WO2025045628A1 - Autonomous microfluidic cell culture platform - Google Patents

Abstract

A cell culture platform for integration into detection systems, in particular microscopy systems, wherein the cell culture platform comprises: a microfluidic system; at least one cell culture component; and a drive system at least comprising a micropump, a control unit and an energy supply unit.

Description

Autonomous microfluidic cell culture platform

The invention relates to an autonomous microfluidic cell culture platform.

background

In vitro cell culture systems are essential in science and industry, for example to investigate basic cellular processes, diseases or the effectiveness of drugs. Cells are not cultivated statically as usual, but dynamically under an applied flow, for example to reconstruct blood flow and create physiologically relevant conditions. The flow induces shear forces that lead to a change in cellular processes such as cell differentiation, membrane organization, cell orientation, etc. This has an impact on the toxicity and effect of substances such as drugs and materials for medical products. Dynamic cell culture systems therefore show great potential in drug testing in the pharmaceutical industry, as they better depict the pharmacology and pharmacokinetics of an active ingredient, such as the absorption of active ingredients into the cell. In this way, on the one hand, ethically questionable animal testing can be significantly reduced and, on the other hand, drug discovery and development can be accelerated. On the other hand, the use of human cells within dynamically cultivated cell culture systems could bring the problem of limited transferability from animals to humans closer to a solution. This also creates immense economic benefits, as drug candidates that are ineffective in humans can be sorted out early and the failure of drug candidates after costly clinical phases can be reduced. In addition to drug research, dynamically cultivated cell culture systems can also be used for the basic investigation of diseases. For example, complex infection processes caused by viruses or bacteria that are influenced by an adjacent current can be better investigated and understood.

Despite the enormous potential of dynamically cultivated cell culture systems, these are by no means established and are mainly found as proof of concept systems in scientific research. They usually consist of so-called microfluidic systems and require precise pumps and microscopes with special Top-mounted incubators that lead to complex experimental setups and require a high level of expertise in this field. Access to these is difficult for research groups or companies that only have expertise in general cell culture, but not in the use or even development of dynamically cultivated cell culture systems.

The monitoring and analysis of the cell culture within the cell culture systems is usually carried out using standard microscopes in combination with top-mounted incubators or on-stage incubators. For classic, static cell culture in tissue culture plates in well-plate format and in the absence of flow, so-called live cell imaging systems (“real-time cell imaging systems”) are increasingly being used, which enable fully automated, continuous monitoring of cells and thus accelerate work processes and significantly increase the data yield and knowledge gained. Numerous different commercial systems are available, which are equipped with everything from inexpensive light microscopes to expensive confocal fluorescence microscopes. All systems have in common that they can either be operated in a conventional incubator or serve as an incubator themselves. However, the integration of dynamic cell culture systems into live cell imaging systems is a major challenge, since tubes or cables that connect the cell culture system to peripherals such as pumps and control electronics are usually difficult or even impossible to integrate into live cell imaging systems.

The present invention is therefore based on the object of providing an autonomously usable, portable cell culture platform for dynamic cell cultivation that is compatible with common microscopy systems.

Summary of the Invention

The solution to this problem in a cell culture platform of the type mentioned at the beginning consists in particular in that the cell culture platform comprises: a microfluidic system, a cell culture component and a drive system comprising at least one micropump, a control unit and a power supply unit. The invention describes a preferably rechargeable or battery-operated and thus portable cell culture platform, the external dimensions of which are based on a standardized tissue culture plate in the so-called well-plate format and can thus be operated within live cell imaging systems. The system consists of a microfluidic chip, which contains access for, for example, four cell culture components. Each platform contains at least one media circuit, which is operated separately by a micropump, which is located directly on the microfluidic chip as part of the drive system. The pumps are operated independently via a suitable controller and a replaceable battery, so that hoses and cables can be dispensed with. In contrast to cell culture platforms already available for purchase, the use of the platform in commercial live cell imaging systems is made possible.

