Abstract
The present invention relates to a microfluidic cell culture system comprising at least one microfluidic structure, wherein the at least one microfluidic structure comprises a cell culture chamber, a first and second reservoir in fluid communication with each other via the cell culture chamber, wherein the microfluidic cell culture system further comprises a detachable seal for sealing the at least one microfluidic structure and wherein the microfluidic cell culture system is configured such that the first and second reservoir of the at least one microfluidic structure are in fluid communication with each other via a communication channel that does not comprise the cell culture chamber.
Claims
1. A microfluidic cell culture system comprising at least one microfluidic structure, wherein the at least one microfluidic structure comprises: a cell culture chamber for holding a medium for culturing cells, the cell culture chamber comprising a microfluidic inlet opening and a microfluidic outlet opening; a first reservoir in fluid communication with the cell culture chamber via the microfluidic inlet opening; and a second reservoir in fluid communication with the cell culture chamber via the microfluidic outlet opening, wherein the microfluidic cell culture system further comprises a detachable seal for sealing the at least one microfluidic structure, and wherein the first and second reservoir of the at least one microfluidic structure are in fluid communication with each other via the cell culture chamber, characterised in that the microfluidic cell culture system is configured such that the first and second reservoir of the at least one microfluidic structure are in fluid communication with each other via a communication channel that does not comprise the cell culture chamber, and in that the communication channel is bidirectional.
2. The microfluidic cell culture system according to claim 1, wherein the fluidic resistance through the communication channel between the first and second reservoir is lower than the fluidic resistance through the cell culture chamber between the first and second reservoir, preferably wherein the fluidic resistance through the communication channel is at least 5 times lower than the fluidic resistance through the cell culture chamber, more preferably at least 10 times lower, even more preferably at least 50 times lower.
3. The microfluidic cell culture system according to claim 1, wherein the communication channel is configured to exchange a gaseous medium, such as nitrogen, oxygen or air, between the first reservoir and the second reservoir.
4. The microfluidic cell culture system according to claim 1, wherein the microfluidic structure comprises one or more capillary pressure barriers, such as phaseguides, in order to provide one or more subvolumes, and wherein the one or more capillary pressure barriers are arranged in the cell culture chamber, thereby providing one or more subvolumes.
5. (canceled)
6. (canceled)
7. The microfluidic cell culture system according to claim 4, wherein the one or more subvolumes are in fluid communication with the first and second reservoir of the at least one microfluidic structure or wherein the one or more subvolumes are in fluid communication with one or more further reservoirs comprised by the at least one microfluidic structure, wherein each of said one or more further reservoirs of the at least one microfluidic structure are in fluid communication with another reservoir of the at least one microfluidic structure via a further communication channel.
8. (canceled)
9. (canceled)
10. The microfluidic cell culture system according to claim 1, wherein the cell culture chamber comprises one or more cells and/or tissues.
11. The microfluidic cell culture system according to claim 1, wherein the first and second reservoirs and/or the cell culture chamber are provided with a reversible solidifying medium comprising a solidifying agent and an aqueous medium.
12. (canceled)
13. (canceled)
14. The microfluidic cell culture system according to claim 1, wherein the communication channel comprises a passage provided in the at least one microfluidic structure or a recess provided in a surface of the at least one microfluidic structure facing the inner side of the detachable seal wherein the passage and the recess are configured for connecting in fluid communication the first reservoir with the second reservoir of the at least one microfluidic structure.
15. The microfluidic cell culture system according to claim 1, wherein the detachable seal is configured to airtight seal the at least one microfluidic structure and wherein the detachable seal is configured to be located relative to the at least one microfluidic structure such that the inner side of the seal is located at some distance from the reservoirs of the at least one microfluidic structure.
16. The microfluidic cell culture system according to claim 1, wherein the microfluidic cell culture system comprises a plurality of microfluidic structures and wherein one or more of the reservoirs of one microfluidic structure is in fluid communication with one or more reservoirs of one or more of the other microfluidic structures.
17-20. (canceled)
21. The microfluidic cell culture system according to claim 1, wherein at least a part of the detachable seal comprises: a semipermeable barrier configured to allow exchange from the microfluidic cell culture system to its external environment and/or vice versa, wherein the semipermeable barrier is configured to be impermeable for a one or more predefined substances or quantities such as a gaseous medium, humidity, heat, moisture, a particle, a microbe, electricity, radiation and/or a virus; and/or a vent, preferably a one-way vent, for providing fluid communication from the reservoirs of the at least one microfluidic structure to the external environment of the microfluidic cell culture system and/or vice versa.
