Abstract
A cell culture apparatus including a cell culture device and a movement system for moving the cell culture device. The cell culture device includes at least two reservoirs for holding a liquid cell medium, one or more chambers for culturing of living cells, tissues or living organoids and at least two perfusion channels connecting the reservoirs. The chambers are separated from at least one of the perfusion channels by a semipermeable barrier for selective transport of cell media and/or for selective growth or migration of living cells. The movement system is configured to move the cell culture device with a movement axis in a tilted orientation in order to move the lowest point of a flow loop through all points within the flow path of the flow loop and thereby generate gravity driven circulation of the liquid cell medium around the flow loop.
Claims
1. A cell culture apparatus comprising: a cell culture device; and a movement system for moving the cell culture device; the cell culture device comprising: at least two reservoirs for holding a liquid cell medium, one or more chambers for culturing of living cells, tissues or living organoids and at least two perfusion channels connecting the reservoirs; wherein the one or more chambers are separated from at least one of the perfusion channels by a semipermeable barrier for selective transport of cell media and/or for selective growth or migration of living cells; and wherein the reservoirs and perfusion channels are in fluid communication with one another in order to form a flow loop having a flow path for a one-way gravity-driven flow of the liquid cell medium when the cell culture device is tilted and moved by the movement system; a movement axis being defined as an axis of the cell culture device that passes through the cell culture device with the flow loop being located around the movement axis; and the movement system being configured to move the cell culture device with the movement axis in a tilted orientation in order to move the lowest point of the flow loop through all points within the flow path of the flow loop and thereby generate gravity driven circulation of the liquid cell medium around the flow loop, wherein the tilted orientation places the movement axis at an angle in the range 5 to 85 away from the vertical.
2. A cell culture apparatus as claimed in claim 1, wherein the movement in a tilted orientation is a movement of the device whilst the axis is at an angle of 20 to 80 away from the vertical.
3. A cell culture apparatus as claimed in claim 1, wherein the barrier acts as a semi-permeable and/or selectively permeable barrier between the perfusion channel and the chamber, allowing for passage of certain cell media to provide a way of connecting the liquid cell medium to the at least one chamber.
4. A cell culture apparatus as claimed in claim 1, wherein the barrier is an extra cellular matrix (ECM) barrier, and/or wherein the barrier includes a biomaterial or extracellular natural material and comprises one or more of Matrigel, Geltrex, Collagen, a synthetic hydrogel, and/or a polymer membrane.
5. (canceled)
6. A cell culture apparatus as claimed in claim 1, comprising two barriers at two locations about the chamber, wherein the two barriers each provide for transport of certain cell media between the chamber and the two perfusion channels.
7. A cell culture apparatus as claimed in claim 1, wherein a part of the flow loop is configured to allow contact of the liquid cell medium with a gas from outside of the flow loop.
8. A cell culture apparatus as claimed in claim 1, the movement axis for the cell culture device being defined as an axis normal to a plane of the device, wherein during movement of the device by the movement system the movement axis is tilted in the sense that it is angled to the vertical thereby positioning lifting one section of the flow loop higher than another section of the flow loop, and wherein the position of the tilted axis moves in order to change the section(s) of the flow loop that are higher and lower relative to one another.
9. A cell culture apparatus as claimed in claim 1, wherein the tilted movement axis is moved by the movement system in a rotational fashion so that a reference point on the movement axis traces around the circumference of a two dimensional shape.
10. A cell culture apparatus as claimed in claim 1, wherein the movement in a tilted orientation includes a rotation of the cell culture device about the movement axis, such that a reference point on the flow loop moves in a circle about the tilted movement axis.
11. A cell culture apparatus as claimed in claim 1, wherein the flow path includes a flow reversal restriction feature comprising one or more of: a change in depth; a capillary stop valve; and/or a surface treatment promoting decreased wetting.
12. A cell culture apparatus as claimed in claim 1, wherein the flow loop comprises a looped flow path that passes, in sequence, from a first reservoir, then through a first perfusion channel and past a first barrier at a first side of the chamber, then to a second reservoir, then from the second reservoir, through a second perfusion channel and past a second barrier at a second side of the chamber, then returning to the first reservoir.