Preferably, the microfluidic system, the at least one cell culture component and the drive system are coupled to the cell culture platform in a form-fitting and/or force-fitting manner. A form-fitting coupling describes in particular that all components are one functional component. A force-fitting coupling has the advantage that the individual components can be retrofitted or plugged in. A form-fitting and force-fitting coupling is present, for example, when the platform is first cast during production and then, for example, the drive system is plugged on and then soldered.

Preferably, the dimensions of the cell culture platform correspond to the size standard according to the ANSI/SBS 1-2004 standard for microtiter plates. In particular, the cell culture platform has a length of 120-130 mm, a width of 80-90 mm and a height of 50 mm. In one embodiment, these dimensions are maximum dimensions. This makes the cell culture platform compatible with standardized systems, such as automated imaging systems, incubators and robotic systems.

In one embodiment, the microfluidic system is designed to be detachably connected to the drive system. This allows, for example, the sterilization of the microfluidic Systems separated from the drive system. This makes the microfluidic system interchangeable and suitable for single-use applications.

In one embodiment, the cell culture component is part of the microfluidic system, wherein in particular the cell culture component is a cell culture chamber with an inlet and an outlet that is closed when coupled. This enables simple manufacture of the microfluidic system together with the predefined cell culture component.

In a further embodiment, the cell culture component comprises a device, in particular a connection, for cell culture inserts. External cell culture inserts can thus be connected to the device without tubes, which further simplifies the autonomous operation of the cell culture platform and makes it usable in a modular manner.

In one embodiment, the cell culture platform comprises at least one medium reservoir (reservoir for cell culture medium), which is arranged in particular laterally adjacent to the cell culture component on the cell culture platform and/or which is arranged in particular vertically adjacent to the cell culture component. This increases the compactness of the system and thus its throughput or operating time.

In one embodiment, the drive system is designed such that the cell culture platform can be operated for at least 24 hours, in particular at least 72 hours, and in particular at least 100 hours. This allows investigations of cell cultures to be carried out over a long period of time without interruption. Furthermore, the (in particular first) battery module can be easily replaced after, for example, one day.

In one embodiment, the drive system is covered by a lid. In another embodiment, the lid can be connected to the cell culture platform by means of a screw connection, plug connection and/or magnetic connection. The screw connection, plug connection and/or magnetic connection can be connected in a form-fitting manner to the cell culture platform by means of a seal. This means that the drive system, in particular electronic components of the drive system, can be protected from liquids, for example from cell culture medium or rinsing medium. Furthermore The drive system, in particular electronic components of the drive system, can be sealed against the high humidity of, for example, 95% or more present in incubators and protected against corrosion.

In one embodiment, the at least one micropump of the drive system can be operated by direct current. In this way, extensive electronics, which are required for alternating current-operated pumps, are avoided and the necessary compactness is achieved.

In a further embodiment, the control electronics comprise communication modules for wireless communication with the platform. This enables wireless operation in closed imaging systems, preferably for an operating period of 1 or 3 days.

In one embodiment, the energy supply comprises a replaceable and rechargeable first battery module and a second battery module that is permanently connected to the cell culture platform. The first battery module preferably has a greater storage capacity than the second battery module. The first battery module can also preferably be used for the long-term supply of the system and the second battery module can be used to operate the cell culture platform when the first battery module is replaced. This makes it possible to bridge the time required to replace the first battery module with the second battery module.

A second aspect of the present invention describes a method for implementation in a detection system, in particular in a microscopy system. In the method, an examination of a cell culture is carried out using an autonomously operable cell culture platform, in particular the cell culture platform described above. The method allows the examination of cell cultures in common detection and microscopy systems without the need for special accessories. This is made possible in particular by the autonomous operation of the cell culture platform.

A third aspect of the present invention describes a use of a drive system on a cell culture platform, in particular the one described above Cell culture platform, wherein the drive system is designed such that the cell culture platform can be operated autonomously by means of the drive system. This allows the cell culture platform to be used in common detection systems, such as microscopy systems, without the need for special, complex and often expensive connection systems or external drive systems.

