DK202430110A1 - Removal of Fluorocarbon Oil from Oil-Water Emulsions by Step Gradient Centrifugation - Google Patents

Abstract

The invention provides a method for separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation. The method is particularly useful for breaking and releasing encapsulated matter of both single-emulsion (W/O) and double-emulsion (W/O/W) droplets, without the use of toxic breaking solutions. The invention also provides a density gradient medium and a kit of parts, to facilitate the execution of the method in practice.

Description

DK 2024 30110 A1 1

Removal of Fluorocarbon Oil from Oil-Water Emulsions by Step Gradient

Centrifugation

FIELD OF THE INVENTION

The present invention relates to the field of water and fluorocarbon-oil comprising emulsions.

Specifically, the invention pertains to methods and compositions for separating at least part of the fluorocarbon oil from the aqueous components of the emulsions. These methods and compositions are valuable in preparing more homogeneous water-oil-water emulsions, as well as releasing encapsulated particles and cells from the droplets of the emulsions.

BACKGROUND OF THE INVENTION

Emulsions comprise a discontinuous liquid phase of small droplets dispersed in a continuous liquid phase.

The immense potential of small (typically nano- to micro-litre seized) emulsion droplets initially led to their utilization as miniaturized reaction vessels for conducting biochemical assay systems. However, the true breakthrough in harnessing these droplets as reaction vessels occurred with advancements in fluoride chemistry and microfluidics, enabling the development of techniques to generate stable emulsions comprising discrete monodisperse droplets in sufficient quantities. In this technology, immiscible phases are passed through microchannels, leading to homogeneous shearing of the liquids and resulting in the formation of emulsions composed of discrete monodisperse droplets.

The potential applications of these discrete monodisperse droplets are fueled by their capacity to encapsulate a variety of reactants, particles, and/or cells within them, thereby unlocking the vast potential for high-throughput screening, single-cell analysis, DNA-based diagnostics, and drug screening.

The field of microfluidically manufactured emulsions deals with single-emulsion droplets and double-emulsion droplets. Single-emulsion (SE) droplets are droplets generated within an immiscible continuous phase, like water-in-fluorocarbon oil droplets (W/O). Conversely, double-emulsion (DE) droplets comprise two nested layers of immiscible liquids, typically water-in-oil-in-water (W/O/W) or oil-in-water-in-oil (O/W/O).

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Importantly, microfluidically manufactured emulsions, are plagued by a range of issues some of which are related to the fluorocarbon-oil phase, namely:

Breakage of droplets.

When it comes to releasing the content of single-emulsion droplets or double-emulsion droplets, the conventional approach in the field is to treat the emulsion with a so-called break- solution, such as 1H,1H,2H,2H-Perfluoro-1-octanol (PFO) or nonafluoro-tert-butyl alcohol (NFTBA). However, besides being moderately to severely toxic, PFO and NFTBA are harmful to cultured cells and difficult to separate from encapsulated cells or particles, often necessitating multiple washing steps.

Removal of undesirable oil drops from double-emulsions.

When DE droplets are formed by conventional microfluidic techniques such as described by

Madsen et al. (2020) Human Mutation 41, 1671-79 (doi: 10.1002/humu.24063) the continuous water-phase contains a variable amount of oil-droplets in addition to the double- emulsion droplets.

Such oil droplets are a undesirable factor that frequently disrupts downstream FACS analysis of the double-emulsion droplets.

SUMMARY OF THE INVENTION

Surprisingly, both the problem of non-toxic breakage of single-emulsion and double-emulsion droplets as well as the removal of confounding oil drops from double-emulsions can conveniently be solved by a brief step gradient centrifugation using a suitable density gradient medium.

Thus, in a first aspect, the invention pertains to a method for separating at least part of the fluorocarbon oil (fluorous/fluorophilic oils) from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation.

The actual density gradient forming composition used should preferably be carefully selected not to influence the contents of the droplets negatively.

Accordingly, a second aspect of the invention relates to the density gradient forming composition used to separate at least part of the fluorocarbon oil from a water and

DK 2024 30110 A1 3 fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation characterized in that the composition comprises a low osmolarity, density gradient medium.

A third aspect of the invention provides a kit of parts for separating at least part of the fluorocarbon oil (fluorous/fluorophilic oils) from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation of an emulsion. The kit comprises a composition according to the second aspect, at least one centrifuge tube and directions for performing the method of the first aspect.

DEFFINITIONS

Prior to a discussion of the detailed embodiments of the invention a definition of specific terms related to the main aspects of the invention are provided.