13. A cell culture apparatus as claimed in claim 1, wherein the reservoirs each comprise a volume of 40 to 900 L for receiving liquid cell medium.
14. A cell culture apparatus as claimed in claim 1, wherein the perfusion channels each extend from the outlet of one reservoir to the inlet of another reservoir, passing by at least one chamber with a barrier providing a link to the chamber at a mid-portion of the channel; and wherein the length of each perfusion channel is in the range 10 to 30 mm.
15. A cell culture apparatus as claimed in claim 1, wherein the chamber comprises a volume or 2.5 to 120 L for receiving the living cells or living organoids and the chamber is protected from shear force of the perfusion channels via the barrier(s).
16. A cell culture apparatus as claimed in claim 1, wherein the cell culture device includes the liquid cell medium therein and/or the chamber includes therein a living cell or living organoid.
17. A cell culture apparatus as claimed in claim 16, wherein the cell culture device includes a volume of 50 to 600 L of the liquid cell medium.
18. A cell culture apparatus as claimed in claim 1, wherein the dimensions of the cell culture device as well as the tilting speed and angle of movement by the movement system are configured to provide flow rates within the range of physiological flow rates such that the apparatus is configured to provide a flow rate of 15 to 80 L/s of the liquid cell medium in the flow path.
19. (canceled)
20. A cell culture apparatus as claimed in claim 1, wherein the dimensions of the cell culture device as well as the tilting speed and angle of movement by the movement system are configured to provide shear stresses within the range of physiological shear stresses such that the apparatus is configured to provide a shear stress in the range of 0.1 to 100 dyne/cm.sup.2 for flow of the liquid cell medium in the flow path.
21. (canceled)
22. A cell culture device for the cell culture apparatus of claim 1, the cell culture device comprising: at least two reservoirs for holding a liquid cell medium, one or more chambers for culturing of living cells or living organoids and at least two perfusion channels connecting the reservoirs; wherein the one or more chambers are separated from at least one of the perfusion channels by a semipermeable barrier for selective transport of cell media and/or for selective growth or migration of living cells; and wherein the reservoirs and perfusion channels are in fluid communication with one another in order to form a flow loop having a flow path for a one-way gravity-driven flow of the liquid cell medium when the cell culture device is tilted and moved by the movement system; wherein a movement axis is defined as an axis of the cell culture device that passes through the cell culture device with the flow loop being located around the movement axis; and wherein the cell culture device is configured such that when it is moved with the movement axis in a tilted orientation in order to move the lowest point of the flow loop through all points within the flow path of the flow loop then this will generate gravity driven circulation of the liquid cell medium around the flow loop.
23. A method for cell culture, the method comprising: using the cell culture apparatus as claimed in claim 1, providing a suitable liquid cell medium, cells and/or other components in the flow loop, providing a suitable living cell and/or suitable living cells/organoids/tissue in the chamber, tilting and moving the cell culture device to move the cell culture device with the movement axis in a tilted orientation in order to move the lowest point of the flow loop through all points within the flow path of the flow loop, and thereby generating gravity driven circulation of the liquid cell medium around the flow loop.
Description
[0073] Certain example embodiments of the invention will now be described by way of example only and with reference to the accompanying drawings in which:
[0074] FIG. 1 is a schematic plan view of a proposed cell culture device;
[0075] FIG. 2 shows a similar cell culture device in perspective view;
[0076] FIG. 3 illustrates a sequences of steps of a tilting movement for one-way gravity circulation of fluid in a cell culture device;
[0077] FIGS. 4a to 4e show varying designs for a fluid loop on the cell culture device;
[0078] FIGS. 5a to 5d are schematic diagrams for four possible layouts for an extracellular matrix (ECM) barrier of a cell culturing chamber;
[0079] FIGS. 6a to 6g illustrate a simple flow path as well as various options for restricting reverse flow; and
[0080] FIG. 7 shows a flow curve illustrating the flow rate achievable by an exemplary cell culture device.
[0081] FIGS. 8a to 8c show alternative cell culture device configurations comprising one or two flow loops, each of the flow loops comprising one reservoir and one perfusion channel.