In one embodiment, the drive system comprises at least one micropump, a control unit and a power supply unit.

In a further embodiment, the cell culture platform can be operated by the drive system for a running time of at least 24 hours, in particular at least 72 hours, further in particular at least 100 hours. This enables the uninterrupted examination of cell cultures over long periods of time.

The invention is described in more detail below using several preferred embodiments.

It shows:

Fig. 1 a cell culture platform;

Fig. 2 a microfluidic system with direct integration of the cell culture component in side view;

Fig. 3 a microfluidic system with direct integration of the cell culture component in top view;

Fig. 4 a microfluidic system with indirect integration of the cell culture component in side view;

Fig. 5 a microfluidic circuit with several cell culture chambers (shown here in the same way);

Fig. 6 shows a section of a cell culture platform with a medium reservoir arranged vertically to the cell culture component in side view;

Fig. 7 shows a section of a cell culture platform with a medium reservoir arranged vertically to the cell culture component in top view;

Fig. 8-10 Possibilities for attaching a housing of the drive system to a cell culture platform; Fig. 11 a drive system on a cell culture platform.

Identical or similar features are provided with the same reference symbols.

The following examples serve to illustrate the invention without limiting its scope, but may themselves represent specific embodiments of the invention.

Cell cultures include biological cells of any kind, including eukaryotic cells, but also microorganisms and viruses, for example.

Figure 1 shows an autonomously operable cell culture platform 100 according to the invention with eight cell culture components 104, each of which is connected to the drive system 106 via a microfluidic system 102 with eight medium reservoirs 114. The drive system 106 comprises, by way of example, eight micropumps 108, a control unit 110, a first battery module 118 and a second battery module 120. The microfluidic system 102 connects in particular each of the eight cell culture components 104 to a micropump 108 via a medium reservoir 114. Each micropump 108 is communicatively connected to the control unit 110 by means of a cable connection inside the drive system 106. The two battery modules 118, 120 supply the control unit 110 and the micropumps 108 with energy.

Exemplary, preferred embodiments of the individual components of the cell culture platform 100 are described below. Unless otherwise stated, all components can be combined with one another. Preferred component combinations are marked as such.

The microfluidic system 102 comprises a channel system and connects the cell culture components 104 with the medium reservoir(s) 114 and the drive system 106, in particular with the micropumps 108. The microfluidic system 102 thus comprises connection elements or interfaces to the drive system 106. Optionally, the microfluidic system 102 can additionally comprise valves and/or sensors The microfluidic system 102 contains all the cell culture medium for dynamic cultivation of cells in one or more medium circuits.

The microfluidic system 102 can be manufactured, for example, by milling the microchannels, injection molding of plastics or by subsequent sealing by plastic welding. Alternatively, the microfluidic system 102 can be manufactured using lithographic processes or additive manufacturing or 3D printing.

Preferably, the microfluidic system 102 is separable from the drive system 106, so that in particular a separate sterilization of the microfluidic system 102 is possible.

The microfluidic system 102 consists of one or more types of plastic. The material is preferably biocompatible, for example in accordance with ISO EN 10993 (version 2023), with animal cells. This enables use in animal cell culture. The microfluidic system 102 is preferably optically transparent and not or only weakly absorbent for wavelengths between 300 nm and 900 nm and has low autofluorescence (at least in the area of the cell culture(s)) to allow the cells to be examined using different microscopy techniques.

Preferably, the microfluidic system 102 can be sterilized by gamma or beta irradiation. Alternatively, the microfluidic system 102 can be sterilized by heat sterilization (dry or steam) or chemical sterilization (ethylene oxide, hydrogen peroxide, chlorine dioxide, peroxyacetic acid, nitrogen dioxide). Combinations of several methods are possible.

