Device and method for cultivating cells

Abstract

The present invention relates to a device for cultivating cells, in particular tissue, comprising a carrier plate unit which has a central axis of rotation, at least one access opening arranged proximally to the axis of rotation, at least one cultivation chamber arranged distally to the axis of rotation, and at least one channel connecting the access opening to the cultivation chamber, and also a method for cultivating cells in a device according to the invention and a method for producing the device according to the invention.

Claims

1. A device for cultivating cells, comprising a carrier plate unit which has a central axis of rotation and has a plurality of access openings, wherein each access opening is arranged proximally to the axis of rotation, a plurality of cultivation chambers, wherein each cultivation chamber is arranged distally to the axis of rotation, and a plurality of channels, wherein at least one channel of the plurality of channels is associated to each access opening connecting the access opening to at least one cultivation chamber, wherein the carrier plate unit comprises at least one first carrier plate and a second carrier plate arranged above or below it, wherein the first carrier plate has the plurality of access openings, the plurality of cultivation chambers, and the plurality of channels connecting the access openings and the cultivation chambers, wherein the second carrier plate has a plurality of media openings, a plurality of media chambers, and at least one media channel connecting the media openings to the media chambers, wherein the access openings have a diameter of 0.2 to 20 mm, and wherein both, the media openings and the access openings, are accessible from outside the carrier plate unit.

2. The device as claimed in claim 1, wherein the carrier plate unit has a central region having at least one connecting device for a rotational device.

3. The device as claimed in claim 1, wherein the device has at least one locking device for a rotational device.

4. The device as claimed in claim 1, wherein the at least one channel is a branched or unbranched channel.

5. The device as claimed in claim 1, wherein the at least one channel connects the access opening to at least two cultivation chambers.

6. The device as claimed in claim 1, wherein the channel has at least two cultivation chambers directly adjoining the channel at least over a part of its length.

7. The device as claimed in claim 1, wherein the channel is curved at least over a part of its length.

8. The device as claimed in claim 7, wherein the channel has a static or angle-dependent curvature.

9. The device as claimed in claim 1, wherein the access openings are designed as loading chambers, which have at least two access openings.

10. The device as claimed in claim 1, wherein the at least one media channel connects at least two media openings to at least one media chamber.

11. The device as claimed in claim 1, wherein at least one separating device is arranged between the first carrier plate and the second carrier plate.

12. The device as claimed in claim 1, wherein the cultivation chambers of the first carrier plate and the media chambers of the second carrier plate are formed overlapping and have a fluidic connection.

13. The device as claimed in claim 1, wherein the carrier plate unit additionally comprises a reservoir for liquids.

14. The device as claimed in claim 1, wherein the carrier plate unit has the form of a disk.

15. The device as claimed in claim 1, wherein the carrier plate unit is designed as a micro-titration plate.

16. The device as claimed in claim 1, wherein the carrier plate unit is constructed from glass or a polymer material.

17. The device as claimed in claim 1, wherein the carrier plate unit is constructed from polydimethyl siloxane (PDMS) or cycloolefin copolymers (COC).

18. The device as claimed in claim 2, wherein the at least one connecting device is a through opening or an anchoring device.

19. The device as claimed in claim 3, wherein the locking device is peripherally arranged relative to said device.

20. The device as claimed in claim 11, wherein the at least one separating device is a membrane.

21. A method for cultivating cells, wherein the cells are cultivated in a device as claimed in claim 1.

22. The method as claimed in claim 21, wherein the cultivation is performed by: a) providing the cells and a device, b) introducing the cells into the device through at least one access opening, c) introducing the device into a rotational device enabling a rotation of the device, d) setting the device into rotation, e) receiving cells in at least one cultivation chamber, and f) cultivating the cells in the at least one cultivation chamber.

23. The method as claimed in claim 22, further comprising the steps of g) introducing cell culture medium into at least one media opening, h) setting the device into rotation, and i) receiving cell culture in at least one media chamber to supply the cells in the cultivation chamber are carried out.

24. The method as claimed in claim 21, wherein a continuous or pulsed flow through the media channels and media chambers with cell culture medium is enabled by the generation of a pressure gradient, external or integrated pumps, or by rotation of the device.

25. The method as claimed in claim 21, wherein a cell complex results due to the cultivation of the cells in the at least one cultivation chamber.

26. A method for producing a cell complex, wherein a method for cultivating cells as claimed in claim 21 is carried out and a cell complex is obtained.

