As the train of droplets advances, it generates a fluid flow that carries away non-bound agents while leaving the magnetic bead-bound analytes intact by magnetophoresis. The washing buffer droplets effectively push non- bound agents towards the edge of the droplet train, clearing them from the vicinity of the magnetic bead-bound analytes (Figure 3H). This process ensures that the magnetic beads remain within the fluid and are not forced out into the surrounding oil or air. By employing a train of droplets for sample manipulation, this scenario minimizes the risk of displacing the magnetic beads and ensures the integrity of the assay.

By subsequently releasing the magnetic beads in both scenarios, subsequently, the droplet comprising the magnetic beads with capture agent comprising target bound therewith and e.g. a detection agent, can be imaged to quantify the detection agent, thereby quantifying the target.

Example 10 - Immunoassay in EWOD microfluidic device with magnetic magnetic beads

A calibration curve was made by measuring the mean particle fluorescence from samples prepared using different antigen standard concentrations. A serial dilution of interferon gamma (IFN-y) was made to obtain the samples with different concentrations. The operation tree in Figure 17 displays the sequential microfluidic operations performed to execute the immunoassay for each of the samples.

In brief, the immunoassay consists of the sequential addition of the following reagents: magnetic beads bound to an anti-IFN-y capture antibody, the corresponding IFN-y dilution, anti-IFN-y detection antibody, and the R-phycoerythrin—streptavidin (SAPE) fluorophore.

Washing steps are performed in between the addition of each reagent using 2x2 electrodes.

Note that in this non limiting example the detection antibody is functionalized with biotin, which has affinity to streptavidin to allow binding with SAPE fluorophore molecules. The use of streptavidin-biotin in this example is optional, the use of magnetic particles covalently bound with capture antibodies, and detection antibody covalently bound with a fluorophore group, may be preferred. All droplet manipulations were performed on-chip using EWOD.

First, a 12ul droplet of a magnetic bead solution was loaded onto the chip. Then, the droplet was split into two-6 HI droplets and each was taken into a different magnet site. The beads were collected by activating the magnet to coalesce them and remove the solvent buffer to dispose of it as waste.

Subsequently, the corresponding IFN-y dilution was brought to the magnetic module where one of the magnetic bead droplets is located. Simultaneously, the magnet is deactivated to allow mixing the magnetic beads and binding of the IFN-y protein to the capture anti-IFN-y antibody. Then, a washing step is executed to discard all unbound IFN-y. After washing, the magnet is activated to coalesce the magnetic bead-IFN-y complex into the electrode, and discard the washing buffer as waste. The same process described was performed concurrently with the second magnetic bead droplet. However, in this case a free-IFN-y buffer was used as negative control.

Then, two 3ul droplets of anti-IFN-y detection antibody were brought to the magnetic module. Once again, the magnets were deactivated to mix each magnetic bead solution with the detection antibody solution to form a magnetic bead-IFN-y-detection antibody complex. Then, a washing step is executed to discard all unbound anti-IFN-y detection antibody. After washing, the magnet is activated to coalesce the magnetic bead-IFN-y complex into the electrode, and discard the washing buffer as waste.

Finally, two Sul droplets with SAPE fluorophore were brought to the magnetic module. Once again, the magnets were deactivated to mix each magnetic bead solution with the SAPE fluorophore and allow its binding to the biotinylated detection antibody molecules attached to the beads in the presence of IFN-Y.

After performing one washing step, each sample is mixed with Sul of reading buffer. Then, each droplet is split into three 2 pl droplets ready to be imaged. Using red fluorescent microscopy we imaged them and measured the mean particle fluorescence signal.

A calibration curve was created using different concentrations of an antigen standard, which in this case was Interferon gamma (IFN-y). The concentrations used were 0.06, 0.24, 0.97, 1.21, 2.43, 4.85, and 9.7 ng/mL of the antigen. The mean particle fluorescence intensity was measured, and the values were plotted against the concentrations used. The best fitting trend was a strong logarithmic correlation, with an R squared value of 0.945 (Figure 13). In addition, a strong linear correlation was found in the concentration range of 0.06, 0.24, 0.97, 1.21, 2.43, and 4.85 ng/mL, but the R squared value (0.856) was found to be lower than in the logarithmic trend (Figure 14). Therefore, the logarithmic trend was chosen as the final calibration curve.

This method proved that it is possible to accurately quantify the concentration of IFN-y from the mean particle intensity. The calibration curve obtained can be used to quantify the concentration of unknown samples based on their fluorescence signal. For each analyte and antibody pair, a new correlation curve must be established.

Figure 15 shows the fluorescence values measured during the experiment for visualization purposes only.

To determine the concentrations where the mean particle fluorescence values varied significantly, a two-tailed unpaired T-test was conducted. Table 3 presents the results of the

T-test, indicating that most of the samples were found to be significantly different with a p- value of less than 0.05.

