HK1262352A1

HK1262352A1 - Method for preparing population of stem cell spheroids

Info

Publication number: HK1262352A1
Application number: HK19122484.9A
Authority: HK
Inventors: 田边刚士; 须藤健太
Original assignee: 株式会社可乐丽; 爱平世
Priority date: 2015-12-29
Filing date: 2016-12-28
Publication date: 2020-01-10

Description

Method for preparing population of stem cell aggregates

Technical Field

The present invention relates to methods of preparing populations of stem cell aggregates.

Background

A method of forming an embryoid body by aggregating pluripotent stem cells is known (patent document 1). In the above method, the cells are substantially individualized by using an enzyme for an Embryoid Body (EB) (claim 9). The individualized cells are re-aggregated (claim 18). The above method is suitable for differentiating pluripotent stem cells into endothelial cells.

Documents of the prior art

Patent document

Patent document 1: japanese Kohyo publication No. 2012-519005

Non-patent document

Non-patent document 1: kenji Osafune, Leslie Caron, Malgorzara Borowiak, Rita JMartinez, Claire S Fitz-Gerald, Yasunori Sato, channel A Cowan, Kenneth R Chien & Douglas A Melton, "Marked differences in differentiation amplification step lines", Nature Biotechnology, Published one: 17 library 2008,26,313 and 315

Disclosure of Invention

Problems to be solved by the invention

In the above method, a large number of embryoid bodies are prepared, the embryoid bodies are crushed to form a plurality of cell masses, and the cell masses are cultured to form new embryoid bodies. However, typically, the embryoid body also contains cells that have begun to differentiate. Therefore, the above-mentioned method is not suitable as a method for increasing the number of pluripotent stem cell aggregates while substantially maintaining the undifferentiated state of the pluripotent stem cell aggregates.

The inventors have obtained the following findings in the course of the invention. Non-patent document 1 shows that the progression of differentiation differs depending on the culture period of the cells (supplementary fig. 1 of non-patent document 1). If cells with different differentiation progresses are directly passaged, the progress of differentiation is inherited by aggregates formed after the passage. It is therefore expected that the homogeneity in an undifferentiated state between aggregates is reduced every 1 passage repeated. This is considered to be due to the change in the orientation of differentiation and non-differentiation depending on the size of the aggregates. Further, as a reason for the above phenomenon, it is considered that the nutrient necessary for the survival of the cells is not properly diffused into the aggregate, and therefore the nutrient is not supplied to the central portion of the aggregate. Further, the diffusion of the unused gas or unnecessary substance also hinders the survival of the cells in the aggregate. In addition, when the aggregates become too large, there is a fear that not only differentiation from the inside but also cell death is caused. On the other hand, if the aggregates are always small, the efficiency of the scale-up culture is poor. Therefore, the inventors considered that it is important to prepare an aggregate of undifferentiated cells to maintain the size of the aggregate at a constant level and to make the timing of passaging uniform.

The present invention has been made in view of the above-mentioned findings, and an object of the present invention is to improve the uniformity of an undifferentiated state between aggregates when a population of stem cell aggregates is prepared.

Means for solving the problems

[1] A method of preparing a population of stem cell aggregates, which is a method comprising:

allocating more than two cell blocks in more than two partitions with equal size,

bringing the two or more cell blocks close to each other in each of the partitions,

aggregating and culturing the two or more cell masses close to each other to form an aggregate,

the aforementioned cell masses of the aforementioned distribution are separated from each other and mixed with each other,

the cell masses are each composed of stem cells.

[2] The method for producing a population of stem cell aggregates according to [1], wherein,

the aggregates formed as described above are decomposed to produce cell masses,

mixing the aforementioned cell masses produced from different aforementioned aggregates with each other,

distributing more than two mixed cell blocks in more than two subareas respectively,

bringing the two or more mixed cell masses into close proximity to each other in each of the partitions,

the two or more cell masses close to each other are again aggregated.

[3] The method for producing a population of stem cell aggregates according to [2], wherein,

the aggregate is decomposed at a time when the diameter of the aggregate is 1mm or less.

[4] The method for producing a population of stem cell aggregates according to [2], wherein,

in the case of the incubation, the aggregate is incubated for a period of 2 to 14 days.

[5] The method for producing a population of stem cell aggregates according to [2], wherein,

in the case of the incubation, the aggregate is incubated for a period of 3 to 7 days.

[6] The method for producing a population of stem cell aggregates according to [2], wherein,

further repeating the following process for 1 or more times:

breaking down the aggregates, mixing the cell clumps, bringing them closer together, distributing them and aggregating them again.

[7] The method for producing a population of stem cell aggregates according to [1], wherein,

the stem cells are plated to form colonies,

the colonies are disintegrated to produce the cell masses,

mixing the aforementioned cell masses produced as described above with each other,

the aforementioned cell blocks were used for the aforementioned distribution.

[8] The method for producing a population of stem cell aggregates according to [7], wherein,

the aforementioned disintegration of the aforementioned colonies is carried out by physical disruption,

the colonies were not treated with enzyme.

[9] The method for producing a population of stem cell aggregates according to [7], wherein,

the aforementioned colony is subjected to the aforementioned disintegration by enzyme treatment alone,

the aforementioned colonies were not physically disrupted.

[10] The method for producing a population of stem cell aggregates according to [7], wherein,

when the colonies are decomposed, the cells are separated,

the aforementioned colonies were subjected to enzymatic treatment and physical disruption.

[11] The method for producing a population of stem cell aggregates according to [1], wherein,

the aforesaid partitions are formed by holes provided in the plate,

the hole is a through hole or a concave part,

the hole has a top opening on the top surface side of the flat plate,

the top openings have areas equal to each other between the partitions,

the diameter of the top opening is 1.5mm or less.

[12] The method for producing a population of stem cell aggregates according to [1], wherein,

the partition is formed by a through hole of the flat plate,

the through hole has a bottom opening on the bottom surface side of the flat plate,

the diameter of the bottom opening is less than 1mm,

recovering the aggregate from the plate by passing the aggregate through the bottom opening.

[13] The method for producing a population of stem cell aggregates according to [12], wherein,

the cell mass is cultured in a culture medium disposed in the partition,

the aforementioned culture solution forms a droplet,

the liquid droplets adhere to the bottom opening and protrude so as to hang down from the bottom opening,

the bottom surface of the partition is formed by the meniscus of the droplet.

[14] The method for producing a population of stem cell aggregates according to [1], wherein,

the diameter of the inscribed sphere of the subarea is 5 multiplied by 10¹1 × 10 μm or more³The particle size is less than the micron range,

the inner tangent ball is tangent to the bottom surface of the subarea.

[15] The method for producing a population of stem cell aggregates according to [1], wherein,

the cell mass is cultured in a culture medium disposed in the partition,

the culture medium is connected to the culture medium disposed in the storage partition through the top of the partition,

the culture medium in the storage partition is not provided with cells.

[16] The method for producing a population of stem cell aggregates according to [1], wherein,

the aforesaid partitions are formed by holes provided in the plate,

the hole is a through hole or a concave part,

the hole has a top opening on the top surface side of the flat plate,

the top surface is covered with a suspension of the cell clumps as the dispensing is performed.

[17] The method for producing a population of stem cell aggregates according to [16], wherein,

the suspension contains a unit area (1 cm) with respect to the top surface²) Is a cell block of 1 or more and 5000 or less.

[18] The method for producing a population of stem cell aggregates according to [1], wherein,

the cell mass is cultured in a culture medium disposed in the partition,

extracellular matrix is suspended or dissolved in the culture medium.

[19] A cell culture method is as follows:

the aggregate is formed from the stem cells and,

differentiating the stem cells while performing suspension culture or adherent culture on the aggregate,

when the aggregate is formed,

separating and mixing the cell clumps with each other prior to the dispensing,

the cell masses are each composed of stem cells.

[20] The cell culture method according to [19], wherein,

further, in the above partition, the cells in the above aggregate are differentiated into any of ectoderm, mesoderm and endoderm.

[21] A population of aggregates, wherein,

selecting 1 of the aforementioned aggregates from the aforementioned population;

selecting more than 10 cells from the selected aggregates;

measuring a positive rate by determining whether at least any one of pluripotent stem cell markers of Nanog, Oct3/4 and TRA-1-60 is positive for the aforementioned 10 or more cells;

when the population is measured for 3 times of the above positive rate;

the average value of the 3-time positive rates was 80% or more.

[22] The population of aggregates according to [21], wherein,

from the population described above 10 aggregates were selected,

when it is determined whether at least any one of pluripotent stem cell markers of Nanog, Oct3/4 and TRA-1-60 is positive for the 10 aggregates selected as described above,

the positive rate of the marker is 80% or more.

[23] The population of aggregates according to [21], wherein,

the ratio of the embryoid bodies induced from the aggregates by the in-vitro differentiation induction system is 80% or more,

the aforementioned embryoid bodies are cell aggregates mixed with the tissue of the three germ layers.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, the size of aggregates can be equalized in the preparation of a population of stem cell aggregates. The present invention can therefore improve the homogeneity of the undifferentiated state between aggregates. The present invention is therefore suitable for the preparation of undifferentiated cell aggregates.

Drawings

FIG. 1 is a flow chart of a method of making a population of aggregates.

FIG. 2 is a sectional view showing the incubator.

FIG. 3 is an enlarged cross-sectional view of a cell block and plate.

FIG. 4 is an enlarged cross-sectional view of an aggregate and a flat plate.

Fig. 5 is a diagram showing separation of the container and the tray.

Fig. 6 is a diagram illustrating separation of the container and the aggregate.

Fig. 7 is an enlarged view showing separation of the container and the aggregate.

Fig. 8 is a graph showing the distribution of aggregate sizes.

Fig. 9 is an observation image 1 of the aggregate of the example.

Fig. 10 is an observation image 2 of the aggregate of the example.

Fig. 11 is an observation image 3 of the aggregate of the example.

Fig. 12 is an observation image 4 of the aggregate of the example.

Figure 13 is a2 parameter histogram of FACS.

FIG. 14 is an observation image of cells obtained from the aggregate of example.

Fig. 15 is an observation image of the population of aggregates of the example.

