JP7833271B2 - Method for manufacturing adhesive stem cell preparations - Google Patents

Description

This invention relates to a method for producing an adhesive stem cell preparation.

In recent years, the development of various regenerative medicine products using stem cells has progressed. However, in order to provide these as therapeutic preparations, a preservation method that can maintain high and stable cell quality over long periods is essential. Generally, cryopreservation is used to preserve therapeutic cell preparations, their intermediate products, and raw cell materials. However, freezing and thawing cells causes significant damage, leading to a decline in cell quality. Therefore, components that protect cells from damage caused by freezing and thawing (cryoprotective agents) are used during freezing, and solutions containing these cryoprotective agents are commercially available as cryopreservation solutions. Furthermore, as an example of improving cell preservation stability, the addition of various antioxidants during freezing has also been reported (Patent Documents 1 and 2).

Special publication 2009-521949 Special publication 2020-516257

Antioxidants remove harmful substances from cells, such as reactive oxygen species, but they can also damage cells, and their effectiveness is not always guaranteed. In fact, when the inventors added ascorbic acid, which is exemplified as an antioxidant in Patent Document 1, to a commercially available cryopreservation solution to confirm its effects, it was found that it did not improve cell viability.

Therefore, the present invention aims to provide a manufacturing method that can improve the quality of adherent stem cell preparations by minimizing damage to cells during and before/after cryopreservation, by devising the composition of the preservation solution used during cryopreservation.

The inventors investigated the types and appropriate concentrations of additives that can protect cells and improve their viability when used during cryopreservation. As a result, they found that by using an ascorbic acid derivative instead of ascorbic acid, which showed no effect, and by using an aqueous solution containing the ascorbic acid derivative in the range of 0.5 to 7 mM as the cryopreservation solution, the decrease in cell viability after cryopreservation was suppressed, and the quality of adherent stem cells was dramatically improved. In other words, the present invention provides the following method.
(1) A method for producing an adherent stem cell preparation, comprising the step of cryopreserving adherent stem cells in a solution containing 0.5 to 7 mM of an ascorbic acid derivative.
(2) The method for producing the product according to (1), wherein the ascorbic acid derivative is an ascorbic acid phosphate ester or a salt thereof.
(3) The method for manufacturing according to (1) or (2), further comprising a cryoprotectant in the solution.
(4) The method for producing the product according to (3), wherein the freeze-protecting agent contains 2 to 10% by volume of dimethyl sulfoxide in the solution.
(5) The method for producing the product according to (4), wherein the cryoprotectant further contains 4 to 10% by mass of hydroxyethyl starch in the solution.
(6) The method for producing the cryoprotective agent according to (4) or (5), further comprising 0.1 to 5% by mass of human serum albumin in the solution.
(7) The method for producing the adhesive stem cells according to any one of (1) to (6), wherein the adhesive stem cells are amniotic membrane-derived cells.
(8) A cell therapy agent comprising an adherent stem cell preparation obtained by any one of the methods described in (1) to (7) as an active ingredient.

By using the manufacturing method of the present invention, it is possible to maintain a high survival rate of adherent stem cells after freezing and thawing, and to enhance the proliferation of cells after freezing and thawing. Therefore, the present invention is expected to contribute to improving the quality of regenerative medicine products utilizing adherent stem cells and to promote the use of such products.

Figure 1 is a graph showing the changes in the viability of cells preserved in each cryopreservation solution for Examples 1-3 and Comparative Examples 1-4. Figure 2 is a graph showing the specific growth rate of cells preserved in each of the cryopreservation solutions for Example 4 and Comparative Examples 1 and 5. Figure 3 is a graph showing the adhesion rates of cells preserved with each of the cryopreservation solutions in Example 4 and Comparative Examples 1 and 5.

[1] Explanation of Terms In this specification, "adherent stem cells" refers to stem cells that satisfy the following definitions i) and ii), and "mesenchymal stromal cells" and "mesenchymal stem cells (MSCs)" are also included in the adherent stem cells of the present invention.

In this specification, adherent stem cells are defined as: i) stem cells that exhibit adhesion to plastic under standard culture conditions. Standard culture medium here refers to a culture medium to which serum, serum substitute reagent, or growth factor (e.g., human platelet lysate, which is a serum substitute reagent) has been added to a basal medium (e.g., αMEM medium).
ii) Surface antigens CD73 and CD90 are positive, while CD45 and CD326 are negative.

The origin of the adherent stem cells in this invention is not particularly limited; those derived from fetal appendages (amnion, umbilical cord, placenta, etc.), bone marrow fluid, adipose tissue, dental pulp, etc., can be used, but those derived from amnion are preferred.

In this specification, "proliferative capacity" refers to the ability of cells to increase in number through cell division. In this specification, "high proliferative capacity" and "high proliferation rate" can be used interchangeably. The proliferative capacity of an adherent stem cell population can be evaluated using specific growth rate, doubling count, doubling time, and/or passage count. The method for measuring specific growth rate is described later in this specification.

In this specification, "ascorbic acid" includes not only L-ascorbic acid, which is denoted by the IUPAC name (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one, but also its isomers and salts. For example, ascorbic acid, rhamno-ascorbic acid, arabo-ascorbic acid, gluco-ascorbic acid, fuco-ascorbic acid, glucohepto-ascorbic acid, xylo-ascorbic acid, galacto-ascorbic acid, gulo-ascorbic acid, allo-ascorbic acid, erythro-ascorbic acid, 6-desoxyascorbic acid, etc., in their L-form, D-form, racemic form, or salts thereof. However, it does not include "ascorbic acid derivatives," which are variations of ascorbic acid with substituted functional groups, as described later.

