Method for producing pluripotent stem cell stock

Abstract

The present invention relates to a production method for obtaining a high-quality pluripotent stem cell stock in large quantity.　This method for producing a cell stock involves the steps of: A) recovering a cultured pluripotent stem cell aggregate from a culture vessel into a collection vessel; B) washing the recovered cell aggregate; C) applying a biological stimuli to the washed cell aggregate in an enzyme solution; D) re-recovering the cell aggregate; E) applying a physical stimuli to the re-recovered cell aggregate to dissociate the cell aggregate into single cells; F) concentrating the single cells; G) suspending the concentrated single cells in a cryopreservation solution; and H) freezing the suspension of the single cells.

Description

Method for producing pluripotent stem cell stock

[0001] The present invention relates to a method for mass production of high-quality pluripotent stem cell stock.

[0002] Pluripotent stem cells such as ES cells and iPS cells have the ability to proliferate indefinitely and differentiate into various somatic cells. The practical application of cell transplantation therapy using somatic cells differentiated from pluripotent stem cells has the potential to fundamentally revolutionize treatment methods for intractable diseases and lifestyle-related diseases. For example, techniques have already been developed to induce differentiation of pluripotent stem cells into a wide variety of somatic cells including neurons, cardiomyocytes, blood cells, and retinal cells in vitro.

[0003] In addition, attempts are underway to mass-culture HLA homozygous iPS cells that do not cause immune rejection in most people, and gene-edited universal iPS cells that do not cause immune rejection in all people, to prepare clinical-grade iPS cell stocks.

[0004] For the preparation of such clinical pluripotent stem cell stock, efficient large-scale culture of pluripotent stem cells is first and foremost indispensable. For example, cell therapy for heart disease requires approximately 1×10 ８ to 1×10 ９ cardiomyocytes per patient, and a larger number of pluripotent stem cells, which serve as the raw material for this, is required when considering differentiation induction efficiency.

[0005] Methods for culturing pluripotent stem cells are broadly classified into adherent culture, in which cells are cultured by adhering to a flat substrate, and suspension culture, in which cells are cultured suspended in a liquid medium. For large-scale culture, compared with adherent culture, in which the number of cells obtained depends on the surface area of the culture vessel where cells can adhere and proliferate, suspension culture is advantageous in terms of operability and productivity, and is also realistic from the perspective of healthcare economics (Non-Patent Document 1). In fact, in Patent Document 1 and other documents, attempts have been made to prepare cell stocks from suspension-cultured pluripotent stem cells.

[0006] When preparing cell stocks, it is necessary to pay attention to cell death and quality deterioration at each stage, from harvesting cultured pluripotent stem cells to separating them into single cells and filling and storing them in appropriate containers.

[0007] Pluripotent stem cells obtained in suspension culture form aggregates, and these aggregates need to be separated into single cells to create cell stocks. It is known that when pluripotent stem cells are separated into single cells, signals that induce cell death are activated, and that the longer they remain in the single-cell state, the more likely they are to die. Therefore, it is preferable to use ROCK inhibitors (such as Y-27632) to inhibit these cell death signals and to perform culture operations that minimize cell death as much as possible (Patent Document 1, Non-Patent Document 2).

[0008] The culture and harvesting of pluripotent stem cells is difficult to perform properly and reliably without skilled personnel who have mastered the prescribed techniques (Non-Patent Literature 3). Technological development is ongoing to expedite the process of separating cell aggregates into single cells, reduce the time cells are exposed to the single-cell state, and simplify culture procedures.

[0009] For example, in Patent Document 1, in order to quickly separate cell aggregates into single cells, an enzyme is added to the aggregates as a biological stimulus, and then pipetting is performed immediately afterward to rapidly separate them into single cells, and the separated cells are recovered by centrifugation. This is also the case in adherent culture. In Non-Patent Document 4, after adding an enzyme as a biological stimulus to colony-like pluripotent stem cells, the cell aggregates are detached from the culture vessel with a scraper, and then rapidly separated into single cells by pipetting.

[0010] The conventional method of unicellularization involves applying physical stimuli, such as pipetting, to cell aggregates after biological stimulation, thereby rapidly separating them into single cells.

[0011] WO2023 / 120420

[0012] Cell Culture Technology and Industrial Development in Regenerative Medicine, CMC Publishing, ISBN 978-4-7813-1480-8; Ohguchi S. et al., Cell Stem Cell 7, 225-239, August 6, 2010; Biotechnology Vol. 100 No. 5 (2022); M. Nakagawa et al., Scientific Reports, 4:3594 (2014)

[0013] The inventors obtained a large number of aggregates of pluripotent stem cells by suspension culture and prepared a cell stock by separating these into single cells using conventional methods. The viability of the resulting cell stock itself was not a problem. However, when the cell stock was subjected to culture (adherent culture and suspension culture), the adhesion rate in adherent culture and the aggregate formation rate in suspension culture were very low, making it difficult to commercially utilize such a cell stock.

[0014] Various attempts were made to produce a cell stock suitable for commercial use (including changes and improvements to the cryopreservation solution, and speeding up the recovery and single-cell separation of aggregates), but none yielded satisfactory results. In other words, there was a need for a technology to produce a cell stock suitable for commercial use from large quantities of aggregates of pluripotent stem cells.

[0015] As a result of further diligent research by the inventors to solve the above problems, they have found that, when separating pluripotent stem cell aggregates into single cells, instead of applying physical stimulation immediately following enzyme addition as in the conventional method, even if the operation time from enzyme addition to single-cell collection is extended, by first collecting the cell aggregates without applying physical stimulation after enzyme addition, and then applying physical stimulation to the collected cell aggregates to separate them into single cells, it is possible to stably produce a cell stock that is suitable for commercial use (high quality).

[0016] In other words, the present invention encompasses the following: [1] A method for producing a cell stock, comprising the following steps: A) a step of recovering cultured pluripotent stem cell aggregates from a culture vessel to a recovery vessel; B) a step of washing the recovered cell aggregates; C) a step of applying biological stimulation to the washed cell aggregates in an enzyme solution; D) a step of recovering the cell aggregates again; E) a step of applying physical stimulation to the recovered cell aggregates to convert them into single cells; F) a step of concentrating the converted single cells. [2] The production method according to [1], further comprising the following steps: G) a step of suspending the concentrated single cells in a cryopreservation solution; H) a step of freezing the single-cell suspension. [3] The production method according to [1] or [2], wherein step D includes a centrifugation treatment. [4] The production method according to [1] to [3], wherein step D reduces the amount of enzyme solution coexisting with the cell aggregates to 1 / 10 or less. [5] The total number of cells in the cultured pluripotent stem cell aggregates in step A is 1 × 10 ９ The manufacturing method according to any one of [1] to [4] above, wherein the number of cells is 5 × 10 ８ The manufacturing method according to any one of [1] to [5], wherein the number of cells is 100 or more. [7] The manufacturing method according to any one of [1] to [6], wherein the recovery container used in step A is a flexible container. [8] The manufacturing method according to any one of [1] to [7], wherein the physical stimulus described in step E is provided by delivering a suspension of cell aggregates into a tube. [9] The manufacturing method according to any one of [1] to [8], wherein the recovery in step A is carried out by utilizing the difference in gravity or by pressurizing the inside of the culture vessel.

[10] The cell stock production per hour, expressed as (number of cells recovered at the end of step F) / (time required for steps A to H), is 5 × 10 ８ A manufacturing method according to any of [2] to [9] above, wherein the cell / hr is greater than or equal to [2]. This specification includes the disclosures of Japanese Patent Application No. 2025-055434, which forms the basis of the priority of this application.

[0017] According to the present invention, a highly efficient and high-quality cell stock can be produced from a large number of pluripotent stem cells obtained by suspension culture.

[0018] This figure shows an example of cell aggregates recovered from the culture medium (after step A), re-recovered cell aggregates (after step D), and single-celled cells (after step E) in the method of the present invention.

[0019] 1. Overview In the method for producing a pluripotent stem cell stock according to the present invention, pluripotent stem cells are cultured in suspension, and a large amount of pluripotent stem cell aggregates are collected in a primary container and washed. Then, an enzyme is added to the washed cell aggregates to provide biological stimulation and carry out a reaction. Next, the cell aggregates are collected again and then physically stimulated to separate the cell aggregates into single cells and concentrate the single-celled pluripotent stem cells. Then, a cryopreservation solution is added to the concentrated pluripotent stem cells and freezing is performed.

