Method for producing aggregates of cells, population of aggregates, and method for culturing cells

Abstract

In one embodiment, cells 12 are suspended inside a hollow capsule 11 including a semipermeable membrane, and the hollow capsule 11 is incubated in a culture medium to allow the cells 12 to aggregate inside the hollow capsule 11. The cells 12 are a mixture of dissociated target cells 14 and auxiliary cells 15 for assisting the aggregation of the target cells 14. The target cells 14 more easily aggregate in the presence of the auxiliary cells 15 than in the absence of the auxiliary cells 15. In another embodiment, dissociated cells 12 are suspended inside a hollow capsule 11 including a semipermeable membrane, and the hollow capsule 11 is incubated in a culture medium to allow the cells to aggregate inside the hollow capsule 11. The cells 12 include at least stromal cells. Thus, an aggregate 21 of the cells 12 is produced.

Description

Method for producing cell aggregates, aggregate population, and cell culture method

[0001] The present invention relates to cell aggregation.

[0002] Figure 1a of Patent Document 1 and Figure 1a of Patent Document 2 disclose culturing cardiomyocytes in an isotonic aqueous solution wrapped in an outer layer of hydrogel. Non-Patent Documents 1 to 3 disclose culturing pluripotent stem cells in hollow capsules. Patent Document 3 shows culturing a mixture of any type of cell and undifferentiated cells on a gel-like support. The entire contents of Patent Documents 1 and 2 and Non-Patent Documents 1 to 3 are incorporated herein by reference.

[0003] International Publication No. WO 2022 / 058615 International Publication No. WO 2022 / 058617 International Publication No. WO 2015 / 129822

[0004] NEWS LETTER, Division of Biotechnology, The Chemical Society of Japan, Vol. 24, No.1 (2020.08.01), pp.17 to 20, [retrieved on 2023-06-26]. Retrieved from<https: / / bio.chemistry.or.jp / _userdata / NL24_1.pdf> Horiguchi, I., Chowdhury, MM, Sakai, Y. and Tabata, Y. (2014), Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production. Biotechnol Progress, 30: 896-904. [retrieved on 2023-06-26]. Retrieved from<https: / / doi.org / 10.1002 / btpr.1891> .Horiguchi, I., Sakai, Y. Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle. J. Vis. Exp. (101), e52835, (2015) [retrieved on 2023-06-26]. Retrieved from<https: / / doi:10.3791 / 52835> .

[0005] The present invention provides a means for efficiently agglutinating specific cells.

[0006] <1> A method for producing a cell aggregate, comprising suspending cells in a hollow capsule made of a semipermeable membrane and incubating the hollow capsule in a culture medium to aggregate the cells in the hollow capsule, wherein the cells are a mixture of dispersed target cells and auxiliary cells that assist in the aggregation of the target cells, and the target cells are more likely to aggregate in the presence of the auxiliary cells than in the absence of the auxiliary cells. <2> A method for producing the above cell aggregate, wherein the target cells are any of cardiomyocytes, liver cells, and other parenchymal cells, and / or the auxiliary cells are at least any of mesenchymal stem cells, fibroblasts, vascular endothelial cells, and other interstitial cells.

[0007] <3> A method for producing a cell aggregate, comprising suspending dispersed cells in a hollow capsule made of a semipermeable membrane and incubating the hollow capsule in a culture medium to aggregate the cells in the hollow capsule, wherein the cells consist of at least interstitial cells. <4> A method for producing the above cell aggregate, wherein the interstitial cells are at least one of mesenchymal stem cells, fibroblasts, vascular endothelial cells, and other interstitial cells. <5> A method for producing the above cell aggregate, wherein the cells further consist of any one of cardiomyocytes, liver cells, and other parenchymal cells.

[0008] <6> A method for producing the cell aggregate, comprising enveloping a gel containing the cells, the gel being made of a material different from the semipermeable membrane, with the semipermeable membrane, dissolving the gel, and eluting the dissolved gel through the semipermeable membrane to produce the hollow capsule, leaving the cells in the hollow capsule. <7> A method for producing the cell aggregate, wherein the gel is made of alginate containing a divalent cation, and dissolving the gel by allowing a chelating agent to act on the gel. <8> A method for producing the cell aggregate, comprising extruding the cell-containing gel and enveloping it in a gas flow to form beads, and enveloping the beaded gel with the semipermeable membrane. <9> A method for producing the cell aggregate, comprising passing the hollow capsule through a tube having an inner diameter smaller than the outer diameter of the hollow capsule to rupture the hollow capsule, and separating and recovering the aggregated cells from the ruptured hollow capsule. <10> A method for producing the cell aggregate, wherein the semipermeable membrane is made of poly-L-lysine. <11> The method for producing the cell aggregate, comprising incubating the hollow capsule with rotary stirring. <12> The method for producing the cell aggregate, comprising further incubating the hollow capsule to grow the aggregated cells.