The term “droplet” as used herein refers to a small volume of liquid, typically in a spherical shape, surrounded by an immiscible fluid such as a continuous phase of an emulsion.

Throughout the present disclosure, the term "droplet" and "micro-droplet" are used synonymously. It refers to droplets forming an emulsion of droplets each of which is comparable with the dimensions of a microfluidic device.

In the present context all droplets, they be single- or double-emulsion droplets, large or small, spherical or elongated, are referred to as droplets. The term droplet is thus used in this patent to include spherical droplets, plugs, and slugs.

An “emulsion” is a dispersion of small droplets in a continuous phase, stabilized by a third compound, typically comprising a surfactant.

The term “double-emulsion droplet” refers to a water-in-oil-in-water droplet (also named

W/O/W droplet) and consists of an aqueous droplet inside an oil droplet, i.e. an aqueous core and an oil shell, surrounded by an aqueous carrier fluid. Preferably, the double-emulsion is a monodispersed emulsion, i.e. an emulsion comprising double-emulsion droplets of approximately the same volume. Typically, the W/O/W droplet has a volume of less than 1000 pL, preferably of less than 200 pL. Preferably, a W/O/W droplet has a volume ranging from 0,1 pL to 150 pL.

For some applications, such as when mammalian cells are encapsulated within the droplets, the W/O/W droplet may have a volume of between 1 pL and 500 pL. Preferably, a W/O/W droplet has a volume ranging from 2 pL to 200 pL, more preferably from 30 pL to 150 pL. ,

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For biochemical analysis that do not involve encapsulated cells smaller W/O/W droplets, e.g. 0,1 pL to 150 pL, preferably from 0,25 pL to 25 pL, more preferably from 1 pL to 10 pL, and in particular from 1,5 pL to 5 pL, are frequently preferred.

Inthe present context the term “single-emulsion droplet” preferably refers to an isolated portion of an aqueous phase that is completely surrounded by an oil phase (also named water-in-oil single-emulsion droplets or W/O droplets). Droplets formed by an isolated portion of an oil phase that is completely surrounded by a water phase are referred to as “oil-in-water droplets” or simply as "oil drops”. Preferably, the water-in-oil emulsion is a monodispersed emulsion, i.e. an emulsion comprising droplets of the same volume. Techniques for producing such a homogenous distribution of diameters are well-known by the skilled person (see for example Madsen et al. (2020) or WO 2004/091763). “Fluorocarbon oil”, “fluorinated oil” and “fluorous or fluorophilic oils” are used synonymously. lt is organofluorine liquids typically perfluorocarbons with a density higher than water and with an “oily” appearance Examples of usable oils include Fluorinert™ FC-40, Sigma-Aldrich, St.

Louis, MO, USA; KrytoxTM, Chemours, Wilmington, DE, USA; and NovecTM Oil, 3M Co.,

Maplewood, MN, USA.

As used herein the term “fluorophilic continuous phase” refers to a continuous liquid phase having a chemical affinity for fluorocarbons, e.g. a fluorocarbon oil.

The term "microfluidic" implies that at least a part of the respective device/unit comprises one or more fluid conduits being in the microscale, such as having at least one dimension, such as width and/or height, being smaller than 1 mm and/or a cross-sectional area smaller than 1 mm?. The smallest dimension, such as the height or width, of at least one part of the fluid conduit network, such as a conduit, an opening, or a junction, may be less than 500 um, such as less than 200 um, for example, less than 20 um. "Particle" is defined as a unit of matter, including, but neither limited to beads nor to cells. The term particle includes cells as well as other particles, e.g. beads, nuclei, nucleotides, viruses, protein complexes, or proteins, biochemical or chemical compounds, all of which may be contained in droplets.

As used herein the term "step gradient centrifugation" designates a technique used to separate entities or liquids based on their buoyant density. In this method, a solution,

DK 2024 30110 A1 suspension, or emulsion is layered on top of one or more cushions of increasing density.

When the sample is centrifuged, the various components of the top layer move into or through the cushion(s) according to their buoyant densities. 5 By a “brief” step gradient centrifugation, is referred to a step gradient centrifugation of less than 5 minutes, such as less than 1 minute; preferably less than 30 seconds; more preferably less than 10 seconds; or even ca. 5 seconds. The brief centrifugation may be repeated.

The term “density gradient medium” refers to a composition that is useful for forming a cushion that has a density greater than an overlaying liquid layer. Examples of agents used to make such medium include density agents such as iodixanol, sodium diatrizoate, sodium metrizaoate, metrizamide, sucrose, and other sugars, oligosaccharides, synthetic polymers (e.g. Ficoll), and various salts such as cesium chloride, potassium bromide, and others.

