JP2023050921A - Method for manufacturing adhesive stem cell preparation - Google Patents

Abstract

To reduce damage to a cell during cryopreservation or before/after cryopreservation as much as possible by devising a composition of cryopreservation liquid in cryopreservation, and improve the quality of an adhesive stem cell preparation.SOLUTION: Manufacturing an adhesive stem cell by a manufacturing method having a process of cryopreserving an adhesive stem cell in solution containing 0.5 to 7 mM of ascorbic acid derivative improves the quality of an adhesive stem cell preparation.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for producing an adherent stem cell preparation.

In recent years, the development of regenerative medicine products using various stem cells is progressing, but in order to provide them as therapeutic preparations, a preservation method that can maintain high and stable cell quality for a long period of time is essential. be. In general, cryopreservation is performed as a method for preserving therapeutic cell preparations, their intermediate products, and raw material cells. Therefore, a component (cryoprotectant) that has the effect of protecting cells from damage due to freezing and thawing is used during freezing, and a solution containing the cryoprotectant is commercially available as a cryopreservation solution. Furthermore, addition of various antioxidants during freezing has been reported as an example of improving storage stability of cells (Patent Documents 1 and 2).

Special Table 2009-521949 Special Table 2020-516257

While antioxidants remove substances harmful to cells such as reactive oxygen species, they may damage cells, and their effects cannot always be expected. In fact, the present inventor confirmed the effect of adding ascorbic acid, which is exemplified as one of the antioxidants in Patent Document 1, to a commercially available cryopreservation solution, and found that the effect of improving the survival rate of cells. turned out not to be seen.

Therefore, in the present invention, by devising the composition of the preservation solution during cryopreservation, the damage to cells during, before, and after cryopreservation can be reduced as much as possible, and the quality of the adhesive stem cell preparation can be improved. The purpose is to provide a method.

The present inventors examined the types and appropriate concentrations of additives that can protect cells and improve viability when used during cryopreservation. By using a derivative of and using an aqueous solution containing the ascorbic acid derivative in the range of 0.5 to 7 mM as a cryopreservation solution, the decrease in cell viability after cryopreservation is surprisingly suppressed, and further, It was found that the quality of adherent stem cells was dramatically improved. That is, according to the present invention, the following method is provided.
(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 ascorbic acid derivative.
(2) The production method according to (1), wherein the ascorbic acid derivative is an ascorbic acid phosphate or a salt thereof.
(3) The production method according to (1) or (2), wherein the solution further contains a cryoprotectant.
(4) The production method according to (3), wherein 2 to 10% by volume of dimethyl sulfoxide is contained in the solution as the cryoprotectant.
(5) The production method according to (4), further comprising 4 to 10% by mass of hydroxyethyl starch in the solution as the cryoprotectant.
(6) The production method according to (4) or (5), further comprising 0.1 to 5% by mass of human serum albumin in the solution as the cryoprotectant.
(7) The production method according to any one of (1) to (6), wherein the adherent stem cells are amnion-derived cells.
(8) A cell therapy agent comprising, as an active ingredient, an adhesive stem cell preparation obtained by the method according to any one of (1) to (7).

By using the production method of the present invention, the viability of adherent stem cells after freezing and thawing can be maintained at a high level, and the proliferation of cells after freezing and thawing can be enhanced. Therefore, the present invention can be expected to contribute to improving the quality of regenerative medicine products using adherent stem cells, and to promote the use of regenerative medicine products.

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

[1] Description of Terms The term "adherent stem cells" as used herein refers to stem cells that satisfy the following definitions i) and ii), and includes "mesenchymal stromal cells" and "mesenchymal stem cells ( Mesenchymal stem cells (MSCs)” are also included in the adhesive stem cells of the present invention.

Definition of Adherent Stem Cells Herein i) Shows adherence to plastic under culture conditions in standard medium. The standard medium as used herein is a medium obtained by adding serum, a serum-replacement reagent or a growth factor (eg, human platelet lysate, which is a serum-replacement reagent) to a basal medium (eg, αMEM medium).
ii) Positive for surface antigens CD73 and CD90, and negative for CD45 and CD326.