22-25. (canceled)
26. A method for transporting microfluidic cell culture systems, the method comprising the steps of: providing one or more microfluidic structures, wherein the one or more microfluidic structures optionally comprise an extracellular matrix and/or cells or cell aggregates, and wherein the microfluidic structures optionally comprise a solidified solidifying medium sealing the one or more microfluidic structures to form the microfluidic cell culture system according to claim 1; and transporting the sealed microfluidic cell culture system
27. The method according to claim 26, wherein the solidifying medium replaces all or part of any cell culture medium present in the microfluidic structure.
28. The method according to claim 26, wherein the method further comprises the step of: after transporting the microfluidic cell culture system, unsealing the one or more microfluidic structures; optionally, allowing the solidified solidifying medium inside the one or more microfluidic structures to liquefy; and optionally, adding fresh cell culture medium to the reservoirs.
29. A kit of parts comprising one or more microfluidic structures and a seal, wherein the one or more microfluidic structures and the seal are configured such that in assembled form the microfluidic cell culture system according to claim 1 is formed.
Description
EXAMPLES
[0054] FIG. 1. Modelling intestinal tubules using the OrganoPlate platform (Trietsch et al. NComms, 2017)
[0055] The OrganoPlate® platform encompasses 40 microfluidic cell culture structures embedded in a standard 384-well microtiter plate format (FIGS. 1a and b, Trietsch et al. NComms, 2017) Trietsch et al. Lab Chip, 2013, Wevers et al. Sci. Rep., 2016. Each microfluidic channel structure is comprised of three lanes that are connected to corresponding wells of a microtiter plate that function as inlets and outlets to access the microfluidic culture. The lanes join in the centre of the structure where two capillary pressure barriers are present called phaseguides (Vulto et al. Lab Chip, 2011). FIG. 1c-j, Trietsch et al. NComms, 2017 shows a schematic representation of vertical and horizontal cross-sections of the centre of a microfluidic structure and the method of growing a tubular structure. First, an ECM gel is introduced in the central lane (FIG. 1c, d, Trietsch et al. NComms, 2017). The phaseguides are used to selectively pattern the ECM gel in the central lane by meniscus pinning. The meniscus stretches beyond the phaseguide, leading to a curved shape. After ECM gelation, epithelial cells are seeded in one lateral lane, allowing them to sediment directly against the ECM gel by placing the titre plate in a vertical position, i.e., standing on one side (FIG. 1e-h, Trietsch et al. NComms, 2017). Upon attachment of the cells, the plate is horizontally placed on an interval rocker that induces flow by reciprocal levelling between reservoirs. Upon application of flow, cells proliferate and start lining all surfaces of the perfusion channel, forming a confluent tubular structure (FIG. 1i, j, Trietsch et al. NComms, 2017). The tubules have a lumen that is connected to the in- and outlet of the respective lanes, making them accessible for perfusion with medium and for apical compound exposure. The basal side of the epithelium is facing the ECM gel and can be accessed by the second perfusion lane on the opposite side of the ECM gel lane. FIG. 1k Trietsch et al. NComms, 2017 depicts an artist impression of the 3D configuration of the tube, showing that the tube is grown directly against the ECM, without the presence of artificial membranes. For modelling of the intestinal barrier, the human intestinal colorectal adenocarcinoma cell line (Caco-2) was used. FIG. 1l-p, Trietsch et al. NComms, 2017 shows phase-contrast pictures of tube formation taken from the observation window well at day 0, 1, 4, 7, and 11, respectively. On day 0, cells are seeded against the ECM and start colonizing the glass walls to form a confluent tube (FIG. 1n-p, Trietsch et al. NComms, 2017). Perfusion was crucial for tube formation.
[0056] FIG. 2. Shipment of CaCo-2 cultures in an OrganoPlate® from Mimetas Leiden to Mimetas US located in Maryland, United States.