Preferably, the microfluidic system 102 is implemented as a single-use component that can be exchanged. This enables the cell culture platform 100 to be used quickly and easily. Furthermore, the risk of damaging the cell culture platform 100 during cleaning is avoided. In addition, potential residues and contamination from previous cell examinations are avoided, so that a higher reproducibility of experimental results can be achieved. A channel system connects all subcomponents of the microfluidic system 102 to one another and thus serves to distribute cell medium and additives. The channel system consists of at least one closed circuit that connects a cell culture chamber or a cell culture component 104, a medium reservoir 114 and the interface(s) to a micropump 108 to one another, as shown in Figure 2 or 3, for example. Alternatively, a circuit can also connect several cell culture components 104 on a chip, as shown in Figure 5. The several cell culture components 104 can be of the same or different types. Such a circuit serves for parallelization or the possibility of examining interactions between different cell culture components 104.

The cell culture components 104 contain the cells that are cultured and examined under flow and the effect of shear forces. The cell culture components 104 are preferably arranged according to the dimensions of standardized cell culture plates. The positioning and arrangement of the cell culture components 104 preferably follows commercially available 6-, 12-, 24-, 48-, 96-, or 384-well plate systems. This ensures integration of the cell culture platform 100 for the use of automated microscopy by standardized microscopes.

The cell culture component 104 consists of a cell culture chamber that is connected to an inlet and outlet or multiple inlets and outlets of the channel system of the microfluidic system 102. The cell culture component 104 can be integrated directly or indirectly into the microfluidic system 102. Figures 2 and 3 show exemplary arrangements of a direct integration of the cell culture component 104 as part of the microfluidic system 102 in side view and top view. The cell culture component 104 is designed in the form of a closed cell culture chamber with an inlet and outlet. This embodiment enables simple manufacture of the microfluidic system 102 together with a predefined cell culture component 104.

In an alternative embodiment, the cell culture component 104 can be designed to be indirectly connectable to the microfluidic system 102, as shown in Figure 4. In this embodiment, the cell culture component 104 contains a connection or a device for connecting external cell culture inserts. These external Cell culture inserts can then be connected to the device without tubes, for example. In this embodiment, there is a seal between the inlets and outlets of the microfluidic system 102 and the cell culture insert.

The cell culture platform 100 preferably comprises at least one medium reservoir 114. The medium reservoir 114 serves as an access point for adding or removing the medium from the microfluidic system 102. Such a medium reservoir 114 consists of at least one outlet and inlet for the connection to the channel system and an upward opening for adding or removing the medium. The at least one medium reservoir 114 is arranged according to the dimensions of standardized cell culture plates, with arrangements of 6, 12, 24, 48, 96, or 384 cell plates being possible. This enables the automation of the medium change by pipetting robots.

The at least one medium reservoir 114 can be adjacent to the cell culture component 104 or arranged vertically above it. A vertical arrangement of a medium reservoir 114 above a cell culture component 104 is shown in Figures 6 and 7 in side view and top view. Here, the bottom of the reservoir is not directly open to the cell culture component 104, but is connected to the cell culture component 104 via the channel system. In this case, the medium reservoir 114, like the cell culture component 104, meets all (in particular above-mentioned) material requirements for microscopy (transparency, low autofluorescence). In this case, medium reservoirs 114 and cell culture components 104 can, for example, be made from two different injection-molded parts and then connected by plastic welding.

The drive system 106 of the cell culture platform 100 according to the invention comprises at least one micropump 108, a control unit 110 and at least one energy supply unit 112 as shown in Figure 1. In addition, the drive system 106 can contain sensors and/or valves and corresponding interfaces to these components.

The service life of the cell culture platform 100 according to the invention is at least 24

hours, preferably at least 72 hours, particularly preferably at least 100

hours. The service life depends on the capacity of the power supply unit 112, in particular the capacity of the first battery module 118 described in more detail below, and the energy consumption of the cell culture platform 100. The consumption of the cell culture platform 100 is primarily determined by the energy consumption of the micropumps 108. Furthermore, the control unit 110 or communication unit or other sensors can contribute to the energy consumption. The service life can be approximately determined from the product of the operating time of the energy supply unit 112 and the number of micropumps 108. Due to the compact design of all electronic components and the arrangement on the microfluidic system 102, the product of autonomous operating time and the number of continuously and independently operated micropumps 108 can be at least 100 hours in one embodiment. For example, a cell culture platform 100 with 4 independent continuously operated micropumps 108 can be operated for a period of at least 25 hours. This ensures a sufficiently long cultivation period within closed systems without an external power supply.