In this specification, "ascorbic acid derivative" refers to an ascorbic acid in which the functional groups of the above-mentioned "ascorbic acid" are substituted with fatty acids, phosphoric acid, sugars, glycerol, etc. The above-mentioned ascorbic acid derivative is not particularly limited, but examples include fatty acid esters such as ethyl ascorbic acid, ascorbic acid palmitate, and ascorbic acid stearate; phosphate esters such as ascorbic acid-2-phosphate ester, ascorbic acid-3-phosphate ester, ascorbic acid-6-phosphate ester, and ascorbic acid-2-polyphosphate ester; ascorbic acid esters such as ascorbic acid-2-sulfate ester; and ascorbic acid-2-glucoside. These may be L-isomers, D-isomers, or racemic mixtures. Furthermore, ascorbic acid derivatives in this specification also include salts thereof. Examples of ascorbic acid derivative salts include sodium salt, potassium salt, magnesium salt, calcium salt, barium salt, ammonium salt, monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, and triisopropanolamine salt. In the present invention, ascorbic acid derivatives are preferably ascorbic acid phosphate esters or their salts, more preferably ascorbic acid-2-phosphate esters or their salts, and even more preferably L-ascorbic acid-2-phosphate esters or their salts.

In this specification, “cryoprotective agent” refers to a chemical substance that facilitates the cryoprotection process by reducing cell damage during freezing and thawing. Cryoprotective agents may be cell-permeable or non-permeable. Examples of cryoprotective agents include, but are not limited to, dehydrators, osmotic agents, and vitrifying solutes. Examples of the above cryoprotective agents include acetamide, agarose, alginate, l-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, dextran, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, α-glycerophosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, and magnesium sulfate. Cium, maltose, mannitol, mannose, methanol, methylacetamide, methylformamide, methylureas, phenols, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine-N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, ureas, valine, and xylose can be used, but are not particularly limited.

In this specification, "cryopreservation solution" refers to a solution used to suspend cells during cryopreservation, and is preferably an aqueous solution. While "the cryopreservation solution of the present invention" is a solution containing the above-mentioned ascorbic acid derivative, as described later, when simply referred to as "cryopreservation solution," it includes not only the cryopreservation solution of the present invention but also solutions that are not the cryopreservation solution of the present invention.

The "adherent stem cell preparation" of this invention is not particularly limited as long as it is a preparation mainly composed of adherent stem cells. Its uses may include medical applications such as treatment and prevention, as well as research and testing applications. It may also be an intermediate raw material (such as cell stock) for manufacturing cell products used in these applications.

[2] Cryopreservation process for adherent stem cells of the present invention The present invention is a method for producing an adherent stem cell preparation comprising at least one step of cryopreserving adherent stem cells in a solution containing 0.5 to 7 mM ascorbic acid derivative. That is, in the production method of the present invention, a solution containing 0.5 to 7 mM ascorbic acid derivative is used as the cryopreservation solution. The cryopreservation solution of the present invention is not particularly limited as long as it contains 0.5 to 7 mM ascorbic acid derivative, but preferably it is an ascorbic acid derivative dissolved in water or an aqueous solution. Examples of aqueous solutions used in this case include buffer solutions, isotonic solutions, hypotonic solutions, and hypertonic solutions. From the viewpoint of reducing damage to tissue, buffer solutions and isotonic solutions are more preferred. For example, buffer solutions such as phosphate-buffered saline (PBS), equilibrium salt solutions such as Hanks equilibrium salt solution (HBSS) and Earl equilibrium salt solution (EBSS), infusion solutions such as Ringer's solution, lactated Ringer's solution, and physiological saline, and culture media are preferred examples.

The concentration of the ascorbic acid derivative in the cryopreservation solution of the present invention is not particularly limited as long as it is between 0.5 and 7 mM, but is preferably 1.0 mM or higher, more preferably 1.2 mM or higher, even more preferably 2 mM or higher, and even more preferably 3 mM or higher. The upper limit is preferably in the range of 5 mM or less, where cell quality is further improved.

The cryopreservation solution of the present invention preferably further contains a cryoprotectant in addition to the ascorbic acid derivative.

The above cryoprotective agent is not particularly limited, but dimethyl sulfoxide is preferred. The concentration of dimethyl sulfoxide added to the cryopreservation solution of the present invention is not particularly limited, but preferably 2% by volume or more, more preferably 4% by volume or more, shows a higher cryoprotective effect. Preferably 10% by volume or less, even more preferably 8% by volume or less, and more preferably 6% by volume or less can further reduce cytotoxicity.

The cryopreservation solution of the present invention may further contain hydroxyethyl starch as a cryoprotective agent. While the concentration of hydroxyethyl starch is not particularly limited, 4% by mass or more is preferred. More preferably, a concentration of 10% by mass or less, and more preferably 8% by mass or less, can reduce the viscosity of the cryopreservation solution and improve its handling properties.

The cryopreservation solution of the present invention may further contain human serum albumin as a cryoprotective agent. The concentration is not particularly limited, but is preferably 0.1% by mass or more, more preferably 3.5% by mass or more, with an upper limit of preferably 5% by mass or less. Within this range, the quality will be further improved.

In the manufacturing method of the present invention, the method of suspending adherent stem cells in the cryopreservation solution of the present invention is not particularly limited. A cryopreservation solution containing an ascorbic acid derivative and, optionally, a cryoprotective agent and other components may be prepared, and adherent stem cells may be directly suspended therein. Alternatively, an aqueous solution containing a cryoprotective agent and a cell suspension containing an ascorbic acid derivative may be mixed in any proportion to adjust the solution to fall within the scope of the present invention. A commercially available cryopreservation solution may also be used as the aqueous solution containing a cryoprotective agent.

The cell concentration (final concentration) in the above cryopreservation solution is not particularly limited, but preferably it is 2 × ^10⁸ cells/mL or less, and more preferably 5 × ^10⁷ cells/mL or less, so that the quality improvement effect of the cryopreservation solution of the present invention is more effectively exhibited. Furthermore, from the viewpoint of productivity efficiency, it is preferable that the lower limit of the cell concentration is 5 × ^10⁴ cells/mL or more.