[0009] <13> A population of aggregates, wherein the average diameter of the aggregates in the population is 70 to 150 μm, and the standard deviation of the diameters of the aggregates in the population is 7 to 20 μm, wherein the aggregates are formed by aggregation of target cells with the intervention of auxiliary cells, and wherein the target cells are more likely to aggregate when the auxiliary cells are present than when the auxiliary cells are not present, or the aggregates are formed by aggregation of interstitial cells. <14> The population of aggregates according to <13>, wherein the aggregates are individually encapsulated in hollow capsules made of semipermeable membranes, and the aggregates are suspended in liquid contained in the hollow capsules. <15> A cell culture method comprising suspending cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium at least until the cells aggregate in the hollow capsule, wherein the cells are a mixture of dispersed target cells and auxiliary cells that assist the aggregation of the target cells, and the target cells are more likely to aggregate in the presence of the auxiliary cells than in the absence of the auxiliary cells. <16> A cell culture method comprising suspending dispersed cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium at least until the cells aggregate in the hollow capsule, wherein the cells consist of at least stromal cells.

[0010] The present invention provides a means for efficiently agglutinating specific cells.

[0011] Schematic of cell aggregation within a capsule Schematic of cell aggregation within a capsule Schematic of beading Schematic of encapsulation Schematic of aggregate recovery Schematic of aggregate population Microscopic image of hollow capsule Microscopic image of formed organoids Histogram of organoid size Histogram of capsule and organoid size Histogram of capsule and organoid size Microscopic image of released organoids Microscopic image of fluorescently stained organoids Microscopic image of cardiomyocyte aggregation Microscopic image of cardiomyocyte and mesenchymal stem cell aggregation Microscopic image of cardiac markers on cardiac spheroids Microscopic observation of myocardial markers on organoids Graph of fluorescence intensity of each marker in Figures 16A and 16B Histogram of expression intensity of CD90 marker Graph of secretion amount of TGF-β1 and VEGF Analysis results of contractile force of myocardial organoids Analysis results of contractile force and contraction speed amplitude Analysis results of cell viability in a hypoxic environment Microscopic observation of myocardial organoids on day 2 of culture Microscopic observation of myocardial organoids on day 8 of culture Counting results of the number of organoids contained in the well Microscopic observation of mesenchymal stem cell spheres derived from adipose tissue Microscopic observation of liver organoids

[0012] <Aggregating target cells that are difficult to aggregate>

[0013] Figure 1 shows a schematic diagram of one embodiment of cell aggregation within a capsule. As shown in the upper part, cells 12 are suspended in a hollow capsule 11 made of a semipermeable membrane. As shown in the lower part, the hollow capsule 11 is incubated in a culture medium, causing the cells 12 to aggregate within the hollow capsule 11. As a result, an aggregate 16 is obtained within the hollow capsule 11.

[0014] In Figure 1, aggregate 16 may be constructed from multiple cell types and may be a three-dimensional tissue structure that functions as an organ. Such a tissue structure is referred to as an organoid in this embodiment. Similarly, a three-dimensional tissue structure constructed from a single cell type is referred to as a sphere / spheroid in this embodiment.

[0015] As shown in the upper part of Figure 1, cells 12 are a mixture of target cells 14, represented by circles, and auxiliary cells 15, represented by squares with rounded corners. The target cells 14 are dispersed before incubation and are cells that are desired to aggregate after incubation. The target cells 14 are cells that are relatively resistant to aggregation. The auxiliary cells 15 are cells that help the target cells 14 aggregate during incubation. The target cells 14 according to this embodiment are more likely to aggregate when auxiliary cells 15 are present than when auxiliary cells 15 are not present.

[0016] In one embodiment shown in Figure 1, the target cells 14 may be any of cardiomyocytes, liver cells, and other parenchymal cells, and the accessory cells 15 may be at least any of mesenchymal stem cells, fibroblasts, vascular endothelial cells, and other interstitial cells.

[0017] As shown in Figure 1, aggregates 16 are encapsulated in hollow capsules 11 made of a semipermeable membrane. Aggregates 16 consist of target cells 14 and accessory cells 15. Aggregates 16 are suspended in a culture medium or buffer solution that fills hollow capsule 11. Aggregates 16 may be released from hollow capsule 11 as described below.

[0018] In one embodiment shown in Figure 1, hollow capsules 11 are incubated, but the aggregated cells are not allowed to grow. In other words, the purpose of incubation is not the proliferation and growth of the individual cells in aggregates 16. That is, incubation is carried out for a short period of time with the sole purpose of cell aggregation. This reduces the variation in size of aggregates 16. Furthermore, the ratio of the number of target cells 14 to the number of auxiliary cells 15 in aggregates 16 is not significantly changed. During incubation, the hollow capsules 11 may be subjected to rotational agitation. Aggregates 16, protected by hollow capsules 11, are less susceptible to physical forces, such as shear forces, that arise during rotational agitation.