By “low osmolarity” or “low osmotic concentration” is referred to solute concentration of approximately 290 mOsm or less. 0,150 M NaCl in water has an osmolarity of approximately 300 mOsm. Normal plasma osmolarity is maintained within a narrow range, 275-290 mOsm/L (Moritz, Textbook of Nephro-Endocrinology (Second Edition), 2018)

The term "dMDA" as used herein refer to the multiple displacement amplification (MDA) technique described by Blanco et al (1989) Chem. 264: 8935-40; and Zanoli et al (2013)

Biosensors 3, 18-43. but performed in droplets.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate the design and utility of embodiments. These drawings are not necessarily drawn to scale. The drawings may only depict typical embodiments and may therefore not be considered limiting of the scope of the invention.

Figure 1: Shows the result of a typical production of double-emulsion droplets as described in example 1. Both double-emulsion droplets and oil droplets are shown.

Figure 2: Shows a double-emulsion droplet production loaded on top of the density gradient medium in a centrifuge tube before and after a brief centrifugation.

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Figure 3: Panel A is a microscopic view of the heterogeneous mixture comprising double- emulsion droplets and oil droplets before the separation workflow. Panel B is a microscopic view of the upper phase (see Fig. 2). After the centrifugation separation process oil drops were removed resulting in a homogenous water-in-oil-in-water double-emulsion droplet preparation (Figure 3 B).

Figure 4: lllustrating that different iodixanol concentrations resulted in different separation qualities of a double-emulsion comprising double-emulsion droplets with encapsulated cells.

The result with varying concentrations of the iodixanol density gradient medium (60, 65, 70, 75, and 80 %w/v) is shown.

Figure 5: Åre microscopic views. Panel A is a microscopic view of the heterogeneous mixture comprising double-emulsion droplets, double-emulsion droplets with encapsulated cells, and oil droplets before the separation workflow. Panel B is a microscopic view of the upper phase (see Fig. 4) after the centrifugation separation process over a 60 %w/v iodixanol step gradient. Panel C, D, E and F show the results with 65, 70, 75, and 80 %w/v iodixanol.

Figure 6: Panel A is a microscopic view of the heterogeneous mixture comprising double- emulsion droplets with and without encapsulated cells and oil droplets before the separation workflow. Panel B is a microscopic view of the upper phase (see Fig. 4) after the centrifugation separation process over a 80 %w/v iodixanol step gradient.

Figure 7: Panel A is a series of Eppendorf tubes with the single-emulsion (SE85) droplets containing a non-solidified gel matrix.

Panel B is a graphical representation of the method. The illustration on the left depicts the scenario where single-emulsion droplets with solidified gel beads are layered on top of a suitable density gradient medium, such as iodixanol. The middle illustration shows the situation after 5 seconds of centrifugation. At the top, a layer with the harvested gel beads, in the middle the density gradient medium, and at the bottom, the fluorocarbon oil of the emulsion. If the density gradient medium is not clear, the centrifugation process is repeated another 5 seconds.

Figure 8:The left panel shows the gel beads inside single-emulsion droplets before centrifugation. Figure 8 right panel shows the released gel beads after centrifugation. On both panels cells are clearly visible inside the gel beads.

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DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a method for separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation.

The situation where the droplets of the emulsion are aqueous droplets surrounded by a fluorocarbon oil phase is of particular interest because most biochemical processes proceed in an aqueous milieu.

As shown in the examples both aqueous droplets dispersed in a fluorophilic continuous phase (single-emulsion or water-in-oil droplets) and aqueous droplets, each encapsulated within a larger droplet of fluorophilic liquid, that are dispersed in a continuous aqueous phase (double- emulsion or water-in-oil-in-water droplets) are readily braked and their contents released by a brief step gradient centrifugation without use of toxic “break-solutions” such as 1H,1H,2H,2H-

Perfluoro-1-octanol (PFO) or nonafluoro-tert-butyl alcohol (NFTBA).

During production of double-emulsion (W/O/W) droplets on standard microfluidic equipment a variable amount of single-emulsion (o/w, oil-in-water droplets or oil drops) are formed. This results in a heterogeneous mixture, which is often fully acceptable. However, in certain cases, such as downstream FACS analysis, it can negatively impact the analysis. In example 2 oil-in- water droplets (oil drops) dispersed in the continuous aqueous phase are selectively removed from the emulsion of double-emulsion droplets by a brief step gradient centrifugation.

Utilizing droplet technology for encapsulating particles, such as cells, is particularly attractive.