[0057] Plates were transported the industry standard way with microtiter plate seals. Brightfield images were used to capture before and after shipment state of the cultures. The CaCo-2 cultures were grown in the OrganoPlate® as previously described (adapted from Trietsch et al. NComms, 2017). The tube cultures were captured with brightfield imaging using an automated imaging system (Molecular Devices, ImageXpress Pico) and then prepared for shipment. This required preparing a 2.5% gelatine-medium solution by dissolving 2.5 g of gelatine powder (Sigma G9391) per 100 mL of CaCo-2 medium, and filtering with 0.22 μ filter once dissolved. All medium in the inlets/outlets of the OrganoPlate® was aspirated, 40 μL of the warmed gelatine-medium was added to each inlet/outlet. An adhesive clear seal (VWR catalogue 391-1251) was placed on top and pressed to complete sealing of all individual wells across the plate. The plate was then placed into a box for shipment. Upon arrival, brightfield images were taken at Mimetas US with an automated imaging system (BioTek, Cytation 1 Cell Imaging Reader) and compared with those taken prior to packaging at Mimetas Leiden (FIG. 2A). A closer view at single chip images show the difference in tube morphology prior to and after shipment (FIG. 2 B). With this packaging method, there were a number of CaCo-2 tubes that were damaged, visible by tube collapse or tube expansion into the ECM lane as indicated by the arrows (FIG. 2A-B).
[0058] FIG. 3. Creation of a secondary route of fluid communication for the microfluidic chips.
[0059] The secondary rout of communication allows uniform equilibration of potential pressure differences during transport. To simulate shipment via air cargo transport, a low-pressure chamber was set up using a vacuum pump and airtight Tupperware. The tube cultures were generated as previously described and prior to shipment simulation their morphology was assessed visually with brightfield imaging and their functionally was assessed using the barrier integrity assay (Trietsch et al. NComms, 2017, WO2017/007325A1). To do this, 4.4 kDa TRITC-Dextran (Sigma-Aldrich T1037) diluted into medium at 0.5 mg/mL was added to all top channels and the observation window was imaged with TRITC microscope filter after t=15 minutes on an automated imaging system. To process for packaging, all inlets/outlets were aspirated, and different medium solutions were added to each OrganoPlate® inlets/outlets as follows (FIG. 3A): Row 1: 40 μL of 2.5% gelatine-medium prepared as described above, Row 2: 40 μL 5% gelatine-medium prepared as above with 5 g/100 mL of medium, Row 3: 120 μL 2.5% gelatine-medium prepared as above, Row 4: 120 μL 5% gelatine-medium prepared as above with 5 g/100 mL medium, Row 5: 120 μL medium. The observation windows of the plates were brightfield imaged using transmitted light on the ImageXpress Pico automated microscope, prior to shipment. One OrganoPlate® was sealed with an adhesive clear seal as done previously, while the other was sealed only at the perimeter of the lid, without separating individual wells. Both plates were then placed into the vacuum chamber. The shipping simulation was run by decreasing the pressure in the chamber to 800 mBar for 18 minutes to induce pressure changes, and then returned to atmospheric pressure and room temperature overnight to simulate the continued time in ground transit. The following morning, brightfield images were captured and another barrier integrity assay was run with the ImageXpress Pico as previously described. Both the brightfield (FIGS. 3 B-E) and t=15 minute TRITC fluorescent images (FIG. 3 F-G) acquired before and after shipment were compared. The images of the plate with individually sealed wells (FIG. 3 B, D, F) from after simulated shipment show both damaged Caco-2 tubes and bubbles in the chips (FIG. 3 B, D) in addition to decrease barrier function observed by permeation of TRITC-dextran to the ECM channel (FIG. 3 F). The images of the plate with perimeter seal showed no visible displacement or damage to the Caco-2 tube structure (FIG. 3 C, E), and little change to the barrier function (FIG. 3 G). These observations are quantified as a percentage of functional tubes relative to the number of functional tubes on that plate prior to shipment simulation (FIG. 3 H), where there is a clear decrease in both morphologically intact and functional barrier tubes with the adhesive sealed plate. This study confirms that the disruption of the CaCo-2 culture in the microfluidic plate under a change in pressure is due to the seal, where fluid communication is only allowed through the microfluidic channels themselves. With only a perimeter seal, fluid communication is possible through the top of the well and any pressure difference can equilibrate via this route to maintain tissue structure in the chip.
[0060] FIG. 4. Creation of a secondary route of fluid communication for the microfluidic chip.