Fig. 16 is an observation image 5 of the aggregate of the example.

Fig. 17 is an observation image 6 of the aggregate of the example.

Figure 18 is a2 parameter histogram of FACS.

Figure 19 is a1 parameter histogram of FACS.

FIG. 20 is a graph showing the expression intensity of TRA-1-60.

Fig. 21A is a fluorescence observation image of the aggregate of the example.

Fig. 21B is a fluorescence observation image of the aggregate of the example.

Fig. 21C is a fluorescence observation image of the aggregate of the example.

FIG. 21D is a view showing the numerical expression of fluorescence observation of the aggregate of example.

Detailed Description

[ terms ]

The term aggregate in this specification means a spherical cell block (cells) consisting of pluripotent stem cells. The aggregates may also be spherical. The aggregates may also be spheres. Aggregates can also be so-called spheroids (sphenoids). Spheroids are sometimes denoted as clumps (pellets). The aggregates are preferably formed by suspension culture. Aggregates are clumps of cells that contain undifferentiated pluripotent stem cells. Aggregates are clumps of cells that have the ability to grow when cultured to produce a variety of cell types. The aggregate is particularly preferably a cell mass composed of 100 or more and 50000 or less cells.

In the present specification, a cell block (cells) refers to a state in which cells are aggregated and bound to each other. In the following, when the term of the cell mass is used, the treatment is performed as follows unless otherwise specified. The term cell mass refers to a state smaller than the size of an aggregate. The term cell mass refers to irregularities in size and shape. The term cell clump includes clumps (aggregates) formed by dividing colonies (colony) or aggregates.

In the present specification, the term of population (population) means a collection of cell masses or aggregates. The term population includes their collection held in a volume of liquid. The population has a defined density. The specified density is a value obtained by dividing the number of cell clumps or aggregates by the volume of the liquid.

[ summary ]

Fig. 1 is a flowchart showing a method for producing a population of pluripotent stem cell aggregates according to the present embodiment. In the method, in step 21, two or more cell blocks (aggregators) are respectively allocated to two or more equal-sized partitions (components). Thereby bringing more than two cell blocks closer together within each partition. More than two cell clumps that are close to each other are aggregated (clustering or organizing) in step 22. The above method can provide a population of aggregates having an equalized size, and can provide a population of aggregates homogenized in an undifferentiated state.

The aggregate is then obtained through steps 23-24 shown in fig. 1. Sometimes a new cell mass is obtained by breaking up the aggregates in step 25. Further, the cell block may be reassigned via step 26 and returning to step 21. Thereby allowing cell proliferation within the aggregate, as well as disruption of the enlarged aggregate, and cycling of these steps to proceed further. Therefore, a large number of aggregates having an equalized size can be obtained, and as a result, a large number of aggregates homogenized in an undifferentiated state can be obtained.

[ incubator ]

Figure 2 shows an incubator 20 suitable for carrying out the series of steps described above. The incubator 20 includes: a container 50 having a plate 30 and a support 45, and a tray 55. When the cells are cultured in the incubator 20, the incubator 20 may be left standing.

The plate 30 shown in fig. 2 is provided with holes represented by holes 31a and 31 b. The holes 31a and 31b in the figure are through holes. The holes 31a, 31b may also be recesses without bottom openings. The holes represented by the holes 31a and 31b form a lattice when the flat plate 30 is viewed in plan. The lattice may be a hexagonal lattice, a square lattice, and other lattices. In the figure, the holes 31a and 31b are filled with a culture solution 35. The culture solution 35 may be any solution as long as it is suitable for culturing pluripotent stem cells.

The support 45 shown in fig. 2 is provided with a side wall 46 and a flange 47, and the side wall 46 surrounds the inner cavity of the plate 30 and the support 45. The plate 30 is located below the interior cavity of the support 45. The plate 30 has a top surface facing the interior cavity of the support 45. The underside of the side wall 46 contacts the plate 30. Preferably, the lower end of the side wall 46 is in contact with the plate 30.

The flat plate 30 shown in fig. 2 is integrated with the support 45 to form a container 50. Preferably, the flat plate 30 is in contact with the support 45 without a gap. The plate 30 is integral with the support 45 and surrounds the inner cavity of the container 50. It is also possible to form the flat plate 30 integrally with the support body 45.

The interior of the container 50 shown in fig. 2 constitutes the storage partition 37. The storage section 37 accumulates the culture solution 35. The top surface of the plate 30 and the inner side surface of the side wall 46 of the support 45 are in contact with the culture solution 35. The reservoir partition 37 forms a continuous space together with the inner cavities of the holes 31a, 31 b.

The side wall 46 and the plate 30 shown in fig. 2 may be inserted as a unit into the interior cavity of the tray 55. The flange 47 is located on the outside of the side wall 46. The tray 55 is provided with a side wall 56 and a bottom 57. The side wall 56 supports the flange 47. The flange 47 preferably contacts the upper end of the side wall 56. The tray 55 supports the flange 47. The tray 55 supports the support 45. The tray 55 supports the container 50. The bottom 57 is opposite the plate 30. A space 58 is provided between the bottom 57 and the plate 30.

The flat plate 30 shown in fig. 2 is preferably a resin molded product. The resin to be molded is preferably any one of an acrylic resin, polylactic acid, polyglycolic acid, a styrene resin, an acrylic/styrene copolymer resin, a polycarbonate resin, a polyester resin, a polyvinyl alcohol resin, an ethylene/vinyl alcohol copolymer resin, a thermoplastic elastomer vinyl chloride resin, a silicone resin, and a silicone resin. These resins may be combined for molding. The flat plate 30 may be a molded article of an inorganic material such as metal or glass. The same applies to other components of the incubator 50.

The surfaces of the holes 31a and 31b shown in fig. 2 are preferably subjected to a modification treatment. The modification treatment is preferably at least any one of plasma treatment, corona discharge and UV ozone treatment. The functional group can be formed on the surface by modification treatment. The functional group is preferably hydrophilic. The hydrophilic surface allows the cell mass to flow smoothly into the holes 31a, 31 b. The modification treatment is particularly preferable when the openings of the holes 31a and 31b are small. The modification treatment is particularly preferable when the resin is hydrophobic. The same applies to the top and bottom surfaces of the plate 30.

The surfaces of the holes 31a and 31b shown in fig. 2 may be covered with a predetermined substance. The substance may be inorganic. The substance may also be a metal. The substance may be one obtained by polymerizing 2, 3 or 4 or more molecules. The surface may be covered with a substance obtained by combining them. The covered surface preferably has some hydrophobicity. Since the surface has a certain hydrophobicity, a droplet described later is easily formed even when a medium having a small surface tension is used. The same applies to the top and bottom surfaces of the plate 30.

The fine structure may be provided on the surface of the holes 31a and 31b shown in fig. 2. The fine structure is preferably a so-called nano-scale. The size of the structural unit of the fine structure is preferably 0.1nm or more and 1 μm or less. The surface may be provided with irregularities to form a fine structure.

[ division ] of

FIG. 3 is an enlarged view of the cell mass and plate 30. The cell mass was cultured in the prescribed partition. The partitions represented by partitions 32a, 32b are formed by holes represented by holes 31a, 31b, respectively. The holes represented by the holes 31a, 31b have equal sizes to each other. The partitions 32a and 32b may be formed by only the holes 31a and 31b in this embodiment, but the present invention is not limited thereto.

The plate 30 shown in fig. 3 has partition walls 29. The holes are separated from each other by partition walls 29. The partition walls 29 are tapered from the bottom toward the top of the flat plate 30. The holes 31a, 31b are tapered from the top toward the bottom of the plate 30.

The holes 31a and 31b shown in fig. 3 have top openings 33a and 33b on the top surface side of the flat plate 30. The holes 31a and 31b have bottom openings 34a and 34b, respectively, on the bottom surface side of the plate.

The top openings 33a, 33b shown in fig. 3 preferably have equal areas to each other. The top openings 33a and 33b are not limited thereto, and a plurality of top openings, preferably all of the top openings, of each hole preferably have the same area. By making the top openings have mutually equal areas, the number of cells per partition can be equalized. Therefore, when an aggregate is formed from a partition, the aggregate can be made uniform in size.

The top openings 33a, 33b shown in fig. 3 may be circular. The diameter of the top openings 33a, 33b is preferably 2.00mm, 1.5mm, 1.4mm, 1.3mm, 1.2mm, 1.1mm, 1.0mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm, 0.5mm, 0.4mm, 0.3mm, 0.2mm, and 0.1mm or less.

The diameter of the top openings 33a and 33b shown in FIG. 3 is preferably 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or less, 50 μm or less, 60 μm or more, 70 μm or less, 80 μm or less, and 90 μm or less.

The top openings 33a, 33b shown in fig. 3 may be triangular, quadrilateral, pentagonal, hexagonal and other polygonal and elliptical shapes. The diameters of the inscribed circles of the top openings 33a, 33b may be set in the same range as the above-described diameters.

The top openings 33a, 33b may also be used in the case of holes 31a, 31b shown in fig. 3 without bottom openings 34a, 34 b. In this case, the top openings 33a and 33b can also exhibit the effect. The top openings 33a, 33b are preferably larger than the bottom openings 34a, 34b, respectively.

[ distribution of cell clumps ]

In step 21 shown in FIG. 1, the population 41 of cell blocks shown in FIG. 3 is allocated among two or more partitions represented by partitions 32a, 32 b. The population 41 comprises a plurality of cell masses comprising cell masses 42a-42 c. Population 41 is preferably contained in suspension 38. The suspension 38 has cell masses 42a-42c dispersed therein without deviation. The small cell masses 42a and the large cell masses 42c are mixed in the population 41. Suspension 38 may also contain single cells (singlecell (s)) that are substantially individualized while containing population 41. The ratio of the number of cells in the single-cell state to the total number of cells in the single-cell state in the suspension 38 may be 10% or more, 30% or more, 50% or more, 80% or more, or 90% or more.

When dispensing, the suspension 38 shown in FIG. 3 is preferably spread over the top surface of the plate 30. The top surface of plate 30 is preferably covered when spreading suspension 38. The top surface of the plate 30 is preferably covered with the suspension 38 without deviation.