[0020] (Cells) The pluripotent stem cells according to the present invention are cells that possess the ability to differentiate into all types of cells that make up an organism (pluripotency), and that can continue to proliferate indefinitely while maintaining pluripotency in in vitro culture under appropriate conditions. More specifically, pluripotency means the ability to differentiate into germ layers that make up an individual (in vertebrates, the three germ layers: ectoderm, mesoderm, and endoderm).

[0021] Examples of such cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells). Among these, ES cells and iPS cells are preferred, with iPS cells being even more preferred.

[0022] The origin of pluripotent stem cells is not particularly limited and can be any cells derived from a multicellular organism, preferably animal-derived cells, more preferably mammalian-derived cells. Examples include rodents such as mice, rats, hamsters, and guinea pigs; domesticated or pet animals such as dogs, cats, rabbits, cattle, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Human-derived cells are particularly preferred.

[0023] The pluripotent stem cells according to the present invention may be commercially available cells or cells obtained through distribution, or cells newly created by introducing reprogramming factors into somatic cells may be used.

[0024] (Cell stock) The cell stock according to the present invention refers to a state in which a large quantity of pluripotent stem cells of the same strain and / or derived from the same human are divided into any amount as needed and stored.

[0025] 2. Suspension Culture In the manufacturing method according to the present invention, the aforementioned pluripotent stem cells are cultured in suspension in a liquid medium to increase the number of cells.

[0026] (Culture vessel) For suspension culture, a container with a surface treatment that suppresses protein adsorption is preferred. The shape of the container is not particularly limited and examples include dish-shaped, flask-shaped, well-shaped, bag-shaped, spinner flask-shaped, and shapes with stirring blades.

[0027] Culture vessels equipped with stirring blades and various ports are also called bioreactors, and examples include the BioBLU 1c Single-Use Vessel (Eppendorf) or the BioBLU 10c Single-Use Vessel (Eppendorf).

[0028] The capacity of the culture vessel used can be selected as appropriate and is not particularly limited, but it is preferable that the lower limit of the volume capable of containing and culturing the culture medium is 100 mL, 200 mL, 300 mL, or 500 mL, and the upper limit is 1000 L, 100 L, 50 L, or 20 L. To produce a large cell stock, a large culture scale is preferable, and it is particularly preferable that the volume of culture medium for one passage period be 500 mL or more. For large-scale culture of pluripotent stem cells, 1 L or more, 2 L or more, or 3 L or more are generally preferred, and although the upper limit is not particularly limited, for example, 50 L or less, 20 L or less, or 10 L or less are preferred.

[0029] In this specification, the volume of culture medium actually contained in a culture vessel and used for cell culture will be referred to as the culture volume or culture medium volume.

[0030] (Culture medium and medium exchange) For suspension culture, basal media commonly used in animal cell culture can be used, such as BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI Examples include 1640 medium, Fischer's medium, and mixed media thereof (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)).

[0031] In particular, the DMEM / F12 medium used is a mixture of DMEM medium and Ham F12 medium in a weight ratio preferably in the range of 60 / 40 to 40 / 60, for example, 58 / 42, 55 / 45, 52 / 48, 50 / 50, 48 / 52, 45 / 55, or 42 / 58. In addition, media used for culturing human iPS cells and human ES cells can also be suitably used.

[0032] In addition to the above, the culture medium used for suspension culture is preferably a liquid medium containing L-ascorbic acid, insulin, transferrin, selenium, and / or sodium bicarbonate. Furthermore, it is preferable that the liquid medium contains at least one growth factor, and more preferably that the growth factor contains FGF2 and / or TGF-β1. Particularly preferable is a DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, as well as FGF2 and TGF-β1, and free of serum.

[0033] In addition, it is preferable that the culture medium used for suspension culture contains additives such as ROCK inhibitors. More preferably, by including additives such as PKCβ inhibitors and / or WNT inhibitors in the culture medium used for suspension culture, it is possible to further suppress the spontaneous differentiation and deterioration of quality of pluripotent stem cells, and in some cases improve quality.

[0034] The ROCK inhibitor concentration in the culture medium according to the present invention can have a lower limit of 0 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, or 10 μM as the final concentration in the liquid medium at the start of culture. The upper limit of the ROCK inhibitor concentration in the culture medium is not particularly limited and can be determined according to the range that does not cause cell death, the range that does not cause undifferentiated cell migration, the solubility of the ROCK inhibitor, etc. For example, the upper limit as the final concentration in the culture medium can be 50 μM, 40 μM, 30 μM, or 20 μM.

[0035] The lower limit of the PKCβ inhibitor concentration in the culture medium according to the present invention can be 0 μM, 0.2 μM, 0.4 μM, 0.6 μM, or 0.8 μM as the final concentration in the liquid medium at the start of culture. The upper limit of the PKCβ inhibitor concentration in the culture medium is not particularly limited and can be determined according to the range that does not cause cell death or undifferentiated cell migration, the solubility of the PKCβ inhibitor, etc. For example, the upper limit of the final concentration in the culture medium can be 10 μM, 5 μM, 2 μM, 1.5 μM, or 1 μM.

[0036] The WNT inhibitor concentration in the culture medium according to the present invention can have a lower limit of 0 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, or 10 μM as the final concentration in the liquid medium at the start of culture. The upper limit of the WNT inhibitor concentration in the culture medium is not particularly limited and can be determined according to the range that does not cause cell death or undifferentiated cell migration, the solubility of the WNT inhibitor, etc. For example, the upper limit of the final concentration in the liquid medium at the start of culture in this step can be 50 μM, 40 μM, 30 μM, or 20 μM.

[0037] Furthermore, the method of changing the culture medium in suspension culture according to the present invention is preferably carried out by perfusion. By changing the culture medium by perfusion, the culture environment can be continuously controlled. When using the perfusion method, the composition of the culture medium used does not have to be constant. Specifically, the composition of the culture medium at the start of the culture in this step and the composition of the culture medium used for culture medium exchange by perfusion during the culture in this step may be different. In addition, multiple types of culture media may be used for culture medium exchange by perfusion, and the culture medium used for culture medium exchange by perfusion may be changed to one with a different composition at any point during the culture.

[0038] By switching the composition of the culture medium in this way, it becomes possible to continuously control the concentration of any additive or medium component in the culture system to match various culture medium perfusion schemes, and to achieve appropriate concentration transitions. Furthermore, in perfusion methods, it is preferable to increase the flow rate of the culture medium from any point in time in accordance with cell proliferation.

[0039] When performing suspension culture using the perfusion method, it is preferable that the culture medium used for perfusion be kept refrigerated until immediately before being used for culture by perfusion. Refrigeration can suppress the decomposition and deterioration of protein components such as growth factors in the culture medium. The lower limit of the refrigeration temperature should be, for example, a temperature at which the culture medium does not freeze, preferably 0°C, 1°C, or 2°C, and the upper limit should be, for example, 12°C, 10°C, 8°C, 7°C, or 6°C. Particularly preferred temperatures are 5°C, 4°C, or 3°C.

[0040] (Seeding Density) In suspension culture, the density of cells seeded into a fresh medium (seeding density) can be appropriately adjusted in consideration of the state of the cells used for seeding, the cell yield in the preceding adherent culture step, the culture time in the present step, and the number of cells required after culture. Although not particularly limited, in general, the lower limit only needs to be a seeding density at which cells can form aggregates and the cell state does not become unstable, for example, 0.01×10 ５ cells / mL, 0.1×10 ５ cells / mL, 0.5×10 ５ cells / mL, 1×10 ５ cells / mL, 1.5×10 ５ cells / mL, or 2×10 ５ cells / mL.

[0041] The upper limit only needs to be a cell density at which excessive aggregation or damage of cells and rapid consumption of medium components do not occur, for example, 100×10 ５ cells / mL, 50×10 ５ cells / mL, 10×10 ５ cells / mL, 8×10 ５ cells / mL, 6×10 ５ cells / mL, or 4×10 ５ cells / mL.