The origin of the adhesive stem cells in the present invention is not particularly limited, and those derived from fetal appendages (amniotic membrane, umbilical cord, placenta, etc.), bone marrow fluid, adipose tissue, dental pulp, etc. can be used, but are preferably derived from amniotic membrane. is.

As used herein, the term "proliferative ability" refers to the ability of cells to increase through cell division. In the present specification, "high proliferative potential" can be used interchangeably with "high proliferative potential". The expansion potential of adherent stem cell populations can be assessed using specific growth rate, doubling number, doubling time, and/or passage number. A method for measuring the specific growth rate is described later in this specification.

The term "ascorbic acid" used herein is represented by the IUPAC name (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one. , L-ascorbic acid as well as its isomers and salts thereof. 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, L-, D-, or racemic forms of erythro-ascorbic acid, 6-desoxyascorbic acid, etc., or salts thereof. However, these "ascorbic acid derivatives", which are those in which the functional group of ascorbic acid is substituted, are not included.

The term "ascorbic acid derivative" used herein refers to a functional group of the above "ascorbic acid" substituted with fatty acid, phosphoric acid, sugar, glycerin, or the like. Examples of the ascorbic acid derivative include, but are not limited to, ethyl ascorbic acid, ascorbic acid palmitate, fatty acid esters such as ascorbic acid stearate, ascorbic acid-2-phosphate, ascorbic acid-3-phosphate, and ascorbic acid. Phosphate esters such as acid-6-phosphate and ascorbic acid-2-polyphosphate, ascorbic acid esters such as ascorbic acid-2-sulfate, and ascorbic acid-2-glucoside. These may be L-forms, D-forms or racemic forms. Further, ascorbic acid derivatives herein also include salts thereof. Salts of ascorbic acid derivatives include sodium salts, potassium salts, magnesium salts, calcium salts, barium salts, ammonium salts, monoethanolamine salts, diethanolamine salts, triethanolamine salts, monoisopropanolamine salts, and triisopropanolamine salts. is mentioned. In the present invention, the ascorbic acid derivative is preferably an ascorbic acid phosphate or a salt thereof, more preferably an ascorbic acid-2-phosphate or a salt thereof, and an L-ascorbic acid-2-phosphate or a salt thereof. is more preferred.

As used herein, "cryoprotectant" refers to a chemical that facilitates the cryoprotection process by reducing cell damage during freezing and thawing. Cryoprotectants can be cell-permeant or non-permeant. Examples of cryoprotectants include, but are not limited to, dehydrating agents, osmotic agents and vitrifying solutes. The above cryoprotectants include acetamide, agarose, alginate, l-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, dextran, diethylene glycol, dimethylacetamide, dimethylformamide, dimethylsulfoxide (DMSO). , erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, α-glycerophosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl Acetamide, methylformamide, methylureas, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine-N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sulfuric acid Sodium, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine, xylose and the like can be used, but are not particularly limited.

As used herein, the term "cryopreservation solution" refers to a solution used to suspend cells during cryopreservation, preferably an aqueous solution. As described later, the "cryopreservation solution of the present invention" is a solution containing the above-mentioned ascorbic acid derivative, but when it is simply described as "cryopreservation solution", not only the cryopreservation solution of the present invention Those that are not cryopreservation solutions of the present invention are also included.

The "adherent stem cell preparation" of the present invention is not particularly limited as long as it is a preparation containing adhesive stem cells as a main component, and its use is not only medical use such as treatment and prevention, but also research and test use. It may also be an intermediate raw material (such as cell stock) for manufacturing cell products used in these applications.

