Cell culture device, method for producing cell spheroids for culturing the cell spheroids with the cell culture device, and method for using the cell culture device

Abstract

The present invention provides a cell culture device for culturing cell spheroids, comprising n compartment walls defining a compartment bottom region and a volume V above the compartment bottom region. K and at least one compartment having p upper ends and a volume V M and a plurality of microwells each having a volume V, the microwells being arranged in the compartment bottom region such that the compartment bottom region outside the microwells simply has an inclined surface. The number of microwells is selected so that the entire compartment bottom region is covered with microwells, and all the microwells have the same volume V. M wherein the microwells have the shape of a pyramid or a cone with a rounded tip to accommodate cells, and the pyramid or the cone is further rounded between the tip and the base region.

Description

Technical Field

[0001] The present invention relates to an apparatus for aggregating cells in a test chamber. The apparatus is characterized in that it includes a cavity of a special shape that enables aggregation of individual cells and forms cell spheroids when a cell suspension is seeded onto the apparatus. The present invention further relates to a method for cell aggregation and the use of the apparatus according to the present invention for cell aggregation.

Background Art

[0002] Stem cell research studies the principles of the regeneration process in tissues in order to develop methods of regenerative medicine. Here, a very important element of stem cell biology is the regular communication between the stem cells themselves and the surrounding tissue, the so-called stem cell niche. These cells gather to form tissue units called cell clusters, cell spheroids, or "organoids", which are large and complex structures that ultimately form entire organs.

[0003] There are various experimental environments for analyzing such processes, but here it is standard to use the "hanging drop". In this case, the growth of stem cells is simulated by dropping a certain amount of stem cells and other cells to form analyzable cell clusters. The disadvantages of this widely used technique are that the number of cell clusters that can grow is limited by this method and the medium exchange is limited. However, this point is important because the differentiation of stem cells depends on the presence of specific cytokines at a certain time, and these cytokines can be added during medium exchange.

[0004] In a clinical setting, in various treatment approaches such as pancreatic islet transplantation, for example, the possibility of mass-producing defined-sized cell clusters with the potential for medium exchange is highly desirable. In this approach, at the initial stage after transplantation, the supply of nutrients and oxygen based on diffusion is limited, and large islets die of oxygen deficiency after transplantation, so smaller islets are considered better than larger ones. As a result, 2 to 3 transplants are required until the patient has functionally sufficient islets and no longer needs insulin injections (Lehmann R. et al., Diabetes. 2007 Mar; 56 (3):594-603). Therefore, it is desirable to create many small islets rather than a few large ones. Numerically, one transplant would be sufficient to guarantee that diabetes is functionally cured and insulin injections are no longer necessary. To grow small islets and succeed in clinical application, it is necessary to break down the islets into individual cells and then re-aggregate them to form small "pseudo-islets". Approximately 1 million pseudo-islets are required for transplantation, and this number cannot be achieved by the hanging drop method.

[0005] WO 2008 / 106771 describes an apparatus for preparing cell aggregates. This apparatus is sold under the trade name Aggrewell (Stemcell Technologies, Vancouver, BC, Canada V5Z 1B3). However, this apparatus has only been developed as a laboratory product and not as a medical device, and should only be used for application to the human body on the premise that the user bears the risk.

[0006] Furthermore, the described device has limited practicality for stem cell cluster production because the microwells have a purely inverted pyramid shape or the shape of a flat-bottomed pyramid. However, in any case, the sides of the pyramid are not rounded. Therefore, instead of being pushed into a round spheroid / cluster shape, the cultured cells will be pushed into a pointed pyramid shape or the corresponding side edges. This contradicts the physical basic principle of "minimization of free energy" that all cell processes undergo. Also, with this microwell shape, the cells are not supported to form circular cell spheroids and are instead pushed into an unnatural structure.

[0007] Furthermore, in such a device, different concentrations of oxygen and signaling molecules exist for individual cells within the cell clusters formed by the irregular shape, thus interfering with the natural cell communication mechanism. Therefore, this device is not suitable for cell therapy because the shape of the microwells causes disruption of the signaling pathway, which may lead to incorrect programming and ultimately tumor formation in subsequent stem cell-based applications. Additionally, this device requires a solution to suppress cell adhesion. Since this solution is not clinically approved, for this reason too, it is not suitable for clinical use of this device.