When spreading the suspension 38, it is preferable that the suspension 38 contain a unit area (1 cm) with respect to the top surface²) Is a cell block of 1 or more and 5000 or less. The number of cell blocks per unit area is preferably any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 and 5000.

The method of spreading the suspension 38 shown in fig. 3 is effective compared to the method of separately dispensing the suspension 38 into each partition. The suspension 38 enters the partitions 32a, 32b due to gravity settling. The suspension 38 containing the cell clumps 42a-42c is thus randomly distributed among the partitions. And the cell mass settles within the partitions 32a, 32 b. The dispersed cell mass leaves the storage partition 37 by settling and accumulates within each partition 32a, 32 b. Thereby bringing the cell masses closer to each other.

The partition walls 29 shown in fig. 3 preferably narrow as they approach the top of the flat plate 30. The partition wall 29 may have a cross section in a shape convex upward near the top of the flat plate 30. The shape may be semicircular or triangular.

The dispersion medium constituting the suspension 38 shown in fig. 3 fills the partitions 32a, 32b and is disposed in the reservoir partition 37. The dispersion medium of the suspension 38 may be a culture solution having the same composition as the culture solution 35. After dispensing, the dispersion medium in the storage compartment 37 may be further filled with a suitable culture medium. After dispensing, the dispersion medium in storage compartment 37 may also be replaced with a suitable culture medium.

In the population 41 shown in fig. 3, the assigned cell blocks are separated from each other. These cell masses are mixed with each other. The population 41 includes a cell mass 42b that is smaller than the cell mass 42 a. The population 41 includes a cell mass 42c that is larger than the cell mass 42 a. Cell masses of different sizes are mixed with each other in the population 41.

The cell masses 42a to 42c are each composed of a pluripotent stem cell (a pluripotent cell). The pluripotent stem cells may be ES cells or iPS cells. Examples of the animal species of the pluripotent stem cells include mammals including humans and mice, but are not limited thereto. Examples of somatic cells that are sources of iPS cells include, but are not limited to, fibroblasts. Somatic cells can also be obtained from any tissue in the body of the individual from which they are derived.

As shown in FIG. 3, the cell mass is cultured in a culture solution 35 disposed in the partitions 32a, 32 b. The cell blocks 42a, 42b are assigned to these partitions as representative of the cell blocks described above. The culture liquid 35 forms droplets 36a, 36 b. Droplets 36a, 36b adhere to bottom openings 34a, 34b, respectively, and protrude in a manner that depends from the bottom openings. Droplets 36a and 36b protrude toward the bottom surface of plate 30. In this embodiment, a so-called hanging drop type culture is performed.

The partitions 32a, 32b shown in fig. 3 may be understood as defined by top openings 33a, 33b, respectively; inner cavity surfaces of the holes 31a, 31 b; and rounded interfaces of droplets 36a, 36 b. The interface faces the space on the bottom surface side of the plate 30. The bottom surfaces of the partitions 32a, 32b are formed by the above-described interfaces of the droplets 36a, 36b, respectively. The rounded corners at the interface are due to the surface tension of the culture solution 35. I.e. the interface of droplets 36a, 36b becomes a meniscus (meniscus).

As shown in FIG. 3, the partitions 32a and 32b are filled with the culture solution 35. Partitions 32a, 32b can be understood to be comprised of culture fluid 35 and droplets 36a, 36b located within wells 31a, 31b, respectively. In other words, the partitions 32a, 32b of cultured cell mass 42a, 42b continue to the droplets 36a, 36b outside the plate 30.

The partitions 32a, 32b shown in fig. 3 are preferably sized as follows. That is, the diameter of the inscribed sphere inscribed in the partitions 32a and 32b is set to a predetermined range. The specified range is 5X 10¹1 × 10 μm or more³And is less than μm. The inscribed sphere is a hypothetical solid. The inscribed sphere is preferably tangent to the bottom surface of the sections 32a, 32 b. By setting the sizes of the partitions 32a, 32b as described above, the formation of aggregates can be promoted.

The size of droplets 36a, 36b shown in fig. 3 can be determined at will as long as the droplets are not broken. It is also possible to culture the cell clumps 42a, 42b only within the droplets 36a, 36 b. That is, the cell masses 42a, 42b may not be cultured in the wells 31a, 31 b.

Droplets 36a, 36b shown in fig. 3 may not be formed. The partitions 32a, 32b may also be located within the apertures 31a, 31b, respectively. This corresponds to the case where the bottom openings 34a and 34b are not provided.

The partitions 32a and 32b shown in fig. 3 are connected to the storage partition 37 via top openings 33a and 33b, which are the tops of the partitions 32a and 32 b. The culture solution 35 in the partitions 32a and 32b is connected to the culture solution 35 disposed in the reservoir partition 37 through the top portions of the partitions 32a and 32 b. The storage partition 37 is not provided with cells represented by the cell masses 42a and 42 b.

As shown in FIG. 3, the following advantages are obtained by integrating the culture medium between the partitions 32a and 32b and the storage partition 37. The movement of the culture liquid 35 is first performed between the partitions 32a, 32b and the storage partition 37. Therefore, the cell masses 42a and 42b can be sufficiently nourished even in the case of hanging drop type culture.

Since no cell is present in the reservoir section 37 shown in FIG. 3, the culture solution 35 can be easily replaced when culturing is performed as described later. In addition, since the culture solution 35 is present in a larger amount than in the case where the culture solution is disposed only in the partitions 32a and 32b, changes in pH and temperature are less likely to occur in the culture solution 35.

Returning to fig. 2. The incubator 20 is usually installed in an incubator, but sometimes has to be moved in the outside air due to transportation. The oxygen concentration and temperature in the incubator are different from those in the outside air. Therefore, the culture solution 35 in the incubator 20 is sometimes affected by the oxygen concentration and temperature of the outside air.

In the conventional pendant drop method, the reservoir partition 37 shown in FIG. 2 is not used, and therefore the influence is strongly transmitted to the drop of the culture solution coating the cells. Therefore, the pH and oxygen concentration of the culture medium change rapidly. The rapid change affects the proliferation and function of cells. Furthermore, since the medium is difficult to replace, the nutrient components are insufficient, waste products cannot be removed, and the growth and survival of cells are affected. The incubator 20 of the present embodiment can reduce such an influence.

The effect exerted by the incubator 20 shown in FIG. 2 depends on the plate 30 forming the storage section 37. The culture liquid 35 in the incubator 20 is not easily affected by changes in the external environment. Thus also reducing the effect on aggregates formed by the cell mass.

[ proximity of cell masses to each other ]

The individual cell blocks that are separated from each other in the population 41 shown in fig. 3 are allocated within the individual partitions 32a, 32b, but are close to each other. As described above, the holes 31a, 31b are tapered from the top toward the bottom of the flat plate 30, and thus the approach can be promoted. By bringing them close to each other, the cell mass can be efficiently aggregated.

[ aggregation of cell clumps ]

In step 22 shown in FIG. 1, two or more cell masses are aggregated within each of the partitions 32a, 32b shown in FIG. 3. As an example, more than two cell masses comprising cell mass 42a are aggregated within partition 32 a. As another example, more than two cell masses comprising cell mass 42b are aggregated within partition 32 b.

Fig. 4 is an enlarged cross-sectional view of the aggregate 40 and the plate 30. The result of the aggregation of the clumps of cells may form an aggregate 40 within each partition 32a, 32 b.

More than two cell clumps including cell clump 42c shown in FIG. 3 may also be aggregated in any partition. The cell blocks allocated in each partition are substantially irregular in size. Thereby clustering populations of cell blocks each having an irregular size in each partition. For example, a population of cell blocks comprising any of cell blocks 42a-42c may also be clustered within a partition.

As described above, in step 21 shown in fig. 1, the population 41 is divided into small portions and distributed in each partition by spreading the suspension 38 as shown in fig. 3. These clumps are aggregated after the above-described distribution. Thus, the variation in the size of the cell mass before aggregation can be reduced. In addition, as described above, the distribution of the cell block sizes of each partition can be equalized. Therefore, as shown in fig. 4, the aggregate 40 formed in each partition by aggregation is homogenized. In addition, the size of the aggregate 40 is homogenized.

The cell blocks are separated from each other prior to being allocated to the partitions 32a, 32b shown in figure 3. In addition, the cell blocks are brought closer together by the allocation. Thus allowing the time period for the clumps to begin to aggregate to coincide with the end of the dispensing described above. In order to separate the cell pieces from each other and mix them with each other, pipetting before dispensing is suitable. Other methods may also be used.

[ incubation of aggregate and recovery of aggregate ]

The aggregate 40 shown in fig. 2 is incubated in step 23 shown in fig. 1. Step 23 is performed before the decomposition of the aggregates shown in step 25 is performed. Fig. 4 is a diagram showing the aggregate formed and the flat plate 30 in an enlarged manner. The aggregate 40 is made larger by incubation in each of the partitions 32a, 32 b. An aggregate is formed by step 23 and step 24.

For the above formation, steps 22, 23 shown in fig. 1 may also be performed simultaneously within the partitions 32a, 32b shown in fig. 3. The cell masses 42a, 42b can also aggregate during the incubation process to form the aggregate 40 shown in FIG. 4. The aggregate 40 may also be incubated after the cell masses 42a, 42b have rapidly aggregated with one another to form the aggregate 40.

In step 23 shown in fig. 1, the aggregate 40 shown in fig. 4 is incubated for a period of 2 days or more and 14 days or less. The above period is preferably 3 to 7 days. It is preferable that: when the diameter of the aggregate is equal to or smaller than the predetermined value, the growth of the aggregate is stopped and the aggregate is collected as shown in step 24.

The diameter of the aggregate, including 40 shown in fig. 4, represents the diameter of the circumscribed sphere of the aggregate. The predetermined value of the aggregate diameter is 3/4 or less, preferably 2/3 or less, of the diameter of the bottom openings 34a, 34 b.

The specified value of the aggregate diameter is preferably any of 1mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm, 0.5mm, 0.4mm, 0.3mm, 0.2mm and 0.1 mm. By recovering the aggregates as described above, it is possible to prevent the aggregates which have a fear of becoming larger and becoming more differentiated in the interior from being mixed.

