Abstract
The invention relates to a carrier which is suitable for the cultivation of cells and is preferably a plate-shaped carrier, e.g. made of glass, silicon or plastic or a combination thereof, in which carrier at least one first recess is formed which extends over a first thickness portion of the carrier, wherein an array of second recesses extends from the first recess into a second thickness portion of the carrier adjacent to the first thickness portion. The first thickness portion may have a thickness of a few micrometres to several centimetres. The second recesses, which extend from a first recess into the second thickness portion and each form a second array, form cups which are suitable for receiving cells and/or synthetic particles, e.g. made of plastic or glass.
Claims
1. A carrier for cultivating cells, comprising at least one first recess extending over a first thickness section from a first surface up to a second thickness section adjacent to the first thickness section, an arrangement of second recesses in the second thickness section and extending from the first recess, wherein the second recesses comprise an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, the carrier comprising an outlet whose cross-section opens in or adjacent to the first thickness section.
2. The carrier according to claim 1, wherein the outlet comprises a cross-section projecting into the carrier (and open to the plane of the first surface.
3. The carrier according to claim 1, comprising a connection conduit connected to the outlet and extending to a second surface that lies opposite the first surface.
4. The carrier according to claim 3, wherein the connection conduit extends along an end wall of the carrier or through the carrier.
5. The carrier according to claim 3, wherein the outlet and the connection conduit are adjacent each other and comprise a hydrophilic surface, and wherein regions of the first surface and/or regions of the second surface are hydrophobic.
6. The carrier according to claim 1, wherein the carrier is defined in a single piece of glass.
7. The carrier according to claim 1, consisting of a second partial carrier extending over the second thickness section and of a first partial carrier extending over the first thickness section, the first and second partial carriers being arranged in a liquid-tight manner relative to one another.
8. The carrier according to claim 7, wherein the first and the second partial carriers are arranged displaceably against one another.
9. The carrier according to claim 7, wherein one or both of the first and second partial carriers consists of glass, plastic and/or silicon.
10. (canceled)
11. The carrier according to claim 1, comprising a supply conduit in the first surface or in the first thickness section, the supply conduit opening into the first recess.
12. The carrier according to claim 1, comprising at least two first recesses, each of which is enclosed by circumferentially arranged connection conduits extending through the carrier.
13. The carrier according to claim 1, comprising at least two first recesses, at least one of which is enclosed by an outlet in the form of a trench that opens into another one of the at least two first recesses, which is enclosed by circumferentially arranged connection conduits extending through the carrier.
14. The carrier according to claim 1, comprising a controlled valve arranged in an outlet, in a connection conduit, and/or in a supply conduit.
15. The carrier according to claim 7, wherein the second partial carrier is arranged at least partially within the first recess of the first partial carrier, the second recesses each having at least one through hole boring in their bottoms, so that each second recess is connected to the first recess through at least one through hole boring.
16. The carrier according to claim 7, wherein the first recesses and second recesses are formed as spaced-apart through holes in the first partial carrier and are connected to one another by transition conduits formed by a bottom recess, which is formed as a region-wise spacing between the first partial carrier and the second partial carrier.
17. The carrier according to claim 16, wherein the first thickness section is equal to the second thickness section and the second partial carrier has a bottom recess extending into the second partial carrier by the height of the cross-section of the transition conduit, the first partial carrier being connected to the second partial carrier along regions of the second partial carrier projecting beyond the bottom recess.
18. A method for cultivating cells, comprising introducing liquid in which cells are suspended into second recesses of the carrier according to claim 7, and subsequent remaining of liquid from the first thickness section along the outlet and the connection conduit.
19. The method according to claim 18, wherein the first partial carrier is formed only by the surface of the second partial carrier by which the second recesses are spaced apart, and the first recess adjacent to the arrangement of second recesses is formed solely by a liquid layer generated by surface tension.
20. (canceled)
21. The method according to claim 18, wherein the carrier has only second recesses and medium forms a layer projecting above the plane of the cross-sectional openings of the second recesses, which layer forms the volume of the first recess.
22. The method according to claim 18, wherein the carrier comprises recesses whose cross-sectional openings are spaced apart by surface regions of the carrier, these surface regions and the cross-sectional openings of the recesses lying in a common plane, wherein liquid is applied onto this plane and the liquid is moved along the carrier by tilting the carrier against the horizontal, by vibrating the carrier, and/or by applying pressurized gas to the plane.