[0008] In the prior art, only one device is known (WO 2010 / 142755 A2) that can culture cell clusters on the required scale. This device has a plurality of designed microwells. However, here, due to technical limitations, the microwells do not cover the entire bottom region of the device, leaving a substantially horizontal edge along the partition wall where individual cells can potentially deposit. To avoid this, WO 2010 / 142755 A2 proposes the use of inserts placed on the protruding edges.

[0009] However, in this system, in the case of careless handling, individual cells can reach below the edge of the insertion part. In the case of stem cells, they can proliferate in an uncontrollable state, and in the worst case, there is a risk of forming tumors in the patients treated with this cell product.

[0010] Therefore, there is still a need for a cell culture device designed to prevent individual cells from settling outside the microwells and differentiating disorderly there.

Summary of the Invention

Problems to be Solved by the Invention

[0011] An object of the present invention is to provide a cell culture device designed such that all introduced cells are cultured under defined conditions and the growth of individual cells outside the microwells is prevented.

Means for Solving the Problems

[0012] At least one compartment having n compartment walls defining a bottom region of the compartment, and a plurality of microwells having p upper ends and a volume V M are included. The microwells are arranged in the bottom region of the compartment such that the bottom region outside the microwells simply has an inclined surface. The number of microwells is selected such that the entire bottom region of the compartment is covered with microwells. All the microwells have the same volume V M and the microwells have a shape of a pyramid or a cone having a rounded tip for accommodating cells, and there is a rounded portion between the tip of the pyramid or the cone and the bottom region. The above object is achieved by a cell culture device for culturing cell spheroids.

[0013] The above object is further achieved by a method for manufacturing cell spheroids for culturing the cell spheroids in the cell culture device of the present invention.

Brief Description of the Drawings

[0014]

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DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to a cell culture device including at least one compartment having a plurality of microwells with n partitioning walls defining a compartment bottom region and p upper ends and a volume V. M Here, the microwells are arranged in the compartment bottom region such that the compartment bottom region outside the microwells simply has an inclined surface, the number of microwells is selected such that the entire compartment bottom region is covered with microwells, and all the microwells have the same volume V. M

[0016] According to this definition, the cell culture device is suitable for culturing eukaryotic cells, particularly stem cells, and in particular, an assay plate is different in terms of materials used, surface treatment, adhesiveness, sterility, gas permeability, light permeability, refractive index, etc.

[0017] In a preferred embodiment, the cell culture device is composed of a material that prevents cell adhesion. Such materials specifically include suitable plastics (e.g., polystyrene, polycarbonate) and glass, whether or not they are coated to reduce cell adhesion. In a particularly preferred embodiment, the cell culture device is made of a cycloolefin copolymer.

[0018] ​The cell culture device according to the present invention includes at least one compartment having n partition walls. Therefore, the cell culture device according to the present invention is partitioned in all directions by the partition walls, and liquids such as cell culture solutions can be retained within the compartments. The partition walls can all have the same height or can have different heights. A typical height of the partition walls is from 1 to 100 mm, preferably from 10 to 30 mm. In any case, the partition walls are smooth, that is, they have no protrusions.

[0019] The partition walls define a compartment bottom region. This compartment bottom region extends across the entire region partitioned by the partition walls and is partitioned by the partition walls. In a preferred embodiment, the compartment of the cell culture device according to the present invention is open at the top, that is, the side opposite to the compartment bottom region is not closed, so that the compartment is directly accessible. The compartment volume V K is above the compartment bottom region.

[0020] The compartment of the cell culture device according to the present invention has an n-sided opening. n can be any natural number. In a preferred embodiment, n is large such that the opening of the compartment is substantially circular. In a further preferred embodiment, n is 3, 4, or 6, particularly preferably 4. Such geometric shapes facilitate production.

[0021] In a further embodiment, in each case, the walls of two compartments facing each other have equal lengths. As a result, the compartments have symmetric openings. In a preferred embodiment, the walls of all compartments have equal lengths such that the compartments have openings in the shape of an equilateral triangle, a hexagon, or particularly preferably a square.

[0022] In a preferred embodiment, the partition walls are substantially perpendicular to the compartment bottom region outside the microwells. Here, each wall angle β of 90° to 110° with respect to the compartment bottom region is said to be substantially perpendicular.