27. A cell culture, produced according to a method as claimed in claim 21.

28. A method for producing a device as claimed in claim 1, wherein in a first method step, at least one material forming the carrier plate unit is provided and this is formed into a device in a method providing shape and stability.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be explained in greater detail on the basis of the following example and the associated figures.

(2) In the figures:

(3) FIG. 1 schematically shows a carrier plate unit (110) according to the invention.

(4) FIG. 2 schematically shows the structure of a first carrier plate (111).

(5) FIG. 3 shows a detail of the first carrier plate (111).

(6) FIG. 4 shows the cultivation chambers (140) of the first carrier plate (111) and media chambers (170) of the second carrier plate (112), which are arranged overlapping.

(7) FIG. 5 shows a cross section through the device according to the invention.

(8) FIG. 6 schematically shows the structure of a first carrier plate (111).

(9) FIG. 7 shows a first carrier plate (111) having colored cultivation chambers (140) and channels (150).

(10) FIG. 8 shows possible cultivation chamber geometries a) round, b) rectangular, and c) dumbbell-shaped.

(11) FIG. 9 schematically shows the structure of a second carrier plate (112).

(12) FIG. 10 schematically shows the structure of the membrane (135).

(13) FIG. 11 shows a device used according to the invention having first and second carrier plate (111, 112) and membrane (135) in between.

(14) FIG. 12 shows the required steps for filling the cultivation chambers (140).

(15) FIG. 13 shows a round cultivation chamber (140) filled with cells.

(16) FIG. 14 shows a dumbbell-shaped cultivation chamber (140) filled with cells.

(17) FIG. 15 shows a laser cut cultivation chamber (140) filled with cells.

(18) FIG. 16 shows a channel (150) inclined at the angle α having cultivation chambers (140).

(19) FIG. 17 schematically shows the filling of the cultivation chambers effectuated by rotation.

(20) FIG. 18 shows eight cultivation chambers (140) filled with cells.

(21) FIG. 19 schematically shows a possible structure of the first carrier plate (111).

(22) FIG. 20 schematically shows a further possible structure of the first carrier plate (111).

(23) FIG. 21 schematically shows an embodiment in which the access opening (130) is formed as a loading chamber (190).

(24) FIG. 22 schematically shows the device according to the invention in micro-titration format.

(25) FIG. 23 shows the structure of a reservoir (200) for cell culture medium or active ingredients to be studied.

(26) FIG. 24 shows the experimentally determined and computed volume flow in a device according to the invention as a function of the rotational velocity.

DESCRIPTION OF EXAMPLE EMBODIMENTS

Example

(27) Production of a Device Used According to the Invention

(28) The production of an exemplary organ disk is described hereafter. The disk according to the invention, i.e., the carrier plate unit, is produced from polydimethyl siloxane (PDMS, purchased from Dow Corning as Sylgard 184). The individual carrier plates are produced by soft lithography, wherein firstly a mold template of the respective carrier plate is manufactured in the required channel height on a silicon substrate (wafer) by means of UV lithography from the photoresist SU-8. The mold templates have the following properties for the described disk:

(29) The first carrier plate of the exemplary disk contains 45 cultivation chambers arranged at a radial distance of 4.5 cm from the disk center point, i.e., the central axis of rotation, having a diameter of 2 mm. FIG. 6 shows the carrier plate design used. In addition, 4 through openings for fastening the disk on the motor are applied in the middle of the carrier plate unit. The chambers are filled via 12 access openings through channels of the height of 50 μm. In each case ⅓ of the channels connecting the access opening to the cultivation chamber has a channel width of 50 μm, 100 μm, and 150 μm. There are 4 different types of symmetrical branches per channel width: 0 branches (access opening connected directly to the chamber), 1 branch (access opening connected to 2 chambers), 2 branches (access opening connected to 4 chambers), 3 branches (access opening connected to 8 chambers). The access opening diameter and the diameter of the passage openings are 3 mm.

(30) The second carrier plate contains media chambers which are arranged matching with the cultivation chambers of the first carrier plate. There is one media channel per media opening which supplies all media chambers connected to the media opening. All media channels have a width of 80 μm and a height of 50 μm. Each media channel has two media openings, wherein one media opening functions as a media outlet and is located outside the channel of the first carrier plate. The media openings have a media opening diameter of 1 mm.