23. (canceled)
Description
[0049] The invention is now described in more detail by means of examples with reference to the figures, which show schematically in
[0050] FIG. 1 an embodiment of the invention,
[0051] FIG. 2 an embodiment of the invention with supply conduit from the second surface of the carrier,
[0052] FIG. 3 an embodiment of the invention with supply conduit and outlet from the second surface of the carrier,
[0053] FIG. 4 embodiments of the invention in top view,
[0054] FIG. 5 an embodiment of the invention with supply conduit and outlet,
[0055] FIG. 6 a further embodiment of the invention with supply conduit and outlet,
[0056] FIG. 7 a further embodiment of the invention,
[0057] FIG. 8 a further embodiment of the invention,
[0058] FIG. 9A in cross-section and 9B in plan view on a carrier an embodiment,
[0059] FIG. 9C a first partial carrier in plan view,
[0060] FIG. 10 a further embodiment of the invention,
[0061] FIGS. 11A and 11B a further embodiment of the invention and
[0062] FIG. 12 a further embodiment.
[0063] FIG. 1 shows a carrier 1 in cross-section, consisting of a first partial carrier 2 and an adjoining second partial carrier 3, wherein in the first partial carrier 2 a first recess 4 is formed which covers the cross-sectional openings 5 of the adjoining second recesses 6. Therefore, the inner volumes of the second recesses 6 are in contact with the inner volume of the first recess 4 along their cross-sectional openings 5, which contact allows for a mass transfer between them, in particular by diffusion. The second recesses 6 extend over a second thickness section 8 of the carrier 1, in this case over a second thickness section 8 in the second partial carrier 2. In the first partial carrier 2, the first recess 4 extends over the first thickness section 7, which therein is equal to the thickness of the first partial carrier 2. In its first thickness section 7, here equal to the first partial carrier 2, the carrier 1 has an outlet 9 through which liquid F whose level reaches the cross-section of the outlet 7 can exit. A supply conduit 10 opens at a distance above the plane over which the outlet 9 extends. The supply conduit 10 is not necessarily connected to the carrier, but can be guided independently of the carrier, for example by means of a traversing device (not shown).
[0064] The large aspect ratio of the second recesses 6 allows movement of the liquid in the first recess 4 without flushing cells Z located at the bottom 14 or in the volume of the second recess 6 out of the second recesses, respectively, while minimizing any influence of turbulence on the cells.
[0065] FIG. 2 shows a carrier 1 in cross-section which, like the carrier 1 shown in FIG. 1, consists of a first partial carrier 2 having a first recess 4 and a second partial carrier 3 attached thereto, in which second recesses 6 adjacent to the first recess 4 extend over the second thickness section 8. An outlet 9 is formed in the first partial carrier 2. The outlet 9 shown in FIGS. 1 and 2 extends along an outer end surface 11 of the carrier 1. A supply conduit 10 extends from the second surface 12 of the carrier 1 or its second partial carrier 3 into the first recess 4. As preferred, the supply conduit 10 comprises a controlled valve 13 to control the supply of liquid F into the first recess 4.
[0066] FIG. 3 shows an embodiment of the carrier 1 in which a connection conduit 15 forming an outlet 9 and, in opposite flow forming a supply conduit 10, extends through the carrier 1 from the first recess 4 to the second surface 12 thereof. Such a connection conduit 15 forming both a supply conduit 10 and an outlet 9, preferably has a controlled valve 13, which further preferably has two or more connected conduits, of which e.g. one is connected to a storage container for medium and another is connected to a collecting container for liquid F discharged from the first recess 4. This embodiment can also be provided with a lid covering the first recess. Preferably, the lid then has a valve by which the composition of the gas atmosphere can be controlled.
[0067] FIG. 4 shows embodiments of the invention in a one-piece carrier 1 in plan view onto the first surface 20 of the carrier 1 and onto the cross-sectional opening of a respective first recess 4. The first recess 4, here rectangular, extends over the cross-sectional openings of the adjacent second recesses 6, which are formed as an arrangement of 3 × 3 second recesses 6 per first recess 4.