In the manufacturing method of the present invention, the time from suspending the adherent stem cells in the cryopreservation solution to the start of freezing is not particularly limited. However, if the time to start freezing is preferably within 24 hours, more preferably within 12 hours, and even more preferably within 6 hours, the quality of the cells after thawing can be further improved. The lower limit is not particularly limited, and a shorter time is preferable within the range permissible in the manufacturing process. However, when using the cryopreservation solution of the present invention, cell viability and quality can be maintained to an acceptable degree even for about 0.5 hours.

In the manufacturing method of the present invention, cryopreservation can be carried out, for example, by a slow freezing method using a programmable freezer or a simple freezing method using a deep freezer. In the slow freezing method using a programmable freezer, cooling can be started at a planned rate, for example, -1 to -3°C/min. On the other hand, in the simple freezing method, for example, cryovials or freezing bags filled with cell suspension can be placed in a cell freezing container or polystyrene container and then placed in a deep freezer for slow freezing. Alternatively, after freezing using the above freezing method, the cells may be moved to liquid nitrogen for storage. Examples of containers for filling cell suspension for cryopreservation include, but are not particularly limited to, Nunc Cryotube 1.8 mL inner cap (manufactured by Thermo Fisher Scientific), internally threaded cryogenic vial with barcode (manufactured by Corning), Frozetube T-1.5 (manufactured by Nipro Corporation), and Frozebag (manufactured by Nipro Corporation). For cell freezing containers used in the simplified freezing method, for example, BICEL (manufactured by Nippon Freezer Co., Ltd.) or CoolCell (manufactured by Corning) can be used.

Furthermore, adherent stem cells cryopreserved using the cryopreservation solution of the present invention may be used directly after thawing, or they may be cultured one or more times after thawing and then used as an adherent stem cell preparation, or they may be sold or provided to medical institutions, etc., as an adherent stem cell preparation in their frozen state. From the viewpoint of fully demonstrating the objectives and effects of the present invention, it is preferable that the cryopreservation process using the cryopreservation solution of the present invention be carried out as an intermediate step in the manufacturing of cell preparations. Also, the cryopreservation process using the cryopreservation solution of the present invention may be carried out multiple times in the manufacturing process of adherent stem cell preparations.

Since the period of freezing and storage in the manufacturing method of the present invention has little effect on the present invention, any desired freezing and storage period can be selected in the process. Therefore, it is not particularly limited, but for example, it could be 1 day or more, 2 days or more, 3 days or more, 1 week or more, 10 days or more, 20 days or more, 1 month or more, or for example, 5 years or less, 3 years or less, 2 years or less, 1 year or less, or 6 months or less.

In the manufacturing method of the present invention, the thawing method for adherent stem cells cryopreserved by the above cryopreservation method can be carried out, for example, by placing a container filled with the frozen cells into a water bath heated to 35-37°C. Alternatively, thawing may be performed using a cell thawing device. Examples of cell thawing devices include ThawSTAR (manufactured by BIOLIFE SOLUTIONS), VIA Thaw CB1000 (manufactured by cytiva), and the YS series cryopreservation device (manufactured by STREX Corporation).

[3] Method for producing the adherent stem cell preparation of the present invention The method for producing the adherent stem cell preparation of the present invention is characterized by having a cryopreservation step of the adherent stem cells, and no other conditions or configurations are limited. For example, the method for producing the adherent stem cell preparation of the present invention may include any steps other than the cryopreservation step of the adherent stem cells described above, such as a cell population acquisition step and a culture step.

In the manufacturing method of the present invention, the cell population acquisition step may be, for example, a cell population acquisition step in which a cell population containing adherent stem cells is obtained by enzymatic treatment of fetal appendages such as the amniotic membrane or adipose tissue. The cell population acquisition step may also include a step of obtaining the amniotic membrane by cesarean section. Alternatively, the cell population acquisition step may include a step of obtaining adipose tissue by aspirating or excising it from a living organism. Furthermore, the cell population acquisition step may also include a step of washing the biological sample containing adherent stem cells.

The cell population containing adherent stem cells in this invention is preferably a cell population obtained by treating a biological sample containing an epithelial cell layer and an adherent stem cell layer, collected from fetal appendages, or adipose tissue, with enzymes.

For example, specifically, the enzymatic treatment of a biological sample collected from a fetal appendage (preferably a biological sample containing an epithelial cell layer and an adherent stem cell layer) preferably involves treatment with an enzyme (or a combination thereof) that can release adherent stem cells contained in the extracellular matrix layer of the fetal appendage without degrading the epithelial cell layer. Such enzymes are not particularly limited, but examples include collagenase and/or metalloproteinases. Examples of metalloproteinases include thermolysin and/or dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids, but are not particularly limited.

The activity concentration of collagenase is not particularly limited, but is preferably 50 PU/ml or higher, more preferably 100 PU/ml or higher, and even more preferably 200 PU/ml or higher. The upper limit is also not particularly limited, but for example, it could be 1000 PU/ml or less, 900 PU/ml or less, 800 PU/ml or less, 700 PU/ml or less, 600 PU/ml or less, or 500 PU/ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 ug of FITC-collagen in 1 minute at pH 7.5 and 30°C.

The activity concentration of the metalloproteinase (e.g., thermolysin and/or dispase) is not particularly limited, but is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, and even more preferably 200 PU/ml or more. The upper limit is preferably 1000 PU/ml or less, more preferably 900 PU/ml or less, even more preferably 800 PU/ml or less, even more preferably 700 PU/ml or less, even more preferably 600 PU/ml or less, and even more preferably 500 PU/ml or less. Here, in embodiments using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that releases an amino acid equivalent to 1 ug of tyrosine per minute from lactate casein at pH 7.5 and 30°C. Within the above range of enzyme concentrations, adherent stem cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of the epithelial cell layer contained in the fetal appendage's epithelial cell layer. The preferred combination of collagenase and/or metalloproteinase concentrations can be determined by microscopic observation of fetal appendages after enzyme treatment or by flow cytometry of the acquired cells.