However, the final steps in such an assay often require the recovery of material from the droplets produced by the microfluidic workflow. The standard method for recovering material from the droplets is to break the emulsions chemically using potent demulsifiers, such as chloroform, perfluorooctanol (PFO), or nonafluoro-tert-butyl alcohol (NFTBA). These demulsifiers displace surfactants from the oil-water interfaces of the droplets, leading them to become unstable and break. However, these chemicals also interact with cell membranes and hydrophobic regions of proteins and other biomolecules, resulting in varying degrees of toxicity and inhibition of many enzymatic processes.

Surprisingly, the present inventors found that a brief step gradient centrifugation sufficed to release encapsulated particles from both single- as well as double-emulsion (W/O/W) droplets.

This is illustrated in examples 4 and 5.

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Although it is preferred to avoid break-solutions for the reasons mentioned above, it is contemplated that in certain instances a break-solution may advantageously be added to the continuous aqueous phase of double-emulsion (W/O/W) droplets with encapsulated particles shortly before the onset of the step gradient centrifugation. It is anticipated that it may partly destabilise the droplets resulting in an improved release of the particles and that the detrimental effects of the break-solution will be minimal because of the minimal time the break-solution is in contact with the biomolecules or cells.

As further demonstrated in examples 4 and 5 the encapsulated particles that can be released by the present comprise both cells, gel beads, and even gel beads that comprise cells. This opens an interesting avenue for producing spheroids and organoids. It also suggests that other types of particulate or dissolved material such as proteins and nucleic acids can be released from droplets by a brief step gradient centrifugation.

While the method is remarkably simple to perform, one must carefully select the density gradient medium that is optimal for the specific intended application. In most experiments with biomolecules or cells this direct use of a low osmolarity, density gradient medium. In particularly low osmolarity, non-ionic iodinated density gradient media such as iodixanol (5- [acetyl-[3-[N-acetyl-3,5-bis(2,3-dihydroxypropylcarbamoyl)-2,4,6-triiodoanilino]-2- hydroxypropyllamino]-1-N,3-N-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-1,3- dicarboxamide; CAS No. 92339-11-2) or lohexol (5-[acetyl(2,3-dihydroxypropyljamino]-1-N,3-

N-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-1,3-dicarboxamide; CAS No. 66108-95-0) are preferred.

The density of the gradient step density gradient medium is of paramount importance to the functionality of specific assays. This is illustrated in examples 2 and 3. In example 2 a density gradient medium with a density only slightly over 1,3 g/ml (1,32 g/ml) works very well for the separation of oil droplets from W/O/W droplets with an aqueous solution. But as shown in example 3, a density gradient medium with a density over 1,4 g/ml (1,442 g/ml) appears optimal when the W/O/W droplets contain cells.

In one embodiment of the present invention, the density gradient medium comprises a colour for easier recognition of phases. The density gradient may also comprise a salt. The salt may be used to obtain an isotonic osmolarity, or it may be used as a specific gravity-modifying means to increase the density of the density gradient medium or to form the density-forming

DK 2024 30110 A1 9 component of the density gradient medium in itself. CsCl for instance has been used for this purpose.

In a preferred embodiment of the invention the step gradient centrifugation involves a density gradient medium that comprises a low osmolarity, non-ionic density gradient component, a salt, and a color and is further characterised by a density of over 1,3 g/ml and a final osmolarity of approximately 290 mOsm.

In a further preferred embodiment of the invention the density gradient medium is pH- adjusted to a pH between 5 and 8,5; preferably between 5,5 - 7,5; more preferably between 7,2 - 7,4: or even ca. 7,3.

Various density gradient media have found widespread commercial use. Therefore, a preferred embodiment of the invention is a composition specifically designed to form the step gradient cushion used in the present method of separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation.

One such composition is characterized in that the composition comprises a low osmolarity, density gradient medium and further comprises a colour for easier recognition of phases.

In particularly density gradient compositions which comprise a low osmolarity, non-ionic iodinated density gradient medium have been found to perform well in the method, see experiment 2 — 5. But other types of density agents, such as sodium diatrizaote, sodium metrizaoate, metrizamide, sucrose, and other sugars, oligosaccharides, synthetic polymers (e.g. Ficoll), and various salts such as cesium chloride, potassium bromide, and others may be used to make density gradient compositions as well.

In further embodiments the density gradient forming composition has a density over 1,3 g/ml; such as over 1,4 g/ml. Such high-density cushions, are favourable to recovering encapsulated particulate matter, such as cells, from droplets.