[0082] A cell culture device 10 is proposed herein, whereby a one-way gravity driven flow about a flow loop can be obtained via a tilting movement of the cell culture device 10. Example configurations for the cell culture device 10 are shown in FIGS. 1 and 2, with FIGS. 4a to 4c showing further variations. FIGS. 5a to 6d show additional detail of possible features for the cell culture device 10. The tilting movement may advantageously be provided by a cell culture system in which the cell culture device 10 is combined with a movement system 12, such as that shown schematically in FIG. 2. The movement system 12 can be a tilting platform of known type, with the cell culture device 10 hence being usable with typical laboratory equipment absent the need for additional investment in expensive devices. Alternatively, if appropriate, the cell culture device 10 might be provided for use with a bespoke movement system 12, that might be supplied separately.
[0083] As seen in FIGS. 1 and 2 the cell culture device has three main elements. Two reservoirs 4 are provided for holding and conveying a liquid cell medium. The reservoirs 4 provide fluid volumes as well as flow paths between two perfusion channels 8. The two reservoirs 4, together with the two perfusion channels 8, provide a flow loop for one-way gravity driven flow of the liquid cell medium. With a suitable tilting movement, as discussed further elsewhere herein, the liquid cell medium may flow from the first reservoir 4 into the first perfusion channel, through the first perfusion channel 8 to the second reservoir 4, through the second reservoir 4 and from the second reservoir 4 into the second perfusion channel, through the second perfusion channel 8 and then back to the second reservoir 4, before continuing on multiple circuits of that fluid loop. The perfusion channels 8 are separated from the chamber 6 via barriers 16, in this case on two sides of a single chamber 6, with the barriers 16 allowing for selective transport of cell media, such as transport of fluids, whilst blocking movement of other elements of the system, e.g. to retain cells or organoids within the chamber 6.
[0084] In the example of FIG. 3 the movement system 12, which may be a known laboratory tilting platform device as noted above, applies a tilting motion to the cell culture device 10 causing it to pass sequentially through positions I, II, III and IV. For example, the tilting platform device may raise and lower respective tilting actuators to cause the device to move between the four tilted positions. Optionally it may first move from a horizontal position to a first one of positions I, II, III and IV at the start of the use of the cell culture device 10. Similarly, optionally it may move to a horizontal position from a last one of positions I, II, III and IV at the end of the use of the cell culture device 10. This can be a movement of a constant speed or it may have a varying speed, including use of pauses, to provide a pulsatile flow pattern. The tilting movement causes the lowest point (i.e. lowest in height with reference to gravity) of the flow loop to move around the flow loop made of the reservoirs 4 and perfusion channels 8, and that in turn drives the one-way flow about the flow loop. The same one-way flow will occur with cell culture devices 10 having variations on the arrangement of the reservoirs and perfusion channels, for example as in FIGS. 1, 2 and 4a to 4c, since in each case there is a flow loop for fluid flow where the flow paths are in a circuit about a central point of the device, and hence the tilting movement will prompt one-way flow around the flow loop.
[0085] The movement of the cell culture device 10 by the movement system 12 can be envisaged with reference to a suitable movement axis, such as the axis 20 shown in FIGS. 2 and 3. This is an axis 20 normal to a plane of the cell culture device 10 and passing through it so that in this example the flow loop, i.e. the sequence of reservoirs 4 and channels 8, passes around the axis 20. With the tilting movement shown in FIG. 3 then the movement axis would have a rotating stirring motion wherein a reference point on the axis 20 (e.g. above the cell culture device 10) would trace around an outline of a square. Other movements could also be used, such as to tilt and rotate the movement axis 20 with a circular movement, and/or to rotate the cell culture device 10 about the movement axis 20 whilst the movement axis 20 is tilted.
[0086] It will be appreciated that the speed of the tilting movement and the maximum angle of the tilt can be controlled/adjusted as discussed elsewhere herein. That can allow for control/optimisation of performance such as in terms of the speed of flow, the rate of circulation, and/or the WSS within the flow paths.