[0019] <Aggregating interstitial cells>

[0020] Figure 2 shows a schematic representation of another embodiment of cell aggregation within a capsule. As shown in the upper diagram, cells 17 dispersed within a hollow capsule 11 made of a semipermeable membrane are suspended. As shown in the lower diagram, the hollow capsule 11 is incubated in a culture medium, causing the cells 17 to aggregate within the hollow capsule 11. At this time, the hollow capsule 11 may be rotated and stirred. As a result, an aggregate 21 is obtained within the hollow capsule 11. The aggregate may be a three-dimensional tissue structure with organ functions, i.e., an organoid.

[0021] As shown in the upper part of Figure 2, cells 17 are composed of at least interstitial cells 19, represented by squares with rounded corners. Interstitial cells 19 may be at least one of mesenchymal stem cells, fibroblasts, vascular endothelial cells, and other interstitial cells. Cells 17 may further be composed of parenchymal cells 20. Parenchymal cells 20 may be any of cardiomyocytes, liver cells, and other parenchymal cells. Cells 17 may also be composed only of interstitial cells.

[0022] In Figure 2, incubation of cells 17 placed in hollow capsule 11 can be performed by the method described with reference to Figure 1. Aggregates 21 are enclosed in hollow capsule 11 made of a semipermeable membrane. Aggregates 21 are composed of at least interstitial cells 19. Aggregates 21 are suspended in a culture medium contained in hollow capsule 11. As will be described later, aggregates may be released from hollow capsule 11. Aggregates 21 may be organoids or spheroids.

[0023] <Placing cells in hollow capsules>

[0024] An example of an encapsulation process that can be used in any of the above aggregation modes will now be described. Here, we will use the example of cells 12 shown in Figure 1, which are composed of target cells 14 and auxiliary cells 15. The following method can also be applied to cells 17, which are composed of stromal cells, shown in Figure 2.

[0025] 3 is a schematic diagram showing one embodiment of the process for forming cells into beads for cell encapsulation. In this process, beads 24 are continuously generated by extruding a gel 13 containing cells 12 from a nozzle 22 while being enveloped in a gas flow 23. Increasing the number of nozzles 22 allows for higher throughput in the production of beads.

[0026] 3, the cross-sectional diameter of the nozzle 22 is preferably 100 μm to 1000 μm, and may be any of 200, 300, 400, 500, 600, 700, 800, and 900 μm. Unless otherwise specified, in this embodiment, the term "diameter" refers to the diameter.

[0027] 3, the gas in the gas flow 23 is an inert gas, such as nitrogen. The pressure of the gas flow 23 may be higher than atmospheric pressure (101.325 kPa if atmospheric pressure is standard pressure). The difference between the gas pressure and atmospheric pressure is preferably 0 kPa to 100 kPa, and may be any of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and 90 kPa.

[0028] As shown in Figure 3, target cells 14 and accessory cells 15 are dispersed in gel 13. Nutritional factors 18 necessary for the culture of these cells are also dissolved in the gel. The gel base material is preferably a material with low cytotoxicity, such as alginate. When gel 13 is composed of an alginate / water system, beads 24 composed of gel 13 can be produced by, for example, dropping a mixture of sodium alginate and a cell suspension into liquid 25. The concentration of sodium alginate may be 0.5% to 5.0% by weight, or any of 0.75, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, and 3.0% by weight.

[0029] In FIG. 3, the diameter of the beads 24 can be adjusted based on, for example, the cross-sectional diameter of the nozzle 22, the pressure of the gas flow 23, or the concentration of the gel 13.

[0030] In FIG. 3, the density of cells 12 per volume of beads 24 is 0.1×10 ６ / ml to 10 x 10６ / ml, and may be 0.2, 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, and 4.0 x 10 ６ / ml.

[0031] As shown in the lower part of Fig. 3, the beads 24 are held in a liquid 25 suitable for maintaining their shape. For example, when the gel 13 is made of alginate, an ionic crosslinking agent, such as Ca, is used to maintain the shape of the beads 24. ２＋ The liquid 25 is, for example, CaCl ２ Alternatively, a salt of a divalent cation such as methyl methyl ketone may be dissolved in water. In the liquid 25, the sodium in the alginate is replaced by the divalent cation. This results in beads 24 made of cross-linked alginate.

[0032] 3, a large number of beads 24 having a uniform size distribution can be produced by using a gas flow 23. This allows the hollow capsules having a uniform size distribution, as described below, to be produced quickly and in large quantities.