[0042] Since the proliferation efficiency in the early stage of culture is affected by the seeding density, particularly preferably, the lower limit of the seeding density is 1×10 ５ cells / mL, and the upper limit is 2×10 ５ cells / mL.

[0043] (Culture Conditions) There are no particular limitations on culture conditions such as culture temperature and culture time. The culture may be carried out within the range of conventional methods in the art. For example, the lower limit of the culture temperature may be 25°C or 35°C, and the upper limit may be 45°C or 40°C, but the culture temperature is preferably 37°C.

[0044] Furthermore, the culture time can be appropriately adjusted depending on the desired number of cells to be obtained as a cell stock, the proliferation rate of the cell line, and the condition of the cells recovered by adherent culture. For example, if the lower limit of the passage period is 24 hours, 48 ​​hours, 60 hours, 72 hours, or 96 hours, the cells can be sufficiently proliferated, and if the upper limit is 168 hours, 144 hours, 120 hours, or 100 hours, the culture can be performed while suppressing the deterioration of quality such as viability and undifferentiation due to excessive cell aggregate formation.

[0045] (Culture Method) In the suspension culture according to the present invention, any method can be used for gas supply, and any standard method used in general culture methods may be used. Although not limited to this, for example, the gas may be supplied by passing it over the liquid surface of the cell culture medium, or by bubbling it in the culture medium using a sparger, or by filling the area around the culture medium with the desired gas and supplying it by natural diffusion. More preferably, the gas is supplied by passing it over the liquid surface of the culture medium.

[0046] Regarding the amount of gas supplied, if cells are cultured in a culture device such as an incubator, the amount should be sufficient to adequately fill the inside of the device. If cells are cultured using a container such as a bioreactor, gas will be supplied through a gas supply port on the container, and the amount should be appropriately determined considering the culture volume, the surface area of ​​the culture medium, the gas requirements of the cultured cells, and the gas transfer rate in the culture medium.

[0047] In the suspension culture according to the present invention, it is preferable to appropriately maintain the culture environment by varying the supplied carbon dioxide concentration within a range of 10% to 0% as the culture progresses, thereby enabling the production of cell stocks with high quality, such as viability. The lower limit of the carbon dioxide concentration in the supplied gas is preferably 0%, 0.5%, or 1%, and the upper limit is preferably 10%, 9%, 8%, 7%, 6%, or 5%.

[0048] The timing for starting to reduce the carbon dioxide concentration is arbitrary. Unlike the timing for starting perfusion of the culture medium, which will be discussed later, the timing for starting to reduce the carbon dioxide concentration can be before the cells form aggregates, or it can be started from the beginning of culture. For example, the carbon dioxide concentration can be started to be reduced when the pH of the culture medium falls below an arbitrary standard, and this standard pH can be, for example, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 7, 6.9, or 6.8.

[0049] The timing for starting to increase the oxygen gas concentration is arbitrary. The oxygen gas concentration may be increased before the cells form aggregates, or it may be increased from the start of culture. For example, the timing for starting to increase the oxygen gas concentration can be when the amount of dissolved oxygen in the culture medium falls below an arbitrary standard. If the dissolved oxygen concentration in the atmosphere is set at 100%, the upper limit can be 200%, 150%, 100%, or 90%, and the lower limit can be 0%, 10%, 20%, 30%, 40%, or 50%.

[0050] In this suspension culture process, the number of cells obtained through proliferation can be arbitrarily set. The desired number of cells and cell state can be appropriately determined according to the cell line being cultured, the type of culture medium and conditions, and the desired number of cells required for stock preparation. For example, the degree of cell proliferation during one passage period is not particularly limited to the initial cell seeding amount, but the lower limit should be 2, 3, 5, 6, 7, 8, 9, or 10 times.

[0051] On the other hand, there is no particular upper limit, but it can be, for example, 100 times, 50 times, 40 times, 30 times, 20 times, or 10 times. In particular, it is preferable to be able to increase the cell volume by 10 times or more. Furthermore, by repeating subculturing and culturing multiple times in suspension culture, the cell volume can be increased to, for example, 110 times, 150 times, 200 times, 300 times, 400 times, 500 times or more compared to the seeded cell volume.

[0052] Furthermore, in this suspension culture process, a portion of the pluripotent stem cells during culture can be removed to confirm the cell number and aggregate size. The aggregates of pluripotent stem cells removed during culture can be broken down into single cells by, for example, biological stimulation in an enzyme solution followed by physical stimulation, and then the number of viable cells can be measured using methods such as the trypan blue method. Alternatively, the cell number can be estimated from the number and size of the aggregates of pluripotent stem cells removed during culture. The aggregate size or volume can be measured by methods such as laser size measurement, image acquisition, or calculation of size from images, although these methods are not particularly limited.

[0053] The size of the cell aggregates produced in this suspension culture process is not limited, but when observed under a microscope, the average diameter of the maximum width of the cell aggregates in the same culture system can be 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm at the lower limit, and 500 μm, 400 μm, 300 μm, 250 μm, or 200 μm at the upper limit. Cell aggregates within this range are preferable as a cell proliferation environment because oxygen and nutrients are easily supplied to the cells inside.

[0054] The size of the cell aggregates is preferably between 40 μm and 250 μm. Furthermore, the size of the aggregates formed after seeding cells in suspension culture, for example, after 24 hours, is preferably small, and particularly preferably 100 μm or less, in order to enable maximum growth with high quality and efficiency during the subsequent passage period of culture. Note that it is not necessary for the size of all aggregates in the culture medium to be within the above range; for example, it is sufficient if the average size by number is within the above range.

[0055] In the group of cell aggregates produced in this suspension culture process, it is preferable that 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of the volume-average size of the cell aggregates fall within the above size range.

[0056] In this suspension culture process, the concentrations of nutrients and metabolites in the culture medium can be measured using the medium removed from the culture system by perfusion. For example, although not limited to this, it is possible to measure glucose concentration, lactic acid concentration, etc., in the removed medium using a culture medium component analyzer that uses an enzyme electrode reaction.

[0057] Furthermore, the glucose concentration in the culture medium removed from the culture system by perfusion is not particularly limited, but it is preferable that the lower limit is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM, and the upper limit is 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, or 15 mM. Particularly preferable is a lower limit of 4 mM and an upper limit of 16 mM.

[0058] Furthermore, the lactate concentration in the culture medium removed from the culture system by perfusion is preferably such that the lower limit is 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM, and the upper limit is 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, or 6 mM. Particularly preferred is a lower limit of 0 mM and an upper limit of 12 mM.

[0059] 3. Manufacturing of Cell Stocks In the present invention, cell stocks are manufactured by performing the following steps A to H) on the cell aggregates obtained by the suspension culture. A) Step of recovering the cultured pluripotent stem cell aggregates from the culture vessel to the recovery vessel B) Step of washing the recovered cell aggregates C) Step of applying biological stimulation to the washed cell aggregates in an enzyme solution D) Step of recovering the cell aggregates again E) Step of applying physical stimulation to the recovered cell aggregates to make them single cells F) Step of concentrating the single cells G) Step of suspending the concentrated single cells in a cryopreservation solution H) Step of freezing the single cell suspension Each step will be described in detail below.

[0060] A) The process of recovering cultured pluripotent stem cell aggregates from the culture vessel to a recovery vessel. This process involves recovering the culture medium containing the cell aggregates after suspension culture into a recovery vessel, and further separating and removing liquid components such as culture medium from the recovered culture medium.

[0061] Methods for recovering the culture medium containing cell aggregates from the culture vessel to the recovery container include, for example, opening the lid of the culture vessel and directly recovering the medium into the recovery container, withdrawing the medium from the culture vessel with a syringe, or connecting the culture vessel and the recovery container with a tube and transferring the culture medium from the culture vessel to the recovery container. Transfer methods may include using a peristaltic pump, utilizing gravity, or pressurizing the culture vessel during transfer.

[0062] In particular, it is preferable to transfer the culture medium containing cell aggregates while maintaining a closed system by connecting the culture vessel and the collection vessel with a tube. Furthermore, as a method of collection without causing physical damage to the cell aggregates, it is preferable to transfer them using the difference in gravity or to transfer them by pressurizing the inside of the culture vessel. These methods are also preferable because they can be carried out efficiently in a closed system even with large volumes of culture medium. It is also preferable to maintain a closed system in each of the above steps B) to E). A closed system means a system in which all steps, or specific steps, are isolated from the outside air and environment (in a sealed container).