[2] Step of Cryopreservation of Adherent Stem Cells of the Present Invention The present invention provides the production of an adherent stem cell formulation comprising at least one step of cryopreserving adherent stem cells in a solution containing 0.5 to 7 mM ascorbic acid derivative. The method. That is, in the production method of the present invention, a solution containing 0.5 to 7 mM ascorbic acid derivative is used as a cryopreservation solution. The cryopreservation solution of the present invention is not particularly limited as long as it is a solution containing 0.5 to 7 mM ascorbic acid derivative, but preferably an ascorbic acid derivative dissolved in water or an aqueous solution. Examples of the aqueous solution used in this case include buffer solutions, isotonic solutions, hypotonic solutions, and hypertonic solutions. Buffers such as acid-buffered saline (PBS), balanced salt solutions such as Hank's balanced salt solution (HBSS) and Earle's balanced salt solution (EBSS), transfusions such as Ringer's solution, lactated Ringer's solution, and physiological saline, culture solutions, etc. Preferred examples include:

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

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

Although the cryoprotectant is not particularly limited, dimethylsulfoxide is preferred. The concentration when adding dimethyl sulfoxide to the cryopreservation solution of the present invention is not particularly limited. %, more preferably 8% by volume or less, more preferably 6% by volume or less, the cytotoxicity can be further reduced.

The cryopreservation solution of the present invention may further contain hydroxyethyl starch as a cryoprotectant. Although the concentration of hydroxyethyl starch in this case is not particularly limited, it is preferably 4% by mass or more. More preferably 10% by mass or less, more preferably 8% by mass or less, the viscosity of the cryopreservation solution can be reduced and the operability can be improved.

The cryopreservation solution of the present invention may further contain human serum albumin as a cryoprotectant. Although the concentration is not particularly limited, it is preferably 0.1% by mass or more, more preferably 3.5% by mass or more, and the upper limit is preferably 5% by mass or less. If it is this range, quality will improve more.

In the production method of the present invention, the method of suspending adherent stem cells in the cryopreservation medium of the present invention is not particularly limited. A cryopreservation solution containing an ascorbic acid derivative and, if necessary, a cryoprotectant and other components may be prepared, and adherent stem cells may be directly suspended therein, or an aqueous solution containing a cryoprotectant and the like and ascorbic acid may be prepared. Cell suspensions containing derivatives can be mixed in arbitrary proportions to prepare solutions within the scope of the present invention. A commercially available cryopreservation solution can also be used as the aqueous solution containing a cryoprotectant and the like.

The cell concentration (final concentration) in the cryopreservation solution is not particularly limited, but is preferably 2×10 ⁸ cells/mL or less, more preferably 5×10 ⁷ cells/mL or less, and the cryopreservation solution of the present invention The quality improvement effect by is more effectively exhibited. Moreover, from the viewpoint of productivity efficiency, the lower limit of the cell concentration is preferably 5×10 ⁴ cells/mL or more.

In the production method of the present invention, the time from suspension of adherent stem cells in the cryopreservation solution to initiation of freezing is not particularly limited, but the time to initiation of freezing is preferably within 24 hours, more preferably. Within 12 hours, more preferably within 6 hours, the quality of the thawed cells can be further improved. The lower limit is not particularly limited, and it is preferable that it is as short as possible in the manufacturing process, but when using the cryopreservation solution of the present invention, if it is about 0.5 hours, there is no problem with cell viability and quality. can be maintained reasonably well.

In the production method of the present invention, cryopreservation can be performed, for example, by a slow freezing method using a program freezer or a simplified freezing method using a deep freezer. In the slow freezing method using a program freezer, cooling can be started at a planned rate of -1 to -3°C/min, for example. On the other hand, in the simple freezing method, for example, a cryovial or a freezing bag filled with a cell suspension can be placed in a cell freezing container or a styrofoam container and placed in a deep freezer for slow freezing. Alternatively, after freezing by the freezing method described above, the cells may be transferred to liquid nitrogen and stored. . Containers filled with cell suspensions for cryopreservation include Nunc cryotube 1.8 mL inner cap (manufactured by Thermo Fisher Scientific), inner screw cryogenic vial with barcode (manufactured by Corning), flow tube T- 1.5 (manufactured by Nipro Corporation), and Flo's Bag (manufactured by Nipro Corporation), etc., but are not particularly limited. BICELL (manufactured by Nippon Freezer Co., Ltd.) and CoolCell (manufactured by Corning), for example, can be used as a cell freezing container used in the simple freezing method.