To recover living cells from the droplets, it is crucial to avoid subjecting the cells to unphysiological pH or osmolarity values. According to the FDA (Reference ID: 4154992), isotonic 0,9% NaCl solution for injection has an osmolarity of 308 mOsmol/L (calculated) and a pH of approximately 5,6 (ranging approximately from 4,5 to 7,0).

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Thus a density gradient forming composition adjusted to a pH between 4,5 and 8,5; preferably between 5,5 - 7,5; more preferably between 7,2 - 7,4; or even ca. 7,3, is preferred.

Cell culture medium is designed to have osmolarity in the range of 260 and 320 milliosmoles (mOsm), basically to mimic the osmolarity of serum at 290 mOsm/kg (Ozturk, Biotechnology and Bioengineering, 37, 989-993 (1991)).

Thus a composition with a density over 1,3 g/ml, e.g. over 1,4 g/ml, which comprises a salt, has a final osmolarity of approximately 290 mOsm and a final pH between 5 and 8,5, is considered particularly advantageous for recovery of encapsulated cells from droplets.

The invention further provides a kit of component parts suitable for separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation or suitable for releasing encapsulated particles or cells the droplets by a brief step gradient centrifugation.

The kit of parts comprises a density gradient forming composition as described above, at least one centrifuge tube and directions for performing the method of the invention.

In additional embodiments the kit of component parts also comprise a break solution.

The Invention Presented in the Form of Embodiments

Preferred aspects and embodiments of the invention may be presented as items of the specification. These are given below. 1. A method for separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation. 2. The method according to item 1, wherein the droplets of the water and fluorocarbon-oil comprising emulsion are aqueous droplets dispersed in a fluorophilic continuous phase (water-in-oil single-emulsion droplets). 3. The method according to item 1, wherein the droplets of the water and fluorocarbon-oil comprising emulsion are aqueous droplets, each encapsulated within a larger droplet

DK 2024 30110 A1 11 of fluorophilic liquid, that are dispersed in a continuous aqueous phase (water-in-oil-in- water double-emulsion droplets). 4. The method according to item 3, wherein oil-in-water droplets dispersed in the continuous aqueous phase are selectively removed from the double-emulsion droplets. 5. The method according to item 3, wherein the continuous aqueous phase of the double-emulsion comprises water-in-oil-in-water droplets with encapsulated particles, and wherein the encapsulated particles are released from the droplets by a brief step gradient centrifugation. 6. The method according to item 5, wherein a break-solution is added to the continuous aqueous phase of the double-emulsion comprises water-in-oil-in-water droplets with encapsulated particles, and are subsequently subjected to brief step gradient centrifugation to obtain the release of the particles. 7. The method according to item 5 or 6, wherein the particles are cells, nucleic acids, or proteins.

8. The method according to item 2, wherein the fluorophilic continuous phase of the single-emulsion comprise water-in-oil droplets with encapsulated particles, and wherein the encapsulated particles are released from the droplets by a brief step gradient centrifugation.

9. The method according to item 8, wherein the particles are gel beads. 10. The method according to item 9, wherein the gel beads comprise cells. 11. The method according to any of the preceding items, wherein the step gradient is formed by a composition which comprises a low osmolarity, density gradient medium. 12. The method according to item 11, wherein the density gradient medium comprises a low osmolarity, non-ionic iodinated density gradient medium.

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13. The method according to item 12, wherein the density gradient medium comprises iodixanol (iodixanol).

14. The method according to any of items 11 - 13, wherein the density gradient medium has a density over 1,3 g/ml.

15. The method according to any of items 11 - 13, wherein the density gradient medium has a density over 1,4 g/ml.

16. The method according to any of items 11 - 15, wherein the density gradient medium comprises a color for easier recognition of phases.

17. The method according to any of items 11 - 16, wherein the density gradient medium in addition to a low osmolarity density gradient forming composition also comprises a salt.

18. The method according to item 17, wherein the density gradient medium comprises a low osmolarity, non-ionic density gradient component, a salt, a color and a buffer and is characterised by a density of over 1,3 g/ml and a final osmolarity of approximately

290 mOsm.

19. A density gradient forming composition for the use of separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation characterized in that the composition comprises a low osmolarity, density gradient medium.

20. The composition according to item 19, wherein low osmolarity, density gradient medium comprises a color for easier recognition of phases.

21. The composition according to items 19 or 20, wherein the composition has a density over 1,3 g/ml.

22. The composition according to any of items 19 - 21, wherein the composition comprises a low osmolarity, non-ionic iodinated density gradient medium.

