JP2015165783A - Method for producing cell mass consisting of pluripotent stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing cell mass consisting of human pluripotent stem cells.SOLUTION: The method for producing cell mass consisting of pluripotent stem cells is characterized by culturing pluripotent stem cells. The method comprises at least a process of adjusting constituents of culture fluid via semipermeable membrane, in a culture fluid constituent adjustment process where closed system is substantially maintained in the presence of culture fluid containing a Rho-associated kinase (ROCK) inhibitor.

Description

  The present invention relates to a method for producing a cell cluster composed of pluripotent stem cells, particularly human pluripotent stem cells.

Human pluripotent stem cells such as ES cells and iPS cells are expected to be an important cell source for realizing regenerative medicine. However, in order to artificially construct a transplantable organ / tissue, it is first necessary to prepare a large number of cells. If the left ventricle of the heart is taken as an example, it is estimated that a cell number of the order of 10 ⁹ is required. In order to prepare such a large amount of cultured cells, the current technical means requires a lot of labor and cost.

  Therefore, one of the challenges for full-scale realization of regenerative medicine is the development of means for efficiently culturing a large amount of human pluripotent stem cells.

  Culture methods for obtaining large amounts of human pluripotent stem cells include culturing cells called feeder cells in plastic dishes or on dishes coated with proteins so that pluripotent stem cells can easily adhere. A method of monolayer culture and a method of forming a cell mass of pluripotent stem cells and suspension culture thereof are generally used.

  When these are compared, suspension culture is preferred because the ratio of the number of cells that can be cultured and the amount of the culture solution is generally more efficient in suspension culture, and expensive substances such as support cells and coat proteins are unnecessary. it is conceivable that. However, human pluripotent stem cells are commonly used for suspension culture because they cause cell death as soon as they are separated into pieces by enzyme treatment, etc., and are susceptible to shear stress during suspension culture. It is not supposed to be.

  In order to overcome the property of easily causing cell death, a method of adding a ROCK (Rho-associated coiled-coil forming kinase) inhibitor to the culture medium at the start of suspension culture has recently been developed. There are reports of suspension culture of human pluripotent stem cells (for example, Non-Patent Document 1 and Non-Patent Document 2). However, even if these techniques are used, the cell growth rate is about 5 times at maximum from 5 days to 1 week, and a more efficient culture method has been demanded.

  In addition, it is necessary to arrange cells in a three-dimensional structure in order to build organs and tissues even after the required number of cells is obtained.

  In order to construct a three-dimensional structure, there is a method of forming a scaffold of a desired shape with a porous body and seeding cells there, but seeding cells uniformly from the inside to the outside of a three-dimensional shape Obviously it is difficult.

  On the other hand, cell clusters with a diameter of several hundreds of μm can be easily confirmed with the naked eye, settled in a short time even in solution, are easy to handle, and are easier to construct a desired three-dimensional structure artificially than separate cells. There is an advantage.

  Recently, the development of “Regenova” (Cyfuse Co., Ltd.), a bio 3D printer device that builds a three-dimensional structure while skewering cell clumps, has been promoted. With the advancement of such technology in the future, the cell mass of pluripotent stem cells may become the basic structural unit for tissue and organ construction.

  However, as the cell mass of human pluripotent stem cells grows larger, the shape tends to collapse, and as the cell mass grows, oxygen and nutrients are less likely to penetrate into its central region, making it difficult to produce a large amount thereof.

  As a technique for producing a cell cluster of pluripotent stem cells, a cell is seeded on a plate having a plurality of wells having a concave curved bottom, and one cell cluster is formed in one well (Patent Document 1). However, the number of cell masses that can be produced by such a technique is defined by the number of wells, and in order to produce a large amount of cell masses, cells are seeded individually in that number of wells. However, it is effective for producing a small amount of cell mass, but is not suitable for producing a large amount of cell mass at a time.