The adherent stem cells that have been cryopreserved using the cryopreservation medium of the present invention may be used as they are after thawing, or may be cultured one or more times after thawing to prepare an adhesive stem cell preparation. It may also be sold or provided to medical institutions or the like as an adherent stem cell preparation in a frozen state. From the viewpoint of sufficiently exhibiting the objects and effects of the present invention, the cryopreservation step using the cryopreservation medium of the present invention is preferably carried out in an intermediate step in the production of cell preparations. In addition, the cryopreservation step using the cryopreservation solution of the present invention may be performed multiple times in the manufacturing process of the adhesive stem cell preparation.

Since the period of cryopreservation in the production method of the present invention hardly affects the effects of the present invention, any desired cryopreservation period can be selected in terms of the process. Therefore, it is not particularly limited, but for example, 1 day or more, 2 days or more, 3 days or more, 1 week or more, 10 or more days, 20 days or more, 1 month or more, for example, 5 years or less, 3 years or less, 2 years or less, 1 year or less, 6 months or less.

In the production method of the present invention, the method for thawing adherent stem cells cryopreserved by the cryopreservation method is, for example, by placing a container filled with frozen cells in a hot water bath heated to 35 to 37°C. can be implemented. Alternatively, the cells may be thawed using a cell thawing apparatus, such as ThawSTAR (manufactured by BIOLIFE SOLUTIONS), VIA Thaw CB1000 (manufactured by Cytiva), and frozen cell thawing apparatus YS series (Strex Inc.). can be used.

[3] Method for Producing Adhesive Stem Cell Preparation of the Present Invention The method for producing the adhesive stem cell preparation of the present invention is characterized by including the step of cryopreservation of the adherent stem cells, and other conditions and configurations are not limited at all. For example, the method for producing an adherent stem cell preparation of the present invention can include a cell population obtaining step, a culturing step, and the like as optional steps in addition to the adherent stem cell cryopreservation step described above.

In the production method of the present invention, the cell population obtaining step may be, for example, a cell population obtaining step of obtaining a cell population containing adherent stem cells by enzymatically treating fetal appendages such as amniotic membranes or adipose tissue. good. The above cell population acquisition step may include a step of obtaining an amniotic membrane by caesarean section. Alternatively, the step of obtaining a cell population may include a step of aspirating or excising adipose tissue from a living body. Furthermore, the above cell population acquisition step may include a step of washing a biological sample containing adherent stem cells.

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

For example, specifically, the enzymatic treatment of a biological sample (preferably a biological sample containing an epithelial cell layer and an adherent stem cell layer) taken from a fetal appendage is preferably performed in the extracellular matrix layer of the fetal appendage. treatment with enzymes (or a combination thereof) that are capable of releasing adherent stem cells and that do not degrade the epithelial cell layer. Examples of such enzymes include, but are not limited to, collagenase and/or metalloproteinase. Metalloproteinases include, but are not limited to, thermolysin and/or dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids.

Although the activity concentration of collagenase is not particularly limited, it is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, and still more preferably 200 PU/ml or more. The upper limit is also not particularly limited, but is, for example, 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 (eg, thermolysin and/or dispase) is not particularly limited, but is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, still more preferably 200 PU/ml or more. Further, the upper limit is preferably 1000 PU/ml or less, more preferably 900 PU/ml or less, still more preferably 800 PU/ml or less, still more preferably 700 PU/ml or less, still more preferably 600 PU/ml or less, still more preferably 500 PU/ml. It is below. Here, in the embodiment using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that releases an amino acid equivalent to 1 μg of tyrosine per minute from lactic casein at pH 7.5 and 30°C. be. Within the above enzyme concentration range, adherent stem cells contained in the extracellular matrix layer can be efficiently released while preventing contamination with epithelial cells contained in the epithelial cell layer of the fetal appendage. Preferred concentration combinations of collagenase and/or metalloproteinases can be determined by microscopic observation of fetal appendages after enzymatic treatment and by flow cytometry of the obtained cells.