[0033] 4 is a schematic diagram of the process for obtaining hollow capsules from beads. First, as shown in the upper part of the figure, beaded gel 13, i.e., beads 24, are wrapped in a semipermeable membrane 27. In one embodiment, semipermeable membrane 27 is made of a material different from that of gel 13. When viewed from the gel 13 side, gel 13 is made of a material different from that of semipermeable membrane 27. Semipermeable membrane 27 may be poly-L-lysine (PLL). For example, the surface of beads 24 may be coated with semipermeable membrane 27 made of poly-L-lysine by incubating beads 24 in an aqueous poly-L-lysine solution.

[0034] The concentration of poly-L-lysine may be from 0.005% to 5% by weight, and may be any of 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 2.0% by weight.

[0035] The incubation atmosphere may be a condition suitable for cell culture, for example, 37°C, 5% CO ２The atmosphere is also good.

[0036] Next, as shown in the middle part of Figure 4, the gel 13 is dissolved. The beads 24 disappear, leaving behind the cells 12. The components of the dissolved gel 13 are eluted to the outside through the semipermeable membrane 27. This results in a hollow capsule 11 made of the semipermeable membrane 27. The cells 12 are left in the space 28 within the hollow capsule 11. The gel 13 is then replaced with a culture medium or buffer solution.

[0037] 4, when the gel 13 is made of alginate containing divalent cations, it is preferable to dissolve the gel by applying a chelating agent to the gel 13. At this time, the chelating agent removes the divalent cations from the gel 13. The gel 13 that has lost the divalent cations dissolves in the buffer solution inside and outside the semipermeable membrane 27. The type of chelating agent is not limited, but for example, sodium EDTA or sodium citrate may be used.

[0038] Next, as shown in the lower part of Figure 4, cells 12 are incubated and aggregated to obtain aggregates 16 in hollow capsules 11. This process is as described with reference to Figures 1 and 2. Organoids with a uniform size distribution can be rapidly formed in hollow capsules 11.

[0039] In Figure 4, uniform capsules can be easily produced by setting the target diameter of hollow capsule 11 to 100, 200, 300, or 400 μm or more. The target diameter of hollow capsule 11 may also be 1000, 900, or 800 μm or less. The target diameter of hollow capsule 11 may also be 500, 600, or 700 μm. The target diameter of hollow capsule 11 may be the major axis of the hollow capsule measured under a microscope. The size of hollow capsule 11 can be adjusted by the size of beads 24.

[0040] 4, the target diameter of aggregates 16 may be 10 to 1000 μm, or any of 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, and 500 μm. The target diameter of aggregates 16 can be controlled by the density of cells 12 in beads 24.

[0041] <Detachment from the hollow capsule>

[0042] FIG. 5 is a schematic diagram illustrating the recovery of aggregates 16. As shown in FIG. 5, aggregates 16 are released from hollow capsules 11. Hollow capsules 11 are ruptured by passing them through tube 30. The shape of tube 30 is not particularly limited. For example, tube 30 may be a syringe needle. It is preferable that tube 30 has an inner diameter smaller than the outer diameter of hollow capsules 11. A preferred method for passing hollow capsules 11 through tube 30 is for hollow capsules 11 to pass through tube 30 only once. For example, the hollow capsules 11 may be sucked into a syringe (not shown) using a tube thicker than tube 30, and the tube connected to the syringe may be changed to tube 30, and the hollow capsules 11 may be discharged from the syringe through tube 30.

[0043] The aggregates 16 shown in Figure 5 may be filtered to separate and recover them from the ruptured semipermeable membrane 27. By using a filter with an appropriate mesh size, the aggregates 16 can be collected in the filtrate, and the ruptured semipermeable membrane 27 can be captured on the filter. The above method can be applied to both the aggregates 16 shown in Figure 1 and the aggregates 21 shown in Figure 2.

[0044] <Collections of aggregates>

[0045] 6 is a schematic diagram of a population 31 of aggregates 16. The aggregates 16 are produced in hollow capsules 11. The average diameter of the aggregates 16 may be 10 to 1000 μm, and may be any of 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, and 500 μm. The standard deviation of the diameter of the aggregates 16 may be 1 to 50 μm, and may be any of 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, and 40 μm.

[0046] In one embodiment shown in Figure 6, aggregate 16 is an aggregate of target cells interposed with auxiliary cells, as described with reference to Figure 1. Here, target cells are more likely to aggregate when auxiliary cells are present than when auxiliary cells are not present. In another embodiment, aggregate 16 is an aggregate of at least interstitial cells, as described with reference to Figure 2.

[0047] 6, aggregates 16 are individually encapsulated in hollow capsules 11 each comprising a semipermeable membrane 27. Aggregates 16 are suspended in a liquid contained in hollow capsules 11. In another embodiment, aggregates 16 are not encapsulated in hollow capsules 11 each comprising a semipermeable membrane 27, and may be exposed.