[0063] The flow rate when collecting from the culture vessel to the collection vessel via a tube is not particularly limited, but the lower limit should be a speed that does not cause cell aggregates to clog the tube, and is preferably 0.01 L / min, 0.03 L / min, 0.05 L / min, 0.1 L / min, 0.3 L / min, or 0.5 L / min. The upper limit should be a speed that does not cause significant damage to the cell aggregates, and is not particularly limited, but is preferably 100 L / min, 80 L / min, 50 L / min, 10 L / min, 5 L / min, or 3 L / min.

[0064] The inner diameter of the tube is not particularly limited, as long as it is large enough for the cell aggregates to pass through. For example, it may be 1.6 mm, 3.2 mm, 4.8 mm, 6.4 mm, 9.6 mm, or 12.7 mm, but preferably 6.4 mm. The length of the tube is not particularly limited, but the lower limit may be 1 cm, 2 cm, 5 cm, 7 cm, 10 cm, or 15 cm, and the upper limit may be 300 cm, 200 cm, 150 cm, 100 cm, or 50 cm.

[0065] The shape of the collection container is not particularly limited, but examples include syringes, centrifuge tubes, bottles, or bags. Furthermore, the collection container is preferably a flexible container. Examples of flexible container materials include polyethylene, polypropylene, polyvinyl chloride, and ethylene-vinyl acetate copolymers, with polyolefin resins such as polyethylene and polypropylene being preferred. For example, closed-system single-use centrifuge bags (Life Technologies Japan Co., Ltd.) or Terumo blood bags (Terumo Corporation) may be used as collection containers.

[0066] The capacity of the collection container is not particularly limited, but it is preferable that the lower limit is 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, or 500 mL, and the upper limit is 100 L, 50 L, 20 L, 10 L, 5 L, 3 L, or 1 L. The culture medium containing cell aggregates may be collected entirely in one collection container, or it may be collected in multiple collection containers.

[0067] Before collecting cultured pluripotent stem cell aggregates from the culture vessel to the collection vessel, the liquid components of the supernatant, such as the culture medium, may be removed by methods such as static separation (allowing the cell aggregates to settle by standing), centrifugation, counterflow separation, or by using a filtration filter or hollow fiber separation membrane. In this case, it is not necessary to completely remove the liquid components of the supernatant. For example, the container containing the culture medium can be left standing for about 5 minutes, and the supernatant can be removed while leaving the settled cells and cell aggregates.

[0068] Furthermore, centrifugation should be performed using centrifugal force and processing time that does not damage the cells. For example, the lower limit of centrifugal force is not particularly limited as long as the cells can be sedimented, but it could be, for example, 50 x g, 100 x g, 200 x g, 300 x g, 800 x g, or 1000 x g. On the other hand, the upper limit should be a speed at which the cells are not damaged or are less likely to be damaged by the centrifugal force, for example, 1200 x g, 1500 x g, or 2000 x g. The lower limit of the time for applying centrifugal force is not particularly limited as long as the cells can be sedimented by the above centrifugal force, but it could be, for example, 30 seconds, 1 minute, 3 minutes, or 5 minutes. The upper limit should be a time at which the cells are not damaged or are less likely to be damaged by the above centrifugal force, for example, 20 minutes, 10 minutes, 8 minutes, 6 minutes, or 5 minutes.

[0069] B) Washing the recovered cell aggregates This step involves washing the cell aggregates recovered in step A using a washing solution to reduce the protein content in the culture medium containing the cell aggregates, thereby making it easier to apply the biological stimulus in the next step. The washing solution used in this step is preferably one with a low protein content, and for example, phosphate buffer, physiological saline, or a serum-free basal culture medium can be used.

[0070] After washing the cell aggregates with a washing solution, the washing solution is removed. The method for removing the washing solution is not particularly limited, but examples include static separation, centrifugation, counterflow separation, separation using a filtration filter, or separation using a hollow fiber separation membrane. Washing may be performed once or multiple times. It is preferable to perform the above washing operation in a recovery container.

[0071] C) Step of applying biological stimulation to the washed cell aggregates in an enzyme solution This step involves applying biological stimulation to the cell aggregates washed in the previous step in an enzyme solution.

[0072] The type of enzyme used in this process is not particularly limited, but examples include trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available Accutase (trademark registered), Accumax (trademark registered), TrypLE. ＴＭYou can use Express Enzyme (Life Technologies Japan Co., Ltd.), TrypLETMSelect Enzyme (Life Technologies Japan Co., Ltd.), Dispase (trademark registered), etc.

[0073] Regarding the amount of enzyme used, for example, when using trypsin as the enzyme, there are no particular limitations as long as it is at a concentration that can disperse the cell aggregates, but the lower limit of the concentration in the solution may be, for example, 0.15% by volume, 0.18% by volume, 0.20% by volume, or 0.24% by volume. On the other hand, there are no particular limitations on the upper limit of the concentration in the solution as long as it is at a concentration that does not cause the cells themselves to be dissolved or otherwise affected, but it may be 0.30% by volume, 0.28% by volume, or 0.25% by volume.

[0074] Furthermore, while the duration of biological stimulation depends on the type and amount of enzyme used, there is no particular limit to the duration as long as the cell aggregates are sufficiently dispersed; for example, 1 minute, 3 minutes, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes may be used. The upper limit to the duration of biological stimulation also depends on the type and amount of enzyme used, but there is no particular limit to the duration as long as the cells are not excessively affected, such as being lysed; for example, 30 minutes, 28 minutes, 25 minutes, 22 minutes, 20 minutes, or 18 minutes may be used. When using commercially available enzymes, use them at the concentrations specified in the attached protocol.

[0075] Enzymes are preferably used in combination with chelating agents. While not particularly limited, chelating agents such as EDTA and EGTA can be used. For example, when using EDTA as a chelating agent, concentrations of 0.01 mM, 0.1 mM, or 0.5 mM are preferred, although not particularly limited. Conversely, concentrations of 100 mM, 50 mM, 10 mM, or 5 mM are preferred, although not particularly limited.

[0076] In this process, it is preferable to add any solution after applying a biological stimulus for a predetermined time. The solution is not particularly limited, but can be culture medium, serum, buffer (including PBS buffer), or physiological saline, but it is preferable to use a culture medium containing a ROCK inhibitor.

[0077] D) Step to recover cell aggregates This step involves recovering the cell aggregates after applying biological stimulation in an enzyme solution, prior to applying physical stimulation to the cell aggregates.

[0078] The method of re-recovery is not particularly limited and any method of separating the cell aggregates from the liquid is acceptable. For example, the cell aggregates may be separated from the supernatant by centrifugation, the supernatant may be removed and then the cell aggregates may be re-recovered; the supernatant may be removed by decantation or aspiration and then the cell aggregates may be re-recovered; or the precipitate may be removed by aspiration and then the cell aggregates may be re-recovered. Separation and re-recovery using centrifugation are preferred because they reduce variations in operation and allow for efficient processing.

[0079] The lower limit of the centrifugal force for centrifugation is not particularly limited as long as the cells can be sedimented, but for example, it could be 50 x g, 100 x g, 200 x g, 300 x g, 800 x g, or 1000 x g. On the other hand, the upper limit should be a speed at which the cells are not damaged, or are less damaged, by the centrifugal force, for example, 1200 x g, 1500 x g, 2000 x g, 3000 x g, or 4000 x g.

[0080] The centrifugation time is not particularly limited to a minimum, as long as it allows the cells to settle; for example, 1 minute, 3 minutes, 5 minutes, or 10 minutes would suffice. The upper limit should be a time during which the cells are not damaged, or are less likely to be damaged; for example, 30 minutes, 25 minutes, or 20 minutes would suffice.

[0081] In the above recovery operation, the cell aggregates do not need to be completely separated from the enzyme solution, but it is preferable that the amount of enzyme solution coexisting with the cell aggregates be 1 / 10 or less, and more preferably 1 / 20 or less, or 1 / 30 or less.