(31) PDMS is firstly mixed in the ratio of 10:1 from the two components base:agent and degassed in the desiccator for 30 minutes under vacuum. 21 g PDMS are poured in each case into the lithographically produced mold template for the first and second carrier plate and cured for 14 hours at 60° C. The silicon substrate used having the structures to be molded has a diameter of 10 cm. Draining off of the PDMS is avoided by an additional acrylic ring, which is clamped on the silicon substrate, and the disk is molded to a final disk diameter of 9.5 cm. To avoid irregularities in the carrier plate thickness in the edge region of the disk, the acrylic ring is filled to the top with PDMS, which results in a height of the first and second carrier plate of 3 mm.

(32) After the curing of the two carrier plates, the through holes preformed in the first and second carrier plate are punched out. Additional access openings having a diameter of 3 mm and media openings having a diameter of 1 mm are stamped out in the second carrier plate. Both carrier plates are now flushed out using isopropanol and dried using nitrogen. The carrier plates are additionally covered with adhesive tape, which is pulled off again to remove contaminants on the surface.

(33) For the illustrated example, a membrane based on epoxy photoresist (1002F) is used. The membrane is produced in carrier plate size for the entire disk. The design of the membrane used for the complete disk is shown in FIG. 10. In this case, the disk membrane is divided into 4 quadrants having the pore sizes of 3 μm (I), 3 μm (II), 5 μm (III), 3 μm (IV) and the porosities 12.7% (I), 5.6% (II), 5.6% (III), 3.2% (IV). The permeable pores are seated arranged in a hexagonal grid only in the edge region, having radial distance between 4.0 and 4.6 cm, i.e., at the overlap between the cultivation chambers and the media chambers to ensure the diffusion in this region. In addition, the membrane is permeable at the point of the access openings. The approximately 10 μm thick membrane is located after the production by means of lithography in the clean room on a silicon wafer and can be detached in H.sub.2O.

(34) For the assembly of the organ disk, the individual layers are bonded on one another with the aid of N.sub.2 plasma, i.e., connected to one another. For this purpose, firstly the first carrier plate is activated in N.sub.2 plasma, for which parameters of 50 W, 90 seconds, and a flow of 0.2 Nl/h are used. The activated carrier plate is aligned on the membrane located on the silicon substrate, weighted using weight, and the bond is cured at 60° C. overnight, i.e., for at least 14 hours in the furnace. Subsequently, the combined layer made of carrier plate and membrane is deposited in H.sub.2O (Milli-Q ultrapure water). After approximately 5 minutes, the soap layer dissolves under the membrane, so that the combined layer can be detached from the silicon substrate. The second carrier plate is now also activated using the above-mentioned parameters in a N.sub.2 plasma, aligned on the free membrane side, weighted, and the bond is cured in the furnace with the above-mentioned parameters. After this step, the disk is finished and ready for use.

(35) Application of the Disk

(36) In the present example, the disk is used to enrich fibroblasts in the cultivation chambers and later to cultivate them. For application of the disk, it is firstly activated in O.sub.2 plasma to achieve better wetting of the channels upon filling. 50 W, 60 seconds, and an O.sub.2 flow of 0.2 Nl/h are used as parameters.

(37) 10 μl of the cell culture medium for fibroblasts (DMEM with 10% FBS and 1% penicillin/streptomycin) are pipetted into the access openings. The organ disk is subsequently closed using a cover and screwed onto the motor via the through holes.

(38) The disk is set into rotation for three minutes at 2000 RPM so that the channels and cultivation chambers of the first carrier plate are filled with medium.

(39) The disk is subsequently removed from the motor again, the cover is opened, and the remaining medium volume in the access openings is suctioned off using a pipette. In the next step, the cell suspension is pipetted into the access openings. For this purpose, in the example 10 μl of a cell suspension of fibroblasts having a concentration of 10.sup.5 cells/10 μl are used

(40) The disk is closed again and attached to the motor. The subsequent disk rotation at 2000 RPM for three minutes conveys the decanted cells into the cultivation chambers. This corresponds to an acceleration, which is routine for the centrifugation of fibroblasts, of 200 g (g for the outer edge of the chambers at r.sub.2=0.045 m:a=5*10.sup.−5*rpm.sup.2 g at a speed of rotation of w=2000 RPM).

(41) The cultivation chambers are now filled with fibroblasts, so that the disk can be removed from the motor and used at a standstill. For this purpose, a defined medium flow is provided with the aid of external pumps via the media openings.

(42) Production of a Cell Complex

(43) For the production of a cell complex by means of the method according to the invention, a device having a channel (150) inclined in relation to the direction of the centrifugal force F.sub.C generated by rotation is used, along the length of which eight cultivation chambers (140) are arranged.