From the viewpoint of efficiently recovering viable cells, it is preferable to treat fetal appendages with a combination of collagenase and metalloproteinase. More preferably, the fetal appendages are treated simultaneously and in a single batch using this combination. In this case, thermolysin and/or dispase can be used as the metalloproteinase, but are not limited to these. Adhesive stem cells can be easily obtained by treating the fetal appendages only once with an enzyme solution containing both collagenase and metalloproteinase. Furthermore, simultaneous, batch treatment reduces the risk of contamination by bacteria, viruses, etc.

Enzymatic treatment of fetal appendages is preferably carried out by immersing the amniotic membrane, which has been washed with a washing solution such as physiological saline or Hanks equilibrium salt solution, in an enzyme solution and treating it while stirring with a stirring means. As such a stirring means, a stirrer or shaker can be used, but is not limited to these, from the viewpoint of efficiently releasing adherent stem cells contained in the extracellular matrix layer of the amniotic membrane. The lower limit of the stirring speed when using a stirrer or shaker is not particularly limited, but if the stirring speed is too slow, the enzyme treatment efficiency will decrease, so it is preferably 5 rpm, more preferably 10 rpm. The upper limit is not particularly limited, but if the stirring speed is too fast, cells may be damaged, so it is preferably 100 rpm, more preferably 60 rpm. The lower limit of the enzyme treatment time is not particularly limited, but if it is too short, adherent stem cells contained in the amniotic membrane cannot be sufficiently separated, so it is preferably 30 minutes, and even more preferably 45 minutes. The upper limit of the enzyme treatment time is not particularly limited, but if it is too long, the viability of the separated cells may decrease, so it is preferably 6 hours, more preferably 3 hours, and even more preferably 90 minutes. The lower limit of the enzyme treatment temperature is not particularly limited, but to ensure efficient enzymatic reaction, it is preferably 15°C, more preferably 25°C, and even more preferably 35°C. The upper limit of the enzyme treatment temperature is also not particularly limited, but since excessively high temperatures can cause the death of adherent stem cells and enzyme inactivation, it is preferably 40°C.

When isolating adherent stem cells from tissues other than the amnion, the method described above can be used, or by known methods.

In the manufacturing method of the present invention, free adherent stem cells can be separated and/or recovered from the enzyme solution containing the free adherent stem cells by known methods such as filters, centrifugation, hollow fiber separation membranes, and cell sorters, if desired. Preferably, the enzyme solution containing the free adherent stem cells is filtered by a filter. In the embodiment in which the enzyme solution is filtered by a filter, only the free cells pass through the filter, and the epithelial cell layer that has not been degraded remains on the filter without passing through it. This not only allows for easy separation and/or recovery of the free adherent stem cells, but also reduces the risk of contamination by bacteria, viruses, etc. The filter is not particularly limited, but for example, a mesh filter can be used. The pore size (mesh size) of the mesh filter is not particularly limited, but for example, it is 40 μm or more, 60 μm or more, 80 μm or more, or 90 μm or more. Furthermore, the pore size of the mesh filter is not particularly limited, but for example, it may be 200 μm or less, 180 μm or less, 160 μm or less, 140 μm or less, 120 μm or less, or 100 μm or less. While the filtration speed is not particularly limited, by setting the pore size of the mesh filter within the above range, the enzyme solution containing adherent stem cells can be filtered by gravity, thereby preventing a decrease in cell viability.

Nylon is preferred as the material for the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm, or 100 μm nylon mesh filters, such as those commonly used in research applications like Falcon cell strainers, are available. Medical mesh cloths (nylon and polyester) used in hemodialysis are also usable. Furthermore, arterial filters (polyester mesh filters, pore size: 40 μm to 120 μm) used during extracorporeal circulation are also available. Other materials, such as stainless steel mesh filters, can also be used.

When filtering adherent stem cells, gravity (free fall) is preferred. While forced filtering using a pump or similar device is possible, it is desirable to use the lowest possible pressure to avoid damaging the cells.

The adherent stem cells, after being filtered, can be recovered by centrifugation after diluting the filtrate with twice the volume or more of culture medium or equilibrium salt buffer. Suitable equilibrium salt buffers include, but are not limited to, physiological saline, Dulbecco's phosphate buffer (DPBS), Earl's equilibrium salt solution (EBSS), Hanks' equilibrium salt solution (HBSS), and phosphate buffer (PBS).

The culture step in the method for producing the adherent stem cell preparation of the present invention may be a step of culturing a cell population containing adherent stem cells obtained in the above step, or it may be a step of culturing a cell population containing adherent stem cells obtained by another method.

The cell seeding density in the step of culturing the cell population including the above-mentioned adherent stem cells is not particularly limited, but can be seeded at a density of, for example, 500 to 10,000 cells/ ^cm² . The lower limit of the seeding density is preferably 550 cells/ ^cm² , more preferably 750 cells/ ^cm² , and even more preferably 1,000 cells/ ^cm² . The upper limit of the seeding density is also not particularly limited, but is preferably 7,000 cells/ ^cm² , and even more preferably 5,000 cells/ ^cm² .

Furthermore, the above culture process may include a subculturing process, or it may include a process of repeating the culture multiple times under different culture conditions.

The culture period for one culture cycle can be, for example, 2 to 15 days, and more specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

The culture medium used for the above-mentioned culture can be prepared by using any liquid culture medium for animal cells as the base medium and adding other components (serum, serum substitute reagents, growth factors, etc.) as needed. In the embodiment where growth factors are added to the base medium, the culture medium may be prepared by adding a reagent for stabilizing the growth factor in the medium (such as heparin) to the growth factor, or by pre-stabilizing the growth factor with a gel or polysaccharide and then adding the stabilized growth factor to the base medium.