From the viewpoint of efficiently collecting living cells, it is preferable to treat fetal appendages with a combination of collagenase and metalloproteinase. More preferably, the fetal appendages are treated all at once by the combination. As the metalloproteinase in this case, thermolysin and/or dispase can be used, but not limited thereto. Adherent stem cells can be conveniently obtained by treating fetal appendages only once with an enzyme solution containing both collagenase and metalloproteinase. In addition, the simultaneous batch processing can reduce the risk of contamination with bacteria, viruses, and the like.

The 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 Hank's balanced salt solution, in the enzyme solution and stirring the enzyme solution with a stirring means. From the viewpoint of efficiently liberating the adhesive stem cells contained in the extracellular matrix layer of the amniotic membrane, a stirrer or shaker, for example, can be used as such a stirring means, but is not limited thereto. The lower limit of the stirring speed when using a stirrer or shaker is not particularly limited, but is preferably 5 rpm, more preferably 10 rpm, because if the stirring speed is too slow, the enzymatic treatment efficiency decreases. The upper limit is not particularly limited, but it is preferably 100 rpm, more preferably 60 rpm, because too high stirring speed may damage cells. The lower limit of the enzymatic treatment time is not particularly limited, but if it is too short, the adherent stem cells contained in the amniotic membrane cannot be sufficiently separated, so it is preferably 30 minutes, and more preferably 45 minutes. In addition, 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. is. Although the lower limit of the enzymatic treatment temperature is not particularly limited, it is preferably 15°C, more preferably 25°C, and still more preferably 35°C in order to allow the enzymatic reaction to proceed efficiently. The upper limit of the enzymatic treatment temperature is not particularly limited, but if the temperature is too high, the adherent stem cells will die and the enzyme will be deactivated, so the temperature is preferably 40°C.

Adhesive stem cells can also be isolated from tissues other than amniotic membrane according to the above method or by known methods.

In the production method of the present invention, if desired, the released adhesive stem cells are separated and/or collected from the enzyme solution containing the released adhesive stem cells by a known method such as a filter, centrifugation, hollow fiber separation membrane, cell sorter, or the like. can do. Preferably, the enzyme solution containing adherent stem cells released by a filter is filtered. In the embodiment in which the enzyme solution is filtered through a filter, only the released cells pass through the filter, and the undegraded epithelial cell layer remains on the filter without passing through the filter. Not only can separation and/or recovery be possible, but the risk of contamination with bacteria, viruses, etc. can also be reduced. Examples of filters include, but are not limited to, mesh filters. The pore size (mesh size) of the mesh filter is not particularly limited, but is, for example, 40 μm or more, 60 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but is, for example, 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. The filtration rate is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzymatic solution containing the adherent stem cells can be filtered by gravity, thereby preventing a decrease in cell viability. can be done.

Nylon is preferably used as the material of the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm or 100 μm nylon mesh filters are available, such as the Falcon cell strainer commonly used for research. In addition, medical mesh cloths (nylon and polyester) used in hemodialysis and the like can be used. Furthermore, an arterial filter (polyester mesh filter, pore size: 40 μm or more and 120 μm or less) used for extracorporeal circulation can also be used. Other materials such as stainless steel mesh filters can also be used.

When the adherent stem cells are passed through a filter, natural fall (free fall) is preferred. Forced passage through the filter, such as suction using a pump or the like, is also possible, but it is desirable to use as weak a pressure as possible in order to avoid damaging the cells.