[0068] In an embodiment A of FIG. 4, the first recess 4 is surrounded by connection conduits 15 passing through the carrier 1 and connected to an outlet 9 or forming the outlet 9. The connection conduits 15 extend from the first recess through the carrier 1 to its second surface 12 opposite the cross-sectional opening of the first recess 4. The connection conduits 15 passing through the carrier 1 selectively guide liquid emerging from the first recess to the opposite second surface 12 of the carrier 1, so that the liquid can be selectively removed there and also does not contaminate another first recess 4 formed in the same carrier 1.
[0069] In an embodiment B of FIG. 4, a first recess 4 is connected to an outlet 9 which is formed in the carrier 1 in the form of a trench whose cross-section extends into the carrier 1. At the circumference of the carrier 1 the outlet 9 is connected to a connection conduit 15, which extends along the circumferential end wall of the carrier 1 onto its second surface 12.
[0070] In an embodiment C of FIG. 4, a first recess 4a is enclosed by a circumferential outlet 9 which by a conduit section 9a forms an inlet into a further first recess 4b so that liquid F can flow through the outlet 9 and its conduit section 9a from a first recess 4a into a further first recess 4b. The first recess 4b is enclosed by connection conduits 15 through which liquid F emerging from this first recess 4b is directed to the opposite second surface 12 of the carrier 1. This embodiment also shows, as an example of a storage recess 21, a first recess 4a connected by a supply conduit 10 to another first recess 4b.
[0071] In an embodiment C of FIG. 4, cells producing certain biomolecules, e.g. cytokines, antibodies, proteins, can be cultivated in the second recesses 6 allocated to a first recess 4. Through the conduit section 9a, these molecules can be transported to another first recess 4 and be detected there or act on other cells.
[0072] In an embodiment D of FIG. 4, as also described with reference to embodiment B, a first recess 4 is connected to an outlet 9 formed in the carrier 1 in the form of a trench whose cross-section extends into the carrier 1. At the circumference of the carrier 1 the outlet 9 is connected to a connection conduit 15 which along the circumferential end wall 11 of the carrier 1 extends onto the second surface 12 thereof. On the wall of the first recess 4 opposite the outlet 9, a supply conduit 10 is formed in the carrier 1, also in the form of a trench.
[0073] The carrier 1 can have one or more first recesses 4 of the same or different shape, e.g. of the FIGS. 4A to 4D. In the case of multiple first recesses 4, different liquids or the same liquids in different concentrations or combination thereof can be contained in different first recesses 4. Different cell types can be contained in second recesses 6 belonging to different first recesses 4.
[0074] FIG. 5 shows an embodiment in which the wall extending over the first thickness section 7 and having or enclosing a first recess 4 can be formed by a first partial carrier 2 adjacent to a second partial carrier 3 in which an arrangement of second recesses 4 is formed. A supply conduit 10, which is preferably connected to a connection conduit 15, can open into the first partial carrier 2 and, preferably opposite to the supply conduit 10, an outlet 9 can be formed, which is preferably connected to a connection conduit 15. The supply conduit 10 shown here and the connection conduit 15 are each provided with a controlled valve 13, which allows control of the supply and discharge of liquid F into or resp. out of the first recess 4.
[0075] As shown in FIG. 6, the first partial carrier 2 can be displaceable relative to the second partial carrier 3, e.g. in that the first partial carrier 2 and the second partial carrier 3 overlap in their edge regions in a displaceable and liquid-tight manner. A seal 16 can be arranged between the first partial carrier 2 and the second partial carrier 3, which seal 16 e.g. is a ridge of plastic or a rubber ring on the first partial carrier 2 or second partial carrier 3 . Further, FIG. 6 shows that the first partial carrier 2 can cover the first recess 4. The covering portion of the first partial carrier 2 can be carried out as a cover which can be recycled.
[0076] FIG. 7 shows a method for separately introducing cell-containing liquid by means of a first metering device 17 and, prior thereto, subsequently or simultaneously, introducing cell-free medium by means of a second metering device 18, into recesses 4, 6 of the carrier 1.
[0077] FIG. 7 shows a carrier 1 whose recesses consist of an arrangement of second recesses 6 and a layer of liquid covering them, which forms the first thickness section 7 that is held in place by surface tension. For this purpose, it is preferred that the enclosure of the arrangement of second recesses 6 is hydrophobic, for example has a hydrophobic coating. The carrier 1 has as outlet 9, for example, a hydrophilic region adjacent to a connection conduit 15 which extends along the end surface 11 of the carrier 1 to its second surface 12 opposite to the cross-sectional openings of the recesses 6. Preferably, the hydrophobic coating enclosing the second recesses 6 is set up such that droplets of a liquid placed on the carrier converge to form a coherent layer and only then run off at the outlet 9. Alternatively, the outlet 9 can be formed by microstructures in the surface of the carrier 1.