DK 2024 30110 A1 13 23. The composition according to any of items 19 - 22, wherein the composition is pH- adjusted to a pH between 5 and 8,5 (preferably 7,0 - 7,5; more 7,2 - 7,4). 24. The composition according to any of items 19 - 23, wherein the composition comprises a salt and a final osmolarity of approximately 290 mOsm. 25. A kit for separating at least part of the fluorocarbon oil from a water and fluorocarbon- oil comprising emulsion by a brief step gradient centrifugation of an emulsion, comprising a composition according to items 20 - 24, at least one centrifuge tube and directions for performing the method of any of claims 1 — 18. 26. The kit according to claim 25 which in addition comprises a break-solution.

The invention is further illustrated in the following non-limiting examples.

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EXAMPLES:

Example 1: Method for producing double-emulsion droplets.

Double-emulsion droplets (water-in-oil-in-water) were generated using Xdrop proprietary droplet production technology as described by Madsen et al., 2020 (Human mutation doi: 10.1002/humu.24063).

Material and methods

The DE50 droplets were produced on an Xdrop instrument-EU (cat no. INO0100-EU, Samplix

ApS, Birkerød, Denmark) using the double-emulsion generating cartridge (Samplix cat. no.

CADE50A100 and standard run parameter for DE50 droplets (production time 8 minutes).

Briefly, an outer aqueous phase consisting of complete RPMI media (Gibco RPMI 1640, cat. no. 52400-025 Thermo Fisher Scientific, Waltham, MA, USA, ;10 % Fetal Bovine Serum (FBS), cat. no. F9665, Sigma-Aldrich, St. Louis, MO, USA; 1 % Pen/Strep, cat. no. 15140-122, Thermo

Fisher Scientific, Waltham, MA, USA), 1x Stabilizing solution for cells (from Samplix, DE stabilizing solution for cells (3x) 8 lanes, cat. no. REDIVSTABSOL1500, and diluted 1x according to instructions), and 10 vol% iodixanol (iodixanol™ Density Gradient Medium, cat. no. D1556, Sigma-Aldrich, St. Louis, MO, USA) was used. Moreover, a fluorocarbon oil (Novec 7500, 3M Co., Maplewood, MN, USA,). The inner aqueous phase consisted of complete RPMI media and 10 vol%w/v iodixanol.

The double-emulsion cartridge was prepared by loading: 1. 400 pl 1x outer aqueous phase in the first well #A, 2. 40 ul 1x outer aqueous phase to the shelf of outlet well #D, 3. 100 pl of inner aqueous phase into well #C, 4. 200 pl of fluorosurfactant mixture into well #B; and then 5. Placing a gasket to the top of the cartridge (from Samplix, cat. no. GADEA100); and 6. Inserting the cartridge into the Xdrop instrument, running the “DE50” program installed on the instrument. 7. When finished the double-emulsion droplets were collected from the outlet well (#D).

Results

The droplet production was analyzed by microscopy (Figure 1). Double-emulsion droplets and fluorocarbon-in water droplets, oil droplets were observed in a heterogeneous mixture.

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Example 2: Separating / removing oil droplets from water-in -oil-in water double-emulsion mixture

During the production of double-emulsion droplets (water-in-oil-in-water) the formation of single-emulsion droplets (oil-in-water, oil droplets) occurs resulting in a heterogenic mixture. In this example the separation of oil droplets is demonstrated that provides the improvement of downstream analysis like microscopy and flow cytometry.

Material and methods

To separate the oil droplets from double-emulsion droplets iodixanol™ Density Gradient

Medium (Merck KGaA, Darmstadt, Germany) was deposited into 2mL reaction tubes (DNA

LoBind® Tubes, Eppendorf SE, Hamburg, Germany) and a double-emulsion production (containing oil droplets), was loaded on the iodixanol, centrifuged (MYFUGE™ mini centrifuge, 2000 rcf, Sayreville, NJ, USA) analyzed by microscopy (Eclipse Ci, Nikon Instruments Inc,

Tokyo, Japan)

The separation followed the protocol, below: 1. 100-300 pl of the double-emulsion droplets production was transferred to 1000 ul pure iodixanol in two 2 mL reaction tubes, 2. Centrifugation for 5 seconds in a mini centrifuge and check separation, 3. Centrifugation for 5 seconds in a mini centrifuge and check separation, 4. Transfer of upper and bottom phases into new 2 mL tubes, 5. Microscope analysis.

Results

Centrifugation resulted in an upper phase, the iodixanol phase, and a lower phase (Figure 2).

Microscopic analysis showed a heterogeneous mixture comprising double-emulsion droplets and oil droplets before the separation workflow (Figure 3 A). After the separation process single oil emulsions were removed resulting in a homogenous water-in -oil-in water double-emulsion droplet population (Figure 3 B).