  A step of suspension culture until the average diameter of the cell mass is about 200 to about 300 μm, and a step of repeating the step of dividing the obtained cell mass into a uniform cell mass having an average diameter of about 80 to about 120 μm; Is disclosed (Patent Document 2). However, in this method, it is necessary to repeat culture and cell mass division in a short time, and there is a problem that the process becomes complicated.

  As described above, there has been a demand for a technique capable of culturing a large amount of pluripotent stem cells and at the same time efficiently producing a homogeneous cell mass of the same cells in large amounts.

WO2013 / 183777 Publication WO2013 / 077423 Publication

Tissue Engineering Part C Methods (2012) 18, 772-784 Stem Cell Research (2013) 11, 1103-1116

  The present invention provides a method for producing a cell mass composed of human pluripotent stem cells for solving the problems described above. That is, it is an object of the present invention to provide a technique capable of culturing a large amount of human pluripotent stem cells in a large amount and simultaneously efficiently producing a homogeneous cell mass of the same cells in large amounts.

  As a result of intensive studies to solve the above problems, the present inventors maintain a state where the ROCK inhibitor is maintained at an effective concentration in the culture solution, and control the concentration of the active ingredient in the culture solution in a closed culture system. Thus, it has been found that a large amount of human pluripotent stem cells can be cultured and at the same time a homogeneous cell mass of the same cells can be efficiently produced in large amounts.

  Furthermore, it has been found that the addition of a ROCK inhibitor is effective not only for inhibiting apoptosis at the initial stage of suspension culture but also surprisingly maintaining the cell mass morphology and the undifferentiated state, and has completed the present invention.

  Moreover, it has also confirmed that the cell which comprises the cell mass obtained by said method has maintained undifferentiation, and has pluripotency.

That is, the present invention is as follows.
(1) A cell mass production method comprising pluripotent stem cells,
In the presence of a culture medium containing a Rho-binding kinase (ROCK) inhibitor,
A method for producing a cell mass composed of pluripotent stem cells, comprising a culturing step of adjusting the components of a culture solution in a state where a closed system is substantially maintained.
(2) The method for producing a cell mass composed of pluripotent stem cells according to (1), wherein the culturing step includes at least a step of adjusting the components of the culture solution via a semipermeable membrane.
(3) a culture tank containing the culture solution;
An adjustment means 1 having a semipermeable membrane independent of the culture tank and connected in a closed circuit, and a storage means for adjusting liquid containing the ROCK inhibitor;
In a culture apparatus comprising the adjusting means 2 independent of the culture tank and the adjusting means 1 and connected by a closed circuit,
The concentration of the substance that passes through the semipermeable membrane among the components of the culture solution is adjusted by the adjusting means 1 and the adjusting solution,
Furthermore, the adjusting means 2 adjusts at least the fibroblast growth factor (FGF) concentration,
The method for producing a cell mass composed of pluripotent stem cells according to (1) or (2), comprising at least a culturing step.
(4) The pluripotency according to (2) or (3), wherein the adjustment liquid contains at least insulin, and the semipermeable membrane of the adjustment means 1 has a pore diameter capable of transmitting insulin. A method for producing a cell mass composed of stem cells.
(5) The method for producing a cell mass composed of pluripotent stem cells according to (4), wherein the semipermeable membrane of the adjusting means 1 has a pore size that does not allow FGF to permeate.
(6) The adjusting means 2 has a storage means containing a mixture of the FGF and a compound having a sulfate group, and the concentration of the mixture is adjusted by adding the mixture to a culture tank ( 3) A method for producing a cell mass comprising the pluripotent stem cells according to any one of (5) to (5).
(7) The method for producing a cell mass composed of pluripotent stem cells according to any one of (1) to (6), wherein the culture is performed while maintaining a closed system during the culture period.
(8) The method for producing a cell mass composed of pluripotent stem cells according to any one of (1) to (7), wherein the amount of the culture solution in the culture tank can be maintained substantially constant.

  According to the method of the present invention, a mass of human pluripotent stem cells maintained in an undifferentiated state can be produced in a large amount at a high density.