[0082] As described above, any solution may be added to the recovered cell aggregates. The solution to be added is not particularly limited, but culture medium, buffer (including aqueous phosphate solution), or physiological saline can be used, however, a culture medium containing a ROCK inhibitor is preferred.

[0083] The enzyme concentration after adding the above-mentioned arbitrary solution is preferably lower than the enzyme concentration in the previous step. The specific concentration is not particularly limited, but for example, the upper limit may be 1 / 5, 1 / 8, 1 / 10, 1 / 20, or 1 / 30 of the enzyme concentration in the previous step, and the lower limit is also not particularly limited, but for example, 1 / 10000, 1 / 1000, 1 / 500, 1 / 100, or 1 / 50 is preferred.

[0084] In the method for producing cell stocks of the present invention, by using step D, it is possible to produce highly efficient and high-quality cell stocks despite the increased operating time from enzyme addition to single-cell separation, thereby dramatically improving production efficiency.

[0085] E) A process to separate the recovered cell aggregates into single cells by applying physical stimulation. This process involves separating the recovered cell aggregates into single cells by applying physical stimulation.

[0086] Single-cell formation refers to a state in which individual cells are present, dispersed from cell aggregates. It is not necessary for all cell aggregates to consist of single, free cells; multiple cells may form small clumps. The individual cells should be dispersed to a degree that allows for the measurement of the number of cells required for the cell concentration adjustment described later.

[0087] There are no particular limitations on the methods for applying physical stimulation to the recovered cell aggregates, but examples include methods such as pipetting the cell aggregate suspension multiple times, creating a Taylor vortex flow in the cell aggregate suspension to apply shear stress, or passing the cell aggregate suspension through a narrow channel, and these methods can also be combined.

[0088] From the viewpoint of ease of operation, one method of passing the cell aggregate suspension through a narrow channel is to apply physical stimulation by delivering the cell aggregate suspension into a tube.

[0089] The inner diameter of the tube in this method is not particularly limited as long as it does not become clogged with cell aggregates, for example, it may be 1.6 mm, 2.4 mm, 3.2 mm, 4.8 mm, 6.4 mm, or 9.6 mm. The fluid delivery rate is not particularly limited as long as it is a flow rate that can apply stress to separate the cell aggregates into single cells without damaging the cells, but the lower limit may be 10 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, or 60 mL / min, and the upper limit may be 800 mL / min, 700 mL / min, 600 mL / min, 500 mL / min, 400 mL / min, 300 mL / min, or 200 mL / min.

[0090] The length of the tube is not particularly limited as long as it is long enough to separate the cell aggregates into single cells at the flow rate described above, but the lower limit should be 1 cm, 2 cm, 5 cm, 7 cm, 10 cm, or 15 cm, and the upper limit should be 200 cm, 150 cm, 100 cm, or 50 cm.

[0091] Furthermore, the method of passing the liquid may be by using a perister pump, by utilizing the difference in gravity, by pressurizing or depressurizing one of the containers, or by using a counterflow centrifuge. Preferably, the method using a counterflow centrifuge is used. Examples of counterflow centrifuges include Rotea (Life Technologies Japan Co., Ltd.) and Ksep 50 (Sartorius Co., Ltd.).

[0092] Furthermore, if necessary, the cells after singestion may be passed through a strainer or mesh. The pore size of the strainer or mesh used in this case is not particularly limited as long as it is large enough for the cells to pass through without damage, but the lower limit is preferably 10 μm, 30 μm, 50 μm, or 70 μm, and the upper limit is preferably 500 μm, 400 μm, 300 μm, or 200 μm.

[0093] F) Concentration of unicellularized cells This step involves concentrating the unicellularized cells from the previous step to a desired cell concentration. The cell concentration after concentration is not particularly limited, but the lower limit is 1 × 10⁻⁶. ５ cells / mL, 5×10 ５ cells / mL, or 1 × 10６ cells / mL, upper limit is 1 × 10 ９ cells / mL, 5×10 ８ cells / mL, or 1 × 10 ８ cells / mL is preferred.

[0094] The concentration method is not particularly limited, as long as it can remove the liquid component from the solution containing single cells. Examples include methods for separating and concentrating cells and liquid by static separation, centrifugation, counterflow separation, separation using a filtration filter, or using a hollow fiber separation membrane. Counterflow separation is preferred, and the counterflow centrifuge exemplified in step E can be used.

[0095] The number of cells recovered at the end of this process will be the number of cells that can be used in subsequent cryopreservation processes (also referred to as the number of cryopreservable cells), i.e., the number of cells that will become the cell stock.

[0096] G) Step of suspending concentrated single cells in preservation solution This step involves adding preservation solution to the concentrated single cell suspension obtained in the previous step and filling it into a container for frozen storage.

[0097] As the preservation solution to be added, buffer solutions, refrigeration solutions, and cryopreservation solutions commonly used for cells can be used, with cryopreservation solutions being preferred. It may be prepared by adding DMSO to any culture medium to a final concentration of about 10%, or a commercially available preservation solution may be used as is or diluted. Examples of commercially available preservation solutions include STEM-CELLBANKER® GMP grade (Xenogen Pharma), CryoStor® CS10 (Hemacare), CP-5E (Kyokuto Pharmaceutical Industry Co., Ltd.), and CP-1 (Kyokuto Pharmaceutical Industry Co., Ltd.). The preservation solution may also contain a ROCK inhibitor.

[0098] The density of cells suspended in the preservation solution should not be such that it significantly reduces the quality of the cells, such as their viability. For example, the lower limit is 0.1 × 10⁻⁶. ６ cells / mL, 0.3×10 ６ cells / mL, 0.5×10 ６ cells / mL, or 1.0 × 10 ６Cells / mL is preferred, with an upper limit of 200 × 10 ６ cells / mL, 150×10 ６ cells / mL, 100×10 ６ cells / mL, 50×10 ６ cells / mL, 10×10 ６ cells / mL, 5×10 ６ cells / mL or 2 × 10 ６ cells / mL is preferred.

[0099] The temperature at which cells are suspended and packed into the preservation solution to prepare the cell stock is preferably low. Low temperature means a temperature at which the solution does not freeze, for example, the lower limit is 0°C, 1°C, or 2°C. The upper limit is not particularly limited, but for the reasons mentioned above, for example, 12°C, 10°C, 9°C, 8°C, 7°C, or 6°C are preferred.

[0100] In this invention, by performing this process while maintaining the container or concentrated single-cell suspension at a low temperature of 10°C or below, or in a low-temperature working environment of 10°C or below, it becomes possible to prepare a high-quality cell stock.

[0101] The container for cryopreservation of single cells and preservatives is not particularly limited, but a container with a surface treatment to suppress protein adsorption is preferred, a sealable container is preferred, and a container that can be stored under liquid nitrogen is preferred. The form can be, for example, vial type, bag type, tube type, etc., and commercially available storage containers can be used. Examples of commercially available containers include Nunc Cryotubes (Thermo Fisher Scientific), Nalgene Cryovials (Thermo Fisher Scientific), Bi. File Jacket Tubes (FCR & Bio, Inc.), Cell Cryopreservation Bags (Nipro Corporation), CryoMax Freezing Bags (Milteny Biotech, Inc.), etc.

[0102] The capacity of the container is not particularly limited, but it should be sufficient to fill it with a sufficient amount of single-cell suspension and preservation solution combined. For example, the lower limit can be 0.2 mL, 0.5 mL, 1.0 mL, or 5.0 mL, and the upper limit can be 1000 mL, 500 mL, 100 mL, 50 mL, 20 mL, or 10 mL.

[0103] H) Step to freeze the single-cell suspension This step involves freezing the cell suspension that was packed into a storage container in the previous step. Freezing methods include, for example, slow freezing and rapid cooling, with slow cooling being more preferable.

[0104] The slow cooling method is a method of freezing by gradually lowering the temperature. The upper limit of the cooling rate is preferably, for example, 3°C / min, 2.5°C / min, or 2°C / min, and the lower limit is preferably 0.5°C / min, 1.0°C / min, 1.5°C / min, or 2°C / min. Furthermore, at the maximum crystal nucleation temperature, the cooling rate may be temporarily faster than the above range in order to prevent melting due to exothermic reaction and subsequent refreezing.