Conclusion

This data demonstrates that the double-emulsion droplets of a heterogenous droplet production comprising both double-emulsion droplets and oil droplets can be separated from oil droplets by a brief step gradient centrifugation. The centrifugation also results in an significant improvement in the imaging quality.

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Example 3: Separating / removing oil droplets from water-in -oil-in water double-emulsion droplets with encapsulated cells.

In this example, the separation of double-emulsion droplets containing cells from oil droplets was performed.

Material and methods

The workflow was performed as described in Example 2, Material and methods. The 100 ul of inner aqueous phase that was loaded into well #C contained 10% K562 cells.

Different concentrations (w/v) of iodixanol based on a stock solution of 80%w/v iodixanol (Serumwerk Bernburg AG, Germany) in ultrapure water (Invitrogen™ UltraPure™

DNase/RNase-Free Distilled Water, Fisher Scientific, Waltham, MA, USA) were used to separate double-emulsion droplets containing cells from oil droplets.

The separation followed the workflow in Example 2, Material and methods.

Results

The use of different iodixanol concentrations resulted in different separation qualities (Figure 4). At 60 %w/v iodixanol the complete double-emulsion droplet production sedimented after centrifugation. With increase of the iodixanol concentration the formation of an upper phase and a bottom phase occurred.

As the concentration of the iodixanol density gradient medium was increased, a distinct upper phase and a bottom phase, separated by an imtermediate phase comprising the iodixanol.

Microscopic analysis of the upper phases showed a removal of oil droplets, while the population of double-emulsion droplets containing cells increased with increasing iodixanol concentrations from 60%w/v to 80%w/v (Figure 5 B-F) compared to before the separation workflow (Figure 5

A).

Conclusion

This example shows that different iodixanol concentrations (60%w/v - 80%w/v) can be used to separate double-emulsion droplets containing cells. The use of 80%w/v iodixanol is a prerequisite to obtain the highest yield of cell-containing droplets.

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Example 4: Release of cells from double-emulsion droplets by centrifugation

This example describes the recovery/release of cells from double-emulsion droplets.

Material and Methods

The workflow essentially followed the procedure described in example 3 material and methods.

Briefly a 80% w/v iodixanol (iodixanol in water) was modified by the addition of sodium chloride to a final 0.9% NaCl (w/v). A preparation of double-emulsion droplets with encapsulated cells was loaded on top of the 80% iodixanol/Nacl step gradient and centrifugated 4 min at 21300 rcf.

Results

Microscope analyses showed cells being encapsulated in double-emulsion droplets before the droplet breakage (Figure 6 A). The centrifugation resulted in the breakage of double-emulsion droplets and recovery of free cells. (Figure 6 B).

Conclusion

Surprisingly, double-emulsion droplets are broken and cells are released and recovered from double-emulsion droplets using 80% iodixanol and a brief 4 minute centrifugation.

Example 5: Breakage of single-emulsion droplets and recovery of encapsulated particles or beads.

In this example an iodixanol/iodixanol density step gradient is used to remove the oil from single-emulsion with an encapsulated gel matrix to obtain a pure preparation of gel beads. The method is useful when encapsulating cells in single-emulsion droplets as it avoids using a break-solution which is toxic to cells.

Cell preparation.

HT29 cells were cultured in T175 flasks in Complete DMEM media. The cells were harvested from the flask by first removing the media and washing with 10 mL dPBS followed by removing this fluid as well. Then 2 mL TrypLE™ Express Enzyme (Gibco) was added to the flask which was incubated for 5-10 min at 37°C in the CO» incubator until the cells started to loosen from the bottom of the flask. 10 mL of Dulbecco's Modified Eagle's Medium (DMEM),

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Complete media was added, followed by transfer of cells and media to a 50 mL tube which was spun at 300 g for 8 min to pellet the cells. The supernatant was removed, and the pellet was resuspended in 10 ml Complete DMEM media. The cells were counted and 20 x10' cells were transferred to a 50 mL tube. The cells were spun at 300 g for 8 min, the supernatant removed, and the cell pellet resuspended in 100 ul. Complete DMEM media to a concentration of 0.2 x10° cell/uL. The cell suspension was transferred to a 1.5 mL Eppendorf tube.

Preparation of gel matrix for droplet loading. 200 ul hydrogel gel matrix was prepared for a full Xdrop SE85 cartridge production of single- emulsion droplets. Reagents Mix A and B (Table 1) were prepared separately and mixed right before cartridge loading as the gel will solidify within minutes. Hydrogel components CB, SG-

Dextran, RGD peptide, and CD Link were obtained from Cellendes GmbH.