The drive system 106 additionally comprises a housing 116 which, after being attached to the microfluidic system 102, protects all internal parts of the drive system 106 from moisture, as shown in Figures 8-10. The housing 116 serves to protect the electronics from corrosion caused by high humidity in an incubator. For this purpose, a seal 122 (Figure 8) is located at the contact points 124 between the housing 116 and the cell culture platform 100. Other elements of the drive system 106 which point outwards through the housing 116 are sealed by gluing or sealing. Examples of such elements are buttons, displays and/or LEDs. The housing 116 can be placed, plugged or slid onto the cell culture platform 100, for example, using a screw connection (Figure 9) or a magnetic connection (Figure 10), or can be removed from the cell culture platform 100. This allows a safe and easy attachment of the drive system 106 after sterilization of the microfluidic system 102.

The drive system 106 further comprises at least one micropump 108. Such a micropump 108 is in direct contact with the microfluidic system 102. In a preferred embodiment, the at least one micropump 108 can be operated by direct current. In this way, extensive electronics, which are required for alternating current-operated pumps, are avoided and the necessary compactness is achieved. The or the pumps can induce the flow within a circuit via a flexible element of the microfluidic system 102, such as integrated hoses or membranes. The micropumps 108 can be designed, for example, as peristaltic pumps, ball bearing squeeze pumps or membrane pumps. The micropumps 108 can be operated continuously and enable an uninterrupted media flow. In other words, the micropumps 108 can be operated, for example, during microscopy in detection systems, during transport by robotics or during the exchange of the medium. As a result, the cells in the cell culture component 104 are exposed to physiological flows at all times. Stopping the flow, as is necessary in other systems, can have an unwanted negative influence on the cultivation conditions and the test results and can be avoided in the cell culture platform 100 according to the invention.

The drive system 106 also includes a control unit 110. Such a control unit 110 includes a controller with a microprocessor and integrated memory that acts as a computer, driver modules for controlling micropumps 108, valves and/or sensors, and communication modules for wireless communication with the cell culture platform 100. The controller processes and issues commands using firmware stored on it, as well as controlling and supplying power to all electronic or mechatronic components of the drive system. Communication takes place, for example, via Bluetooth or wireless LAN (WLAN) and is used to send commands to the cell culture platform 100 and to read data on the cell culture platform 100. Alternatively, a predefined program can be loaded onto the platform, started and executed, or the data can be subsequently downloaded from the platform.

The drive system 106 comprises an energy supply unit 112. The energy supply unit 112 consists of at least one (preferably separable) energy storage device that supplies the electronic components of the drive system 106 with power. Preferably, the energy supply unit 112 consists of a replaceable and rechargeable first, larger battery module 118 for the long-term supply of the system and a second, smaller, permanently installed battery module 120 that enables the operation of the platform when the larger battery module 118 is replaced for a short time. The first, larger battery module 118 can be attached to the drive system 106 while the drive system 106 is already mounted on the microfluidic system 102 and is in operation, as shown in Figure 11. The battery module 118, 120 can be plugged or pushed on, or magnetically attached and secured by snapping into place. The battery module 118, 120 is connected to the drive system 106 by contacts for power transmission. The battery module 118, 120 and the drive system 106 are either sealed separately or the seal is only created when they are attached to ensure protection against moisture. The battery module 118, 120 has a separate power connection so that it can be charged separately from the platform. Preferably, several larger battery modules 118 are used so that an empty battery module can be replaced by a charged one. This allows the battery to be replaced after a certain period of time in order to extend the overall cultivation time. The second, smaller battery module 120 consists of a small battery which is always charged by the larger battery module 118 and is only discharged when the larger battery module 118 is replaced. The terms “larger” and “smaller” battery module refer to a larger or smaller energy storage capacity.