The basal media used include 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 (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, and RPMI. While 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM/F12 medium (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham)) can be used, they are not particularly limited. A preferred basal medium is αMEM medium. Various commercially available serum-free media can also be used. Examples of serum-free media include STK1 and STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), and Corning Stemgro Human Mesenchymal Stem Cell Medium (Corning).

Other components that can be added to the basal culture medium include, for example, albumin, bovine serum, serum substitute reagents, or growth factors. Among these, serum substitute reagents are preferred, and platelet lysates are particularly preferred. In particular, it is preferable to perform the culture in a basal culture medium containing a serum substitute reagent but free from albumin, bovine serum, and growth factors. The lower limit of the platelet lysate concentration in the culture medium can be, for example, 1% by volume or more, preferably 2% by volume or more, and more preferably 3% by volume or more as the final concentration. The upper limit of the platelet lysate concentration in the culture medium is, for example, 20% by volume or less, preferably 10% by volume or less, and more preferably 7% by volume or less.

Cell populations containing adherent stem cells can be cultured using, for example, the following procedure: First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Next, the cells are seeded in a plastic culture vessel and cultured using the aforementioned culture medium at a _CO2 concentration of 3% to 5% and a temperature of 37°C. Cells obtained by the above culture method are cells that have been cultured once.

The cells cultured once as described above can be further subcultured as follows: First, the cells cultured once are detached from the plastic culture vessel using a cell detachment device. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a culture medium. Finally, the cells are seeded in a plastic culture vessel and cultured in a culture medium at a _CO2 concentration of 3% to 5% and a temperature of 37°C until the confluence rate is 95% or less. Examples of the culture medium include, but are not limited to, αMEM, M199, or media based on these. Cells obtained by the above subculturing and culture are cells that have been subculturised once. Cells that have been subculturised n times can be obtained by performing the same subculturing and culture (where n is an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of subculturings n is, for example, 1, preferably 2. From the viewpoint of suppressing cell senescence, the upper limit of the number of subculturings n is, for example, 20 or 10. As the cell detachment device described above, for example, a cell detachment agent may be used. As cell detachment agents, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc., can be used, but are not particularly limited. Commercially available cell detachment agents may also be used. Examples include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technologies), and Accumax (manufactured by Stemcell Technologies). Furthermore, physical cell detachment means may be used as cell detachment means, for example, a cell scraper (manufactured by Corning), but are not limited to this. Cell detachment means may be used individually or in combination.

In the method for producing the adherent stem cell preparation of the present invention, the culture step can be performed any number of times. Furthermore, the combination with the cryopreservation step using the cryopreservation solution of the present invention is also flexible. For example, after culturing the adherent stem cell population obtained in the cell population acquisition step, cryopreservation may be performed using the cryopreservation solution of the present invention, and then the culture step may be performed again. Alternatively, the culture step and cryopreservation step may be repeated multiple times.

[4] Cell Therapeutic Agents The adherent stem cell preparation produced by the manufacturing method of the present invention can be used as an active ingredient in therapeutic agents for intractable diseases. That is, a cell therapy agent containing an adherent stem cell preparation produced by the manufacturing method of the present invention as an active ingredient is also one embodiment of the present invention. Furthermore, the adherent stem cell preparation produced by the manufacturing method of the present invention can also be cultured and used as an active ingredient in the cell therapy agent of the present invention.

The cell therapy agent of the present invention is effective for immune-related diseases, muscular dystrophy, ischemic diseases, lower limb ischemia, cerebral vascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen diseases, stroke, cerebral infarction, intracerebral hematoma, cerebral vascular palsy, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes mellitus, mycosis fungoides, and other conditions. It can be used as a therapeutic agent for diseases selected from the following: skin diseases, diseases resulting from degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscal tears, osteochondrosis dissecans, avascular necrosis, osteoarthritis of the knee, inflammatory arthritis, rheumatoid arthritis, eye diseases, neovascularization-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancer.

The cell therapy agent of the present invention may be diluted with a pharmaceutically acceptable medium. The pharmaceutically acceptable medium is not particularly limited as long as it is a solution that can be administered to a patient or subject. The pharmaceutically acceptable medium may be an intravenous solution, and examples include, but are not limited to, water for injection, physiological saline solution, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, amino acid solution, initiation solution (Solution 1), dehydration replenishment solution (Solution 2), maintenance infusion solution (Solution 3), postoperative recovery solution (Solution 4), Plasma-Lyte A®, etc.

In this specification, "patient or subject" typically refers to a human, but may also refer to other animals. Other animals include, but are not limited to, mammals such as dogs, cats, cattle, horses, pigs, goats, sheep, monkeys (crab-eating macaques, rhesus macaques, common marmosets, Japanese macaques), ferrets, rabbits, and rodents (mice, rats, gerbils, guinea pigs, hamsters), as well as birds such as chickens and quail.

In this specification, "treatment" includes, but is not limited to, significantly improving at least one of the following conditions in a patient or subject: life expectancy, functional prognosis, survival rate, weight loss, anemia, diarrhea, bloody stools, abdominal pain, fever, loss of appetite, malnutrition, vomiting, fatigue, rash, inflammation, ulcers, erosions, fistulas, strictures, intestinal obstruction, internal bleeding, rectal bleeding, convulsions, pain, decreased liver function, decreased cardiac function, decreased lung function, motor function, muscle weakness, or blood test results.

The cell therapy agent of the present invention may contain any components used in the treatment of a patient or subject. Examples of such components include, but are not limited to, salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts) and aqueous solutions containing them (e.g., physiological saline, Ringer's solution, bicarnate infusion), polysaccharides (e.g., hydroxyethyl starch, dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxides, amino acids, and culture medium components (e.g., components contained in RPMI 1640 medium, etc.).