Table 1. Reagents Mix A and B are required to prepare 200 ul of a 2 mM hydrogel.

WE

ML ML jer | 8 er

TT

[oo

Then mix A and B by pipetting up and down.

Single-emulsion droplet generation and gel solidification. 20jL of the gel matrix with cells was loaded per SE85 cartridge (item# CASE85A100, Samplix

ApS, Birkeroed, Denmark) lane by injecting this volume directly into the inlet well using wide bore pipette. After this 75 uL of dMDA oil (Xdrop® SE Oil I, Samplix ApS, Birkeroed, Denmark) was loaded on top of the inlet well. The SE85 cartridge was sealed with a gasket and the cartridge was loaded into the Xdrop Instrument (item# IN00110, Samplix ApS, Birkeroed,

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Denmark) followed by starting the standard dMDA run (~1 minute). The cartridge was taken out of the Xdrop instrument and droplets were pipetted up from the Collection well into a 1.5 mL Eppendorf tube with 3 holes punctured in the lid with a syringe (see Figure 7, panel A). The

Eppendorf tube was transferred to the CO, incubator at 37°C for 1 hour to solidify the gel.

Release of gel beads from single-emulsion droplets

After the incubation, the droplets were transferred with a pipette and layered on top of 1 mL of iodixanol in a 2 mL Lo-Bind Eppendorf tube. 100 ul of Complete DMEM media was added on top and the Eppendorf tube was transferred into a tabletop centrifuge and spun for 5-10 seconds. The oil was now at the bottom of the Lo-Bind Eppendorf tube and the gel beads in the upper media phase with iodixanol in between this upper and lower phase (see Figure 7B).

The gel beads were pipetted into a 24-well culture plate for adherent cells with 1000 pl of

Complete DMEM media. Figure 8, shows pictures of the formed gel beads taken via an inverted microscope. Fig. 8 left panel shows the gel beads inside single-emulsion droplets; Fig. 8 right panel shows the released gel beads after centrifugation. Cells inside the gel beads are clearly visible in both panels.

Conclusion

This experiment shows that single-emulsion droplets are easily broken and that encapsulated particles or beads are released and recovered from the single-emulsion droplets by a simple density step gradient centrifugation of a few seconds in a standard centrifuge.

Claims (11)

DK 2024 30110 A1 20 CLAIMS

1. A method for separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation.

2 The method according to claim 1, wherein the droplets of the water and fluorocarbon- oil comprising emulsion are aqueous droplets dispersed in a fluorophilic continuous phase.

3. The method according to claim 1, wherein the droplets of the water and fluorocarbon- oil comprising emulsion are aqueous droplets, each encapsulated within a larger droplet of fluorophilic liquid, that are dispersed in a continuous aqueous phase.

4, The method according to claim 3, wherein oil-in-water droplets dispersed in the continuous aqueous phase are selectively removed from the double-emulsion droplets.

5. The method according to claim 3, wherein the continuous aqueous phase of the double-emulsion comprises water-in-oil-in-water droplets with encapsulated particles, and wherein the encapsulated particles are released from the droplets by a brief step gradient centrifugation.

6. The method according to claim 5, wherein a break-solution is added to the continuous aqueous phase of the double-emulsion comprises water-in-oil-in-water droplets with encapsulated particles, and subsequently subjected to a brief step gradient centrifugation to obtain the release of the particles.

7. The method according to claim 2, wherein in the fluorophilic continuous phase of the single-emulsion comprises water-in-oil droplets with encapsulated particles, and wherein the encapsulated particles are released from the droplets by a brief step gradient centrifugation.

8. The method according to any of the preceding claims, wherein the step gradient is formed by a composition which comprises a low osmolarity, non-ionic iodinated density gradient medium such as iodixanol and has a density over 1,3 g/ml.

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9. A density gradient forming composition for the use of separating at least part of the fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation characterized in that the composition comprises a low osmolarity, density gradient medium and a colour for easier recognition of phases.

10. The composition according to claim 9, wherein the composition has a density over 1,3 g/ml, comprises a salt, has a final osmolarity of approximately 290 mOsm and a final pH between 5 and 8,5.

11. AKit for separating at least part of a fluorocarbon oil from a water and fluorocarbon-oil comprising emulsion by a brief step gradient centrifugation of an emulsion, comprising: - a composition according to claims 9 or 10, - at least one centrifuge tube, and - directions for performing the method of any of claims 1 — 8.