[0105] Furthermore, if the temperature exceeds the maximum crystal nucleation zone and the material is completely frozen, the subsequent cooling rate may be outside the above range. For example, after freezing to -80°C by slow cooling, the storage container may be immediately moved to liquid nitrogen for rapid cooling.

[0106] The cell stock produced in this manner is stored frozen until use. The storage method is not particularly limited and may be stored in a freezer at -80°C or below, in the gas phase of a container filled with liquid nitrogen, or in the liquid phase of a container filled with liquid nitrogen.

[0107] The time required for steps A to H is preferably, for example, within 7 hours, within 6 hours, within 5 hours, within 4.5 hours, or within 4 hours. The time required for steps A to H refers to the time from the start of transferring the culture medium to the recovery container until the internal temperature of the cryopreservation container containing the single-cell suspension and preservation solution reaches 0°C.

[0108] The method for producing a cell stock according to the present invention involves, in step A described above, the total number of cultured pluripotent stem cells being 1 × 10⁶９ cells or more, 2 x 10 ９ cells or more, 4 x 10 ９ cells or more, 6 x 10 ９ cells or more, or 8 x 10 ９ It is preferable that the number is greater than or equal to cells.

[0109] Furthermore, according to the manufacturing method of the present invention, the number of freezeable cells obtained at the end of step F is usually 50% or more of the total number of cells in the cultured cell aggregate in step A. That is, typically 5 × 10 ８ cells and above, this is 1 x 10 ９ cells or more, 2 x 10 ９ cells or more, 3 x 10 ９ cells or more, 4 x 10 ９ cells or more, or 5 x 10 ９ It is above cells.

[0110] Furthermore, in terms of cell stock production volume, the production volume of frozen stock per hour in the manufacturing method of the present invention, expressed as [number of cells recovered at the end of step F] / [time required for steps A to H (hr)], is 5 × 10 ８ cells / Hr or more, 8×10 ８ cells / Hr or more, 1×10 ９ cells / Hr or more, 1.5×10 ９ cells / Hr or more, 2.0×10 ９ cells / hr or more, or 2.2 × 10 ９ It is greater than or equal to cells / hr.

[0111] The present invention is a manufacturing method characterized by improving both the yield and quality of the resulting cell stock by incorporating a step of re-collecting cell aggregates (step D) between the step of applying biological stimulation (step C) and the step of separating cells into single cells by physical stimulation (step E).

[0112] As described above, the method for producing cell stocks according to the present invention is suitable for the production of large quantities of cell stocks, particularly on a commercial scale. Generally, as the production scale increases, problems such as quality degradation and decreased productivity arise due to increased time required and losses, even with the same operations. However, this production method is an industrialized technology that solves these problems and greatly contributes to the production of cell stocks with quality and productivity suitable for commercial use.

[0113] The method for producing a pluripotent stem cell stock according to the present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0114] In Production Example 1 and Example 1, human iPS cell line QHJI (Kyoto University Center for iPS Cell Research and Application) was used. In Production Example 2 and Example 2, human iPS cell line 1383D6 (Kyoto University Center for iPS Cell Research and Application) was used. In Production Example 3 and Comparative Example 1, human iPS cell line QHJI (Kyoto University Center for iPS Cell Research and Application) was used.

[0115] (Manufacturing Example 1) Culture using human iPS cell line QHJI A large number of cells were cultured according to the following Steps 1 to 4.

[0116] [Step 1] Adhesion culture of human iPS cells After thawing the frozen human iPS cells, add 0.5 μg / cm³ of iMatrix-511MG (Matrixome Corporation). ２ 25cm coated ２ In the culture flask, 6000 cells / cm³ ２ Sow the seeds at 37°C and 5% CO2. ２ Adhesion culture for maintenance was performed under atmospheric conditions. StemFit® AK03N (manufactured by Ajinomoto Co., Inc.) was used as the culture medium. The day of cell seeding was designated as culture day 0, and the entire culture medium was changed on culture days 1, 4, and 5. The amount of medium was 5 mL on culture days 0, 4, and 5, and 10 mL only on culture day 1. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium only at the time of cell seeding to a final concentration of 10 μM. LY333531 (Cayman Inc.) was added to the medium only on culture days 4 and 5 to a final concentration of 1 μM, and IWR-1endo (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 20 μM.

[0117] On the 6th day of culture, 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the TrypLE for subculturing. ＴＭ Cells were treated with Select Enzyme (Life Technologies Japan Co., Ltd.) for 15 minutes, and then dispersed into single cells by pipetting while detaching them from the culture surface. These cells were suspended and collected in StemFit® AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0118] The collected cells were treated with 0.9 μg / cm³ ２ 300cm coated with Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Life Technologies Japan Co., Ltd.) ２ In the culture flask, 12,000 cells / cm³ ２ Sow the seeds at 37°C and 5% CO2. ２ Adhesion and expansion culture was performed under atmospheric conditions. StemFit® AK03N (Ajinomoto Co., Inc.) was used as the culture medium, and the entire volume of medium was changed on the second and third days of culture. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium only at the time of cell seeding to a final concentration of 10 μM, and LY333531 (Cayman Inc.) was added to the medium only on the second and third days of culture to a final concentration of 1 μM, and IWR-1endo was added to a final concentration of 20 μM.

[0119] On day 4 of culture, the culture medium was removed, the cells were washed with phosphate buffer, and then 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the TrypLE cells. ＴＭ Select Enzyme (Life Technologies Japan Co., Ltd.) was added to a culture flask and incubated for 3 minutes. The cells were then detached from the culture surface by tapping and dispersed into single cells by pipetting. These cells were suspended and collected in StemFit® AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0120] [Step 2] Suspension culture of human iPS cells (1st suspension passage) The cells harvested in Step 1 were seeded in a culture vessel and cultured in suspension. A BioBlue 1c Single-Use Vessel (Eppendorf) was used as the culture vessel, and a Bioflo 320 (Eppendorf) was used as the biobioreactor system to control the culture.

[0121] For cell seeding, StemFit® AK03N (manufactured by Ajinomoto Co., Inc.) was used as the culture medium, supplemented with 10 μM Y-27632 and a final concentration of 20 μM IWR-1 endo. The culture medium temperature was 25°C during cell seeding, and the culture temperature during incubation was 37°C. The supply gas rate was maintained at 0.2 L / min, with top aeration of the culture medium. The supply gas was prepared by mixing an arbitrary amount of carbon dioxide with air. The pH sensor and culture medium perfusion pump were calibrated according to the manufacturer's specified method.

[0122] Assuming a total culture medium volume of 500 mL, the cell density at the start of culture is 5.0 × 10⁶. ４ Cells were seeded to a concentration of cells / mL, and suspension culture was initiated. The carbon dioxide concentration in the supply gas was initially set at 5%, and was adjusted within a range of 0% to 5% to maintain the pH of the culture medium at around 7.15 (suppressing a decrease in pH), thus maintaining the pH at approximately 7.15 ± 0.15.

[0123] The oxygen gas concentration in the supply gas was set to 21% at the start of cultivation, and was adjusted within the range of 21% to 100% to maintain the dissolved oxygen concentration in the culture medium at around 50% (suppressing the decrease in dissolved oxygen concentration), thereby maintaining the dissolved oxygen concentration in the culture medium at around 65% ± 30%.

[0124] The stirring speed of the bio-bioreactor system was set to 87 pm until 48 hours of incubation, and then to 79 rpm thereafter.

[0125] Perfusion of the culture medium was started at 24 hours of incubation. The perfusion flow rate F per unit time was controlled every hour to regulate the culture environment. Perfusion flow rate F per unit time at the start of perfusion ０ F is the value obtained by dividing the culture volume of 500 mL by 24 hours. ０= 20.8 mL. The point at which the culture medium flow rate per unit time is changed is set to 64 hours into incubation, and thereafter the culture medium flow rate per unit time is determined from International Publication No. 2022 / 203051 using the formula [F = M × K × F ０ × (C / C ０ Using ), the following settings were configured.