The cell therapy agent of the present invention may contain various additives to increase storage stability, isotonicity, absorption, and/or viscosity, such as emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc. Examples of thickeners include, but are not limited to, hydroxyethyl starch, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, and hydroxypropylcellulose. The concentration of the thickener depends on the selected thickener, but can be arbitrarily set within a range of concentrations that are safe when administered to a patient or subject and achieve the desired viscosity.

The cell therapy agent of the present invention may contain one or more other pharmaceutical components in addition to adherent stem cells. Examples of other pharmaceutical components include, but are not limited to, antibiotics, albumin preparations, vitamin preparations, and anti-inflammatory agents. Examples of anti-inflammatory agents include, but are not limited to, 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, and biological preparations. Examples of 5-aminosalicylic acid preparations include, but are not limited to, sulfasalazine and mesalazine. Examples of steroid preparations include, but are not limited to, cortisone, prednisolone, and methylprednisolone. Examples of immunosuppressants include, but are not limited to, tacrolimus, cyclosporine, methotrexate, azatiprine, and 6-mercaptopurine. Examples of the above-mentioned biological agents include, but are not limited to, infliximab, adalimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, tocilizumab, vedolizumab, filgotinib, golimumab, certolizumab pegol, abatacept, and etanercept.

Furthermore, the other pharmaceutical components mentioned above may be administered in other types of cells. Examples of other types of cells that can be administered include, but are not limited to, blood-derived cells (leukocytes, erythrocytes, mononuclear cells, etc.), vascular endothelial cells, vascular endothelial progenitor cells, pericytes, vascular wall cells, fibroblasts, skeletal muscle blasts, epithelial cells, stromal cells, and mature adipocytes.

The pH of the cell therapy agent of the present invention can be near neutral, for example, pH 5.5 or higher, pH 6.5 or higher, or pH 7.0 or higher, and can also be pH 10.5 or lower, pH 9.5 or lower, pH 8.5 or lower, or pH 8.0 or lower, but is not limited to these values.

The concentration of adherent stem cells in the cell therapy agent of the present invention is such that, when administered to a patient or subject, a therapeutic effect against the disease can be obtained compared to a patient or subject who does not receive the agent. The specific cell concentration can be appropriately determined depending on the form of administration, method of administration, purpose of use, and the age, weight, and symptoms of the patient or subject. The lower limit of the cell concentration of the cell therapy agent of the present invention is not particularly limited, but for example, it is 1.0 × ^10⁵ cells/mL or more, 1.0 × ^10⁶ cells/mL or more, 1.2 × ^10⁶ cells/mL or more, 1.4 × ^10⁶ cells/mL or more, 1.6 × ^10⁶ cells/mL or more, 1.8 × ^10⁶ cells/mL or more, 2.0 × ^10⁶ cells/mL or more, 3.0 × ^10⁶ cells/mL or more, 4.0 × ^10⁶ cells/mL or more, 5.0 × ^10⁶ cells/mL or more, 6.0 × ^10⁶ cells/mL or more, 7.0 × ^10⁶ cells/mL or more, 8.0 × ^10⁶ cells/mL or more, 9.0 × ^10⁶ cells/mL or more, 9.5 × ^10⁶ cells/mL or more, or 1.0 × ^10⁷ cells/mL or more. The upper limit of the cell concentration of the cell therapy agent of the present invention is not particularly limited, but for example, it may be 1.0 × ^10¹⁰ cells/mL or less, 1.0 × ^10⁹ cells/mL or less, 8.0 × ^10⁸ cells/mL or less, 6.0 × ^10⁸ cells/mL or less, 4.0 × ^10⁸ cells/mL or less, 2.0 × ^10⁸ cells/mL or less, or 1.0 × ^10⁸ cells/mL or less.

The cell therapy agent of the present invention is preferably a liquid formulation, and more preferably an injectable liquid formulation. As an injectable liquid formulation, for example, liquid preparations suitable for injection are known in International Publication WO2011/043136 and Japanese Patent Application Publication No. 2013-256510. The cell therapy agent of the present invention can also be an injectable liquid formulation described in the above-mentioned literature.

Furthermore, the above-mentioned liquid preparation may be a cell suspension or a liquid preparation in which cells are dispersed in the liquid. The morphology of the cells contained in the liquid preparation is not particularly limited; for example, they may be single cells or cell aggregates. Needless to say, the above-mentioned liquid preparation can also be used for topical application, patch application, or spray application.

Furthermore, according to one aspect of the present invention, the cell therapy agent of the present invention may be a transplantable formulation. The transplantable formulation may be a solid or gel-like formulation. For example, a solid transplantable formulation may have a sheet-like or pellet-like structure. As a gel-like transplantable formulation, for example, International Publication WO2017/126549 describes a transplantable formulation containing a gel obtained by adhering isolated cells with an adhesive (e.g., fibrinogen). Furthermore, according to one aspect of the present invention, the cell therapy agent of the present invention may be a gel formulation obtained by mixing cells with an arbitrary gel. As a gel formulation, for example, Japanese Patent Publication 2017-529362 describes a cell therapy agent composed of an adherent stem cell-hydrogel composition. The cell therapy agent of the present invention can also be made into a gel formulation, for example, by using the method described in the above-mentioned document.

Furthermore, as sheet-like structured transplant preparations, for example, International Publication WO2006/080434 and Japanese Patent Publication No. 2016-52272 describe cell sheets obtained by culturing cells using a temperature-responsive culture dish (e.g., UpCell® (manufactured by CellSeed Co., Ltd.)), laminates of sheet-like cell cultures and fibrin gel, and cell-coated sheets obtained by coating a cell suspension onto a sheet-like substrate. The cell therapy agent of the present invention can also be made into various sheet-like structured transplant preparations by using, for example, the methods described in the above-mentioned literature.

