DEVICE FOR SEEDING CELLS

Abstract

A device for seeding cells includes a container with a wall, a bottom and a lid. The wall extends between the bottom and the lid. The container can be equipped to be loaded with cells, in particular with cells form a cell suspension. The container defines a rotation axis. The device is further equipped to rotate the container around the rotation axis. The container includes a structured surface that can be arranged at the inner surface of the container. The structured surface has structures equipped to receive the cells. The rotation exerts a (g-)force in direction of the structured surface, such that the g-force acts perpendicular to the structured surface. The exerted force in the direction of the structured surface resembles a g-force required for sedimentation of the cells into the structures.

Claims

1. A device for seeding cells, wherein the device comprises: a container with a wall, a bottom and a lid; wherein the container comprises a rotation axis; wherein the device is equipped to rotate the container around the rotation axis, by means for rotating the container about said rotation axis; wherein: the container comprises a structured surface, wherein the structured surface is equipped to receive cells, and wherein a rotation around the rotation axis exerts a (g-)force in direction of the structured surface; wherein the structured surface is at least one of biologically inert and non-adhesive to cells.

2. The device according to claim 1, wherein the structured surface comprises a hydrogel and/or wherein the structured surface comprises micro wells for receiving cells.

3. The device according to claim 1, wherein the structured surface supports the scaffold-free formation of cell aggregates.

4. The device according to claim 1, wherein the structured surface is arranged at the wall of the device and/or the structured surface is arranged such that the bottom of the device is free of the structured surface.

5. The device according to claim 1, wherein the device is equipped to promote the growth of 3D cell aggregates, in particular the device is equipped such that the structured surface covers essentially the whole wall of the container and/or such that at least the a portion of the wall not covered by the structured surface is coated with a cell repellent.

6. The device according to claim 1, wherein the structured surface is arranged essentially parallel to the rotation axis.

7. The device according to claim 1, wherein the container comprises an inner tubing, wherein cells are homogenously emitted from the inner tubing towards the structured surface.

8. The device according to claim 1, wherein the container is symmetrical with respect to the rotation axis, and wherein the container comprises at least one of the following features: the wall of the container is curved; the structured surface is curved; the container is cylindrical, conical and/or can have another regular or irregular shape; the device comprises a closing mechanism to removable connect the bottom and/or the lid to the wall.

9. A serial reactor, wherein the serial reactor comprises first and second devices according to claim 1, and an adaptor; wherein the adaptor is equipped to connect a first device to a second device.

10. the device according to claim 1, wherein the structured surface comprises a surface, at least a first recession and a second recession for receiving cells for generating and cultivating cell aggregates, wherein the first and the second recessions are spatially separated, and wherein the surface between the first and the second recession is a sloped surface and wherein the first and the second recessions comprise an upper part and a bottom part and wherein the upper part has the shape of a truncated cone with an opening angle of 12° to 60° and a ratio of a depth of the upper part to a diameter of the widest end of the upper part is at least 1.5:1.

11. The device according to claim 1, wherein the structured surface is removably positioned in the container, wherein the structured surface is designed as a cartridge that is mountable to the wall of the container; and wherein the device comprises at least one of the following features: the removable structured surface is positioned at the inner surface of the container; the structured surface is positioned at the wall; the device further comprises means for sterilisation of the container.

12. A cartridge for being mounted in a device according to claim 1, wherein the cartridge is mountable in the container, and wherein the cartridge comprises the structured surface for receiving the cells.

13. Use of the device according to claim 1, for generating, maintaining and/or harvesting of 3D cell aggregates and/or for the production of conditioned medium.

14. A method to generate 3D cell aggregates comprising the steps of: providing a device for seeding cells, said device comprising a container with a wall, a bottom and a lid; wherein the container comprises a rotation axis wherein the device is equipped to rotate the container around the rotation axis, by means for rotating the container about said rotation axis; the container comprises a structured surface, wherein the structured surface is equipped to receive cell, and wherein the rotation exerts a (g-)force on the/in direction of the structured surface; providing a cell suspension to be introduced in the container; loading the device with the cell suspension; rotating the container to exert a (g-)force on the/in direction of the structured surface and collecting cells in the structured surface; culturing the collected cells in the structured surface while further rotating the container.

15. The method according to claim 14, wherein 2D cell aggregate formation is promoted on an inner wall of the container and the structured surface is arranged on top of the 2D cell aggregates.