(44) To deaerate the channel (150), the device was firstly rotated for the duration of 2 minutes at 200 g. A suspension containing 80,000 cardiomyocytes was subsequently placed in the access opening (130). After closing the access opening (130), the device was centrifuged for 10 minutes at 200 g, whereby the cardiomyocytes were conveyed into the cultivation chambers (140).

(45) FIG. 18 shows the formation of a dense three-dimensional cell complex in the eight cultivation chambers (140) of the device. For further cultivation of the obtained cell complex, the cells in the cultivation chambers (140) were subsequently supplied with medium by an external spray pump at a flow rate of 50 μl/h.

(46) Measurement of the Volume Flow Conveyed by Rotation

(47) A measurement of the volume flow conveyed by rotation was performed by gravimetric measurement of the collected conveyance volume. For this purpose, the volume flow of water from a reservoir having constant fill level through a media channel of a device according to the invention was determined at different speeds of rotation (RPM) (Table 1).

(48) TABLE-US-00001 TABLE 1 measured values of the gravimetric flow measurement and theoretically computed flow rates. Number of Theoretical Average Standard measured Speed flow flow deviation values [rpm] [μL/h] [μL/h] [μL/h] [—] 100 64 86 7 12 200 144 118 31 12 300 277 214 35 9 400 463 414 65 12 500 702 715 69 12 600 994 1011 89 12 700 1339 1457 99 8 800 1737 1783 163 11

(49) FIG. 24 illustrates that the volume flow of water determined by gravimetric measurement as a function of the speed of rotation in a device according to the invention nearly corresponds to the theoretically computed volume flows.

(50) The experiment thus shows that it is possible using the device according to the invention to convey liquids, in particular medium, by rotation around a central axis of rotation from the at least one access opening arranged proximally to the central axis of rotation via the channel to the at least one cultivation chamber arranged distally to the central axis of rotation even without external pumps.

DESCRIPTION OF THE FIGURES

(51) FIG. 1 shows a carrier plate unit (110) according to the invention in the form of a disk. Carrier plate unit (110) has a central axis of rotation (120) and a central region (121) enclosing it. An access opening (130), which is connected via channel (150) to cultivation chamber (140), is arranged proximally to the central axis of rotation (120).

(52) FIG. 2 shows a first carrier plate (111) having central axis of rotation (120) and access openings (130) arranged proximally thereto at distance r.sub.1 and cultivation chambers (140) arranged distally at distance r.sub.2 to the central axis of rotation (120), wherein the channel (150) connecting the access openings (130) to the cultivation chambers (140) is a branched channel.

(53) FIG. 3 shows a detail of the central axis of rotation (120) of the first carrier plate (111) and the access opening (130) arranged proximally to the central axis of rotation (120) at distance r.sub.1, the cultivation chamber (140) arranged distally at distance r.sub.2, and the branched channel (150) connecting the access opening (130) and the cultivation chambers (140). The cultivation chambers (140) are filled with cells by rotation of the carrier plate at angular velocity ω.

(54) FIG. 4 shows two cultivation chambers (140) of the first carrier plate, which are arranged overlapping with two media chambers (170) of the second carrier plate and are separated by a membrane. Channel (150) connects access openings (not shown) to the cultivation chambers (140) of the first carrier plate and the media channel (180) connects the media openings (not shown) to a first media chamber (170) and a second media chamber (170) of the second carrier plate. The cultivation chambers (140) and channels (150) of the first carrier plate are shown by dashes.

(55) FIG. 5 shows a cross section of a carrier plate unit (110) according to the invention, consisting of a first carrier plate (111) and a second carrier plate (112), which is arranged above the first carrier plate (111). A separating device, in particular membrane (135), is arranged between the first carrier plate (111) and the second carrier plate (112). It separates the cultivation chambers (140) of the first carrier plate (111) from the media chambers (170) of the second carrier plate (112).

(56) FIG. 6 shows the schematic structure of a first carrier plate (111) having access openings (130), which are connected to one or more cultivation chambers (140) via channel (150), and through openings (125) for connecting the device to an external rotational device.

(57) FIG. 7 shows the structure of a first carrier plate (111), wherein the access openings (130), the channels (150), and the cultivation chambers (140) are colored with ink for highlighting. The through openings (125) are not colored. The carrier plate is sealed using an unstructured PDMS layer.

(58) FIG. 8 shows a schematic illustration of possible cultivation chamber geometries. a) shows a single round cultivation chamber (140), b) shows three rectangular cultivation chambers (140), and c) shows three dumbbell-shaped cultivation chambers (140), which are especially designed for cardiomyocytes.