Table 3. Results of statistical analysis.

Ee P-value of a two-tailed T-test (HO: the samples are equal) between the samples í fluorescence signals oe 77 TGE GTG 777 EV Too

Ee ETE EE Ee

Eb Ta) ed ol on] om 1.21

ELT Tb Tand ed om 0.97

ZT TTF [ed 0.24

ELT TTT Tan

Claims

CONCLUSIONS

1. A method for determining target cell killing by effector cells with an electrowetting on dielectric (EWOD) microfluidic device, comprising the steps of: a) providing an EWOD microfluidic device comprising means for holding fluid comprising cells and/or agents in a plurality of regions configured to generate, move, resuspend, merge, split, and mix droplets with an array of alternating electrodes by applying a voltage to independently addressable electrodes, and comprising means for imaging droplets and cells and/or agents therein using microscopic imaging; b) providing effector cells in culture medium; c) providing target cells in culture medium; d) providing culture medium; e) moving the effector cells and target cells suspended in a culture medium and culture medium each to a separate reservoir region, respectively, on the EWOD microfluidic device; fl) generating droplets of the effector cells and target cells in culture medium, and culture medium, of defined volumes, and optionally, determining the number of effector cells or target cells in each generated droplet using imaging, and then; g) preparing droplets of the effector cells and the target cells in culture medium, respectively, with a defined concentration of cells, and a defined volume, each in a separate sample preparation area, thereby, obtaining prepared droplets of effector cells and of target cells; I} wherein the prepared droplets are obtained by splitting, merging and/or mixing generated droplets to obtain a desired amount or concentration of cells in the prepared droplets; II) wherein the prepared droplets have a volume of preferably at least 0.1 microliter, preferably up to 10 microliters, preferably between 0.5 and 5 microliters, more preferably between 1 and 2 microliters; and II!) wherein the effector cells and the target cells are homogeneously distributed in their respective droplets;

IV) wherein the number of effector cells or target cells in each prepared droplet is preferably between 10 and 1 x 10%4, more preferably between 1 x 10%2 and 1 x 1043; V) optionally, determining the number of effector cells and/or target cells in each prepared droplet using imaging; h) pooling the prepared droplets of effector cells and prepared droplets of target cells in a culture area in which effector cells and target cells are in different ratios of effector cells to target cells, thereby, obtaining pooled droplets; I) wherein preferably the different ratios of effector cells to target cells in the pooled droplets range from 1:10 to 10:1; II) wherein optionally, for each ratio the number of technical replicates is at least 2; II) determining the number of effector cells and target cells, and, preferably, individual location and cell morphology of effector cells and target cells; IV) and, after pooling, the pooled droplet comprising effector cells and target cells is mixed, or, is not mixed; incubating the pooled droplets in the EWOD microfluidic device, preferably in an area with an optically black background; i) imaging the pooled droplets, preferably using fluorescence and/or optically automated microscopy, at least at the end of the incubation; k) determining from the captured images for each pooled droplet the number of at least viable target cells at least at the end of the incubation.

2. The method of claim 1, wherein in step k) for each incubated pooled droplet the number of killed target cells and/or the ratio of viable target cells to total target cells is determined.

The method of claim 1 or claim 2, wherein at least the target cells are provided with a fluorescent label.

4. The method according to any one of claims 1 to 3, wherein in imaging step j} a plurality of images are captured over time, preferably every minute, and wherein preferably location and morphology of individual cells is determined in step k), thereby enabling tracking of individual cells.

5. The method according to any one of claims 1 to 4, wherein individual cells are monitored, and in step k)

- target cell killing is plotted against time; - target cell contact time and/or the frequency of target cell contact is determined for individual effector cells; - motility of individual effector cells is determined; and/or - individual cells are classified as serial killer, monokiller, or non-killer.

6. The method according to any of the preceding claims, wherein after step j) an assay, preferably an immunoassay, is performed with the pooled and incubated droplets on an EWOD microfluidic device, the EWOD microfluidic device further comprising an integrated magnet for magnetophoretic elution of magnetic beads, wherein magnetic beads with a capture agent are provided in a buffer medium, the capture agent is preferably an antibody, the capture agent is capable of binding to a target of interest, the magnetic bead is paramagnetic or superparamagnetic, and wherein a detection agent is provided, preferably an antibody with a detection label, preferably with a fluorescent label, and, comprising means for (fluorescence) imaging in an analysis area, preferably comprising an optically black background, wherein the pooled and incubated droplets are allowed to interact with droplets of the magnetic beads with the capture agent and the detection agent, and Subsequently, magnetic beads with capture agent comprising the target bound to them and detection agent are magnetophoretically eluted and imaged, and the target quantified.