[0087] As the flow is driven by gravity, liquid reservoirs 4 are needed that can fill and release the media volume during the tilting operation. Elongated (rectangular, elliptic, ring-shaped) reservoirs 4 on two sides of the flow loop are suitable because the liquid level is tilted in them as shown in FIG. 3, thus preventing backflow. The image also shows the flow direction (red) and the movement of the platform (black). Additional surface treatment e.g. via laser-engraving can be used to increase the wetting behaviour in the reservoir thus further preventing backflow. The enhanced wetting will keep remaining liquid in the emptied reservoir in place when the platform tilts back so that almost no media will flow back in the opposite direction. Placing the inlets and outlets in the open side channel/reservoirs 4 on different levels can also be used to reduce the amount of liquid flowing back during normal circulation. The flow rate depends on tilting angle, the channel dimensions, the width and the volume in the reservoirs and thus can be tuned by both, the designer and the end-user. Importantly, no pumps are needed to operate the device and several circuits can be run in parallel on one tilting platform allowing a robust scalability. Different organ models can be placed one after the other to study cross-organ toxicity and other effects.
[0088] By controlling tilting speed and angle we can precisely tune the flow and/or pressure in the channels without the need for external pumps. This allows to implement an atrial and a venous channel on one chip with different WSS/flow. An exemplary flow curve is given in FIG. 7. The flow was measured with a modified micro-Particle-Imaging-Velocimetry setup, and 19 degrees centigrade and for revolution rates of 4 rpm (shown in grey) and 8 rpm (shown in black) of the cell culture device. The flow rate is measured at a point within one of the perfusion channels so that during one revolution of the cell culture device one peak in the measured flow rate is seen. The curve demonstrates that fluid flows through the perfusion channel at a rate similar to physiological flow rates and that backflow is effectively prevented. Furthermore, an oxygen rich and an oxygen depleted side (red: arteria/blue: venous can be achieved by actively reducing/increasing the oxygen content in the reservoir, e.g. with a chemical reaction that consumes oxygen or by integrating a barrier (lung-on-chip model, see FIG. 2). Having arterial and venous components in a scalable OoC platform allows to develop far more complex cell, organoid or tissue structures than possible with current gravity based OoC platforms. Between both channels, cultivation chambers are placed that are later filled with endothelial cells and cells/organoids of interest either without or with hydrogel of different compositions to enable vascularization on-chip and building different OoC models depending on end-user needs. The present examples include a perfusion system with two separated circuits (mimicking an arterial and a venous channel) during tilting. The inventors have thus shown that several circuits can be run parallel, which underlines the scalability of the proposed device.
[0089] FIGS. 4a to 4e show further examples of the cell culture device 10. These devices 10 consists of a thick connection plate 1 with the reservoirs 4 having suitable fluidic connections 18 along with cell injection ports 7. The device can have a layered construction with one or more flow layers 2 integrating the perfusion channels 8 and the chamber 6, with a closing layer 3 at the bottom, i.e. at the opposite side to the connection plate 1. The chamber 6 is oriented in the middle of two perfusion channels 8 and separated from direct flow (straight arrow) by the barrier 16, which in this example comprises a layer of extracellular matrix (ECM) 9.
[0090] Different fluidic circuits are proposed:
[0091] A. As shown in FIG. 4a, one or more chambers 6 aligned between an arterial and venous channel 8. The arterial side is achieved by actively adding oxygen (dashed arrow) whereby a venous channel is generated by reducing the oxygen level.
[0092] B. As in FIG. 4b, two separated fluidic circuits, one in the middle and one outside, whereby one is forming an arterial and the other a venous circuit by adding or removing oxygen (dashed arrow). The cell cultivation chambers 6 are aligned between both circuits.
[0093] C. As in FIG. 4c, two separated fluidic circuits in close proximity to each other, one featuring a high oxygen level and flow rate (arterial side) and one with low oxygen level and flow (venous side). The cell cultivation chambers 6 are aligned between both channels.
[0094] D. The layout in FIG. 4d contains two separate circuits as proposed in FIG. 4c with the difference that no cell cultivation chambers, but a (semi)-permeable membrane 9 is added between both circuits thus adding a transport model (organ) to the chip. Different cells e.g. endothelial or epithelial cells can be seeded on one or both sides of the membrane 9 to allow an (active) exchange from one circuit to the other. Both perfusion channels 5a and 5b are placed in different layers and connected to separate reservoirs 4a and 4b and one or more cell cultivation chambers 6a and 6b on both sides. The cell chambers 6a and 6b can be shaped differently as described in FIGS. 5a to 5d and contain several cell loading ports 7. The layout in FIG. 4d can comprise a drainage channel 5c on one or both sides of the device. These can be connected to one or more drain reservoirs 4c.