In step 24 shown in fig. 1, the vessel 50 and tray 55 are first separated as shown in fig. 5. Next, as shown in fig. 6, the bottom surface of the flat plate 30 is immersed in the collected liquid 65 in the tray 60. Tray 60 may also be made of the same material as tray 55.

The aggregate 40 is passed through the bottom surface of the plate 30 as shown in fig. 6. The aggregate 40 is moved from the culture solution 35 into the recovery solution 65. Or the culture liquid 35 is flowed into the tray 60 together with the aggregate 40. The above operation may be performed by gravity or by suction. The aggregate 40 is separated from the flat plate 30 by the above-described process. Thereby recovering the aggregate 40 into the recovering liquid 65. The recovering solution 65 may be a culture medium or a buffer solution.

As described above, the holes 31a and 31b shown in fig. 5 and 6 cannot pass through the bottom surface of the flat plate 30 if they are recesses. The above case also allows recovery of the aggregate 40 by pipetting. The method of passing the aggregate 40 through the bottom surface of the plate 30 has an advantage of less physical irritation to the aggregate 40. The above-mentioned simple method has a low possibility of damaging the undifferentiated state of the aggregates.

Fig. 7 is a diagram showing separation of the container from the aggregate in an enlarged manner. The aggregates 43a-43c are representations that classify the aggregate 40 by size. Aggregate 43a is smaller than aggregate 43 b. Aggregate 43c is larger than aggregate 43 b.

The diameter of the aggregates 43a, 43b is smaller than the diameter of the bottom opening 34a as shown in fig. 7. The aggregates 43a, 43b thus pass through the bottom opening 34 a. The group 44a composed of the aggregates can be obtained by the above separation.

The diameter of the aggregate 43c is greater than the diameter of the bottom opening 34 b. The aggregates 43c do not pass through the bottom openings 34a, 34 b. The group 44b composed of the aggregates is left on the plate 30 by the above separation.

The population of aggregates 40 shown in fig. 5 is divided into population 44a and population 44b shown in fig. 7 by the action of the plate 30 shown in fig. 6. I.e. the plate 30 has a filter function.

Fig. 8 is a diagram showing the distribution of aggregate sizes. The horizontal axis is the size of the aggregate. The vertical axis represents the number of aggregates shown in proportion. The aggregates 43a, 43b contained in the population 44a are smaller in size than the threshold 39. The aggregate 43c contained in the population 44b is greater in size than the threshold 39 of the bottom openings 34a, 34 b.

The threshold value 39 shown in fig. 8 depends on the diameter of the bottom openings 34a, 34 b. The threshold value 39 is equal to the diameter of the bottom openings 34a, 34 b. The size of the aggregates 43a, 43b separated from the plate 30 as shown in fig. 7 can be controlled by the diameter of the bottom openings 34a, 34 b. The plate 30 screens aggregates by a threshold 39.

The diameter of the collected aggregates 43a and 43b shown in FIG. 7 is preferably 1mm or less as described above. The aggregate having the above diameter can be realized by adjusting the incubation period and the incubation condition, for example. The aggregate 43a, 43b having the above-described diameter can be selected by the above-described filter action. The following preferable effects can be expected for the filter action of the flat plate 30.

When cells that proliferate faster than normal cells are contained in the cell masses 42a and 42b shown in fig. 3, the aggregate 40 (fig. 4) formed by the cell masses may be larger than usual. Such a change in the proliferation rate is caused by, for example, an abnormality in the karyotype of the cell.

Cells with karyotypia not only proliferate faster than normal cells, but also have a high survival rate. Therefore, even if the same-sized cell mass is cultured for the same period, the cell mass including the cells having the karyotypia is larger than the normal cell mass. In addition, the frequency of occurrence of such aggregates is also not negligible.

Cells with karyotypic abnormalities are preferably not contained in the aggregate. The reason for this is that the aggregate is likely to be used in various tests, medical treatments, and the like, and therefore the aggregate is preferable to exhibit a normal function. On the other hand, karyotype abnormalities occur with a certain probability even if the incubation period or incubation conditions are adjusted.

Aggregate 43c can be excluded from population 44a by the filter action of plate 30 shown in fig. 7. The aggregate 43c can be regarded as an aggregate larger than usual due to the above karyotype abnormality, for example. Aggregates with karyotypic abnormalities can therefore be excluded from the population 44a by the filter action of the plate 30.

In order to obtain the above-described effects, the diameters of the bottom openings 34a, 34b shown in fig. 7 are preferably 1.0mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm, 0.5mm, 0.4mm, 0.3mm, 0.2mm, and 0.1mm or less.

The bottom openings 34a, 34b shown in fig. 7 preferably have inscribed circle diameters equal to one another. Without being limited to the bottom openings 34a, 34b, the plurality of bottom openings, preferably all of the bottom openings, of each hole preferably have inscribed circle diameters equal to one another. The upper limit of the size of the recovered aggregates can be made uniform by the bottom openings having inscribed circle diameters equal to each other. The bottom openings preferably further have mutually equal areas.

[ decomposition of aggregates and mixing of cell masses ]

The recovered aggregates are decomposed in step 25 shown in fig. 1. It is preferable that the aggregate is decomposed at a time when the diameter of the aggregate is 1mm or less. Thus, when aggregates are further increased as described later, it is possible to prevent the differentiated cells from being mixed into the aggregates. In other words, a homogeneous undifferentiated state between aggregates can be maintained.

The decomposed aggregates are the aggregates contained in the recovered population 44a as shown in fig. 7. The aggregate is broken down to produce a plurality of cell clumps. Decomposition may also be carried out by physical disruption of the aggregates. Physical disruption may be performed by pipetting. The decomposition may also be carried out by enzymatic treatment. The enzyme treated aggregates may also be physically broken up. The physically broken aggregates can also be enzymatically treated to produce a cell mass.

The cell clumps are further mixed with each other in step 26 shown in fig. 1. The mixed cell mass is generated from different aggregates. Mixing can be performed by pipetting. Mixing can also be carried out simultaneously due to the disruption by pipetting.

[ Recycling of Process ]

Returning again to step 21 shown in FIG. 1, the mixed population of cell blocks 41 is distributed among the partitions represented by the two or more partitions 32a, 32b as shown in FIG. 3. More than two mixed cell blocks are allocated in more than two zones, respectively. The incubator shown in FIG. 2 is preferably a newly prepared vessel.

In step 22 shown in fig. 1, the allocated cell blocks are brought closer together again within each partition 32a, 32 b. More than two cell masses close to each other are again aggregated. That is, the steps are performed in the order of (aggregation) - > (decomposition) - > (aggregation). The cell clumps are re-aggregated after the mixed cell clumps are distributed in each partition, thereby enabling homogenization to be maintained between aggregates and increasing homogenized aggregates.

In the flowchart shown in fig. 1, the number of times of returning from step 26 to step 21 is not limited. The cycle of decomposing the increased aggregates, mixing the cell masses obtained by the decomposition, distributing and bringing them close together, and aggregating again can be further repeated 1 or 2 times or more.

In the above-described method, the respective steps are repeated in the order of (aggregation) - > (decomposition) - > (aggregation) - > …. Thereby enabling to maintain homogeneity between aggregates and to increase the homogenized aggregates.

Further, as indicated by arrow 27 shown in fig. 1, step 25 may also be omitted in any cycle. In this case, as described above, the aggregate formed by the re-aggregation in step 22 is not decomposed in step 25. The aggregates are thus mixed with each other by jumping from step 24 to step 26.

Via arrow 27 shown in fig. 1, from step 26, the process returns to step 21. The aggregates mixed in step 26 are distributed among two or more partitions 32a, 32b, respectively, as are the cell blocks 42a-42c shown in FIG. 3. Two or more mixed aggregates are brought close to each other in each partition.

In step 22, two or more aggregates close to each other are aggregated in each of the partitions 32a, 32 b.

In the above-described method, the respective steps are performed in the order of, for example, (aggregation) - > (decomposition) - > (aggregation). With the above method, it is possible to suppress a decrease in the homogenization state and to increase the homogenized aggregates among the aggregates.

In one example, step 25 shown in FIG. 1 may also not be implemented at all. In step 26, the different aggregates formed are mixed with each other. Returning to step 21, the mixed aggregate is distributed among more than two partitions. Two or more mixed aggregates are brought close to each other in each partition. In step 22, two or more aggregates close to each other are further aggregated in each partition.

The steps in the above-described method are performed in the order of (aggregation) - > (aggregation). With the above method, it is possible to suppress a decrease in the homogenization state and to increase homogenized aggregates among aggregates.

In one example, as described above, larger aggregates formed without step 25 of FIG. 1 may also be decomposed at step 25 again. That is, as described above, the aggregate formed by further aggregating the aggregates in step 22 is decomposed in step 25. The cell clumps are generated from the larger aggregates formed.

In step 26 shown in FIG. 1, the cell clumps created by the different aggregates are mixed with each other. Returning to step 21, the mixed cell block is allocated to more than two partitions. Two or more mixed aggregates are brought close to each other in each partition. In step 22, more than two cell blocks close to each other are grouped again in each partition. In the above-described method, the respective steps are performed in the order of, for example, (aggregation) - > (decomposition) - > (aggregation).

[ preparation of initial cell Mass ]

In step 21 shown in FIG. 1, a cell mass which becomes a starting point of aggregate formation can be prepared by any method. As an example, pluripotent stem cells may be plated to form colonies. The colonies are broken up to produce a cell mass according to step 25. The cell clumps are mixed with each other, according to step 26.

The population of the cell blocks was used for distribution in step 21 (FIG. 1) at the 1 st time as population 41 shown in FIG. 3. Using the above method, homogenized aggregates among aggregates can also be obtained from plated cells.

When the colonies are resolved as described above, pipetting may also be performed. Colonies can be broken up by enzymatic treatment alone. It is also possible to carry out only physical disruption. Both enzymatic treatment and physical disruption may also be performed.

[ use of aggregate ]

The resulting aggregates can be cultured by suspension culture or adherent culture, as described above. In the above culture, the pluripotent stem cells in the aggregate may be differentiated according to a predetermined method. As a predetermined method, for example, an in vitro differentiation induction system can be used.