A method according to claim 6, wherein the magnetophoretic elution comprises: - capturing magnetic beads with capture agent having the target bound thereto and a detection agent, - washing away unbound detection agent, and, optionally, target cells and effector cells, if present, from the captured magnetic beads with capture agent having the target bound thereto and detection agent, the washing comprising the steps of: o moving away from the purification region the droplet with unbound detection agent, and, if present, target cells and effector cells; o moving to the purification region a wash droplet, wherein the steps of moving away from and moving to the purification region are performed sequentially or simultaneously, and optionally, repeated one or more times; thereby obtaining magnetophoretically eluted magnetic beads with capture agent comprising the target bound thereto and detection agent.

8. The method according to any of claims 1 to 5, further comprising the steps of: D) providing magnetic beads with a capture antibody in a buffer medium; wherein the capture antibody is capable of binding the target of interest; wherein the magnetic bead is paramagnetic or super paramagnetic; m) providing a detection antibody in a buffer medium with a fluorescent label; wherein the detection antibody has the same target as the capture antibody, preferably wherein the detection antibody and capture antibody are directed to different epitopes of the same target; o optionally wherein the magnetic bead with capture antibody and the detection antibody provided are combined in a single suspension; n) providing buffer medium; o) moving the magnetic beads with the capture antibody and the detection antibody, suspended in buffer medium, each to a separate reservoir, or combined in one reservoir, on the EWOD microfluidic chip/device; p) resuspending the incubated target cells and effector cells of step j) in the droplet and combining and mixing the droplet(s) comprising the magnetic beads with capture antibody and the detection antibody, thereby allowing the capture antibody and detection antibody to interact with their respective targets; q) moving the resuspended droplet to a purification area; wherein the purification area comprises an integrated magnet for magnetophoretic eluting of magnetic beads, and, capturing the magnetic beads; - eluting the magnetic beads with capture antibody comprising the target bound thereto and detection antibody with a wash droplet, - releasing and resuspending the magnetophoretically eluted magnetic beads with capture antibody comprising the target bound thereto and detection antibody; and moving the droplet with magnetophoretically eluted magnetic beads to an analysis area, i) wherein the analysis area comprises an optically black background; ii) wherein the analysis area comprises means for a reduced fluorescent background; r) quantifying the amount of detection antibody using fluorescence imaging, thereby quantifying the amount of target in the droplet.

9. The method according to claim 8, wherein the step of eluting comprises: - washing away unbound detection antibody, and target cells and effector cells from the captured magnetic beads with capture antibody having the target bound thereto and detection antibody, washing comprising the steps of: o moving away from the purification region the droplet with unbound detection antibody, and target cells and effector cells; o moving to the purification region a wash droplet, wherein the steps of moving away from and moving to the purification region are performed sequentially or simultaneously, and optionally, repeated one or more times; thereby obtaining magnetophoretically eluted magnetic beads with capture antibody comprising the target bound thereto and detection antibody.

10. The method of any one of claims 6 to 9, wherein the magnetic force for capturing the magnetic beads with capture antibody having the target bound thereto and detection antibody is orthogonal to the displacement forces exerted by EWOD on the droplet with unbound detection agent/antibody and wash droplet(s).

11. The method according to any of claims 6 to 10, wherein a plurality of (immuno)tests are performed simultaneously or sequentially.

12. The method according to any one of the preceding claims, wherein the effector cells are selected from (engineered) immune effector cells, such as CAR-T cells, CAR-NK cells, CAR-macrophages, or non-cytotoxic immune cells (including regulatory T cells or T helper cells), cytotoxic T cells, and tumor-infiltrating lymphocytes.

13. The method according to any one of the preceding claims, wherein the target cells, wherein the target cells are optionally adherent cells, and wherein the target cells are cancer cells or organoids.

The method of any preceding claim, wherein the EWOD microfluidic device comprises at least one further reservoir comprising an agent in a medium, wherein droplets of the agent are generated with EWOD and a defined amount of agent is further combined with a droplet of the effector cells and/or of the target cells and mixed, I) wherein the agent is mixed with the droplet of effector cells or target cells prior to combining the droplets of effector cells with the target cells in step h) II) wherein the agent is mixed with the combined droplet of target cells and effector cells in step h).

The method of claim 14, wherein the agent is a cell engager, such as a bispecific antibody or a drug.

16. The method according to any one of the preceding claims, wherein the effector cells and target cells are combined simultaneously.

17. The method according to any one of the preceding claims, wherein the droplets in the EWOD microfluidic device are surrounded by an oil, preferably a silicone oil, perfluoroalkyl oil or a liquid hydrocarbon.

The method of any preceding claim wherein the culture region comprises at least two further electrodes on different sides of the electrode for hosting the required volume for merging and subsequent mixing of the droplets or merging without mixing.

19. The method of any preceding claim wherein the reservoir area, sample preparation area, culture area, purification area and/or waste area comprises one or more electrodes for holding a defined volume.