FIG. 1 is a schematic diagram of a culture apparatus used in the production methods of Examples 1 and 2 and Comparative Example 3. FIG. 2 is a schematic diagram of the culture apparatus used in the production method used in Comparative Examples 1, 2, and 4. FIG. 3 is a photomicrograph of a cell mass composed of human pluripotent stem cells obtained by the method of Example 1. FIG. 4 is a photomicrograph of human pluripotent stem cells obtained by the method in Example 2. FIG. 5 is a photomicrograph of a cell mass composed of human pluripotent stem cells obtained by the method of Comparative Example 1. 6 is a photomicrograph of a cell mass composed of human pluripotent stem cells obtained by the method of Comparative Example 2. FIG. FIG. 7 is a photomicrograph of a cell cluster composed of human pluripotent stem cells obtained by the method of Comparative Example 3. FIG. 8 is a photomicrograph of a cell mass composed of human pluripotent stem cells obtained by the method of Comparative Example 4. FIG. 9 is a photomicrograph of cells obtained after differentiation culture in order to confirm that the cells constituting the cell cluster of human pluripotent stem cells obtained in Example 1 have pluripotency.

  Embodiments of the present invention (hereinafter referred to as “present embodiments”) will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

  The pluripotent stem cells to which the method of the present embodiment can be applied are pluripotent cells having self-replicating ability, and are ectoderm (such as nerve cells), mesoderm (such as muscle cells), and endoderm (such as intestinal epithelium). It is a cell that can differentiate into cells. Examples thereof include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), stimulation-induced pluripotency acquisition (STAP) cells, and the like.

  The cell mass refers to a state in which a plurality of pluripotent stem cells are aggregated together. The cell-to-cell association may be in a state in which the cells are directly associated with each other or in a state in which the cells are associated with each other via proteins such as extracellular matrix, peptides, antibodies, synthetic polymers, etc. Even if formed, there is a substance other than the cells constituting the nucleus, and the state in which the cells are associated with each other is also referred to as a cell mass in the present invention.

  As the culture solution, a culture solution having components capable of culturing pluripotent stem cells in an undifferentiated state is used. Many of the culture solutions for culturing pluripotent stem cells are already available on the market. Essential-8 (Life Technologies), StemPro hESC SFM (Life Technologies), mTeSR1 (Veritas), mTeSR2 (Veritas) Etc. can be illustrated. In order to apply the method of the present embodiment, a culture solution containing FGF and insulin is desirable.

  The adjustment solution preferably contains a component having a molecular weight that permeates the semipermeable membrane used in the adjustment means 1 among the components that are lost from the culture solution during the cell culture period. Moreover, what contains insulin as a component is desirable. Furthermore, it is desirable to set the concentration so that the concentration of the above components is not depleted during the culture period.

  The culturing period is preferably 3 days or more and 20 days or less, more preferably 5 days or more and 15 days or less, although it depends on the size of the cell mass required and the volume of the culture solution or adjustment solution used. If the culture period is shorter than this, it is difficult to obtain a sufficient number of cells because the time for forming a cell mass is too short and the period for cell growth is too short. Further, if the culture period is longer than this, nutrient components in the culture solution are easily depleted, and metabolic components produced from the cells are accumulated in the culture system, resulting in a poor culture environment such as pH.

  Moreover, it is desirable to set the amount of the adjustment liquid as much as possible from the viewpoint of preventing accumulation of cell metabolites. Desirably, it is set to 5 times or more of the culture solution, more desirably 10 times or more. However, since the amount of the adjustment liquid affects the culture cost, it may be determined depending on the culture period and the number of necessary cells. In addition, the adjustment solution is designed to have a buffering capacity so that it is easy to maintain a physiological pH like the culture solution, and a pH indicator dye is mixed so that changes in pH can be easily distinguished by color. Etc. are also possible.

  ROCK inhibitors are drugs that are used clinically in the cardiovascular / brain surgery field as vasodilators, and examples thereof include Y-27632 and fasudil hydrochloride.