Adherent stem cells that have passed through the filter can be recovered by centrifugation after diluting the filtrate with a double or greater volume of medium or balanced salt buffer. As the balanced salt buffer, physiological saline, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), phosphate buffer (PBS) and the like can be used. is not limited to

The culturing step in the method for producing an adhesive stem cell preparation of the present invention may be a step of culturing a cell population containing the adhesive stem cells obtained in the above steps, or a step of culturing the adhesive stem cells obtained by another method. It may be a step of culturing a cell population containing.

The cell seeding density in the step of culturing the cell population containing the adherent stem cells is not particularly limited, but can be, for example, 500 to 10,000 cells/cm ² . The lower limit of the seeding density is preferably 550 cells/cm ² , more preferably 750 cells/cm ² , still more preferably 1,000 cells/cm ² . Also, the upper limit of the seeding density is not particularly limited, but is preferably 7,000 cells/cm ² , still more preferably 5,000 cells/cm ² .

In addition, the step of culturing may include a subculturing step, or may include a step of repeating culturing a plurality of times under different culture conditions.

The culture period of the single culture can be, for example, 2 to 15 days, more specifically, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days Days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days can be mentioned.

The medium used for the above culture can be prepared by using any liquid medium for animal cell culture as a basal medium and optionally adding other components (serum, serum replacement reagent, growth factor, etc.). . In the embodiment in which the growth factor is added to the basal medium, a reagent (such as heparin) for stabilizing the growth factor in the medium may be added in addition to the growth factor. Alternatively, the growth factor may be stabilized in advance with gel or polysaccharide, and then the stabilized growth factor may be added to the basal medium.

The basal medium includes 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 Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM/F12 medium ( A medium such as Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham)) can be used, but is not particularly limited. αMEM medium can be exemplified as a preferable basal 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 medium include, for example, albumin, bovine serum, serum-replacement reagents, growth factors, etc. Among them, serum-replacement reagents are preferred, and platelet lysate is particularly preferred. Among others, it is preferable to culture in a basal medium containing a serum replacement reagent and free of albumin, bovine serum and growth factors. The lower limit of the concentration of the platelet lysate in the medium is, for example, a final concentration of 1% by volume or more, preferably 2% by volume or more, more preferably 3% by volume or more. The upper limit of the concentration of the platelet lysate in the medium is, for example, 20% by volume or less, preferably 10% by volume or less, more preferably 7% by volume or less.

Cultivation of a cell population containing adherent stem cells can be performed, for example, by the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and cultured in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less using the medium described above. The cells obtained by culturing as described above are cells that have been cultured once.

For example, the cells that have been cultured once can be further subcultured and cultured as follows. First, the cells that have been cultured once are treated with a cell detachment means to be detached from the plastic culture vessel. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in medium. Finally, the cells are seeded in a plastic culture vessel and cultured in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less using a medium so that the confluency rate is 95% or less. Examples of the medium include, but are not limited to, αMEM, M199, or media based on these. Cells obtained by passage and culture as described above are cells that have been passaged once. By performing similar passages and cultures, cells that have been passaged n times can be obtained (n is an integer of 1 or more). The lower limit of the number of passages n is, for example, 1 time, preferably 2 times, from the viewpoint of mass production of cells. In addition, the upper limit of the passage number n is preferably 20 times or 10 times, for example, from the viewpoint of suppressing aging of cells. For example, a cell detachment agent may be used as the cell detachment means. As the cell detachment agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) and the like can be used, but are not particularly limited. A commercially available cell detachment agent may be used as the cell detachment agent. 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). Moreover, as the cell detachment means, a physical cell detachment means may be used, for example, a cell scraper (manufactured by Corning) can be used, but it is not limited to this. The cell detachment means may be used singly or in combination.

In the method for producing an adhesive stem cell preparation of the present invention, the culture step can be performed any number of times. In addition, the cryopreservation step using the cryopreservation solution of the present invention can be freely combined. For example, after culturing the cell population of adherent stem cells obtained in the cell population obtaining step, the cryopreservation may be performed using the cryopreservation solution of the present invention, and then the culturing step may be performed again. and cryopreservation steps may be repeated multiple times.