[0126] L of lactate produced per cell by metabolism per unit time ０ The amount of lactate L per cell produced by metabolism per unit time at each culture time is set from the value of a typical pluripotent stem cell, and K(=L / L) ​​is given by formula 4 described in International Publication No. 2022 / 203051. ０ The culture medium flow rate F per unit time was determined using the formula described in International Publication No. 2022 / 203051.

[0127] The culture variable is cell density, C ０ The value of the seeding density is 5 × 10 ４ cells / mL, the percentage of cells forming aggregates at 24 hours of culture relative to the expected seeding rate (160%), and the expected specific cell growth rate (0.90 days). －１ The calculations were performed from the above. Similarly, C was calculated from the predicted assumed cell density changes. The constant M, which corrects for the effects of cell lines, etc., was set to 1.

[0128] For perfusion, we used StemFit® AK03N (manufactured by Ajinomoto Co., Inc.) supplemented with Y-27632 at a final concentration of 2.5 μM, IWR-1 endo at a final concentration of 20 μM, and LY333531 at a final concentration of 1 μM. In addition, to remove only the culture medium from the culture solution by removing cell aggregates, the medium was removed by passing it through a sintered wire mesh filter with a mesh size of 20 μm.

[0129] At 96 hours of culture, the entire culture medium from the suspension culture was collected, and the cell aggregates and culture supernatant were separated by centrifugation. The cells were then treated with TrypLETMSelect Enzyme (Life Technologies Japan Co., Ltd.) for 5 minutes while swirling, and the cell aggregates were dispersed into single cells by pipetting. Subsequently, the cells were suspended in StemFit® AK03N (Ajinomoto Co., Inc.) and collected.

[0130] [Step 3] Suspension culture of human iPS cells (2nd suspension passage) The cells harvested in Step 2 were further cultured in suspension. The same BioBlue 1c Single-Use Vessel (Eppendorf) as used for the 1st passage was used as the culture vessel, and the Bioflo 320 (Eppendorf) was used as the biobioreactor system to control the culture.

[0131] The culture medium volume was 682 mL, and the seeding density was 7.5 × 10⁻⁶. ４ cells / mL, the culture medium perfusion rate per unit time at the start of perfusion is F ０ = 28.4 mL. The point at which the culture medium flow rate per unit time was changed was defined as 54 hours into incubation.

[0132] For the second suspension passage, LY333531 was added to the culture medium to a final concentration of 1 μM. Otherwise, the suspension culture was carried out under the same procedures and culture conditions as in Step 2 until 96 hours, and the cells were resuspended and collected in StemFit® AK03N (manufactured by Ajinomoto Co., Inc.).

[0133] [Step 4] Suspension culture of human iPS cells (3rd passage in suspension) The cells harvested in Step 3 (suspension culture: 2nd passage) were further cultured in suspension. A BioBlue 10c Single-Use Vessel (Eppendorf) was used as the culture vessel, and a Bioflo 320 (Eppendorf) was used as the biobioreactor system to control the culture.

[0134] The culture medium volume is 6700 mL, and the seeding density is 10 x 10. ４ cells / mL, the culture medium perfusion rate per unit time at the start of perfusion is F ０ The volume was set to 279.2 mL. The point at which the culture medium flow rate per unit time was changed was set to 48 hours of culture. The supply gas rate was set to 1.5 L / min. The stirring speed of the bioreactor system was set to 63 pm until 48 hours of culture, and to 56 rpm thereafter. StemFit® AK03N (manufactured by Ajinomoto Co., Inc.), supplemented with Y-27632 at a final concentration of 2.5 μM and LY333531 at a final concentration of 1 μM, was used as the perfusion medium. Otherwise, the culture was carried out until 96 hours in the same manner as in Step 4.

[0135] (Example 1: Recovery and Stock Preparation of Human iPS Cells) Pluripotent stem cells cultured in Production Example 1 (Steps 1-4) were recovered by the method described below, unicellularized, and then packed into appropriate containers to prepare a cell stock. All steps A through F of the following example were performed in a closed system. The bioreactor system was stopped, and the culture vessel was connected to a CentriPAK BPC 6×1.7L manifold (Life Technologies Japan Co., Ltd.). Then, all other lines were stopped with forceps, and all the culture medium was recovered into the CentriPAK BPC 6×1.7L manifold by pressurizing it by passing air through the top of the culture medium at a rate of 2.0 L / min.

[0136] Furthermore, 1 L of PBS was added to the culture vessel, and all the PBS was recovered into a CentriPAK BPC 6 × 1.7 L manifold by pressurizing the culture medium by passing air through it from the top surface at a rate of 2.0 L / min. Subsequently, six CentriPAK BPC single bags were separated using a sterile joining machine, and the cell aggregates were precipitated by centrifugation at 1000 × g for 5 minutes under conditions of accelerator 9 and brake 7. The supernatant was aspirationed and the liquid volume in the single bags was removed until it was approximately 70 mL or less (Step A).

[0137] After removing the supernatant from all six single bags, the cell suspensions in the single bags were collected into a 1000 mL Terumo isolation bag (manufactured by Terumo Corporation) using a 100 mL syringe. After collecting the cell suspensions from all six single bags, 50 mL of PBS was added to each single bag using a 100 mL syringe to wash the bags, and the contents were collected into the Terumo isolation bag. Furthermore, another 50 mL of PBS was added to each single bag to wash the bags, and the contents were collected into the Terumo isolation bag.

[0138] The cell suspension was collected in a Terumo isolation bag and centrifuged at 1000 x g for 5 minutes under the conditions of accelerator 9 and brake 7 to precipitate the cell aggregates. The supernatant was aspirationed and the liquid volume in the Terumo isolation bag was removed until it was approximately 100 mL or less (Step B).

[0139] 0.5×TrypLE heated to 37℃ ＴＭ 424 mL of Select Enzyme (Life Technologies Japan Co., Ltd.) (diluted with 0.5 mM EDTA solution) was added to a Terumo separation bag and left standing in a 37°C incubator. After 3 minutes, 424 mL of StemFit® AK03N (manufactured by Ajinomoto Co., Inc.), to which Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) had been added to a final concentration of 10 μM, was added to the Terumo separation bag (Step C).

[0140] The cells were precipitated by centrifugation at 2000 x g for 5 minutes under the conditions of accelerator 9 and brake 7. The supernatant was then removed by aspiration, and the cell aggregates were collected again in a Terumo separation bag until the volume was approximately 100 mL (Step D).

[0141] Subsequently, 424 mL of StemFit® AK03N (manufactured by Ajinomoto Co., Inc.), to which Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a Terumo isolation bag to a final concentration of 10 μM. Next, the Terumo isolation bag was connected to a CTS Rotea Single-Use Kit (Life Technologies Japan Co., Ltd.) using a sterile bonding machine. Furthermore, a new Terumo isolation bag with a 200 μm filter attached was connected to a separate line of the CTS Rotea Single-Use Kit using the sterile bonding machine, and the CTS Rotea Single-Use Kit was installed in an automated washing and concentrating device Rotea®.

[0142] Subsequently, the cell suspension was transferred from the Terumo isolation bag to a new Terumo isolation bag connected to a separate line (φ3.2 mm) at a rate of 100 mL / min, providing physical stimulation to the cell aggregates and causing them to become single cells (Step E).

[0143] Using the Rotea® automated washing and concentrating device, a 90 mL cell suspension was concentrated to 7 mL, resulting in a cell concentration of 1.5 × 10⁻⁶. ９ The concentration was set to cells / mL (step F).

[0144] The concentrated single-cell suspension was collected in a 20 mL syringe and filled into a Froze Bag F-050 (Nipro Corporation). All filled Froze Bags were left to stand at 4°C. After filling all the suspensions, 14 mL of CP-1 (Kyokuto Pharmaceutical Co., Ltd.) was added to the Froze Bags that had been left to stand at 4°C. The Froze Bags were kneaded until the cell suspension and CP-1 were uniformly mixed, then placed in aluminum protectors and stored at 4°C (Step G).

[0145] All freeze bags placed in aluminum protectors were placed in a programmable freezer and frozen at a cooling rate of 1°C / min to produce cell stocks (Process H).

[0146] In Example 1, the total time required for steps A to H was 3.6 hours, and the time required for each step was as follows: Step A: 40 minutes, Step B: 37 minutes, Steps C to E: 52 minutes, Step F: 35 minutes, Step G: 35 minutes, Step H: 14 minutes (time until the internal temperature reaches 0°C: the same applies to the following examples).