FGF
Fibroblast growth factor (FGF) is a protein having a molecular weight of 16,000 to 20,000 that promotes proliferation of fibroblasts and endothelial cells. FGF includes basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), keratinocyte growth factor (KGF), and more than 20 types of FGF are known in humans and mice. It has been. Of these, bFGF is generally added to commercial cultures of pluripotent stem cells.

  FGF can also be used by mixing with a compound containing a sulfate group such as sulfated polysaccharide known to act for the purpose of protecting its degradation, denaturation, inactivation and the like. Examples of the compound containing a sulfate group include sulfated polysaccharides such as heparin, dextran sulfate, and carrageenan. By mixing these compounds and FGF in the use form of the present invention, FGF can be maintained during the culture period without using additional means such as refrigeration equipment.

  The state in which the closed system is substantially maintained means that a state in which bacteria or the like cannot substantially enter the system is maintained. For example, a closed system is maintained substantially in a state in which the air is communicated with the outside through an air filter having a pore diameter of about 0.2 μm that does not allow the most recent passage, and a state in which gas or liquid is exhausted in one way from the inside of the system to the outside. That state. However, even in an environment where a clean environment such as a clean bench is maintained, it is not said that the closed system is substantially maintained when bacteria or the like can substantially enter from the outside. .

  The adjustment of the culture solution component means that the initial concentration of each component in the culture solution set at the start of the culture is maintained or adjusted to be closer to the initial concentration. During the culture period, the nutrient components in the culture solution and the components for maintaining the undifferentiated state gradually decrease in concentration, while the components produced by the cells gradually increase in concentration.

  For this reason, for example, the adjusting means 2 can hold the components that decrease in the culture tank in advance in a space independent of the culture tank, and adjust the initial concentration or close to it by adding it in a timely manner. it can.

  Moreover, in the adjustment means 1, the culture solution and the adjustment solution are brought into contact with each other through a semipermeable membrane, so that the nutrient component in the adjustment solution or the component for maintaining the undifferentiated state is semi-permeable. The components of molecular weight that permeate the membrane are replenished from the adjustment solution to the culture solution through the membrane, and the components with higher concentrations diffuse from the culture solution to the adjustment solution, each close to the initial concentration. It is adjusted to become.

  A semipermeable membrane refers to a semipermeable membrane in which the membrane permeability of a culture solution component depends on its molecular weight. The adjustment solution contacts the culture solution through the semipermeable membrane. The pore size of the semipermeable membrane is designed according to the molecular weight of the component to be retained in the culture tank. That is, it is selected so that the minimum molecular weight substance does not permeate the membrane among the components to be retained in the culture tank. For the purposes of the present invention, it is desirable that the pore size of the semipermeable membrane has a size that allows insulin to permeate. More preferably, insulin (molecular weight: 5807) permeates but FGF (molecular weight: 16,000-20,000) does not permeate. Since the concentration of insulin is likely to decrease during the culture period, the culture tank is replenished with the insulin in the adjustment liquid throughout the culture period by using a semipermeable membrane that contains insulin in the adjustment liquid and permeates the insulin. In addition, since the concentration of FGF tends to decrease during the culture period, it is possible to prevent FGF in the culture medium from diffusing into the adjustment solution by selecting a pore size that does not allow FGF to permeate.

  A flat membrane shape or a hollow fiber shape can be used as the shape of the semipermeable membrane of the culture solution component adjusting means 1, but the hollow fiber shape is preferable for the purpose of perfusing the culture solution or the adjustment solution.

  The material of the semipermeable membrane is not particularly limited, but a material that does not adsorb, decompose, etc. the components that are desired to be retained in the culture tank is preferably used. Further, when the material has a property of easily adsorbing them, it is possible to suppress the adsorption by coating them with another material or using a surface modifier.

  The liquid feeding circuit according to the present embodiment refers to a tube having a lumen structure that is installed between the culture tank and the adjusting means 1 and can aseptically perfuse the culture liquid or the adjusting liquid. As the material, silicon, urethane, fluororesin, polyvinyl chloride or the like is used.

  The means for perfusing the culture solution and / or the adjustment solution according to the present embodiment refers to a means that is installed so as to be in contact with the liquid feeding circuit and can continuously feed the liquid in the circuit using power. Any general pump can be used, and examples include a peristaltic pump and a diaphragm pump.