[0078] In the method, the running off of the liquid over the outlet 9 can be controlled by tilting the carrier 1 or by bringing the liquid into contact with a device having capillary action.
[0079] FIG. 8 shows a carrier 1 in whose first partial carrier 2 optionally there is arranged no outlet 9, but the inlet 10 simultaneously serves as a metering device 17 and as an outlet 9. Alternatively, inlet and outlet can be realized as two metering devices 17, 18. The first recess is subdivided in perpendicular to the cross-sectional opening of the first recess by a membrane 19, which is preferably semi-permeable, whereby the common liquid volume of the first recess 4 is subdivided above the second recesses 6. The membrane 19 can also be combined in an analogous manner with other embodiments and there subdivide the first recess 4 or the first thickness section 7.
[0080] FIG. 9A shows a two-pieced embodiment of a carrier 1 of a first partial carrier 2 and a second partial carrier 3 attached thereto in a liquid-tight manner. In the case of first and second partial carriers 2, 3 made of glass or silicon, these can be joined together, for example, by bonding. In this embodiment, the second recesses 6 are spaced from a first recess 4 and connected thereto by means of the transition conduit 23. The first recess 4 can have any diameter independent of the second recesses 6. Therein, the transition conduit 23 is formed by a bottom recess 24, which forms a region-wise spacing between the first partial carrier 2 and the second partial carrier 3. The first recess 4 and the second recesses 6 are formed as through holes in the first partial carrier 2, the bottoms of the first and second recesses 4, 6 are each formed by the second partial carrier 3. Therein, the first thickness section 7 is preferably equal to the second thickness section 8. The second partial carrier 3 has a bottom recess 24 which at the height of the cross-section of the transition conduit 23 extends into the second partial carrier. The first partial carrier 2 is connected to the second partial carrier 3 along the sections of the second partial carrier 3 protruding beyond the bottom recess 24. The bottom recesses 24 form a connection conduit having a diameter smaller than the diameter of the cells in order to retain them in the second recesses 6. Through the bottom recesses 24, the exchange of the liquids takes place, whereby the interference with the cells is minimized.
[0081] This embodiment has the advantage that the cross-sectional openings 5 of the second recesses 6 are directly open and accessible, and the second recesses 6 are also in liquid communication with the first recess 4 through the transition conduit 23.
[0082] FIG. 9B shows a carrier 1, in plan view onto the cross-sectional openings 5 of second recesses 6 and onto the cross-sectional opening of a first recess 4. The transition conduits 23 shown here in hatched lines are concealed by the first partial carrier 2. The cross-section along A-A through the first partial carrier 2 is shown in FIG. 9A in the first partial carrier 2.
[0083] FIG. 9C shows a top view of a first partial carrier 2 in which both the first recess 4 and the second recesses 6 are formed as spaced-apart through holes.
[0084] The second partial carrier 3 has one or more bottom recesses 24 which extend into a constant depth and which are covered by the cross-sections of at least one first recess 4 and at least two second recesses 6 and which forms a transition conduit 23 connecting a first recess 4 to the second recesses 6.
[0085] FIG. 10 shows an embodiment in which the carrier 1 consists of a first partial carrier 2, preferably made of plastic, and a second partial carrier 3, preferably made of glass, the first partial carrier 2 being connected to the second partial carrier 3 along the circumference thereof. A first recess 4 extends over second recesses 6 formed in the second partial carrier 3. The first partial carrier 2 has an outlet 9 at a distance from the region of its first recess 4, preferably at a distance from the region covered by the second partial carrier 3. A supply conduit 10 opens at a distance above the first recess 4 to meter drops of liquid F into the first recess 4. Preferably, the first partial carrier 2 opposite to the second partial carrier 3 has a cross-sectional opening, into which or against which the supply conduit 10 is directed. Preferably, a connection conduit 15 is connected to the outlet 9.