[0023] The cell culture device according to the present invention includes at least one compartment, but may also include a plurality, for example, 2, 3, 4, 5, 6, 8, 10, 12, 18, 24, 48, 96, 384, 1536, 3456, or 9600 compartments. Preferably, the cell culture device includes 4, 6, 12, 24, 96, 384, or 1536 compartments. In a particularly preferred embodiment, the cell culture device includes 6 compartments.

[0024] The cell culture device according to the present invention further includes a plurality of microwells having p upper ends c. Here, the microwell is defined as a recess in the bottom region of the compartment where cells can settle, approach each other, and grow as cell clusters. In this specification, the microwell may also be referred to as a picowell, nanowell, microcavity, and well.

[0025] The microwell of the cell culture device according to the present invention has a bottom region having p corners. In this case, the bottom region G is an opening through which cells can slide into the interior of the microwell. The opening has p upper ends c surrounding the opening. p can be any natural number. In a preferred embodiment, p is large enough so that the bottom region G approximates a circle. In a further preferred embodiment, p is 3, 4, or 6, and particularly preferably 4.

[0026] In a preferred embodiment, p is equal to n and is 4. This ensures optimal utilization of the available bottom region. In another embodiment, n is 6 and p is 3. In a further embodiment, n is large enough so that the opening of the compartment approximates a circle, and p is 4 or 6. In yet another embodiment, n and p approach infinity.

[0027] The microwells have a pyramid or cone shape, the tip of the pyramid is located within the bottom region of the compartment, and the bottom region of the pyramid or the bottom region of the cone forms the opening of the microwell. In other words, the volume V of the microwell extends from the surface of the bottom region of the compartment into the bottom region of the compartment. Thus, on the side of the present invention, the tip of the pyramid or the tip of the cone is also referred to as the bottom B of the microwell. Between the tip of the microwell and the bottom region G, specifically between the corner of the opening and the bottom B, there is a triangular side end f that demarcates the side S.

[0028] The tip serves to accommodate cells. The roundness of the tip promotes the formation of regular spheroids. In one embodiment, all the tips of the microwells in the compartment are equidistant from each other. This is important for maintaining communication between the spheroids. Cells communicate with each other through messenger substances. The information transmitted depends on the concentration of the messenger substance. From such principles of morphogenetic gradients and lateral inhibition, for cell spheroids to grow at the same rate, it is necessary for the distance between the cell spheroids to be equal. Safe use in cell therapy is possible only when all cell spheroids are at the same growth stage at the time of transplantation.

[0029] In one embodiment, the microwell has the shape of a pyramid. In a preferred embodiment, the tip of the pyramid has a diameter d B with a rounded shape.

[0030] In an embodiment where p is large enough such that the bottom region G approaches a circle, the microwell has the shape of a cone. In a preferred embodiment, the tip of the cone has a diameter d B with a rounded shape.

[0031] In a preferred embodiment, the tip of the cone or the tip of the pyramid has a diameter d between 2 μm and 500 μm, preferably between 5 μm and 400 μm, and particularly preferably between 70 μm and 200 μm. Bhas. In a preferred embodiment, the diameter d B is 90 μm. In a more preferred embodiment, the diameter d B is 180 μm or 270 μm.

[0032] That is, the tip of the pyramid or the tip of the cone of the microwell has a radius r of 1 to 250 μm, preferably 2.5 μm to 200 μm, particularly preferably 35 μm to 100 μm B has. In a preferred embodiment, the radius r B is 45 μm. In a more preferred embodiment, the radius r B is 90 μm or 135 μm.

[0033] The microwell has a depth h. The depth is 10 μm to 2000 μm, preferably 50 μm to 1000 μm, more preferably 100 μm to 500 μm, most preferably 200 μm to 400 μm. In a more preferred embodiment, the depth is 600 μm to 700 μm, particularly preferably 641 μm.

[0034] The upper end c has a length between 0 μm and 5 mm, preferably 200 μm and 2 mm, most preferably 400 μm and 1200 μm. In a preferred embodiment, all upper ends have the same length.

[0035] In a preferred embodiment, the side edge f of the triangle, i.e., the edge between the sides of the pyramid, is rounded. In a particularly preferred embodiment, the rounding of the side edge f of the triangle gradually increases from the opening towards the tip of the pyramid or the bottom of the microwell, where s (i.e., the diameter d of the rounding near the opening of the microwell f1 ) is different from the diameter d near the tip of the pyramid or the bottom of the microwell f2 . In a particularly preferred embodiment, d f1 is smaller than d f2 . In a preferred embodiment, d f1 is 0, and d f2 is d B .