[0095] E. The layout in FIG. 4e is similar to the configuration of FIG. 4d with the difference that the placement of the reservoirs 4a/b and perfusion channels 5a/b is similar to the one in FIG. 4b and a (semi)-permeable membrane 9 is placed between both circuits 5a/b. Due to the configuration of the reservoirs, the flow in both circuits is in phase and pointing in the same direction and not alternating and reversed like in FIG. 4d. Analog to FIG. 4d, different cells e.g. endothelial or epithelial cells can be seeded on one or both sides of the membrane 9 to allow an (active) exchange from one circuit to the other. The cell chambers can be configured like in FIG. 4d or as highlighted in the FIG. 5. In this particular configuration, a specific model is used. So-called Transwell inserts 30 are introduced into the connection plate 1. Transwells inserts are plastic containers sealed with a semi-permeable membrane at the bottom. Cells can be cultured on the membrane e.g. to generate a transport model 30a to study the uptake of a substance. This can be for instance a skin, lung or kidney model. Moreover, Transwells inserts can include organoids or other 3d-models embedded in an ECM 30b to mimic metabolism or substance uptake of a target organ. Both, several Transwell inserts-based models and specifically structured models as described in FIG. 5 can be mixed in one device.
[0096] Microfluidic cell culture devices 10 as in the current examples can be manufactured by laser structuring of thermoplastic sheets with varying thickness (currently Polymethyl-methacrylate (PMMA)). Besides, other thermoplastics like polycarbonate (PC), Cyclic-Olefin-Copolymers (COC), Polystyrene (PS) or Polyethylene-terephthalate (PET) are possible. The substrates are later UV activated and thermally bonded in a hot-press. Possible other manufacturing technologies include hot-embossing, injection moulding, micro milling or 3D printing. Moreover, the chip can be manufactured using the established soft-lithography process by casting the structures from a master mould in a silicone elastomer like PDMS and later bonding it to a glass slide.
[0097] One major functionality of the proposed cell culture device 10 is the ability to protect the cells from direct flow while allowing modelling of connection to blood vessels (vascularization), interaction with circulating cells such as (see above) and innervation. In contrast to other developments, we are not integrating an artificial barrier 16 but propose four different fluidic layouts (A, B, C, D) to structure a biodegradable ECM 9 between the cell chamber 6 and the perfusion channels 8. The forming of the ECM barrier 9 and loading of cells can be performed in three or four steps (I, II, III, and IV) as illustrated in FIG. 5a, FIG. 5b, FIG. 5c and FIG. 5d. FIG. 5a shows formation of a proposed Layout A, which integrates bridges with different heights 100 in the flow layer(s) 2 between the perfusion channels 8 and the cell chamber 6. In a first step (I), a liquid hydrogel 101 is injected via the loading port 7 at 4 degrees and transported via capillary forces into the bridges. Next (II), the liquid hydrogel is removed with a slight vacuum so that Hydrogel only remains within the bridge 102. The hydrogel sticks due to capillary forces and forms a meniscus. After polymerization, cells or organoids or tissues 103 are loaded through the injection port 7 and the chamber 6 is filled with hydrogel (III). Finally, endothelial cells 104 are loaded on both sides of the perfusion channels 8 and form a vascular network 105 connecting the organoids with the perfusion channels (IV).
[0098] Besides, it is possible to load the hydrogel (101) and the organoids, cells (103) in one step (refer to step III) if only one type of cells is used.
[0099] Another chamber layout Layout B is shown in FIG. 5b. Layout B also integrates bridges with different heights 100 in the flow layer(s) 2 between the perfusion channels 8 and the cell chamber 6. Layout B comprises additional channels 106 within the bridges 102 to allow for the injection of a hydrogel in step I. Due to capillary forces, the hydrogel remains in the small gaps beneath the bridges 102. This allows a similar or different cell/hydrogel composition 107 to be loaded into the middle cell culture chamber 6 in step III. Similar to Layout A, endothelial cells (104) can be seeded to form micro capillaries (105) in step IV.