[0086] FIGS. 11A and 11B show embodiments in which a second partial carrier 3 is arranged at least partially or completely within the first recess 4 of a first partial carrier 2, wherein the cross-sectional openings of the first recess 4 and the cross-sectional openings of the second recesses 6 are arranged in parallel to one another and preferably lie in a common plane. When the first and second partial carriers 2, 3 are arranged horizontally, the cross-sectional openings of the first recess 4 and the cross-sectional openings of the second recesses 6 are horizontal and open at the top. The second recesses 6 each have at least one through hole 6c in their bottoms 6b, so that each second recess 6 communicates with the first recess 4 through at least one through hole 6c even if the second partial carrier 3 protrudes over the cross-sectional opening of the first recess 4 and/or over the level of the liquid F in the first recess 4. The bottoms 6b of the second recesses 6 formed in the second partial carrier 3 are spaced from the first partial carrier 2, preferably spacers 25 are arranged between the bottoms 6b of the second partial carrier 6 and the first partial carrier 2. The through holes 6c preferably each have cross-sections smaller than cells Z. The through holes 6c can be coated, e.g. hydrophobically, to prevent or assist the exchange of certain liquids.
[0087] As shown in FIG. 11B, the first partial carrier 2 can have an outlet 9 arranged, for example, below the second partial carrier 3. A connection conduit 15 can be connected to the outlet 9. Optionally, the connection conduit 15 can also form a supply conduit 10. Alternatively or additionally, a supply conduit 10 can open above the first partial carrier 2 in order to meter liquid F into the first recess 4.
[0088] The embodiments in FIGS. 11A and B are particularly suitable for separating cells and cell products from one another.
[0089] In the method, the second recesses 6 and the first recess 4 are filled with liquid F, which is in particular medium, and are in contact with one another through the through holes 6c. In particular in an embodiment in which a supply conduit 10 opens below the second recesses 6 or below the second partial carrier 3 in the first partial carrier 2, a gas, e.g. inert gas or air, e.g. air or oxygen for cell cultivation, optionally air or O.sub.2 with 5% CO.sub.2, can be introduced through the supply conduit 10 in addition to or as an alternative to liquid F. The gas can be introduced in a bubble-free manner by the volumetric flow of the gas being sufficiently small. Alternatively, the gas can be supplied with a volumetric flow sufficient to form bubbles, wherein liquid F is moved in the second recesses and optionally cells Z are floated by means of the gas bubbles G. Gas bubbles can alternatively be generated by cavitation induced by laser radiation.
[0090] By pressure changes, cells Z can be moved away from the through holes 6c to aid in the exchange of the liquids.
[0091] FIG. 12 schematically shows an embodiment in which the carrier 1 consists of a second partial carrier 3 having second recesses 6, the cross-sectional openings 5 of which and regions of the surface of the carrier 1, 3 between these cross-sectional openings 5 lie in a common plane E. In particular, if these surface regions between the cross-sectional openings 5 have a surface energy that does not permit wetting by the liquid F or permits it only to a small extent, for example surface regions made of plastic or functionalized, coated or hydrophobically structured glass, a liquid F moving across the plane E, for example in the form of a rolling drop Fr, can take up spent medium from the second recesses 6 and at least partially replace it. A rolling drop Fr can be introduced, for example, by applying liquid F in the form of a plurality of drops onto the plane E or into second recesses 6, e.g. through a supply conduit 10 opening into a second recess 6, as e.g. shown in FIG. 2. Liquid F projecting above the plane E, in particular in the form of a rolling drop Fr, can be driven by tilting the carrier 1 against the horizontal, by accelerating and/or vibrating the carrier, by applying a gas flow.
[0092] Generally, in embodiments in which the carrier consists of a second partial carrier 3 having second recesses 6 therein, the second partial carrier can be referred to as the carrier 3 having recesses 6 therein.
TABLE-US-00001 Reference sign 1 carrier 14 bottom of the second recess 2 first partial carrier 15 connection conduit 3 second partial carrier 16 seal 4, 4a, 4b first recess 17 first metering device 5 cross-sectional opening of a second recess 18 second metering device 19 membrane 6 second recess 20 first surface of the carrier 6b bottom of a second recess 21 supply recess 6c through hole 22 material 7 first thickness section 23 transition conduit 8 second thickness section 24 bottom recess in second partial carrier 9 outlet 25 spacer 9a conduit section of the outlet E plane 10 supply conduit F liquid 11 end wall, end surface Fr rolling drop 12 second surface of the carrier Z cell 13 controlled valve G gas bubble