[0048] <Cell conditions>

[0049] Unless otherwise specified, cardiomyocytes were selected as the target cells or parenchymal cells to be aggregated. Cardiomyocytes are sometimes abbreviated as CM. Cardiomyocytes were not thawed from frozen samples, but were collected from cultures. Furthermore, unless otherwise specified, cardiomyocytes were selected as auxiliary cells or interstitial cells for aggregation. Mesenchymal stem cells are sometimes abbreviated as MSC. Mesenchymal stem cells were thawed from frozen samples. Both cardiomyocytes and mesenchymal stem cells were differentiated from iPS cells. Furthermore, when cardiomyocytes and mesenchymal stem cells were mixed, the mixing ratio was 10:1.

[0050] <Conditions for manufacturing hollow capsules>

[0051] The beads were prepared as explained using Figure 3. Sodium alginate was used as the gel base material at a final concentration of 1.5 wt%. Nitrogen was used as the gas at 25 kPa to 75 kPa. The inner diameter of the nozzle used to extrude the gel was 160 μm. The hollow capsules were prepared as explained using Figure 4. Poly-L-lysine was used as the semipermeable membrane.

[0052] <Incubation conditions>

[0053] Unless otherwise stated, cultures were incubated at 37°C, 5% CO under rotary agitation. ２ The cells were incubated in an ambient atmosphere under 5% serum conditions.

[0054] Controlling the gas pressure of the gas stream

[0055] Figure 7 shows microscopic images of hollow capsules made from beads produced using gas flows with various pressures. The black bar at the bottom right of each image represents a scale of 200 μm. No cells were included in the beads or hollow capsules in this experiment. The size of the beads and hollow capsules can be adjusted by the pressure of the gas flow. The higher the pressure, the smaller the size of the beads and hollow capsules. If the diameter of the target hollow capsule size is larger than 400 μm, the size distribution of the hollow capsules can be made more uniform.

[0056] Controlling the density of cells inside the hollow capsule

[0057] Figure 8 shows microscopic images of organoids created with cells at various densities ( / ml). The black bar at the bottom right of each image represents a scale of 200 μm. Hollow capsules were created from beads created with a gas flow of 55 kPa. The diameter of the hollow capsules was greater than 400 μm. The diameter of the organoids is closely related to the cell density. When the cell density was 1×10 ６ / ml to 2 x 10 ６ At a concentration of 1 / ml, a single organoid is more likely to form in a single hollow capsule, and the size distribution of these organoids tends to be uniform.

[0058] The histograms in Table 1 and Figure 9 show the distribution of organoid diameters. The formula attached to the histogram represents (mean organoid diameter) ± (standard deviation of organoid diameter) (μm). In the histogram, the horizontal axis represents the organoid diameter (diameter), and the vertical axis represents the number of organoids (count). Table 1 shows the percentage (%) of the number of organoids for each organoid diameter (diameter) class relative to the total number for each cell density.

[0059]

[0060] <Relationship between hollow capsule size and organoid size>

[0061] The upper part of Figure 10 shows the density of the beads, 1x10 ６This is a histogram of the diameter of hollow capsules (Capsule Diameter) when organoids were produced using cells at 1000 x g / ml. Hollow capsules were produced from beads produced at gas flows of 35 kPA to 55 kPA. Table 2 shows the percentage (%) of the number of hollow capsules in each diameter class relative to the total number.

[0062]

[0063] The bottom of Figure 10 shows these hollow capsules with a density of 1x10 ６ 1 is a histogram showing the diameter of organoids prepared from cells in a 1000 μg / ml culture. Table 3 shows the percentage (%) of the number of organoids in each diameter class relative to the total number.

[0064]

[0065] The upper part of Figure 11 shows the density of the beads, 2x10 ６ This is a histogram of the diameter of hollow capsules (Capsule Diameter) when organoids were produced using cells at 1000 x g / ml. Hollow capsules were produced using beads produced at gas flows of 35 kPA to 55 kPA. Table 4 shows the percentage (%) of the number of hollow capsules in each diameter class relative to the total number.

[0066]

[0067] The bottom row of Figure 11 shows these hollow capsules with a density of 2x10 ６ 1 is a histogram showing the diameter of organoids prepared from cells in a 1000 μg / ml sample. Table 5 shows the percentage (%) of the number of organoids in each diameter class relative to the total number.

[0068]

[0069] <Release of organoids from hollow capsules>

[0070] The top row of Figure 12 shows a microscopic photograph of organoids before release from the hollow capsules. Organoids were obtained by culturing cells in the hollow capsules for 7 days. First, the hollow capsules were drawn into a syringe using an injection needle with an inner diameter larger than the outer diameter of the hollow capsule, e.g., an 18G needle with an inner diameter (mm) of 0.838±0.038. Next, the needle was replaced with an injection needle with an inner diameter smaller than the outer diameter of the hollow capsule. The hollow capsules were then expelled from the syringe through the smaller inner diameter needle. This caused the poly-L-lysine membrane that constituted the hollow capsule to crack, releasing the organoids from the hollow capsule. A 27G needle was used. The inner diameter (mm) of the needle was 0.210±0.019. A 23G needle with an inner diameter (mm) of 0.337±0.019 can also be used. The released organoids were then passed through a 200 μm mesh. This leaves a broken semipermeable membrane on the mesh and a filtrate containing organoids. The middle photo shows a micrograph of organoids contained in the filtrate. The bottom photo shows a micrograph of hollow capsules left on the mesh from the organoids.