In the present embodiment, the aggregate can be obtained as a population. In the present embodiment, the size of the pluripotent stem cell aggregates collected in each cycle is equalized throughout the entire process. Thus, in the above population, a homogeneous undifferentiated state is maintained between aggregates. Therefore, the aggregate of the present embodiment is suitable for achieving homogenization of the differentiation state among pluripotent stem cells when the pluripotent stem cells are differentiated as described above.

Maintaining the undifferentiated state of the population is characterized by a positive rate for the pluripotent stem cell marker. As an example, the aggregate group may be positive for 80% or more of all aggregates. The positive rate was calculated as the proportion of aggregates in the population of aggregates for which the pluripotent stem cell marker was positive.

For example, when 80% or more of aggregates having positive pluripotent stem cell markers in a population are aggregates, it can be determined that the population is in an undifferentiated state.

The measurement method can be performed by the following method. First, 10 aggregates were selected from the population. 100 cells were selected for the 1 aggregate selected. The number of cells selected may be more than 100. For the 100 cells, the positive rate of one aggregate was measured by determining whether the pluripotent stem cell marker was positive. In this determination, if the pluripotent stem cell marker of 3 or more cells out of 100 cells is positive, the pluripotent stem cell marker of the aggregate is determined to be positive. When 1000 or more cells are selected in this determination, if 3% or more of the cells are positive, the pluripotent stem cell marker of the aggregate is determined to be positive.

The above method was used to determine the proportion of aggregates in which the pluripotent stem cell marker was positive among 10 aggregates (positive rate). This positive rate was measured 2 more times for the same population, i.e. 3 times in total. The average value of the positive rates of 3 times was defined as the average value of the positive rates.

The pluripotent stem cell marker may be, for example, TRA-1-60. Whether TRA-1-60 is positive or not can be determined, for example, by examining the presence or absence of a positive cell population as compared with a negative cell population using a flow cytometer. Alternatively, the pluripotent stem cell marker may be detected by PCR. In this case, the pluripotent stem cell marker may be at least one selected from the group consisting of Nanog and Oct 3/4. The expression of these marker genes is detected using, as a control, an unexpressed differentiated cell such as a fibroblast.

Within a population of aggregates, it is preferred that the aggregates are also homogeneous in their function. The condition of being homogeneous in function can be judged by an in vivo differentiation induction method such as the forming ability of teratoma. By transplanting the aggregates or the pluripotent stem cells in the aggregates into a mouse, it is possible to determine whether or not a teratoma is produced in the mouse. The proportion of aggregates forming teratomas differentiating into three germ layers inside aggregates in the population is preferably 80% or more, more preferably 95% or more, and most preferably 100%. In the above case, the population can be functionally determined to be homogeneous.

In the aggregate population, it is preferable that the homogeneity of the differentiation ability of the aggregates is maintained. The case of being homogeneous in differentiation ability can be judged by, for example, whether or not the cells in the aggregate differentiate into cells of three germ layers at the time of differentiation-inducing the aggregate.

For example, 10 aggregates from the population. The number of aggregates selected may be more than 10. The 10 aggregates were induced to differentiate into any of the three germ layers in each tube. Otherwise, the aggregates are induced to differentiate to form embryoid bodies. Embryoid bodies are understood herein to mean fertilized eggs or cell aggregates that, like embryos, contain a variety of differentiated cells. For various aggregates, it is preferred that more than 80% of the embryoid bodies formed express markers for any of the three germ layers. Preferably, all of the selected 10 or more than 10 aggregates satisfy the requirement.

The expression level of the gene of the embryoid body can be determined by measuring the expression level of the gene individually by the PCR method.

In another embodiment, the ratio of embryoid bodies induced from the various aggregates by the in vitro differentiation induction system is preferably 80% or more. It is preferable that: all of the selected 10 or more than 10 aggregates satisfy the requirement. Embryoid bodies are defined herein as aggregates of cells that incorporate the tissue of the three germ layers.

The differentiation marker is preferably a differentiation marker of at least any one of ectoderm, endoderm and mesoderm. The differentiation marker for ectoderm may also be set to at least any one of Pax6, SOX2, PsANCAM, and TUJ 1. Differentiation markers for endoderm may also be set to at least any one of FOXA2, AFP, cytokine 8.18, and SOX 17. The mesoderm differentiation marker may be at least one of brachyury (brachyury) and MSX 1.

The present invention is not limited to the above-described embodiments and the following examples, and can be modified as appropriate without departing from the scope of the invention. For example, in the above embodiment, aggregates are recovered after forming the aggregates from the cell mass. However, it is also possible to collect a large cell mass by aggregating two or more cell masses. The large cell clumps may also not reach the size of the aggregates. I.e. the above cycle can also be repeated to finally obtain aggregates of sufficient size and function. In addition, one embodiment of the present invention is a cell culture method for pluripotent stem cells. In this embodiment, cells may be cultured in the same manner as in the above-described embodiment for the purpose of proliferating pluripotent stem cells and maintaining the survival of pluripotent stem cells.

Examples

[ example 1]

< preparation of iPS cells >

As the pluripotent stem cells, artificial pluripotent stem cells (iPS cells) were used, and it was confirmed that Nanog, OCT3/4, TRA1-60 or an undifferentiated marker similar thereto, which expresses an undifferentiated marker, differentiated into the three germ layers.

(cell culture)

The iPS cells were used as cell masses that become starting points of aggregate formation. iPS cells were first cultured on feeder layer cells in 6-well plates for 5-7 days. After confirming that iPS cells reached 70-80% confluence, the medium was removed from the wells using an aspirator. Human ES/iPS cell Dissociation Solution (Dissociation Solution for human ES/iPS Cells) (CTK Solution, reproCELL Incorporated) was added to each well at 500. mu.L per well. Placing a 6-well plate in CO₂Incubator (37 degree, 5% CO)₂) And incubated for 3 minutes.

After incubation, from CO₂The 6-well plate was removed from the incubator. Feeder cells were exfoliated by gently tapping the well plate or well. However, the device is not suitable for use in a kitchenAfter that, the CTK solution was removed with an aspirator and 1ml of PBS was added to each well.

The detachment of feeder cells from the 6-well plate was confirmed by a microscope. PBS was then removed from the culture dish with an aspirator. Then, 500. mu.l of TrypLE Select Enzyme (1X) (trade name; manufactured by Thermo Fisher scientific; hereinafter, TrypLE Select.) was added to each well, followed by CO₂Incubate in incubator for 5 minutes.

As a culture medium for ES cells or iPS cells, a culture medium Y was prepared in the following manner. First, human ES medium (reprocell) was prepared as a basal medium. Further, 0.2ml of 10. mu.g/ml basic fibroblast growth factor (bFGF) (Thermofisoher PHG0266) was added to the above medium.

After incubation, from CO₂The 6-well plate was removed from the incubator. Media was added to each well at 500. mu.l per well. The iPS cells were suspended 10 to 30 times by using an automatic pipette (P1000). The same suspension was carried out in example 3 and the following examples. A suspension containing a population of cell clumps was thus prepared. The above suspension also contained single cells of iPS cells produced by suspension. The media change was performed and the population of cell clumps was finally suspended in commercial feeder-free culture medium. In this example, the culture solution without a feeder layer is referred to as culture solution A (Medium A).

As a plate for forming the aggregate (hereinafter referred to as a plate unless otherwise mentioned), an < Elplasia > plate manufactured by kuraray co. Plates of the porous Type (Multiple Pore Type) in < Elplacia > plates were used. As shown in fig. 9, the Multiple Pore Type plate has a plurality of holes formed by through holes.

Fig. 9 shows an observation image of the aggregate when the flat plate is viewed from above. The holes are equal in size to each other as shown in fig. 9. The top opening and the bottom opening of the through hole are quadrilateral. Specifically, the top opening and the bottom opening are both square. The top opening and the bottom opening have angles whose directions coincide with each other in a plan view. In addition, they have centers that coincide.

One side of the top opening was 650 μm in length. The length of one side of the bottom opening was 500. mu.m. Relative to a plane having 7cm²The bottom surface of the area (2) of (2) is formed by arranging 680 holes regularly in the flat plate. The number of partitions formed by the holes N is 680. Specifically, the holes are arranged in a tetragonal lattice shape. The unit of the lattice is a square with one side of 500 μm.

The entire surface of the plate was inoculated with a culture medium without variation in order to distribute two or more cell masses to each partition formed by each well. The culture broth spread into each well. To contain 1x 10 per partition⁵Cell blocks were allocated for each cell (number of cells per partition n: 1 × 10)⁵). Since the top openings are uniform in size and the holes are uniformly arranged in a grid pattern, the number of cells distributed in each partition is considered to be uniform.

The number of cells per unit volume in the culture solution a, that is, the cell concentration C [1/ml ], was determined according to the formula C ═ N · N/V. Here, N represents the number of partitions, N represents the number of cells per partition, and V represents the volume of the culture solution A used per plate.

A drop of culture solution A protruded from the bottom opening of each well (FIG. 3). The meniscus which is the interface between the droplet and the atmosphere is formed by the surface tension of the culture solution A. The cell clumps are brought closer together within the zone formed by the well interior surface and the meniscus as they aggregate toward the meniscus. Thereby, the cell mass was cultured in the culture solution A disposed in the partition. As described above, the meniscus in the present embodiment is also a part of the components of the partition.

Cell masses close to each other were aggregated as shown on days 1 and 2 of culture solution a of fig. 9. Day 1 means 1 day after inoculation, and day 2 means 2 days. In other figures, the numbers after days or days indicate the number of days elapsed since the day of initial plating in the plate. Thereby, the cells are cultured while the cell masses are aggregated, thereby obtaining a pluripotent stem cell aggregate.

[ example 2]

In example 1, feeder layer Cells and iPS Cells were detached from the wells using a human ES/iPS cell Dissociation Solution (separation Solution for human ES/iPS Cells). iPS cells were additionally treated with TrypLE Select Enzyme.

On the other hand, in example 2, iPS cells were scraped only with a spatula, and were not subjected to enzyme treatment. iPS cells were suspended less than 10 times. The other steps were carried out in the same manner as in example 1.