[0100] Turning to FIG. 5c, in contrast to Layout A, a proposed Layout C uses small ridges or dead channels 109 placed underneath the place where perfusion channels 8 and cell cultivation chamber 6 should be separated. All channels have here the same height and form a big chamber 108. After cleaning and drying of the device (in step I) a liquid hydrogel 102 containing cells or organoids 103 is injected via the injection port 7 (in step II) and the hydrogel is polymerized. The amount of ECM injected is controlled in that way so that the hydrogel fills the small ridges first thus forming a meniscus separating the cell chamber 6 and the perfusion channels 8. The cells will then be immobilized in the hydrogel and analog to layout endothelial cells 104 are flushed in on both perfusion channels 8 and finally form a vascular network 105 (in step III).
[0101] Another Layout D features a cell separation layer in a multilayer setup with the perfusion channel(s) 110 on-top of the cell culture chamber 112. This is shown in FIG. 5d. The perfusion channel 110 and the cell culture chamber 112 are horizontally separated by a window 111, the window being narrower than the perfusion channel 8 and cell culture chamber 112 as shown. After cleaning and drying of the device (in step I) a liquid hydrogel 102 containing cells or organoids 103 is injected via the injection port 7 (in step II) and the hydrogel is polymerized. The cell culture chamber 112 is shaped as a channel beneath the perfusion chamber 110 and connected to the surface so that the cells/organoids can be placed underneath the perfusion channel by pipetting. The cells will then be immobilized in the hydrogel and analog to Layout A endothelial cells 104 can be flushed in on the perfusion channel and finally form a vascular network 105 (in step III).
[0102] We furthermore propose certain layouts and/or other features for the liquid reservoirs 4 and/or perfusion channels in order to obtain a directed flow and/or to inhibit reverse flow, as shown in FIGS. 6a to 6g. When the reservoirs 4 are tilted by an angle of a, the liquid 200 in the reservoir 4 is tilted by this angle too because it is opened to the atmosphere at the filling ports 8. In FIG. 6a this leads to the effect that the liquid leaves the outlet 205 in the desirable flow direction. Nevertheless, remaining liquid in the reservoir might cause an unwanted backflow through the inlet 204 when the reservoir is tilted in the opposite direction () as shown in FIG. 6b. We propose four different layout improvements to reduce the backflow due to this effect:
[0103] A. As shown in FIG. 6c the inlet 206 is created within the reservoir layout 4 at a higher level than the outlet 205, thus preventing backflow when it is tilted in the opposite direction ().
[0104] B. An alternative or additional feature, as shown in FIG. 6d, is laser engraving of the reservoir surface 201 to decrease wetting in order that the contact angle between the liquid and the surface is increased, resulting in the promotion of a meniscus that will prevent remaining liquid to be tilted back into the inlet 204.
[0105] C. Another possibility, as in FIG. 6e, is to reduce the height of the reservoir 4 so that the meniscus can be further increased to form a so-called capillary stop valve 203 that prevents backflow.
[0106] D. An alternative reservoir design is shown in FIG. 6f and FIG. 6g. Here, the liquid enters and leaves the reservoir through holes 207 from underneath. This alternative inlet/outlet configuration will result in the formation of a capillary-stop valve at the outlet at which a meniscus 202 will be formed at the air-liquid interface which prevents inflow of liquid into the chamber through the outlet.
[0107] All four layouts for the liquid reservoirs 4 and/or perfusion channels/methods for obtain a directed flow and/or to inhibit reverse flow can be combined to promote a fully one-directional flow.