16. The method according to claim 14, wherein the provided device is a first device, the method comprises the steps of: providing a second device, comprising a second container with a second wall, a second bottom and a second lid; wherein the second container comprises a second rotation axis wherein the second device is equipped to rotate the second container around the second rotation axis, by means for rotating the container about said rotation axis; the second container comprises a structured surface, wherein the structured surface is equipped to receive cell, and wherein the structured surface is arranged essentially parallel to the second rotation axis; wherein the rotation exerts a (g-)force on the/in direction of the structured surface; connecting the first device to the second device as a serial reactor; loading the second device with a cell suspension, wherein the cell suspension loaded in the first device and in the second device is identical and/or different from each other; rotating the serial reactor, in particular wherein the container of the first device and the second container rotate at the same and/or different speed.

17. A method for producing conditioned medium produced by 3D cell aggregates comprising the steps of: generating 3D cell aggregates according to claim 12; and maintaining the 3D cell aggregates in the container; and collecting the culture medium surrounding the 3D cell aggregates from the container as conditioned medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0179] The subject matter of the invention will be explained in more detail in the following text with reference to exemplary embodiments which are illustrated in the attached drawings, in which:

[0180] FIG. 1 shows a cross-section of a device for seeding cells;

[0181] FIG. 2 shows a longitudinal section of the device;

[0182] FIG. 3 shows a three-dimensional view of cross-section of the device;

[0183] FIG. 4 shows a cross section of the device immediately after being filled with cell suspension;

[0184] FIG. 5 shows a cross section of the device after compacting of the cells by rotation is complete;

[0185] FIG. 6 shows the process of 3D cell aggregate formation;

[0186] FIG. 7 shows the process of harvesting the 3D cell aggregates;

[0187] FIG. 8 shows a serial reactor including two connected devices;

[0188] FIG. 9 shows the distribution of cells in the structured surface;

[0189] FIG. 10 schematically shows the structured surface; and

[0190] FIG. 11 shows a longitudinal section of the container including an inner tubing.

DETAILED DESCRIPTION OF THE INVENTION

[0191] The reference symbols used in the drawings, and their meanings, are listed in summary form in the list of reference symbols. In principle, identical parts are provided with the same reference symbols in the figures.

[0192] FIG. 1 shows a cross-section of a device for seeding cells with a cylindrical container 10. The cylindrical container 10 includes a wall 100, a bottom 104 and a lid 105. The wall 100 is containing a structured surface 101 with multiple conical recessions 102 or micro-wells. A cavity 103 is formed by the container 10 circumscribed by the wall, the bottom and the lid (see FIG. 2).

[0193] FIG. 2 shows a longitudinal section of a cylindrical device. It is designed as cylindrical tube. One side of the wall 100 is closed by a bottom 104. The bottom might be flat or structured. On the opposite side of the bottom, a lid 105 is serving to close the wall in a way that a junction 106 between wall 100 and lid 105 is air- and waterproof. This is either achieved in a permanent way, e.g., by welding or gluing the lid to the wall, or in a removable way, e.g., by screwing or any other known way of connecting a lid to a cavity, as, e.g., a bayonet lock or flange.

[0194] On one hand, the use of a removable lid may support multiple uses of the wall 100 and the lid. The structured surface attached to the wall 100 would have to be replaced in the multi-use operation. On the other hand, a permanent closure would imply a sterile single use device.

[0195] As shown in FIG. 1, the inner surface of the wall 100 is essentially entirely covered with a structured surface 101 with multiple conical recessions 102 or micro wells. The micro well can have any desired form required to maintain desired 3D cell aggregates. The cavity 103 is accessible via two ports (107 and 108). Each of the two ports carries a closing mechanism as, e.g., thread or a valve, so that both sides of the device may be either connected with a tubing system for gas and liquid transfer or be closed with a plug, e.g., a screw plug.

[0196] FIG. 3 shows a three-dimensional view of cross-section. The entire inner surface of the wall 100 is covered with structured surface 101 including conical micro wells or recessions 102.

[0197] FIG. 4 shows a cross section of the device immediately after being loaded with cell suspension 110. Rotation has not started yet and cells have not sedimented/compacted.

[0198] FIG. 5 shows a cross section of the device after sedimentation/compacting by rotation is complete. All cells have formed aggregates 111 at the bottom of micro wells 102. The cavity 103 of device is now containing essentially cell-free culture medium.

[0199] FIG. 6 shows the process of 3D cell aggregate formation in detail. The 3D cell aggregates can also be called spheroids. Insert A shows the device before filling with cell suspension. Insert B shows the device immediately after being loaded with cell suspension, as depicted in FIG. 4. Insert C shows that the cells have sedimented/compacted into micro-wells by rotation but not yet aggregated. Insert D shows that in each micro-well, cells have formed a 3D cell aggregates or spheroid.

[0200] FIG. 7 depicts the process of harvesting the 3D cell aggregates. A centrifugal force keeps cells in the micro-wells. When medium is removed while device is rotating, cells (C) and 3D cell aggregates/spheroids (B) remain trapped in the micro-wells due to surface tension of the air-liquid interface, even if rotation is stopped. For harvesting, 3D cell aggregates are flushed out of the micro-wells by shaking of device (A).