Claims

claims 1. Cell culture platform (100) for integration into detection systems, in particular into microscopy systems, the cell culture platform (100) comprising: a microfluidic system (102); at least one cell culture component (104); and a drive system (106) comprising at least one micropump (108), a control unit (110) and a power supply unit (112). 2. Cell culture platform (100) according to claim 1, wherein the microfluidic system (102), the at least one cell culture component (104) and the drive system (106) are positively and/or force-fittingly coupled to the cell culture platform (100). 3. Cell culture platform (100) according to claim 1 or 2, wherein the dimensions of the cell culture platform (100) correspond to the size standard according to the ANSI/SBS 1-2004 standard for microtiter plates, in particular wherein the cell culture platform (100) has a length of 120-130 mm, a width of 80-90 mm and a height of 50 mm. 4. Cell culture platform (100) according to one of the preceding claims, wherein the microfluidic system (102) is detachably connectable to the drive system (106). 5. Cell culture platform (100) according to one of the preceding claims, wherein the at least one cell culture component (104) is part of the microfluidic system (102), in particular wherein the at least one cell culture component (104) is a cell culture chamber with inlet and outlet that is closed in the coupled state. 6. Cell culture platform (100) according to one of the preceding claims, wherein the at least one cell culture component (104) comprises a device, in particular a connection, for cell culture inserts. 7. Cell culture platform (100) according to one of the preceding claims, additionally comprising at least one medium reservoir (114), which is arranged in particular laterally adjacent to the at least one cell culture component (104) on the cell culture platform (100). and/or which is arranged in particular vertically adjacent to the at least one cell culture component (104). 8. Cell culture platform (100) according to one of the preceding claims, wherein the Drive system (106) is designed such that the cell culture platform can be operated for at least 24 hours, in particular at least 72 hours, further in particular at least 100 hours 9. Cell culture platform (100) according to one of the preceding claims, wherein the drive system (106) is covered by a housing (116) and/or wherein the housing (116) can be connected to the cell culture platform (100) by means of a screw connection, plug connection and/or magnetic connection and/or wherein the screw connection, plug connection and/or magnetic connection can be positively connected to the cell culture platform (100) by means of a seal (122). 10. Cell culture platform (100) according to one of the preceding claims, wherein the at least one micropump (108) is operable by direct current. 11. Cell culture platform (100) according to one of the preceding claims, wherein the control unit (110) comprises a communication module for wireless communication between the cell culture platform (100) and a computer system. 12. Cell culture platform (100) according to one of the preceding claims, wherein the energy supply unit (112) comprises a replaceable and rechargeable first battery module (118) and a second battery module (120) permanently connected to the cell culture platform (100). 13. Cell culture platform (100) according to claim 12, wherein the first battery module (118) has a greater storage capacity than the second battery module (120) and/or wherein the first battery module (118) can be used for the long-term supply of the cell culture platform (100) and the second battery module (120) can be used for the operation of the cell culture platform (100) when the first battery module (118) is replaced. 14. Method for execution in a detection system, in particular in a microscopy system, wherein an examination of a cell culture is carried out by means of an autonomously operable cell culture platform (100), in particular the cell culture platform (100) from claims 1-13. 15. Use of a drive system (106) on a cell culture platform (100), in particular the cell culture platform (100) from claims 1-13, wherein the drive system (106) is designed such that the cell culture platform (100) can be operated autonomously by means of the drive system (106). 16. Use of the drive system (106) of claim 15, wherein the drive system (106) comprises at least one micropump (108), a control unit (110) and a power supply unit (112). 17. Use of the drive system (106) of claim 15 or 16, wherein the Cell culture platform (100) can be operated by the drive system (106) for a running time of at least 24 hours, in particular at least 72 hours, further in particular at least 100 hours.