The method of administering the cell therapy agent of the present invention is not particularly limited, but examples include subcutaneous injection, intradermal injection, intramuscular injection, lymph node injection, intravenous injection, intra-arterial injection, intraperitoneal injection, intrathoracic injection, direct local injection, direct patch application, or direct local transplantation. According to one aspect of the present invention, the injectable solution can be filled into a syringe and administered intravenously, intra-arterially, intramyocardially, intranodally, intrahepatic artery, intramuscularly, epidurally, gingivally, intraventricularly, subcutaneously, intradermally, intraperitoneally, or into the portal vein via a needle or catheter, but is not limited to these. For more information on the method of administering the cell therapy agent, see, for example, Japanese Patent Application Publication No. 2015-61520, Onken JE, t al. In the American College of Gastroenterology Conference 2006 (Las Vegas, NV, Abstract 121), Garcia-Olmo D, et al. (Dis Colon Rectum 2005;48:1416-23), etc., methods such as intravenous injection, intravenous drip infusion, direct local injection, and direct local transplantation are known. The cell therapy agent of the present invention can also be administered by the various methods described in the above-mentioned literature.

The dose of the cell therapy agent of the present invention is the amount of cells that, when administered to a patient or subject, will produce a therapeutic effect against the disease compared to a patient or subject who does not receive the agent. The specific dose can be appropriately determined depending on the form of administration, method of administration, purpose of use, and the age, weight, and symptoms of the patient or subject. The single dose of adherent stem cells to humans is not particularly limited, but for example, it may be 1 × ^10⁴ cells/kg body weight or more, 1 × ^10⁵ cells/kg body weight or more, 5 × ^10⁵ cells/kg body weight or more, 1 × ^10⁶ cells/kg body weight or more, 2 × ^10⁶ cells/kg body weight or more, 4 × ^10⁶ cells/kg body weight or more, 6 × ^10⁶ cells/kg body weight or more, or 8 × ^10⁶ cells/kg body weight or more. Furthermore, the single dose of adherent stem cells to humans is not particularly limited, but for example, it may be 1 × ^10¹² cells/kg body weight or less, 1 × 10¹¹ cells/kg body weight or less, 1 × 10¹⁰ cells/kg body weight or less, 1 × ^10⁹ cells/kg body weight or less, 5 × ^10⁸ cells/kg body weight or less, 1 × ^10⁸ cells/kg body weight or less, 8 × ^10⁷ cells/kg body weight or less, 6 × ^10⁷ cells/kg body weight or less, 4 × ^10⁷ cells/kg body weight or less, or 2 × ^10⁷ cells/kg body weight or less.

When the cell therapy agent of the present invention is an injectable solution, the single dose of adherent stem cells to humans in the injectable solution is preferably 1 × ^10⁵ cells/kg body weight or more, 5 × ^10⁵ cells/kg body weight or more, 1 × 10⁶ cells/kg body weight or more, 2 × ^10⁶ cells/kg body weight or more, 4 × ^10⁶ cells/kg body weight or more, 6 × ^10⁶ cells/kg body weight or more, or 8 × ^10⁶ cells/kg body weight or more, from the viewpoint of enhancing the therapeutic effect against the disease. Furthermore, the single dose of adherent stem cells to humans in the injectable solution is preferably 1 × 10⁹ cells/kg body weight or less, 5 × ^10⁸ cells/kg body weight or less, 1 × ^10⁸ cells/kg body weight or less, 8 × ^10⁷ cells/kg body weight or less, 6 × ^10⁷ cells/kg body weight or less, 4 × ^10⁷ cells/kg body weight or less, or 2 × ^{10⁷ cells} /kg body weight or less, from the viewpoint of facilitating the preparation ^and administration of the injectable solution.

The administration frequency of the cell therapy agent of the present invention is such that a therapeutic effect can be obtained against the disease when administered to a patient or subject. The specific administration frequency can be appropriately determined depending on the administration form, method, purpose of use, and the patient's or subject's age, weight, and symptoms, but examples include once every four weeks, once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week.

The duration of administration of the cell therapy agent of the present invention is a period that allows for the acquisition of a therapeutic effect against the disease when administered to a patient or subject. The specific duration of administration can be appropriately determined depending on the administration form, method, purpose of use, and the patient's or subject's age, weight, and symptoms, but examples include one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks.

The timing of administering the cell therapy agent of the present invention to a patient or subject is not particularly limited, but examples include immediately after the onset of symptoms, within n days from the onset of symptoms (where n is an integer of 1 or more), immediately after diagnosis, within n days from diagnosis (where n is an integer of 1 or more), before remission, during remission, after remission, before relapse, during relapse, and after relapse.

The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

(Comparative Example 1)
<Step 1: Enzymatic treatment of amniotic membrane and recovery of adherent stem cells>
The amniotic membrane, including the amnion, and the placenta were aseptically collected from pregnant women undergoing elective cesarean section who had given informed consent. The collected amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amnion was manually detached from the cut end of the amniotic membrane. The amnion was washed with Hanks equilibrium salt solution (Ca/Mg-free) to remove any attached blood and blood clots, and then enzymatically treated by immersion in Hanks equilibrium salt solution (Ca/Mg-containing) containing 240 PU/mL collagenase and 200 PU/mL dispase I, and shaking at 10 rpm for 60 minutes at 37°C. Undigested amniotic material was removed from the solution after enzymatic treatment by filtering through a 95 μm nylon mesh, and a cell suspension containing adherent stem cells was recovered.

<Step 2: Culture and harvesting of adherent stem cells>
The cell population, including adherent stem cells, obtained in Step 1 was seeded at a density of 1,000 cells/ ^cm² into a 75 ^cm² U-shaped cant-neck cell culture flask (vent cap) (Corning Corporation), and cultured using αMEM containing human platelet lysate at a final concentration of 5% by volume. After culturing until the cells reached subconfluence, the cells were detached from the flask using trypsin-EDTA solution. The detached cells were washed with physiological saline.

<Step 3: Suspension of cells in cryopreservation solution>
The adherent stem cells obtained in step 2 were suspended in physiological saline to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL. A solution (A) containing 10% by volume dimethyl sulfoxide, 12% by mass hydroxyethyl starch, and 8% by mass human serum albumin was prepared separately and mixed with the cell suspension in a 1:1 ratio.