[0036] The side surface S forms a wall angle α with the virtual space at the bottom region G or the opening of the microwell, or with the partitioned bottom region. α is 35° to 75°, preferably 40° to 70°, more preferably 50° to 60°, and most preferably 54.7°. At this angle, all cells slide down to the bottom or the tip B, and at the same time, it assists the cells to form natural cell spheroids, while there is an optimal condition where the cells are compressed and not pushed into an unnatural structure.

[0037] According to the present invention, the depth and the upper end length of the microwell depend on the radius r B or the diameter d B and the angle α. In a particularly preferred embodiment, α is 54.7°, p is 4, the diameter d B is 90 μm, the length of the upper end c is 500 μm, and the depth h is 320 μm. In a more preferred embodiment, α is 54.7°, the diameter d B is 180 μm, the length of the upper end c is 1000 μm, and the depth h is 641 μm.

[0038] Each section of the cell culture device according to the present invention has a multiplicity of microwells. Here, the multiplicity is defined as a natural number from 1 to 1,000,000. Therefore, one section contains 1 to 1,000,000 microwells.

[0039] In a preferred embodiment, one section contains two or more microwells. In a more preferred embodiment, one section contains exactly one microwell.

[0040] According to the present invention, the bottom region of the section is completely covered with microwells. The microwells are arranged such that there is as little intermediate space as possible between them. In one embodiment, the microwells are arranged in regular rows and columns. On the side surface of the present invention, when the surface of the end between two microwells has a width of less than 15 μm, there is almost no intermediate space between the two microwells.

[0041] In one embodiment, each microwell has at least one common upper end with each adjacent microwell.

[0042] Here, the fact that the upper ends are common means that the openings of the microwells are seamlessly adjacent to each other. Thus, in an embodiment where n equals p and is 4, the microwell x located between four further microwells x1, x2, x3, x4 shares the upper ends located between x and x1, x2, x3, x4 with each of the four adjacent microwells, that is, the upper end c1 for x1, the upper end c2 for x2, the upper end c3 for x3, and the upper end c4 for x4. The microwell y located in a row or column adjacent to the partition wall has a common upper end with the three adjacent microwells in any case. The microwell z located at the corner of the partition bottom region has a common upper end with the two adjacent microwells. The same applies when the values of n and p are other than 4.

[0043] Since the upper ends are common, cells do not remain between the microwells and grow in an uncontrolled manner there. In the cell culture device according to the present invention, due to the specific shape and arrangement of the microwells, all cells are induced into the interior of the microwells, so that each cell can grow correctly under the desired environment.

[0044] The upper ends of the microwells y and z not shared with the adjacent microwells are in the same plane as the partition wall. Here, "in the same plane" means that the shape fits so that no flat ends where individual cells can settle occur at the transition between the upper end and the partition wall.

[0045] In an aspect of the present invention, when the resulting end has a maximum width of 15 μm, it means that the upper end part is in the same plane as the partition wall. In one embodiment, this end is not horizontal, that is, not parallel to the partition bottom region, but inclined.

[0046] Each microwell has at least one common end with each adjacent microwell, and the upper end of the outermost microwell is on the same plane as the partition wall. Therefore, the partition bottom region outside the microwell only has a simple inclined surface where cells cannot settle. Thus, in the device according to the present invention, cells do not grow uncontrollably outside the microwell.

[0047] According to the present invention, all microwells have the same volume V M That is to say, in all microwells, the corresponding upper ends are of the same length respectively. Further, in other words, the cell culture device according to the present invention does not have microwells with their tips cut off. Therefore, the same growth conditions can be ensured in all microwells.

[0048] According to the present invention, all microwells in the partition are in fluid communication with each other. As a result, the same growth conditions prevail in all microwells.

[0049] The present invention further relates to the use of the cell culture device described for culturing cells. Due to the special shape of the microwell, when appropriate starting cells and culture conditions are used, cell clusters can be generated from the cultured cells.

[0050] In this case, an aggregate of cells in a regular or irregular shape extending in all spatial directions is called a cell spheroid, a cell cluster, an organoid, or a 3D cell colony. A spherical cell population is also called a cell spheroid. In this specification, the terms cell cluster, cell aggregation, and cell aggregate are used synonymously.