[0147] (Manufacturing Example 2) A large number of cells were cultured using the culture-frozen human iPS cell strain 1383D6, in the same manner as in Manufacturing Example 1 (Steps 1-4) described above.

[0148] (Example 2: Recovery and stock preparation of human iPS cells) Pluripotent stem cells cultured in Production Example 2 were recovered in the same manner as in Example 1, unicellularized, and then packed into appropriate containers to prepare a cell stock.

[0149] Note that the difference from Example 1 is 0.5 × TripLE ＴＭ The volume of StemFit® AK03N (manufactured by Ajinomoto Co., Ltd.), to which Select Enzyme and Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a final concentration of 10 μM, was set to 502 mL each, and the volume of cell suspension to be concentrated was set to 63 mL.

[0150] In Example 2, the total time required for steps A to H was 4.1 hours, and the time required for each step was as follows: Step A: 35 minutes, Step B: 37 minutes, Steps C to E: 59 minutes, Step F: 65 minutes, Step G: 40 minutes, Step H: 8 minutes.

[0151] (Manufacturing Example 3) A large number of cells were cultured using the human iPS cell QHJI strain in a culture-frozen state, in the same manner as in Manufacturing Example 1 (Steps 1-4) described above.

[0152] (Comparative Example 1: Recovery and Stock Preparation of Human iPS Cells) Pluripotent stem cells cultured in Production Example 3 were recovered by the following method, unicellularized, and then packed into appropriate containers to prepare a cell stock.

[0153] 0.5 x Triple ＴＭ Except for the following differences, in Example 1, the cells were unicellularized and packed into appropriate containers to prepare a cell stock: Select Enzyme and Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to StemFit® AK03N (manufactured by Ajinomoto Co., Ltd.) to a final concentration of 10 μM, the volume of each solution was 469 mL; the volume of cell suspension to be concentrated was 185 mL; and after biological stimulation of the cell aggregates, physical stimulation was applied without re-collecting the cell aggregates (i.e., without performing step D).

[0154] In Comparative Example 1, the time required for each process A to H was as follows: Process A: 41 minutes, Process B: 61 minutes, Process C and Process E (without Process D): 45 minutes, Process F: 75 minutes, Process G: 60 minutes, Process H: 6 minutes.

[0155] For Examples 1 and 2 and Comparative Example 1, the following items were measured and calculated, and the production efficiency and quality of the cell stocks were compared and evaluated.

[0156] (Cell stock production per hour) The number of cells recovered at the end of process F was measured using a cell counter NucleoCounter® NC-200 (ChemoMetec), and this value was divided by the time required for processes A to H (Hr). The value shown as [Number of cells recovered at the end of process F (cells)] / [Time required for processes A to H (Hr)] was calculated as the cell stock production per hour (cells / Hr).

[0157] Furthermore, in order to confirm the cell yield in steps A to F, the number of cells before the start of step A (the number of cells contained in the cell aggregate after the end of culture) was also measured. Specifically, the measurement was performed using the following procedure: 3 mL of culture medium was centrifuged to obtain cell aggregates. These cell aggregates were treated with Accutase (Innovative Cell Technologies, Inc.) for 10 minutes, and then separated into single cells by pipetting. The separated single cells were suspended in StemFit® AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM, and measured using the cell counter described above.

[0158] The results calculated and measured above are shown in Table 1.

[0159] (Survival Rate) After thawing the cell stocks prepared in Examples 1 and 2 and Comparative Example 1, the cell viability was measured using the NC-200. The results of calculating the dead cell rate as (100 - viability rate) are shown in Table 1.

[0160] (Adhesion Rate) After thawing the cell stocks prepared in Examples 1 and 2 and Comparative Example 1, 0.5 μg / cm³ of iMatrix-511 (Matrixome Co., Ltd.) was added to each cell stock. ２ In a 10 cm culture dish coated with [a certain substance], 6000 cells / cm ２ Sow the seeds at 37°C and 5% CO2. ２ Adhesion culture was performed under atmospheric conditions. 12 mL of StemFit® AK03N (Ajinomoto Co., Inc.), to which Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 10 μM, was used as the culture medium. The day on which the cells were seeded was designated as day 0 of culture. On day 1 of culture (after 24 hours of culture), the culture flask was observed, the number of adherent cells in the field of view was counted, and this was converted to the total number of adherent cells in the culture vessel. The adhesion rate (the ratio of adherent cells to the seeded number of cells after 24 hours of culture) was then calculated, and the results are shown in Table 1.

[0161] (Agglomeration rate) After thawing the cell stocks prepared in Examples 1, 2, and 3 and the cell stock prepared in Comparative Example 1, they were each placed in a 30 mL reactor (ABLE) at a rate of 10,000 cells / cm². ２ Sow the seeds at 37°C and 5% CO2. ２Under controlled conditions, suspension culture was performed at 100 rpm. 30 mL of StemFit® AK03N (Ajinomoto Co., Inc.), to which Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 10 μM, was used as the culture medium. The day on which the cells were seeded was designated as day 0 of culture. On day 1 of culture (24 hours after culturing), the entire culture medium from the suspension culture was collected, and the cell aggregates and culture supernatant were separated by centrifugation. The mixture was then treated with Accutase (Innovative Cell Technologies, Inc.) for 10 minutes, and the cell aggregates were dispersed into single cells by pipetting.

[0162] These cells were suspended in StemFit® AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM. The number of viable cells was counted using NC-200 (MS Techno Systems Co., Ltd.), and the aggregate formation rate (the ratio of cells surviving at 24 hours of culture to the number of seeded cells) was calculated. The results are shown in Table 1.

[0163]

[0164] As shown in Table 1, if the cell stock is produced via step D, the cell stock production per hour is 5 × 10 ８ The cell / hr ratio was significantly higher than that of the cell stocks produced by the method of the present invention in Examples 1 and 2. Compared to the cell stock produced by the method of Comparative Example 1, the cell viability was higher, and the adhesion rate and aggregate formation rate were significantly higher, resulting in the production of high-quality cell stocks.

[0165] Based on the above, the present invention provides a method for producing cell stocks that is satisfactory on a commercial scale in terms of both productivity and quality. All publications, patents and patent applications referenced herein are incorporated herein by direct reference in their entirety.

Claims

1. A method for producing a cell stock, comprising the following steps: A) a step of recovering cultured pluripotent stem cell aggregates from a culture vessel to a recovery vessel; B) a step of washing the recovered cell aggregates; C) a step of applying biological stimulation to the washed cell aggregates in an enzyme solution; D) a step of recovering the cell aggregates again; E) a step of applying physical stimulation to the recovered cell aggregates to convert them into single cells; F) a step of concentrating the converted single cells.

2. The manufacturing method according to claim 1, further comprising the following steps: G) suspending concentrated single cells in a cryopreservation solution; H) freezing the single cell suspension.

3. The manufacturing method according to claims 1 and 2, wherein step D includes centrifugal separation.

4. The manufacturing method according to claim 1 or 2, wherein the amount of enzyme solution coexisting with cell aggregates is reduced to 1 / 10 or less by step D of claim 1.

5. The total number of cells in the cell aggregate cultured in step A is 1 × 10⁻⁶. ９ The manufacturing method according to claim 1 or 2, wherein the number of cells is greater than or equal to 1.

6. The number of cells recovered at the end of step F is 3 × 10⁶. ９ The manufacturing method according to claim 1 or 2, wherein the number of cells is greater than or equal to 1.

7. The manufacturing method according to claim 1 or 2, wherein the recovery container used in step A is a flexible container.

8. The manufacturing method according to claim 1 or 2, wherein the physical stimulus described in step E is provided by delivering a suspension of cell aggregates into a tube.

9. The manufacturing method according to claim 1 or 2, wherein the recovery method in step A is carried out by utilizing the difference in gravity or by pressurizing the inside of the culture vessel.

10. The cell stock production rate per hour, expressed as (number of cells recovered at the end of step F) / (time required for steps A to H), is 5 × 10 ８ The manufacturing method according to claim 2, wherein the cells / hr is greater than or equal to 2.

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