  Hereinafter, the present embodiment will be described in more detail by way of examples, but the present embodiment is not limited thereto.

(Cell culture system)
As the culture apparatus, an 8-linked animal culture apparatus BioJr. 8 (BJR-25NA1S-8C, Able Corporation) was used. This device can control eight 100mL culture tanks with one controller, and each culture tank can be controlled independently with stirring / speed, temperature, pH, and dissolved oxygen concentration (DO) as measurement / control items. .

(Preparation of human iPS cells)
Human iPS cells (253G1) were selected as cultured cells. The cells were seeded in a culture dish (Corning) coated with Matrigel (Nippon Becton Dickinson Co., Ltd.) and cultured using Essential-8 as a culture solution. The culture medium was changed every day, and subculture was performed once every 3 to 4 days.

(Manufacture of cell mass)
A dedicated glass tank (Able Co., Ltd.) was used as the culture tank, and the culture solution was 100 mL.

The culture was started with human iPS cells at a density of 2 × 10 ⁵ cells / mL. This time point was regarded as the 0th culture day.

  As a culture solution, the following components were added to Essential-8 medium (Life Technologies) and used.

  That is, Y-27632 (Wako Pure Chemical Industries, Ltd.) was added as a ROCK inhibitor at a concentration of 10 μM and heparin (Sigma Aldrich) at a concentration of 750 ng / mL.

  The culture tank was equipped with a stirring rotor, and the rotation speed was 60 rpm.

  A sensor (Able Co., Ltd.) capable of measuring temperature, pH, and dissolved oxygen concentration was installed in the culture tank.

  Further, the dissolved oxygen concentration was set to be controlled to 40%, and for this purpose, a gas introduction line was installed so that a mixed gas of oxygen, nitrogen, and air could be vented to the culture medium in the culture tank. In addition, a lead-out line for discharging gas from the tank was installed. The temperature was set at 37 ° C.

  In Example 1, a cell culture system as shown in FIG. 1 was produced. That is, the culture tank was provided with a mouth capable of taking out and returning the culture solution at the lid portion of the culture tank.

  Furthermore, as a means for preventing the cell sputum formed in the culture tank from being led to the outside of the culture tank together with the culture solution, a cylindrical shape made of a polyethylene sintered body having a diameter of 8 mm, a length of 37 mm, and an average pore diameter of 30 μm. A membrane was installed.

  A glass sterilization bottle having a capacity of 1 L was used as the adjustment liquid tank.

  An aeration line, a culture solution inlet, and an outlet line were installed in the tank lid.

  In the adjusting means, urethane adhesive is used so that the hollow structure of both ends of the hollow fiber is released so that the effective length is 20 cm by bundling 400 hollow fibers of Asahi Polysulfone Dialyzer APS (Asahi Kasei Medical Co., Ltd.). A fixed one was installed.

  The hollow fiber bundle was disposed inside the adjustment liquid storage means, and both ends of the hollow fiber bundle were connected to the culture solution inlet and outlet lines by a circuit.

  As the adjustment solution, 1 L of Essential-6 (Life Technologies) containing no bFGF and TGF-β was used among the components of Essential-8. Essential-6 contains the same concentration of insulin as Essential-8.

  A silicon tube (inner diameter 1 mmφ, outer diameter 4 mmφ) was used as the liquid feeding circuit.

  Two Perista Pumps RP-23 (Able Co., Ltd.) were used as the culture solution feeding means. Each of the two pumps was placed in contact with a liquid supply circuit connecting the culture tank and the adjusting means 1 so that the liquid could be supplied.

  Culture tank in closed circuit, independent of adjustment means 1, 10 mL syringe (Terumo Corporation), bFGF (Life Technologies) 10 μg / mL, heparin (Sigma Aldrich) 250 μg / mL culture solution 2 mL was dissolved in DMEM / F12 (Life Technologies).