[0051] Thus, the cell culture device according to the present invention is particularly suitable for three-dimensional cell culture aimed at obtaining cell spheroids. Different from conventional three-dimensional culture methods, in the culture method according to the present invention, no additional device, chemical reactor, or special skills of the experimenter are required to reliably obtain cell spheroids. Instead, cells can be seeded like cells on a conventional cell culture plate, and as a result of the special shape of the microwells, cell sterols characterized by a high degree of homogeneity in size and functionality are automatically formed.

[0052] This differentiates the device according to the present invention from known devices in which cells can grow in cell clusters of any shape. As a result, the distribution of oxygen and nutrients within the cell clusters becomes non-uniform, individual clusters die, and the function of the cell clusters as a whole deteriorates.

[0053] A further advantage when using the cell culture device according to the present invention is that it is possible to eliminate individual cells that remain outside the microwells and grow uncontrollably there, so that all cells applied to the cell culture device will necessarily grow in cell clusters. This is particularly important in therapeutic applications where the obtained (stem) cell clusters are transplanted into patients. It is also effective for research to ensure that only cells with the same properties are present.

[0054] Basically, any type of cell can be cultured in the cell culture device according to the present invention. However, it is particularly preferred to culture eukaryotic cells. In the cell culture device according to the present invention, any animal cells can be cultured, particularly mammalian cells, and particularly preferably human cells.

[0055] In a preferred embodiment, the cell culture device of the present invention is used for culturing stem cells. By using the cell culture device according to the present invention, stem cell spheroids suitable for use in regenerative medicine can be obtained from stem cells. Such stem cell spheroids have already been experimentally successful in the regeneration of various organs and tissues such as the heart, lungs, liver, salivary glands, bone tissue, skin, thymus, and nerve cells (Ong CS, Zhou X, Han J, et al. in vivo therapeutic applications of cell spheroids. Biotechnology Advances. 2018 Mar - Apr;36(2):494-505.). However, the transplantation of such stem cell spheroids involves a risk of tumor formation if the cell spheroids are incorrectly programmed. The shape of the microwells and their arrangement within the device according to the present invention can prevent incorrect programming of the stem cells, and the thus obtained stem cell spheroids are safe for therapeutic use.

[0056] In another preferred embodiment, the cell culture device is used for culturing pancreatic islet cells. The term "pancreatic islet cells" is well known to those skilled in the art and relates to a group of insulin-producing cells derived from the human pancreas. By transplanting these cells, diabetes can be functionally cured. Pancreatic islet cell spheroids known from the prior art are usually too large, and most of them die due to oxygen deficiency during transplantation. By using the cell culture device according to the present invention, the size of the spheroids can be standardized, and if the size is optimal, oxygen can diffuse to the center, thus improving the survival rate.

[0057] In a more preferred embodiment, the cell culture device is used for culturing tumor cells. According to current medical knowledge, tumor cells are degenerated stem cells (so-called "cancer stem cells"). Therefore, the cell culture device according to the present invention can also be used to obtain spheroids of tumor cells. Thus, for example, tumor cells isolated from a patient can be cultured as spheroids exhibiting exactly the same characteristics as the patient's tumor. Thus, for example, it is possible to test in vitro which chemotherapy to accurately use for a patient. In addition, these tumor cell spheroids also have a high potential for drug discovery, and as a result, it is also possible to reduce the number of necessary animal experiments.

[0058] The present invention further relates to cell spheroids obtained when using the described cell culture device. Such cell spheroids can be used for medical applications, for example, as drugs. Therefore, the present invention also relates to a method for treating a human body by administering cell spheroids cultured using the device of the present invention. Its scope of application is enormous and consists of treating substantially all organ systems, for example, myocardial infarction, myocardial weakness, liver failure, stroke, wound healing, pulmonary fibrosis or vascular disorders.

[0059] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[0060] In the embodiments shown in FIGS. 1 and 2, the cell culture device has six compartments where n = 4. Here, p of the microwells is 4. The microwells are arranged such that each microwell has a common upper end with each adjacent microwell (see FIG. 3A). Further, the microwells have the same volume V and a rounded tip with a diameter d B or a radius r B The side surface of the microwell forms an angle α with the bottom region. G indicates the virtual bottom region of the pyramid, and c indicates the upper end delimiting the bottom region.