[0061] Results of the proposed method with a real shipment. To confirm the feasibility of real life shipping, without individually sealing all reservoirs of a cell culture device, a shipment of CaCo-2 cultures in an OrganoPlate® was sent from Mimetas Leiden to Mimetas US. CaCo-2 cultures were grown as described, packaged by aspirating all inlet/outlet medium and replacing with 40 μL 2.5% or 4% gelatine-medium. (Columns 1-4 2.5% Columns 5-8 4%). A perimeter seal was applied to the device, which sealed the reservoirs of the device from outside influences, but allowed fluid communication between the wells. Medium compositions were chosen to maintain a gelled solution that would not spill out of the plate during normal transport forces. It should be noted that while the gelled solution is able to withstand inertial forces to prevent spillage, such gelled solution is typically not able to withstand the pressure differences induced by pressure changes associated with shipment of individually sealed reservoirs, as shown above by displacement or other distortion of the ECM and cells. Brightfield images were captured with the ImageXpress Pico automated microscope before at Mimetas Leiden, and after shipment to Mimetas US with the Cytation 1 automated microscope. The package was sent with a pressure data logger (MadgeTech, PRHTemp101A) to record absolute pressure. Comparing the before and after images, there was no damage, displacement, or trapped bubbles found in the plate upon receiving (FIG. 4A-B). The data logger did indicate several fluctuations in pressure and reached a minimum of 818 mBar during the transport (FIG. 4 C). Comparing different plates and shipments from The Netherlands to the US, the proposed method results in a higher percentage of functional tube tissues than the adhesive seal method following a real courier shipment (FIG. 4 D). This confirms that using gelled medium within the reservoirs of a microfluidic cell culture system, where multiple reservoirs are in fluid communication with each other forms a feasible method to ship microfluidic titerplates.
[0062] FIG. 5 shows a schematic view of the microfluidic cell culture system 1 comprising one microfluidic structure 2 sealed by a detachable seal 3. The microfluidic structure 2 comprises a first reservoir 4 and a second reservoir 5. Both reservoirs 4, 5 are in fluid communication via a cell culture chamber 6 as well as via a communication channel 7. The cell culture chamber 6 is filled with a cell culture medium 8. The volume of the cell culture medium 8 is chosen such that the cell culture chamber 6 is completely filled with the medium 8 as such. By providing a communication channel 7 between the first reservoir 4 and the second reservoir 5, a sudden increase in pressure (ΔP) in one of the reservoirs is easily balanced via the communication channel 7, instead of resulting in a sudden increase in pressure onto the cell culture medium 8 comprised in the cell culture chamber 6. The fluidic resistance of the different media used in the cell culture chamber 6 and the communication channel 7 is depicted in FIG. 5 by the thickness of the arrows shown in FIG. 5. The fluid communication lines between the first reservoir 4 and the second reservoir 5 is shown by a first arrow P.sub.1 passing through the cell culture chamber 6 and a second arrow P.sub.2 passing through the communication channel 7. The fluidic resistance of the medium 8 in the cell culture chamber 6 is significantly higher than the fluidic resistance of the medium in the communication channel 7 resulting in a major flow of medium from the first reservoir 4 to the second reservoir 5 via the communication channel 7 by an increased pressure in the first reservoir 4.
[0063] FIGS. 6A and 6B show a schematic view of the microfluidic cell culture system 10 comprising a plurality of microfluidic structures 12 sealed by a detachable seal 13. Each of the microfluidic structures 12 comprise a first reservoir 14 and a second reservoir 15 in fluid communication with each other via a cell culture chamber 16. The first and second reservoirs 14, 15 of the microfluidic structures 12 are further in fluid communication with each other via central communication channel 17 formed by a gap between the inner side of the seal 13 and the upper opening of each of the reservoirs 14, 15. Again any pressure increase in one of the reservoirs 14, 15 is easily balanced by providing a major flow of pressure trough the communication channel 17 instead of through one or more of the cell culture chambers 16 comprised in the microfluidic structures 12. In FIG. 6B the microfluidic cell culture system 10 is further provided with a grid structure 11, wherein the grid structure 11 encloses one or more complete microfluidic structures 12. In FIG. 6N the grid structure 11 encloses one single microfluidic structure 12.
[0064] FIG. 6C shows a perspective view of the microfluidic cell culture system 10 of which the schematic view is shown in FIG. 6B. in FIG. 6C, the grid structure 11 as well as the microfluidic structure 12 are visualised.