  A cell culture system as shown in FIG. 1 was produced by the method described above. Using this system, a cell mass composed of iPS cells was produced by the following method.

  The circulation rate of the culture solution was 100 mL / day from the first day to the fourth day, 400 mL / day from the fourth day to the fifth day, and 800 mL / day from the fifth day to the tenth day.

  The flow rate of the two pumps was finely adjusted so that the liquid volume in the culture tank could be maintained at substantially the same level, and the culture was carried out while continuously perfusing the culture liquid up to the seventh day.

  The mixed solution of bFGF and heparin was administered at 1 mL on the 2nd and 4th days of culture.

  On the seventh day, the cell culture was terminated and the number of cells was measured.

(Measurement of cell number)
On the 7th day of culture, the cell mass was collected from the culture tank, treated with 0.25% trypsin / EDTA (Invitrogen) to form a single cell, and dead cells were stained by the trypan blue method. Were counted using a calculator.

(Measure cell number and equivalent circle diameter)
The number of cell masses was measured while observing using an optical microscope ECLIPSE Ti-U (Nikon Corporation). The circle-equivalent diameter of the cell mass was obtained by taking an image of the cell mass using an optical microscope ECLIPSE Ti-U (Nikon Corporation) and then measuring the outer circumference of the cell mass using software (Nikon Elements D, Nikon Corporation). The equivalent circle diameter in the case of a perfect circle was calculated from the value. The equivalent circle diameter of 100 cell clusters from each sample was measured.

(Undifferentiation rate measurement)
In order to confirm that human iPS cells were maintained in an undifferentiated state, the positive rate of Tra1-60, an undifferentiated marker, was measured by flow cytometry. Anti-Tra1-60 antibody 560380 (Nippon Becton Dickinson Co., Ltd.) was used as the antibody, and Cell Lab QuantaSC (Beckman Coulter Co., Ltd.) was used as the measurement apparatus. The positive rate of Tra-1-60 was defined as the undifferentiated rate.

(Differentiation culture into three germ layer cells)
In order to verify that the cells obtained by the method of this embodiment have pluripotency, differentiation culture into three germ layers was performed by the following method.

  As a culture solution, IMDM (Life Technologies) has 1% MEM (Non-essential amino acid) (Life Technologies), 2-Mercaptoethanol (Life Technologies) 0.1 mM, L-glutamine (Life Technologies) 1 mM, And fetal bovine serum (FBS) (Nichirei Bioscience) added to 20% was used.

  The cells obtained in Example 1 were cultured in a culture solution for 24 days. The medium exchange frequency was once every 3 days.

(Immunostaining)
After culture, β3 Tubulin was used as an ectoderm marker, cTnT (Troponin T Cardiac Isoform) was used as a mesoderm marker, and AFP (αfeto protein) was used as an endoderm marker by immunostaining. As the antibodies, anti-β3 Tubulin antibody (Merck Millipore, 05-559), anti-cTnT antibody (thermo scientific, MA5-12960), and anti-AFP antibody (R & D Systems, MAB1368) were used. As a secondary antibody, an anti-mouse IgG antibody (Life Technologies, A11031) was used.

  The stained cells were photographed with FLUOVIEW FV1200 (Olympus Corporation) with a confocal microscope (40x objective lens). FV10-ASW (Olympus Corporation) was used as software.

  FIG. 9 shows a photograph of the cells obtained in Example 1 stained after differentiation culture. Multipotency to differentiate into all three germ layers was confirmed.

Cultivation was carried out for 7 days in the same manner as in Example 1 except that 4 mL of bFGF and heparin were added in advance to the adjusting means 2 and added on the 3rd, 4th, 5th, and 6th days 1 mL per day.
[Comparative Example 1]

In Comparative Example 1, a cell culture system as shown in FIG. 2 was produced. The same culture tank as in Example 1 was used. The adjustment means 1 retains the culture medium Essential-8 medium (maintains 4 ° C in a refrigerated state), and is added to the culture tank at a rate of 100 mL / day through the liquid supply circuit by the liquid supply means. The culture solution was withdrawn at the same speed by the means, and cultured for 7 days by a method of discarding it in another storage means through the liquid feeding circuit. Y-27632 having the same concentration as in Example 1 was added to the culture solution in the culture tank and the adjusting means 1. The other points were cultured under the same conditions as in Example 1.
[Comparative Example 2]