[0071] Figure 13 shows fluorescence microscopy images of organoids released from hollow capsules. The top row shows organoids before release from the hollow capsule. The bottom three images show organoids after release from the hollow capsule. For organoids larger than 100 μm in diameter, physical damage remained in the cells on the organoid surface after treatment with the needle and mesh. It is preferable to make the inner diameter of the injection needle and mesh size sufficiently large relative to the outer diameter of the target organoid.

[0072] <Assistance of aggregation by mesenchymal stem cells>

[0073] Figure 14 is a microscopic photograph showing the aggregation of only cardiomyocytes. Only cardiomyocytes were placed in a hollow capsule and incubated for 8 days. The cell density was 3 x 10 ６The concentration was set at 1 mL / mL. The photographs at the top left of the photographs taken on day 1 (day 1) and day 8 (day 8) are fluorescence observation photographs. On day 1, the cells did not organize. On day 8, the cells organized. However, dead cells were frequently observed within the spheroids. The cell viability was 60%. Viability was measured by observing live and dead cell markers under a fluorescence microscope. Viability was measured by staining live cells and tissues using the LIVE / DEAD™ Cell Viability Assay Kit (ThermoFisher). In the figures, live cells are represented in green, and dead cells are represented in red. The same applies below.

[0074] Figure 15 is a microscopic photograph showing the aggregation of a mixture of cardiomyocytes and mesenchymal stem cells. Cardiomyocytes and mesenchymal stem cells were placed in a hollow capsule at a ratio of 10:1 and incubated for 8 days. The cell density was 3 x 10 ６ The concentration was set at 1 mL / mL. The photographs in the upper left of the photographs taken on day 1 and day 8 are fluorescence observation photographs. On day 1, the cells were organized. On day 8, the cells were fully organized. Compared to the cardiomyocyte spheroids shown in Figure 14, the organoids shown in this figure showed a lower frequency of dead cells.

[0075] <Evaluation of cardiac organoids>

[0076] Table 6 shows the cell viability (%) for eight trials of the test shown in Figures 14 and 15. First, although cardiomyocytes (CM) were prepared under the same culture conditions each time before culturing in the hollow capsules, their viability on Day 0 varied. In contrast, the viability of mesenchymal stem cells (MSCs) on Day 0 converged at a high level. The already low viability even on Day 0 is likely due to damage caused by treatment of the cells prior to aggregation. Examples of this include damage caused by enzymatic or physical treatment of cells precultured on a petri dish. Furthermore, cardiomyocytes appear to be more sensitive to this damage than mesenchymal stem cells.

[0077] After incubation for six days (Day 6), the viability of the cells in the myocardial organoids was high. Even if the viability of cardiomyocytes was low on Day 0, high viability was observed after Day 6. This tendency was observed, for example, in the fifth and sixth trials. This is thought to be due to the fact that mesenchymal stem cells (MSCs) not only support the aggregation of cardiomyocytes (CMs), but also have the effect of increasing the survival rate within the aggregates.

[0078]

[0079] Figure 16A is a microscopic image of myocardial markers on myocardial spheroids. Figure 16B is a microscopic image of myocardial markers on myocardial organoids. The myocardial marker cTnT (TnT2 gene) and the MSC marker Vimentin were fluorescently stained. Additionally, cell nuclei were stained with DAPI. Figure 17 shows the sum of the fluorescence intensities of each marker in Figures 16A and 16B. Compared to myocardial spheroids, myocardial marker expression was stronger in myocardial organoids. Furthermore, cardiomyocyte-specific structures were clearly observed in myocardial organoids. This indicates that the state of myocardial organoid tissue is better than that of myocardial spheroids.

[0080] Figure 18 shows a histogram of the expression intensity of the MSC marker CD90. Expression intensity was measured by FACS. The myocardial spheres contained 0.9% CD90-positive cells. In contrast, the myocardial organoids contained 10.7% CD90-positive cells. Before organoid production, the CM:MSC ratio was 10:1. After six days of culture, the CM:MSC ratio had changed to 9:1.

[0081] Figure 19 shows a graph of the secretion levels of TGF-β1 and VEGF. Three types of cells were compared: myocardial spheres (CM), myocardial organoids (CM + MSC), and MSC spheres. These secreted factors are markers of myocardial tissue. Data were normalized by cell number. Cells in myocardial organoids secreted more of these secreted factors than cells in myocardial spheres, especially on day 7. Furthermore, the amount of secreted factors tends to increase with increasing culture period.