<Step 4: Freezing>
One mL each of the mixture of the cell suspension and solution (A) prepared in step 4 was filled into 1.8 mL Nunc cryotubes with inner caps (Thermo Fisher Scientific). Immediately thereafter, the cryovials filled with the mixture were placed in CoolCell (Corning) containers and frozen by standing them in a deep freezer at -80°C for 24 hours. After freezing, the cells were transferred to a liquid nitrogen storage container and stored in the liquid nitrogen phase for 10 days.

(Comparative Example 2)
In step 3, the adherent stem cells were suspended in physiological saline containing 2 mM L-sodium ascorbate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL, except that the procedure was the same as in Comparative Example 1.

(Comparative Example 3)
In step 3, the same procedure as in Comparative Example 1 was followed, except that adherent stem cells were suspended in physiological saline containing 8 mM L-sodium ascorbate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL.

(Comparative Example 4)
In step 3, adherent stem cells were suspended in physiological saline containing 10 mM L-sodium ascorbate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL. The procedure was the same as in Comparative Example 1.

(Comparative Example 5)
In step 3, the adherent stem cells were suspended in physiological saline containing 0.4 mM L-ascorbic acid-2-phosphate magnesium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL. The procedure was the same as in Comparative Example 1.

(Example 1)
In step 3, adherent stem cells were suspended in physiological saline containing 2 mM L-ascorbic acid-2-phosphate magnesium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL. The procedure was the same as in Comparative Example 1.

(Example 2)
In step 3, adherent stem cells were suspended in physiological saline containing 8 mM L-ascorbic acid-2-phosphate magnesium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL, except that the procedure was the same as in Comparative Example 1.

(Example 3)
In step 3, adherent stem cells were suspended in physiological saline containing 10 mM L-ascorbic acid-2-phosphate magnesium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL, except that the procedure was the same as in Comparative Example 1.

(Example 4)
In step 3, adherent stem cells were suspended in physiological saline containing 4 mM L-ascorbic acid-2-phosphate magnesium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a cell suspension with a cell concentration of 2 × ^10⁷ cells/mL. The procedure was the same as in Comparative Example 1.

Table 1 below lists the types of antioxidants added to each cryopreservation solution and their final concentrations.

(Evaluation of survival rate in cryopreservation solution)
The adherent stem cells from Examples 1-3 and Comparative Examples 1-4, after cryopreservation, were thawed in a 37°C water bath and left to stand at 22°C. The survival rates of each adherent stem cell were measured at 0, 3, 20, and 26 hours after thawing. The relative survival rates at 3, 20, and 26 hours after thawing, with the survival rate at 0 hours set to 100%, are shown in Table 2 and Figure 1. As shown in Table 2 and Figure 1, in Examples 1-3, the decrease in survival rate was kept within the range of 1-4% even after 26 hours, but in Comparative Examples 1-4, a decrease in survival rate of 9% or more was observed. From these results, it was shown that the cryopreservation solutions in the examples were less toxic than those in the comparative examples.

(Evaluation of proliferation)
The specific growth rate of adherent stem cells frozen using the cryopreservation solutions of Example 4 and Comparative Examples 1 and 5 was evaluated after thawing. Specifically, the frozen adherent stem cells were first thawed in a 37°C water bath and seeded at a density of 1000 live cells/ ^cm² using cell culture medium in a 75 ^cm² U-shaped flask with a canted neck vent cap (Corning). After 148 hours of culture, the adherent stem cells adhering to the flask were collected, the number of collected cells was measured, and the specific growth rate was calculated using the following formula (n=3).

Specific growth rate (h ^{- 1} ) = LN (number of harvested cells / number of seeded viable cells) / total culture time

The results are shown in Figure 2. The asterisks (*) in Figure 2 indicate a statistically significant difference at a significance level of 0.05 compared to Comparative Example 1. The results in Figure 2 demonstrate that the proliferative capacity of adherent stem cells frozen with the cryopreservation solution of Example 4 was higher than that of adherent stem cells frozen with the cryopreservation solution of Comparative Example 1.

(Adhesion evaluation)
The adhesion rate of adherent stem cells frozen using the cryopreservation solutions of Example 4 and Comparative Examples 1 and 5 was evaluated after thawing. Specifically, the frozen adherent stem cells were first thawed in a 37°C water bath and seeded in a 15 cm dish at a rate of 4 × ^10⁶ live cells/dish using cell culture medium. After 22 hours of culture, the adherent stem cells adhering to the dish were collected, the number of collected cells was measured, and the adhesion rate was calculated using the following formula. The results are shown in Table 3 and Figure 3.

Adhesion rate (%) = Number of recovered cells / (4 × ^10⁶ viable cells) × 100

As shown in Table 3 and Figure 3, the adhesion rate of adherent stem cells frozen with the cryopreservation solution of Example 4 was higher than that of adherent stem cells frozen with the cryopreservation solutions of Comparative Examples 1 and 5.

Claims (6)

A method for producing an adherent stem cell preparation, comprising the step of cryopreserving adherent stem cells in a solution containing 0.5 to 7 mM ascorbic acid derivative and 2 to 10 volume percent dimethyl sulfoxide . The production method according to claim 1, wherein the ascorbic acid derivative is an ascorbic acid phosphate ester or a salt thereof. The manufacturing method according to claim 1 or 2 , wherein the solution further contains 4 to 10% by mass of hydroxyethyl starch. The method for producing the product according to any one of claims 1 to 3 , wherein the solution further contains 0.1 to 5% by mass of human serum albumin. The manufacturing method according to any one of claims 1 to 4 , wherein the adherent stem cells are amniotic membrane-derived cells. A cell therapy agent comprising an adherent stem cell preparation obtained by the method described in any one of claims 1 to 5 as an active ingredient.