Using the system shown in FIG. 2, the culture solution Essential-8 medium was added to the culture tank at a rate of 100 mL / day, and at the same time, the culture solution was withdrawn at the same rate and cultured for 7 days. Y-27632 and heparin at the same concentration as in Example 1 were added to the culture solution. The other points were cultured under the same conditions as in Example 1.
[Comparative Example 3]

The culture was carried out for 7 days in the same manner as in Example 1 except that Y-27632 was not added to the adjustment solution.
[Comparative Example 4]

  The culture was performed for 7 days in the same manner as in Comparative Example 1 except that Y-27632 was not added to the culture solution.

The above examples and comparative examples are summarized in the following table.

  From the results, it was shown that the method of this embodiment is effective as a method for producing a cell cluster of human pluripotent stem cells.

  The cell culture method according to the present embodiment can provide a method capable of culturing uniform human pluripotent stem cell mass at low cost, with less labor, and at high density, such as regenerative medicine, drug screening, safety evaluation, etc. Therefore, it can be used in a field where pluripotent stem cells are used in large quantities.

DESCRIPTION OF SYMBOLS 1 Culture tank 2 Culture solution 3 Stirring blade 4 Rotating shaft 5 Filter 6 Ventilation filter 7 Adjustment means 1
8 Adjustment liquid storage tank 9 Adjustment liquid 10 Semipermeable membrane 11 Stirring rotor 12 Liquid supply means 13 Liquid supply circuit 14 Liquid level detection means 15 Information transmission circuit 16 Liquid supply means adjuster 17 Dissolved oxygen detection means 18 Adjustment means 2
19 FGF solution

Claims (8)

A cell mass production method comprising pluripotent stem cells,
In the presence of a culture medium containing a Rho-binding kinase (ROCK) inhibitor,
A method for producing a cell mass composed of pluripotent stem cells, comprising a culturing step of adjusting the components of a culture solution in a state where a closed system is substantially maintained.   The method for producing a cell mass composed of pluripotent stem cells according to claim 1, wherein the culturing step includes at least a step of adjusting a component of the culture solution via a semipermeable membrane. A culture vessel containing the culture solution;
An adjustment means 1 having a semipermeable membrane independent of the culture tank and connected in a closed circuit, and a storage means for adjusting liquid containing the ROCK inhibitor;
In a culture apparatus comprising the adjusting means 2 independent of the culture tank and the adjusting means 1 and connected by a closed circuit,
The concentration of the substance that passes through the semipermeable membrane among the components of the culture solution is adjusted by the adjusting means 1 and the adjusting solution,
Furthermore, the adjusting means 2 adjusts at least the fibroblast growth factor (FGF) concentration,
The method for producing a cell mass composed of pluripotent stem cells according to claim 1, comprising at least a culturing step.   The cell mass composed of pluripotent stem cells according to claim 2 or 3, wherein the adjustment liquid contains at least insulin, and the semipermeable membrane of the adjustment means 1 has a pore diameter capable of permeating insulin. Manufacturing method.   The method for producing a cell mass composed of pluripotent stem cells according to claim 4, wherein the semipermeable membrane of the adjusting means 1 has a pore size that does not allow FGF to permeate.   The adjusting means 2 has a storage means containing a mixture of the FGF and a compound having a sulfate group, and the concentration of the mixture is adjusted by adding the mixture to a culture tank. 6. A method for producing a cell cluster comprising the pluripotent stem cells according to any one of 5 above.   The method for producing a cell mass composed of pluripotent stem cells according to any one of claims 1 to 6, wherein the culture is performed while maintaining a closed system during the culture period.   The method for producing a cell mass composed of pluripotent stem cells according to any one of claims 1 to 7, wherein the amount of the culture solution in the culture tank can be maintained substantially constant.