[0082] Figure 20 shows the results of contraction and motion analysis of myocardial (CM) spheres. Figure 21 shows the results of contraction and motion analysis of myocardial (CM) organoids. au represents arbitrary units. The middle graph in each figure shows the change in contraction over time. The bottom graph in each figure shows the change in contraction velocity over time. The top graph in Figure 22 shows the average contraction amplitude. The bottom graph shows the average motion amplitude. The contraction and motion velocity of myocardial organoids are higher than those of myocardial spheres.

[0083] Figure 23 shows the results of analyzing cell viability in a hypoxic environment. In a hypoxic environment with a partial pressure of 5%, the viability of cardiac organoids tended to be higher than that of cardiac spheres.

[0084] <Comparison with microwell plate culture>

[0085] The upper row of Figure 24 shows a microscopic image of myocardial organoids grown in hollow capsules. The lower row shows a microscopic image of myocardial organoids grown in microwell plates. EZ sphere dishes (trademark) were used as microwell plates. Static culture was performed in the microwell plates.

[0086] The photograph in Figure 24 shows organoids on day 2 of culture. After two days, beating cardiac organoids were formed in both the hollow capsules and the microwell plates. However, no significant difference in the beating rate was observed between these cardiac spheroids. On the other hand, unexpected fusion of cardiac organoids occurred in the microwell plates. While only one cardiac organoid could be accommodated in each hollow capsule, multiple organoids filled each well of the microwell plate. It was believed that these organoids would eventually fuse.

[0087] The top row of Figure 25 shows a microscopic image of myocardial organoids grown in hollow capsules. The bottom row shows a microscopic image of myocardial organoids grown in microwell plates. The image shows organoids on day 8 of culture. After eight days, organoids grown in microwell plates migrated between microwells and fused with other organoids. The organoids likely migrated due to vibration of the container or cell migration. As shown in the bottom row, the organoids were not uniform in size. Some wells were also found to contain no organoids. In contrast, the organoids grown in hollow capsules, shown in the top row, were more uniform in size.

[0088] Figure 26 shows the results of counting the number of organoids per well in a microwell plate. The change in the number of organoids in each well over time was calculated. From the early stages of culture, wells containing two or more organoids appeared. Furthermore, as the culture period increased, the number of wells without organoids increased.

[0089] Table 7 shows the percentage of organoids in each diameter range in each well of the microwell plate relative to the total number of wells. Wells without organoids were also included in the calculation. Organoid diameters ranged widely from 40 μm to 300 μm.

[0090]

[0091] These results suggest that myocardial organoids grown in microwell plates tend to fuse easily, resulting in heterogeneous sizes. In contrast, the size of myocardial organoids grown in hollow capsules was more uniform, as shown in Figures 3 to 5 and Tables 3 and 5.

[0092] Table 8 shows the cell viability of organoids grown in hollow capsules and in microwell plates (EZ sphere dishes). The viability of cardiomyocyte (CM) cell spheres and mesenchymal stem cell (MSC) spheres on Day 0 was also calculated. On Days 9 and 16 of incubation, organoids grown in hollow capsules showed viability comparable to that of organoids grown in microwell plates (EZ sphere dishes).

[0093]

[0094] <Application to other stromal cells>

[0095] Figure 27 shows a microscopic image of adipose tissue-derived mesenchymal stem cells (AD-MSC) spheres. AD-MSCs, a type of stromal cell, were aggregated using hollow capsules. This resulted in AD-MSC spheres. The cells in the spheres highly expressed the MSC marker Vimentin.

[0096] <Application to other target cells>

[0097] Figure 28 shows a microscopic image of organoids composed of liver cells induced from iPS cells and mesenchymal stem cells (MSCs). Liver cells and mesenchymal stem cells (MSCs) were aggregated using hollow capsules. This resulted in liver organoids. Cells in the liver organoids frequently expressed the liver cell marker α-1-antitrypsin.

[0098] <Consideration>

[0099] Enclosing a single or multiple cell types within a hollow capsule allows for efficient cell aggregation. Furthermore, because the hollow capsules isolate the aggregates, the hollow capsule approach offers advantages over microwell plate-based approaches, such as reduced aggregation, the ability to rotate the aggregates, and fewer restrictions on the manipulation and transport of the aggregates.

[0100] The size of the hollow capsules can be changed by controlling the process of forming the hollow capsules. In this case, it is preferable to make the diameter of the hollow capsules larger than 400 μm. The size of the aggregates can also be changed by adjusting the cell density within the hollow capsules. In this case, it is preferable to make the diameter of the aggregates larger than 70 μm. By appropriately matching the diameter of the hollow capsules with the diameter of the target aggregates, highly uniform aggregates can be produced.