In example 2, 80% of the cells contained in the suspension spread (spread) on the plate for forming the aggregate were the cells constituting the cell mass.

[ example 3]

In this example, the number of cells n allocated to one partition was set to 1 × 10⁶Otherwise, cells were cultured in the same manner as in example 1. Fig. 10 shows an observation image of the aggregate when the flat plate is viewed from above. The right column of FIG. 10 shows the assignment of 1 × 10 per partition⁶Results for individual cells. The left column shows the assignment per partition 10 as a control as in the previous example⁵Results for individual cells. Figure 10 shows the results on day 2 and day 4 post inoculation.

It is known that: cells in a partition were assigned at 1X 10⁵1 to 10⁶Within the range of one, an aggregate having a homogeneous size can be produced.

[ example 4]

Fig. 11 shows an observation image of the aggregate when the flat plate is viewed from above. In this embodiment, as shown in fig. 11, the top opening and the bottom opening are formed in a circular shape. The centers of the flat panels are coincident when viewed from above. The length of the left-right direction bar seen in the observation image represents 1000 μm. iPS cells were cultured under the same conditions as in example 1.

The size of the diameter of the top opening is 650 μm. The diameter of the bottom opening is 500 μm. Relative to a plane having 7cm²Has 648 holes regularly arranged on the flat plate.

Aggregates of the state 1, 3, 5, 7 days after seeding of the cell mass are shown in fig. 11. For each date, an aggregate of uniform size between partitions can be obtained. For each partition, approximately 1 aggregate can be obtained. For the rate of aggregate increase with time, no difference between the partitions was observed. Therefore, it was demonstrated that the quality of the cells was uniformly maintained between the partitions.

On day 7, the number of cells per partition is expected to be 2000 or more and 5000 or less.

[ example 5]

Unless otherwise mentioned, cells were cultured in the same manner as in example 4. The resulting aggregates were recovered from the plates on day 7 of culture. In the collection, the aggregate is passed through the bottom opening. Specifically, as shown in FIG. 6, the bottom surface of the plate was brought into contact with the collected solution, thereby eliminating the interface of the culture solution and collecting the culture solution. The above recovery method is hereinafter referred to as a contact method. The recovered solution was recovered in a 15ml tube.

After centrifugation of the tubes at 270G, the supernatant was removed. Add 500. mu.l of TrypLE Select to the tubes and incubate the tubes in a 37 ℃ incubator for 10 minutes. After centrifugation, the supernatant was removed and the cells in the tube were suspended in 1ml of culture solution A. The number of suspended cells was counted using a blood cell counting plate. Based on the calculation results of the number of cells, 2X 10⁵Individual iPS cells were suspended in 2ml of culture solution a. Further, 2. mu.l of a Rho-associated-kinase/Rho-binding kinase inhibitor (ROCK inhibitor) was added, and then iPS cells were plated. The old culture was replaced with 1. mu.l of a medium containing 1ml of the culture medium A supplemented with ROCK inhibitor during the cultureAnd (4) a base. Media changes were performed daily.

The culture broth was re-inoculated against the same shaped plate. The culture broth spread into each well. More than two cell blocks are allocated to be mixed in more than two zones, respectively. The number of cells per partition was the same as in the 1 st inoculation. The cell blocks are brought close to each other within each partition. Thereby causing the cell mass to again aggregate.

The aggregates are disintegrated to give cell masses, the cell masses are mixed, brought close to each other, distributed, and aggregated again, and these steps are repeated to passage the cells. Repeat 2 (P2), 3 (P3), 4 (P4) and 5 (P5) passages. The number of passages was calculated by setting the course (passage) of the initial inoculation of the cell mass obtained from the iPSC colony on the plate as 1 (P1).

Passages were performed 1 time every 7 days. The observed images of aggregates represented by initial plating onto the plates to days 14, 21, 28, and 35 are shown in fig. 12. The length of the left-right direction bar seen in the observation image represents 1000 μm. Even after a long period of 1 month, aggregates having a uniform size between the partitions can be obtained. The pluripotent stem cells after long-term culture were evaluated by flow cytometry.

< flow cytometer and Fluorescence Activated Cell Sorting (FACS) >

Aggregates obtained at the 10 th and 20 th days of culture were recovered by the above contact method and collected in 15ml tubes. After centrifugation of the tubes at 270G, the supernatant was removed. Cells were individualized by adding 500. mu.l of TrypLE Select to the tubes and incubating the tubes in a 37 ℃ incubator for 10 minutes. After incubating the tubes for 10 minutes, 500. mu.l of culture solution A was added to the tubes. And suspending the aggregate and the culture solution A for 10-30 times by using an automatic pipettor to break the aggregate. After 9ml of the culture medium A was added to the tube, the tube was further centrifuged at 270G.

After centrifugation, the supernatant was aspirated from the tube, and the precipitated cells were suspended in 1ml of culture solution A. The number of suspended cells was counted using a blood cell counting plate. Based onCalculation of the number of cells, cells were counted at 1X 10⁶One aliquot was dispensed into a new tube. The tube was again centrifuged at 270G. After centrifugation, the supernatant in the tube was aspirated. 2.5. mu.l of the antibody for detecting TRA-1-60 was suspended in 50. mu.l of PBS. After the antibody suspension was added to the tube, the tube was incubated at room temperature for 30 minutes in the dark.

After 30 minutes, 1ml of PBS was added to the tube. After the tubes were centrifuged at 270G, the supernatant was removed from the tubes. The TRA-1-60 positive rate of iPS cells was determined by flow cytometry Cytoflex.

Histograms of 4 FACS are shown in figure 13. The vertical axis of the histogram represents the intensity of TRA-1-60. The horizontal axis represents the intensity of autofluorescence.

The upper left histogram (Old method) of fig. 13 shows the results of the positive control. In the case of the positive control, the feeder layer cells were used to maintain differentiation pluripotency and subcultured as in the conventional method. The results of the flow cytometer at passage 2 are shown.

The lower left histogram (P2) was obtained from the cells at the time of day 10 in this example. Passage was 2 nd. The lower right histogram (P4) was obtained from the cells at the time of day 20 in this example. Passage was 4 th.

1 point plotted in the histogram (hereinafter referred to as a plot point) represents 1 cell. The upper left region (P4) in the histogram located in the collective part of the red plotted points (the light-colored part) represents the population of cells that maintain the function of iPS cells. The other black-plotted portion (dark-colored portion) indicates cells with a low expression level of iPS cell markers.

The upper right histogram (NC) shows the results of a negative control based on cells that are not iPS cells. Distribution deviating from the region (P4) having iPS cell function (black plot).

As shown in the lower part of fig. 13, iPS cells obtained by culturing on a plate with a partition were mostly TRA-1-60 positive even on day 10 (passage 2) and day 20 (passage 3). The intensity or the percentage of the TRA-1-60 positive cells to the total cells was about the same as that of the positive control.

The results of FACS-based assays show: in the case where it is necessary to perform multiple passages to prepare a population of pluripotent stem cell aggregates, the method of the present example can maintain an undifferentiated state between aggregates in a state of high homogeneity. In addition, even in the case where feeder layer cells were not used, the undifferentiated state of iPS cells could be maintained by using a plate with a partition.

< antibody staining >

The aggregate of iPS cells obtained at the 10 th day of culture was recovered by the above contact method and collected in a 15ml tube. After the aggregates were treated with TrypLE Select to individualize the cells in the same manner as described above, the tubes were centrifuged at 270G, and the supernatant was removed. After iPS cells were suspended using an appropriate medium, iPS cells were seeded on feeder cells previously cultured on 6-well plates.

After 5 to 7 days from cell inoculation, iPS cells on the feeder layer were stained according to the following procedure.

1. Media was removed from each well of the 6-well plate and 1ml of PBS was added to each well.

2. PBS was removed and 500. mu.l of 4% PFA (paraformaldehyde) was added.

3. Cells were reacted with PFA for 15 minutes in a refrigerator at 4 ℃.

4. PFA was removed from the wells and 1ml PBS was added.

5. The primary antibody was diluted 200-fold with PBS containing 5% fortified Calf Serum (CCS: cosmetic Calf Serum) and 0.1% Triton. To the wells, 500. mu.l of the diluted antibody solution was added. The first antibody was anti-OCT 3/4 antibody (C-10, SC-5279, Santacruz) and anti-NANOG antibody (abcam, ab 21624).

6. The antibody was allowed to react with the cells at room temperature for 1 hour.

7. The diluted antibody fluid was removed and the wells were washed with 1ml PBS. The wells were washed again with PBS.

8. The secondary antibody was diluted 1000-fold with PBS containing 5% fortified Calf Serum (CCS: cosmetic Calf Serum) and 0.1% Triton, and the diluted antibody solution was added to the wells. The secondary antibodies were donkey anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 488conjugate and donkey anti-goat IgG (H + L) secondary antibody, Alexa Fluor647 conjugate. Alexa Fluor is a trademark.

9. The antibody was allowed to react with the cells for 30 minutes at room temperature.

10. Wells were washed 2 times with PBS. Cells were observed using fluorescence microscope EVOS (thermo Fisher scientific).

An observation image of the cells stained with the antibody is shown in fig. 14. The length of the left-right direction bar seen in the observation image indicates 400 μm. The upper left is the image of the bright angle. The upper right is the result of staining for Oct 3/4. The lower left is the result of the Nanog staining.

From the dyeing results, it can be seen that: iPS cells cultured on plates with partitions expressed OCT3/4 and NANOG (OCT3/4 and NANOG positive) as marker genes for pluripotent stem cells. The results show that: iPS cells cultured on plates with compartmentalization maintained differentiation pluripotency.

< comparison with plate culture >

In this experiment, the iPS cell lines 1, 2 and 3 described above were used.