[0201] For harvesting of the conditioned medium the 3D cell aggregates 111 remain in the microwells with and/or without rotating the container 10.

[0202] FIG. 8 shows two devices coupled with an adaptor 112 to provide a serial reactor. The adaptor 112 enables the connection between the two devices. In the connected serial reactor culture medium can flow (arrow) from one device into another. It is also possible to couple several devices together. The shown devices are coupled in the lid-bottom manner. It might also be possible to connect the devices in a bottom-bottom or lid-lid manner. The type of coupling between the devices with the adaptor depends of the type of closing mechanism provided at the lid and/or bottom.

[0203] FIG. 9 shows the uniform distribution of cells in the structured surface 101. The 3D cell aggregates are uniformly structured with respect to size and shape. The cells are WiDr cells. 500 cells per microwell were seeded in the container. The 3D cell aggregates 111 were grown for 7 days in culture medium. The obtained 3D cell aggregates 111 are very evenly developed after seeding. The developed/grown 3D cell aggregates 111 reveal a homogenous and/or uniform distribution in terms of size and shape.

[0204] FIG. 10 schematically show the structured surface 101. FIG. 10 shows several structured surfaces 101 for generating and cultivating or cell aggregates 111, which includes a first recession 201 for receiving cells and a second recession 202 for receiving cells. Furthermore, the structured surface 101 includes a partition wall 203, which separates the first and the second recession and a bottom part 213. As can be gathered from FIG. 10, parts a-c, the “upper surface” 212 of the structured surface 101 has a shape that facilitates a transport of cells from a distal end (“upper surface” 212) of the structured surface 101 into at least one of the first recession and the second recessions. FIG. 10, parts a-b show, that the surface can be shaped like a sloped sidewall 205, which causes transport effect. These sidewalls can end in a tip 207. But other shapes are also possible as, e.g., a convex shape or a curved shape. Consequently, the structured surface 101 of FIG. 10 facilitate a high throughput uniform cell distribution within the structured surface 101 and advantageously avoid sedimentation of cells at undesired locations, e.g., the upper surface or recession sidewall. As can be gathered from FIG. 10, the structured surface 101 does not include any “horizontal” section or any “horizontal” portion, where the cells could disadvantageously sediment.

[0205] FIG. 10, part d, shows different possibilities for the bottom part 214 of the recessions. The upper part 206 is preferably always shaped as a truncated cone. The bottom part can have various geometries. For example, the bottom part can just be flat like in FIG. 10, parts a-c. In this case the bottom part is the apical end of the upper part.

[0206] Furthermore, the partition wall 203 provides for a section 206 between the distal end and the base section 104. Section 206 includes a second slope (truncated cone) and it can be seen that the second slope is steeper than the slope of sidewall 205 of the “upper surface” portion 112. Therefore, the cell transport to the bottom of the structured surface is provided. The structured surface 101 of FIG. 10, part a, includes a partition wall with a tapered surface ending in a tip 207 at the distal end of the partition wall 203. Furthermore, the recession can include a tapered bottom part as shown in FIG. 10, part d, (3. and 4. figure from the left). Providing such geometry of the device on the given scale of structured surface 101 facilitates and improves the formation of 3D cell aggregates 111, their cultivation as well as their analysis.

[0207] As can be gathered from FIG. 1, a raster of conical recessions microwells with respective chamfers at the opening, i.e. distal end, of the recessions, and a tapered bottom is presented. The depth of the conical recessions allows for an uncomplicated change of the cell culture medium, the transfer into another culture dish, and the staining of 3D cell aggregates 111 without flushing the aggregates out of the structured surface 101.

[0208] FIG. 11 shows a longitudinal section of the container 10 including an inner tubing 110. The inner tubing reaches though the container 10 from the bottom 104 to the lid 105. Cells can be homogenously emitted from inner tubing 110 towards the structured surface 101.

[0209] The inner tubing 110 is positioned inside the container 10. The inner tubing 110 is surrounded by the wall, the bottom and the lid. In other words: The inner tubing 110 is an additional cavity inside the container 10.

[0210] The inner tubing 110 essentially extends from the bottom 104 to the lid 105. The inner tubing 110 can be punctured and/or perforated. The cells can be injected inside the container 10 via the inner tubing 110.

[0211] The inner tubing 110 can rotate together with and/or in a different speed and/or in a different direction than the container 10. In another embodiment of the invention the inner tubing 110 is stationary and does not rotate.

[0212] While the invention has been described in present preferred embodiments of the invention, it is distinctly understood that the invention is not limited thereto, but may be otherwise variously embodied and practised within the scope of the claims.