[0101] The continuous bead production process allows for the rapid mass production of hollow capsules, making it easy to scale up the agglomerate production process to industrial scale. For example, the use of multiple bead-producing nozzles can further increase throughput.

[0102] Even if the target cells, such as cardiomyocytes, do not have a high viability before being cultured in the hollow capsules, good aggregates can be obtained. This is a secondary effect of fusing the target cells with auxiliary cells, such as MSCs. By using auxiliary cells, highly functional and organized aggregates can be produced in a short time with a high viability.

[0103] By using hollow capsules, aggregates can be created from a variety of cells, including cardiomyocytes, AD-MSCs, and iPS-derived liver cells.

[0104] This application is based on Japanese Patent Application No. 2023-204841, entitled "Method for producing cell aggregates, aggregate populations, and cell culture methods," filed on December 4, 2023, and claims the benefit of priority from this Japanese patent application. The entire contents of this Japanese patent application are incorporated herein by reference.

[0105] 11: hollow capsule, 12: cells, 13: gel, 14: target cells, 15: accessory cells, 16: aggregates, 17: cells, 18: trophic factors, 19: stromal cells, 20: parenchymal cells, 21: aggregates, 22: nozzle, 23: gas flow, 24: beads, 25: liquid, 27: semipermeable membrane, 28: space, 30: tube, 31: population

Claims

1. A method for producing cell aggregates, comprising suspending cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium to cause the cells to aggregate in the hollow capsule, wherein the cells are a mixture of dispersed target cells and auxiliary cells that assist in the aggregation of the target cells, and the target cells are more likely to aggregate in the presence of the auxiliary cells than in the absence of the auxiliary cells.

2. The method for producing a cell aggregate according to claim 1, wherein the target cells are either cardiomyocytes, liver cells or other parenchymal cells, and / or the auxiliary cells are at least any of mesenchymal stem cells, fibroblasts, vascular endothelial cells and other interstitial cells.

3. A method for producing a cell aggregate, comprising suspending dispersed cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium to aggregate the cells in the hollow capsule, wherein the cells consist of at least interstitial cells.

4. The method for producing a cell aggregate according to claim 3, wherein the stromal cells are at least one of mesenchymal stem cells, fibroblasts, vascular endothelial cells and other stromal cells.

5. The method for producing a cell aggregate according to claim 4, wherein the cells further comprise any one of cardiomyocytes, liver cells and other parenchymal cells.

6. A method for producing a cell aggregate described in any one of claims 1 to 5, comprising the steps of: enclosing a gel containing the cells and made of a material different from the semipermeable membrane with the semipermeable membrane; dissolving the gel; and producing the hollow capsule by allowing the dissolved gel to elute out through the semipermeable membrane, wherein the cells remain in the hollow capsule.

7. The method for producing cell aggregates described in claim 6, wherein the gel is made of alginate containing divalent cations, and the gel is dissolved by applying a chelating agent to the gel.

8. The method for producing a cell aggregate according to claim 6, comprising: extruding a gel containing the cells while wrapping it in a gas flow to form beads; and wrapping the beaded gel in the semipermeable membrane.

9. A method for producing a cell aggregate described in any one of claims 1 to 5, comprising rupturing the hollow capsule by passing the hollow capsule through a tube having an inner diameter smaller than the outer diameter of the hollow capsule, and separating and recovering the aggregated cells from the ruptured hollow capsule.

10. The method for producing a cell aggregate according to claim 9, wherein the semipermeable membrane is made of poly-L-lysine.

11. The method for producing a cell aggregate according to any one of claims 1 to 5, wherein the hollow capsules are incubated with rotary stirring.

12. The method for producing a cell aggregate according to claim 1, further comprising incubating the hollow capsule to proliferate the aggregated cells.

13. A population of aggregates, wherein the average diameter of the aggregates in the population is 70 to 150 μm, and the standard deviation of the diameters of the aggregates in the population is 7 to 20 μm, wherein the aggregates are formed by aggregation of target cells with the intervention of auxiliary cells, and wherein the target cells are more likely to aggregate when the auxiliary cells are present than when the auxiliary cells are not present, or the aggregates are formed by aggregation of interstitial cells.

14. The population of aggregates according to claim 13, wherein each of the aggregates is encapsulated in a hollow capsule made of a semipermeable membrane, and the aggregates are suspended in a liquid contained in the hollow capsule.

15. A method for culturing cells, comprising suspending cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium at least until the cells aggregate within the hollow capsule, wherein the cells are a mixture of dispersed target cells and auxiliary cells that assist in the aggregation of the target cells, and the target cells are more likely to aggregate in the presence of the auxiliary cells than in the absence of the auxiliary cells.

16. A method for culturing cells, comprising suspending dispersed cells in a hollow capsule made of a semipermeable membrane, and incubating the hollow capsule in a culture medium at least until the cells aggregate in the hollow capsule, wherein the cells comprise at least interstitial cells.

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