An observation image of the population of recovered aggregates is shown in fig. 15. The upper half of the observation image (KRR Dish ) shows the results of subculturing each cell on the plate with partitions. The lower half of the observed image (Non-adherent culture dish, Non-adherent dish) shows the result of subculturing each cell on the plate culture dish subjected to the low-adhesion treatment of the cell. Feeder layer cells were not used for any subculture. All additional passages were 1 (P1). As shown in fig. 15, it can be seen that: in preparing a population of pluripotent stem cell aggregates, the method of this example is advantageous for equalizing the size of the aggregates.

iPS cells generally have the property of differentiating to a certain size or more. Further, the nutrients in the medium are not easily diffused into the inside of the aggregate. Therefore, the aggregate composed of the cells cultured on the plate culture dish subjected to the low cell adhesion treatment is not uniform in size. In addition, differentiation and cell death were induced in these cells. On the other hand, cells cultured on plates with partitions were not affected by these effects. The culture method of the present example is suitable for culturing iPS cells, compared to conventional plate culture methods.

[ example 6]

In this example, iPS cell lines 1 and 2 were cultured in the same manner as in example 4. The left side of fig. 16 shows an observation image of an aggregate of iPSC line 1. The right side shows an observation image of the aggregate of iPSC line 2. Passage is 1 st. On day 5 from the time of initial plating on the plate. In this example, too, the size of the aggregates was equalized in the case of preparing a population of pluripotent stem cell aggregates in the same manner as in example 4. Therefore, the method of the above example contributes to obtaining aggregates of an equal size regardless of the type of cell line.

[ example 7]

In this example, cell line 4 expressing an undifferentiated marker and having iPS cells differentiated into the three germ layers was used in place of lines 1, 2 and 3, as in the case of these cell lines. The cells were cultured under the same conditions as in example 4.

FIG. 17 is an image of the observation of line 4 seeded on the plate of this example. The upper half of fig. 17 shows line 4 immediately after inoculation. The bottom half shows line 4 recovered from the plate on day 7. As shown in the left column of fig. 17, when line 4 was cultured in the same manner as in example 1, line 4 formed aggregates less efficiently than lines 1, 2 and 3. However, the line survival activity was maintained. The inventor considers that: the composition of culture solution A is insufficient for aggregation of line 4.

The culture was performed using a medium in which extracellular matrix was added to the culture solution a. As shown in the right column of fig. 17, line 4 formed aggregates.

In this example, commercially available Matrigel (trade mark) was added to the culture solution A so that the concentration was 10. mu.L/mL or more. The concentration of extracellular matrix in the medium is considered to be in the range of 10. mu.L/mL or more. The extracellular matrix may be any one of Matrigel, laminin, collagen, fibronectin, vitronectin, and Lamin 551 which is a variant of Lamin, or a combination thereof. Other conditions were the same as in the examples.

The above results show that: even cells that do not form aggregates in a medium that does not contain extracellular matrix can be aggregated by using a medium to which extracellular matrix is added.

[ example 8]

In this example, the culture based on the partition of the present example and the culture on a plate coated with an existing extracellular matrix were compared with each other using the TRA-1-60 expression intensity as a standard.

< preparation of cells >

(w/feeder cells (w/feeder))

As a positive control, a culture dish coated with extracellular matrix was used. Hereinafter, the positive control is referred to as "w/feeder".

1. Preparation of culture vessel: matrigel was used as the extracellular matrix to be coated on the culture dish. The solution was obtained by adding 180. mu.l of Matrigel to 12ml of DMEM on ice. An appropriate amount of the solution was poured into a culture plate using a 12-well petri dish (hereinafter, the culture vessel is simply referred to as culture vessel)The dish. ) In each culture dish. In CO₂The petri dish was left in the incubator for more than 1 hour. Immediately prior to use of the culture dish, the medium is removed. Hereinafter, the above-mentioned culture dish is referred to as an extracellular matrix culture dish.

2. iPS cells were cultured using feeder cells according to the same procedure as in example 1. Then, only feeder layer cells were removed from the culture product. Next, iPS cells were cultured in each well of the 6-well plate. After the culture, 1ml of the medium Y was injected into each well. iPS cells were peeled from the wells with a spatula. Suspension was performed sufficiently to individualize iPS cells into single cells.

3. For extracellular matrix culture dishes prepared as described above, each dish was inoculated with 2X 10⁵And (4) an iPS cell. Plating extracellular matrix onto CO₂Incubating in an incubator. As the medium, a medium obtained by adding 1/1000% of ROCK inhibitor to the medium of culture solution A was used.

4. After 1 day from the inoculation, the extracellular matrix culture dish was taken out from the incubator. Thereafter, medium replacement was performed daily using the same medium as described above, culture solution A, to which the ROCK inhibitor was added.

5. After 7-10 days from inoculation, the medium was removed from the extracellular matrix culture dish. 1ml of PBS was added to the petri dish. PBS was removed with an aspirator. Add 500. mu.l TrypLE Select to the petri dish. Placing the culture dish in CO₂Incubate in incubator for 5 minutes.

6. After incubation, 500. mu.l of medium Y was added to the petri dish. The iPS cells were individualized into single cells by performing suspension using an automatic pipette.

(w/o feeder cells (w/o feeder))

As a comparative example, plate culture was performed. A plate culture dish subjected to a low-adhesion treatment of cells was used. Hereinafter, the comparative example will be referred to as "w/o feeder".

The plate culture dish subjected to the low adhesion treatment of the cells was the same as that shown in < comparison with plate culture > of [ example 5 ]. Culturing on a plate culture dish is carried out 7 to 10 days after inoculation. The number of passages was 1 (P1).

The cell suspension after the culture was recovered in a 15ml tube. After the tubes were centrifuged at 270G, the supernatant was removed by aspiration. After adding 500. mu.l of TrypLE Select to the tubes, the tubes were incubated in an incubator at 37 ℃ for 10 minutes. After incubation, the tubes were supplemented with 500. mu.l of medium Y. The tube and cells were suspended using an automated pipettor, thereby individualizing the iPS cells into single cells.

(KRR)

iPS cells were cultured on the plate with partitions of this example. The above examples are hereinafter referred to as "KRR".

iPS cells were cultured on the plate having a partition of this example after 7 to 10 days from the inoculation according to the same procedure as in example 4. iPS cells were recovered into 15ml tubes according to the same procedure as in example 5. After centrifugation of the tubes at 270G, the supernatant was removed by aspiration. After addition of 500. mu.l of TrypLE Select, the tubes were incubated in an incubator at 37 ℃ for 10 minutes. After incubation, the tubes were supplemented with 500. mu.l of medium Y. The tube and cells were suspended using an automated pipettor, thereby individualizing the iPS cells into single cells.

<FACS>

After preparing the cells of the above-mentioned w/feeder, w/o feeder and KRR, the cells were analyzed as follows.

1. iPS cells, which were individualized single cells, were recovered into 1.5ml tubes. The number of cells was counted using a blood cell counting plate. Then, the tubes were centrifuged at 270G to remove the supernatant.

2. For 5X 10⁵PBS 50. mu.l was added to each iPS cell. To PBS was added 2.5. mu.l of an anti-TRA-1-60 antibody in advance. The anti-TRA-1-60 antibody was chemically treated in advance to emit fluorescenceAnd (6) processing. The tubes were incubated for 30 minutes at room temperature in the dark.

3. After incubation, 1ml of PBS was added to the tube. After centrifugation at 270G on the tubes, the supernatant was removed from the tubes.

4. The fluorescence intensity of TRA-1-60 positive cells was analyzed by a flow cytometer (CytoFlex).

< results >

As described below, iPS cells cultured on plates with compartmentalization were able to maintain higher expression of undifferentiated markers than iPS cells cultured on extracellular matrix culture dishes.

FIG. 18 is a2 parameter histogram of ACS. The vertical axis is the intensity of autofluorescence (Auto fluorescence). The horizontal axis represents the fluorescence intensity of TRA-1-60. KRR can obtain the same pattern as W/feeder.

Figure 19 is a1 parameter histogram of FACS. The horizontal axis represents the fluorescence intensity of TRA-1-60. The count on the vertical axis indicates the number of cells. KRR can obtain the same pattern as W/feeder.

From the results shown in fig. 18 and 19, it can be seen that: KRR shows a histogram equivalent to w/feeder. This can be determined, for example, by the position of the gray part with the darkest color in the drawing.

[ example 9]

The expression rate of the undifferentiated marker was measured for each cell mass cultured on a plate with partitions.

The culture was carried out for 7 to 10 days after inoculation in the same manner as in example 4 (P1). When the aggregates are recovered from the plate, the aggregates are recovered one by one. The above-described aggregate is hereinafter referred to as a monolith (single column) or a briquette (column). A total of 10 to 12 monoliths were collected into a 1.5ml tube previously filled with 300. mu.l of TrypLE Select.

The tubes were incubated at 37 degrees for 10 minutes. After incubation, 700. mu.l of PBS was added to the tube. The cells were suspended 10 to 30 times. The tubes were centrifuged at 270G. Then, treatment was carried out according to the < antibody staining > of [ example 5] steps 8 to 10.

The result of analysis of the obtained stained image is shown in a graph showing the expression intensity of TRA-1-60 in FIG. 20. Among 10 to 12 blocks, 80% or more of the blocks showed a positive rate of TRA-1-60 of 70% or more.

[ example 10]

Each piece cultured on a plate having a partition was tested for differentiation pluripotency.

1. iPS cells were cultured for 3 days in the same manner as in example 4. Medium was replaced with medium Y without bFGF type. Further, the culture was carried out for 7 days. Medium changes were performed 1 time every 2 days.

2. After the 7-day culture, iPS cell masses were recovered from the plates with partitions. iPS cells were seeded on 10cm gelatin-coated culture dishes. Then, the culture was further carried out for 7 days. Medium changes were performed 1 time every 2 days.

3. After 7 days of culture, immunostaining was performed according to the following protocol using the following antibodies.

4. After washing the dishes with PBS, the PBS was removed and 500. mu.l of PBS containing 4% PFA was added to the dishes.

5. PFA was reacted with the cells in a refrigerator at 4 ℃ for 15 minutes.

6. PFA was removed from the culture dish and 1ml PBS was added

7. The primary antibody was diluted with PBS containing 5% CCS and 0.1% Triton. The diluted antibody solution was added to the petri dish in an amount of 500. mu.l. The first antibody was obtained by diluting TUJI-1 antibody, FOXA2 monoclonal antibody, and Brachyury antibody 200-fold.

8. The antibody was allowed to react with the cells at room temperature for 1 hour.

9. The diluted antibody fluid was removed from the culture dish. Cells were washed with 1ml of PBS. Cleaning was performed again.