(59) FIG. 9 shows a schematic illustration of the second carrier plate (112) having connecting devices arranged in the central region (121), in particular through openings (125), media openings (160), media chambers (170), and the channels (180) connecting the media inlets (160) to the media chambers (170). In this case, each two media openings (160) are connected to one media chamber (170) or multiple media chambers (170), which are then connected in series.

(60) FIG. 10 shows the schematic structure of the separating device 135, which is arranged between the first carrier plate (111) and the second carrier plate. The black dots represent permeable pores, which are arranged in a hexagonal grid. The differing density of the pores represents regions of differing porosity of the separating device (135), in particular the membrane.

(61) FIG. 11 shows the device according to the invention consisting of a first carrier plate (111), a membrane (135), and a second carrier plate (112). In the central region (121), the device has four connecting devices, in particular through openings (125) for fastening the device on an external motor. The larger access openings (130) and the smaller media openings (160) can be seen proximally thereto, and also channels (150) of the first carrier plate (111) and media channels (180) of the second carrier plate (112) and the cultivation chambers (140) and media chambers (170), which are arranged overlapping.

(62) FIG. 12 shows the steps required for charging the cultivation chambers (140): a) pipetting cell medium into access opening (130), b) rotating (ω.sub.1) the device for t.sub.1 so that all channels (150) and cultivation chambers (140) are filled with medium and air bubbles are removed, c) pipetting the cell suspension into access opening (130), and d) further rotation (ω.sub.2) for t.sub.2, so that all cells are conveyed into the cultivation chambers (140) and are preferably provided in greater density therein.

(63) FIG. 13 shows a round cultivation chamber (140) of the first carrier plate (111) filled with cells (fibroblasts). The cells were conveyed by rotation of the carrier plate into the cultivation chamber (140) and accumulated therein.

(64) FIG. 14 shows a dumbbell-shaped cultivation chamber (140) of the first carrier plate filled with cells (cardiomyocytes).

(65) FIG. 15 shows a laser-cut cultivation chamber (140) of the first carrier plate filled with cells (fibroblasts).

(66) FIG. 16 shows cultivation chambers (140) arranged along a channel (150). In this case, the channel (150) is inclined by a defined angle α in relation to the direction of the centrifugal force F.sub.C generated by rotation.

(67) FIG. 17 schematically shows the filling of the cultivation chambers (140) by the centrifugal force F.sub.C, which may be split into the down force F.sub.II along the channel (150) inclined by the angle α and the contact pressure force F.sub.⊥, which acts perpendicularly on the peripherally located channel wall of the channel (150) inclined by the angle α.

(68) FIG. 18 shows three-dimensional cell complexes (cardiomyocytes) obtained by means of the method according to the invention in eight cultivation chambers (140) of the device.

(69) FIG. 19 shows an embodiment of the device according to the invention, in which, on a first carrier plate (111), multiple access openings (130) arranged around a central axis of rotation are each connected via an inclined channel (150) to cultivation chambers (140) arranged along the channel (150).

(70) FIG. 20 shows an embodiment of the device according to the invention, in which, on a first carrier plate (111), multiple access openings (130) arranged around a central axis of rotation are each connected via a curved channel (150) to cultivation chambers (140) arranged along the channel (150).

(71) FIG. 21 shows an embodiment of the device according to the invention, in which the at least one access opening is designed as a loading chamber (190), which comprises two access openings (131, 132). The cells are conveyed to the cultivation chambers (140) by the centrifugal force F.sub.C.

(72) FIG. 22 shows an embodiment in which the device comprising a central axis of rotation is designed for the cultivation of cells in the micro-titration plate format. The channels (150), the media channels (180), and the membrane (135) are shown.

(73) FIG. 23 shows an embodiment of the device according to the invention, in which the carrier plate unit (110) additionally comprises a reservoir (200) for liquids, in particular for cell culture medium or active ingredients to be studied, which is arranged above a second carrier plate (112) and comprises separate containers (205) arranged proximally to the centrally located axis of rotation of the carrier plate unit having media outlets (165) and separate containers (206) arranged distally to the centrally located axis of rotation of the carrier plate unit having media inlets (166), wherein the media outlets (165) each have a fluid connection via media openings (160) and media channels (180) of the second carrier plate (112) located underneath to the media inlets (166).

(74) FIG. 24 shows the volume flow determined experimentally by gravimetric measurement in a device according to the invention as a function of the speed of rotation (experimental) in comparison to the values determined by computer (theory).