[0108] Another alternative layout for providing the cell culture device is proposed in FIG. 8a. The layout comprises one flow loop which comprises a single reservoir 41. The single reservoir comprises a first end 50 and a second end 51. A perfusion channel 8 connects the first end 50 of the reservoir to the second end 51 of the reservoir. A flow loop is therefore provided via a single reservoir 41 and a single perfusion channel. The perfusion channel 8 is in contact with a cell culture chamber 6 separated by a barrier 16. Directionality of the flow within the flow loop is achieved by the use of a specifically-shaped reservoir. In the example shown a U-shaped reservoir 41 is provided. As the U-shaped reservoir is tilted as shown in FIG. 6, the 3D tilting motion and the geometric confinement of the fluid in the reservoir forces the fluid to flow in a single direction as dictated by the tilting motion, thus a directed flow is generated. One or more venting holes/filling ports 7 are included in the reservoir 41 to allow a defined filling of the reservoir and to remove the air in the system.
[0109] FIG. 8b shows a further alternative layout for the cell culture device. The layout is an adaptation of that shown in FIGS. 1 and 2 in which two of the single reservoir flow loops as shown in FIG. 8a are provided in order to allow the cell culture chamber to contact two perfusion channels. The device shown comprises two separate flow loops so that the cell culture chamber is contacted by two perfusion channels each belonging to a separate flow loop.
[0110] FIG. 8c shows a further alternative layout for the cell culture device. In this example, two separate flow loops each comprise a single reservoir 42, 43 and a single perfusion channel 8. The second flow loop is disposed radially outwardly of a first flow loop. The layout is an adaptation of that shown in FIG. 4b in which one flow loop is provided in the middle and one outside with a cell culture chamber disposed between the respective perfusion channels. In the example shown, the second flow loop comprises a bigger U-shaped reservoir 42 which is placed around a smaller U-shaped reservoir 43 belonging to the first flow loop. Both reservoirs 42, 43 may comprise one or more venting hole/filling port 7. A cell culture chamber 6 is placed between perfusion channel of the first flow loop and the perfusion channel of the second flow loop. Each perfusion channel 8 is separated from the cell culture chamber 6 by a barrier 16 as described above.
[0111] Analog to the layout with two reservoirs and two connecting channels, the previously mentioned methods to improve directionality of the flow, discussed with reference to FIGS. 6a to 6g, may be applied to the layout shown in FIGS. 8a and 8b also.
[0112] For scalable fabrication of the cell culture devices it is proposed to use CO.sub.2 laser cutting/etching, with subsequent UV activation and thermal bonding of Poly(methyl methacrylate) (PMMA) sheets. PMMA is a polymer widely used in cell cultivation due to its good optical properties, its biocompatibility and due to the fact that it can be easily machined with CO.sub.2 lasers. In contrast to the elastomer Polydimethylsiloxane (PDMS) that is widely used in microfluidics, PMMA has a low adsorption of small molecules and is impermeable for oxygen which makes it suitable for separating arterial and venous flow and hence for creating a more physiological circulation than currently standard. Moreover, PMMA can be processed like other thermoplastics using hot-embossing or injection moulding and is therefore better suited for mass production than PDMS. The inventors have established that endothelial cells (HUVECs) can be cultivated in the device and invade liver organoids that are embedded in an extracellular matrix (ECM).
[0113] For a proper functionality of the HUVECs (alignment, sprouting) a sufficiently high WSS (>1 dyne/cm2) and a uni-directional flow are needed. The inventors have found that, with the proposed cell culture device 10, this can advantageously be achieved using a standard tilting platform as the movement system 12 (for example, a Mimetas OrganoFlow rocker, as provided by MIMETAS B.V., of Leiden, The Netherlands).
[0114] The proposed cell culture device 10, which is moveable on such a tilting platform to achieve one-way flow, thus has the following advantages over other solutions on the market: [0115] Flow directionality. [0116] Precisely controllable flow rate and pressure in the system. [0117] Easy to handle scalable platform that fits with standard laboratory processes. [0118] Only a low amount of cell cultivation media is needed. [0119] Experiments can be parallelized which makes it suitable for drug screening. [0120] Several tissues/organoids can be combined in one circuit. [0121] Platform can be adapted to circulating blood, cancer and immune cells, bacteria and virus particles. [0122] Different flow rates and oxygen levels can be integrated in one device without using pumps. [0123] Only a standard tilting support device is needed (no additional laboratory equipment). [0124] Setup fits in standard incubators without tubes (no risk of contamination). [0125] Air bubbles are automatically trapped in the reservoirs and do not enter the channel.