10. The secondary antibody was diluted 1000-fold with PBS containing 5% CCS and 0.1% Triton. The following were used as the second antibody.

Donkey anti-rat IgG (H + L) secondary antibody, Alexa Fluor 488conjugate

Donkey anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 555conjugate

Donkey anti-goat IgG (H + L) secondary antibody, Alexa Fluor647conjugate

11. Dilutions of the secondary antibody were allowed to react with the cells for 30 minutes at room temperature.

12. Cells were washed 2 times with PBS. Cells were observed using a fluorescence microscope, EVOS.

Fluorescence observation images of the aggregates are shown in fig. 21A-C.

FIG. 21A is TUJI-1, FIG. 21B is FOXA2, and FIG. 21C is the staining pattern of the brachyury gene. In FIGS. 21A-C, the upper left is series 1, the upper right is series 2, and the lower left is series 3. TUJ-1 is a differentiation marker for ectoderm. The results shown in fig. 21A show: the cells in the aggregate have a differentiation-inducing ability to differentiate into cells derived from an ectoderm such as nerve cells. FOXA1 is a differentiation marker for endoderm. FOXA1 is particularly an essential differentiation marker during the earliest stages of liver tissue formation. The results shown in fig. 21B show that: the cells in the aggregate have a differentiation-inducing ability to differentiate into cells derived from endoderm, such as liver. The brachyury gene is a differentiation marker for the primary mesoderm. The results shown in fig. 21B show that: the cells in the aggregate have a differentiation-inducing ability to differentiate into cells derived from mesoderm such as muscle.

FIG. 21D is a graph showing the ratio of the number of cells expressing each type of embryo marker in histograms using the total number of cells as a reference. The graph shows that the ratio of embryoid bodies induced from aggregates by the in vitro differentiation induction system was 80% or more.

[24] The method for producing a population of stem cell aggregates according to [2], wherein,

the aggregates formed by the reaggregation are mixed without being decomposed,

distributing the mixed aggregate in more than two subareas respectively,

bringing two or more of the mixed aggregates into proximity with each other in each of the partitions,

further aggregating the two or more aggregates close to each other.

[25] The method for producing a population of stem cell aggregates according to [2], wherein the following process is repeated 1 or 2 or more times:

before the aggregate is decomposed after the aggregate is formed,

mixing two or more of the aggregates with each other,

distributing the mixed aggregate in more than two subareas respectively,

further aggregating the two or more aggregates close to each other,

forming aggregates larger than the aforementioned mixed aggregates.

[26] The method for producing a population of stem cell aggregates according to [1], wherein,

mixing two or more of the aggregates with each other,

distributing the mixed aggregate in more than two subareas respectively,

further aggregating the two or more aggregates close to each other.

[27] The population of aggregates according to [19], wherein,

when induced to differentiate in vivo, the aggregate forming teratomas differentiating into the three germ layers is 80% or more in proportion.

[28] The population of aggregates according to [19], wherein,

from the population described above 10 aggregates were selected,

in the case of inducing endoderm from the aforementioned aggregate differentiation using an in vitro differentiation induction system,

for the aggregate described above, determining whether at least any one endoderm marker of FOXA2 and AFP is positive,

the positive rate of the endoderm marker is 80% or more.

[29] The population of aggregates according to [19], wherein,

from the population described above 10 aggregates were selected,

in the case of inducing mesoderm from the aforementioned aggregate differentiation using an in vitro differentiation-inducing system,

when the aggregate is determined to be positive for at least one mesoderm marker selected from the group consisting of brachyury gene and MSX1,

the mesoderm marker has a positive rate of 80% or more.

[30] The population of aggregates according to [19], wherein,

from the population described above 10 aggregates were selected,

in the case of inducing ectoderm differentiation from the aggregate using an in vitro differentiation induction system,

when the aggregate is determined to be positive for at least any one of the ectodermal markers Pax6, SOX2, PsANCAM and TUJ1 by measuring the gene expression level of the embryoid body by PCR,

the positive rate of the ectodermal marker is 80% or more.

[31] A method for preparing a population of stem cell aggregates,

allocating two or more pre-aggregation units to two or more partitions having equal sizes, respectively, wherein the pre-aggregation unit is at least one of a cell mass and a single cell,

in each of the sections, the two or more pre-accumulation units are brought close to each other,

aggregating and incubating the two or more pre-aggregation units that are close to each other to form an aggregate,

wherein the dispensed pre-aggregation units are separated from each other and mixed with each other,

the cell masses are each composed of stem cells.

[32] The method for producing a population of stem cell aggregates according to [1] or [31], wherein,

the stem cell is a pluripotent stem cell.

[33] The method for producing a population of stem cell aggregates according to [31] or [32], wherein,

the pre-aggregate units are clumps of cells.

This application claims priority based on U.S. provisional application 62/272524 filed on day 29, 12/2015, the entire disclosure of which is incorporated herein by reference.

Description of the reference numerals

20 incubator, 21-26 steps, 27 arrows, 29 partitions, 30 plates, 31a, 31b wells, 32a, 32b partitions, 33a, 33b open top, 34a, 34b open bottom, 35 broth, 36a, 36b droplets, 37 storage partitions, 38 suspension, 39 threshold, 40 aggregates, 41 population, 42a-42c cell mass, 43a-43c aggregates, 44a, 44b population, 45 support, 46 side wall, 47 flange, 50 vessel, 55 tray, 56 side wall, 57 bottom, 58 space, 60 tray, 65 recovery solution.

Claims

1. A method of preparing a population of stem cell aggregates, which is a method comprising:

bringing said two or more cell blocks into proximity with each other within each of said partitions,

aggregating and incubating the two or more cell masses in proximity to each other to form an aggregate,

the cell pieces distributed are separated from each other and mixed with each other,

the cell masses are each composed of stem cells.

2. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

decomposing said formed aggregates to produce a cell mass,

mixing the cell masses generated from different said aggregates with each other,

bringing said two or more mixed cell masses into proximity with each other within each of said compartments,

the two or more cell masses close to each other are again aggregated.

3. The method for preparing a population of stem cell aggregates according to claim 2, wherein,

decomposing the aggregate at a time when the diameter of the aggregate is 1mm or less.

4. The method for preparing a population of stem cell aggregates according to claim 2, wherein,

the aggregate is incubated for a period of 2 days or more and 14 days or less.

5. The method for preparing a population of stem cell aggregates according to claim 2, wherein,

the aggregate is incubated for a period of 3 days or more and 7 days or less.

6. The method for preparing a population of stem cell aggregates according to claim 2, wherein,

further repeating the following process for 1 or more times:

breaking up the aggregates, mixing the cell clumps, bringing them closer together, distributing them and aggregating them again.

7. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

plating the stem cells to form colonies,

(ii) disaggregating said colonies to produce said cell mass,

mixing the generated cell masses with each other,

using the cell block for the assignment.

8. The method for preparing a population of stem cell aggregates according to claim 7, wherein,

said disintegration of said colonies is carried out by physical disruption,

the colonies were not treated with enzyme.

9. The method for preparing a population of stem cell aggregates according to claim 7, wherein,

the colonies are subjected to the disintegration by enzyme treatment only,

the colonies were not physically disrupted.

10. The method for preparing a population of stem cell aggregates according to claim 7, wherein,

when the colonies are to be broken down,

the colonies were subjected to enzymatic treatment and physical disruption.

11. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the partitions are formed by holes provided in the plate,

the hole is a through hole or a concave part,

the hole has a top opening on the side of the top surface that the plate has,

the top openings have areas equal to each other between the partitions,

the diameter of the top opening is less than 1.5 mm.

12. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the partitions are formed by through holes provided in the flat plate,

the diameter of the bottom opening is less than 1mm,

13. The method for preparing a population of stem cell aggregates according to claim 12, wherein,

the cell mass is cultured in a culture solution disposed in the partition,

the culture liquid forms a liquid drop and,

the liquid droplet adheres to the bottom opening and protrudes in a manner to hang down from the bottom opening,

the bottom surface of the partition is formed by the meniscus of the droplet.

14. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the diameter of the partitioned inscribed sphere is 5 multiplied by 10¹1 × 10 μm or more³The particle size is less than the micron range,

the inscribed sphere is tangent to the bottom surface of the partition.

15. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the cell mass is cultured in a culture solution disposed in the partition,

the culture fluid of the storage partition is not configured with cells.

16. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the partitions are formed by holes provided in the plate,

the hole is a through hole or a concave part,

the hole has a top opening on the side of the top surface that the plate has,

17. The method for preparing a population of stem cell aggregates according to claim 16, wherein,

the suspension contains a unit area (1 cm) relative to the top surface²) Is a cell block of 1 or more and 5000 or less.

18. The method for preparing a population of stem cell aggregates according to claim 1, wherein,

the cell mass is cultured in a culture solution disposed in the partition,

extracellular matrix is suspended or dissolved in the culture solution.

19. A cell culture method is as follows:

the aggregate is formed from the stem cells and,

when the aggregate is formed,

separating and mixing the cell blocks with each other prior to the dispensing,

the cell masses are each composed of stem cells.

20. The cell culture method according to claim 19,

further within the partitions, the cells in the aggregate are differentiated into any of ectoderm, mesoderm, and endoderm.

21. A population of aggregates, wherein,

selecting 10 of said aggregates from said population;

selecting more than 10 cells from the selected aggregate;

measuring a positive rate by determining whether at least any one of pluripotent stem cell markers of Nanog, Oct3/4 and TRA-1-60 is positive for the 10 or more cells;

when the population is measured for 3 times of the above positive rate;

the average value of the 3-time positive rates was 80% or more.

22. The population of aggregates of claim 21, wherein,

selecting 10 aggregates from the population,

in determining whether at least any one of the pluripotent stem cell markers Nanog, Oct3/4 and TRA-1-60 is positive for the selected 10 aggregates,

the positive rate of the marker is more than 80%.

23. The population of aggregates of claim 21, wherein,

the ratio of embryoid bodies induced from the aggregate by the in-vitro differentiation induction system is 80% or more,

the embryoid bodies are aggregates of cells mixed with the tissue of the three germ layers.