[0061] Figure 3 is a cross-sectional view of the device according to the present invention at the transition between the partition bottom region and the partition side wall. There is an angle β of about 90° between the partition bottom region and the partition side wall. There is no edge between the partition side wall and the adjacent microwell.

[0062] Figure 4 shows an embodiment of a single microwell in the form of a pyramid (Figure 4A) or a cone (Figure 4B) having a rounded tip. The pyramid has a rounded end f between the sides S and a diameter d of the rounding near the opening or bottom region G f1 is the diameter d near the pyramid tip f2 which is smaller. d f2 may be approximately the same as d B

[0063] Figure 5 shows various embodiments of the microwells according to the present invention. For example, p = 3 and the microwell has the shape of a triangular pyramid with a rounded tip. In another embodiment, p = 4 and the microwell has the shape of a square pyramid with a rounded tip. In another embodiment, p = 6 and the microwell has the shape of a hexagonal pyramid with a rounded tip. In another embodiment, p is a very large value such that the bottom region G of the microwell is close to a circle and the microwell has the shape of a cone with a rounded tip. As can be seen from Figure 5, in all these embodiments, the triangular side ends f are rounded and the diameter d of the rounding near the opening or bottom region G f1 is smaller than the diameter d near the tip of the pyramid or cone f2

[0064] Figure 6 shows the device according to the present invention for p = 3 and n = 6. In this case, the microwell has the shape of a triangular pyramid with a rounded tip and rounded ends between the sides (see Figure 6A).

[0065] Figure 7 shows the device according to the present invention for p = 4 and n = ∞. In this case, the microwell has the shape of a square pyramid with a rounded tip and rounded ends between the sides (see Figure 7A).​​

[0066] Figure 8 shows a further arrangement of the micro-wells with p = 4 in the device according to the present invention. In this case, the micro-wells have a shape of a square pyramid with a rounded tip and rounded ends between the sides (see Fig. 8A).

[0067] Figure 9 shows the device according to the present invention in the case of p = 6, n = ∞. In this case, the micro-wells have a shape of a hexagonal pyramid with a rounded tip and rounded ends between the sides (see Fig. 9A).

[0068] Figure 10 shows the arrangement of the micro-wells in the device according to the present invention with p = ∞. The micro-wells have a shape of a cone with a rounded tip (see Fig. 10A). Here too, the micro-wells have a common upper end with each adjacent micro-well.

Claims

1. n partition walls surrounding the bottom region of the partition, and at least one partition having a volume V above the bottom region of the partition Ｋ and p upper ends and a volume V Ｍ a plurality of microwells having the same, and The microwells are arranged below the bottom region of the partition such that all of the bottom regions outside the microwells have inclined surfaces, the number of microwells is selected such that the entire bottom region of the partition is covered with microwells, All of the above-mentioned microwells have the same volume V Ｍ and the microwells have a shape of a pyramid or a cone having a rounded tip for accommodating cells below the microwells, and further, there is a round between the tip and the bottom region of the pyramid or the cone, a cell culture device for culturing cell spheroids.

2. The cell culture device according to claim 1, wherein all of the tips of the microwells in the partition are equidistant from each other.

3. The microwells have side surfaces, The cell culture device according to claim 1, wherein the side surfaces form an angle of 54.7° with the bottom region of the partition.

4. The cell culture device according to any one of claims 1 to 3, wherein each partition wall is located on the same plane as at least one upper end of at least one microwell.

5. The cell culture device according to any one of claims 1 to 4, wherein each microwell has a common upper end with each adjacent microwell.

6. The cell culture device according to any one of claims 1 to 5, wherein two opposing partition walls have equal lengths.

7. The cell culture device according to any one of claims 1 to 6, wherein all upper ends of the microwells have the same length.

8. The cell culture device according to any one of claims 1 to 7, wherein n and / or p is 3, 4 or 6.

9. The cell culture device according to any one of claims 1 to 8, wherein n and / or p is large such that the partition and / or the microwell has a substantially circular bottom region or a bottom region.

10. The cell culture device according to any one of claims 1 to 9, wherein n is equal to p.

11. The cell culture device according to any one of claims 1 to 10, comprising 1, 2, 4, 6, 12, 24, 96, 384, 1536, 3456 or 9600 partitions.

12. A method for producing cell spheroids, the method comprising culturing the cell spheroids using the cell culture device according to any one of claims 1 to 11.

13. A method of using the cell culture device according to any one of claims 1 to 11 for culturing stem cell spheroids.

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