[0065] FIGS. 7A to 7F show schematic depictions of the steps in a method for transporting microfluidic cell culture systems. FIG. 7A shows a microfluidic cell culture system 20 comprising a cell culture chamber 26 in which cells are cultured and perfused against an extracellular matrix. The cells, e.g. CaCo-2 cells, are usually cultured in the form of a tubular structure or a tube (not shown). The combination of cells, extracellular matrix and cell culture medium is indicated in FIG. 7 with a dotted pattern fill and has the reference numeral 281. The microfluidic structure 22 also comprises reservoirs 24 and 25 that are filled with cell culture medium 28 for perfusing the tube inside the cell culture chamber 26. It is noted that the schematic drawing of FIG. 7A is highly simplified and does not show how exactly the medium 28 in the reservoirs 24, 25 enters the cell culture chamber 26 comprising the tube and the extracellular matrix. Advantageously, the microfluidic structure 22 has a layout as schematically shown in FIG. 1.
[0066] To prepare the microfluidic cell culture system 20 for transport, in a first step the medium 28 in the reservoirs 24, 25 is aspirated (FIG. 7B).
[0067] In the next step, shown in FIG. 7C, a warm liquefied reversible solidifying medium 29, e.g. a 2.5% gelatine solution, is added to the reservoirs 24, 25, thereby filling the cell culture chamber 26. The reversible solidifying medium 29 is allowed to solidify and indicated with the shingle fill pattern. For simplicity, the contents of the cell culture chamber 26 are shown with a dotted fill pattern, indicating the cells and the extracellular matrix, although the reversible solidifying medium 29 can also be present in the cell culture chamber 26.
[0068] By capping the microfluidic cell culture system 20 with a detachable seal 23 the microfluidic structure is closed from the surroundings and a communication channel 27 between the reservoirs 24 and 25 is created (FIG. 7D). The whole system can now be transported to its destination. Any pressure fluctuations that occur during transport, e.g. as a consequence of the microfluidic cell culture system being at an altitude of 10 km in an airplane cargo bay, will be relieved through communication channel 27 and absorbed by the solidified reversible solidifying medium 29.
[0069] After transport, the detachable seal 23 is carefully removed and the microfluidic cell culture system 20 is heated to re-liquefy the reversible solidifying medium 29. Heating should be done with care so as to not to damage the cell culture inside the cell culture chamber 26. When said medium 29 turned liquid again it is aspirated from the microfluidic cell culture system 20 (FIG. 7E).
[0070] Fresh cell culture medium 28 is added to the reservoirs 24, 25 after which the cell culture chamber 26 and its contents 281 can be perfused again (FIG. 7F). Note that the step depicted in FIG. 7F shows the starting situation depicted in FIG. 7A, on a different location.
[0071] FIGS. 7G and 7H(a)-(c) schematically depict the methods in which the reversible solidifying medium 29 is added to reservoirs 24, 25 in addition to and on top of the cell culture medium 28. In FIG. 7G the situation is depicted where the reversible solidifying medium 29 solidifies quickly and does not mix with cell culture medium 28. FIGS. 7H(a)-(c) show 3 subsequent steps. FIG. 7H(a) shows the reservoirs 24, 25 comprising cell culture 28 and the cell culture chamber 26 comprises a mixture of cells, e.g. in the form of a tube, extracellular matrix and cell culture medium, the mixture denoted 281. The volume of cell culture medium can be reduced to prevent spilling when adding reversible solidifying medium 29. Step ii (FIG. 7H(b)) shows how reversible solidifying medium 29 is added to reservoirs 24, 25, in liquid form, whereas step iii (FIG. 7H(c)) shows the situation where the reversible solidifying medium 29 and the cell culture medium 28 have mixed into mixed medium 291 and solidified. Note that the liquid reversible solidifying medium 29 also mixes with the cell culture medium 28 present in the cell culture chamber 26, leading to a situation where the tube of cells and extracellular matrix are surrounded and infiltrated by mixed medium 291. For clarity reasons, the whole of cells, extracellular matrix and solidified mixed medium 291 is denoted mixture 282. Note that FIG. 7H(c) is a highly simplified schematic. In reality, there will be no clear demarcation line between mediums 291 and mixture 282.