[4] Cell therapy agent The adherent stem cell preparation produced by the production method of the present invention can be used as an active ingredient of a therapeutic agent for intractable diseases. That is, a cell therapeutic agent containing, as an active ingredient, the adhesive stem cell preparation produced by the production method of the present invention is also one aspect of the present invention. The adhesive stem cell preparation produced by the production method of the present invention may be further cultured and used as an active ingredient of the cell therapeutic agent of the present invention.

The cell therapy agent of the present invention can be used for immune-related diseases, muscular dystrophy, ischemic diseases, leg ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, and inflammatory bowel disease. , Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular palsy, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, Epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defect, meniscus injury, deelastic osteochondrosis, atrophic osteonecrosis, Knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye disease, angiogenesis-related disease, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wound, epithelial injury , fibrosis, lung disease, and cancer.

The cell therapeutic agent of the present invention may be diluted with a pharmaceutically acceptable medium. The above 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 infusion formulation, for example, water for injection, physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, amino acid solution, starting solution (No. 1 solution), dehydration replenishment solution (No. 2 solution), maintenance infusion solution (No. 3 solution), postoperative recovery solution (No. 4 solution), Plasma-Lyte A (registered trademark), etc., but are not limited thereto. .

A "patient or subject" as used herein is typically a human, but may be other animals. Other animals include, for example, dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese monkeys), ferrets, rabbits, rodents (mouse, rats, gerbils, guinea pigs, mammals such as hamsters), and birds such as chickens and quails, but are not limited thereto.

"Treatment" as used herein includes, for example, patient or subject life prognosis, functional prognosis, survival rate, weight loss, anemia, diarrhea, melena, abdominal pain, fever, loss of appetite, malnutrition, vomiting, fatigue, rash , inflammation, ulcer, erosion, fistula, stenosis, intestinal obstruction, internal bleeding, rectal bleeding, spasm, pain, decreased liver function, decreased cardiac function, decreased lung function, motor function, muscle weakness, or blood test items. include, but are not limited to, significantly improving the

The cell therapeutic agent of the present invention may contain any component used in the treatment of a patient or subject. Examples of the above components include salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts), aqueous solutions containing them (e.g., physiological saline, Ringer's solution, Bikanate Infusion), polysaccharides (e.g., hydroxylethyl starch). , dextran, etc.), proteins (eg, albumin, etc.), dimethylsulfoxide, amino acids, medium components (eg, components contained in RPMI 1640 medium, etc.), and the like, but are not limited thereto.

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

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

Also, the above other pharmaceutical ingredients may be other cells that can be administered. Other cells that can be administered include blood-derived cells (leukocytes, erythrocytes, mononuclear cells, etc.), vascular endothelial cells, vascular endothelial progenitor cells, pericytes, vascular wall cells, fibroblasts, skeletal myoblasts, and epithelial cells. cells, stromal cells, mature adipocytes, etc., but are not particularly limited.

The pH of the cell therapy agent of the present invention can be around neutral pH, for example, pH 5.5 or higher, pH 6.5 or higher, or pH 7.0 or higher. 0.5 or less or pH 8.0 or less, but are not limited to these.

The concentration of adherent stem cells in the cell therapeutic agent of the present invention should be such that when administered to a patient or subject, a therapeutic effect on the disease can be obtained compared to a patient or subject who has not been administered. concentration. A specific cell concentration can be appropriately determined depending on the dosage form, administration method, purpose of use, age, body weight and symptoms of the patient or test subject. The lower limit of the cell concentration ^{of the} cell therapeutic agent of the present invention ^is not particularly limited. mL or more, 1.4×10 ⁶ /mL or more, 1.6×10 ⁶ /mL or more, 1.8×10 ⁶ /mL or more, 2.0×10 ⁶ /mL or more, 3.0 ×10 ⁶ /mL or more, 4.0 × 10 ⁶ /mL or more, 5.0 × 10 ⁶ /mL or more, 6.0 × 10 ⁶ /mL or more, 7.0 × 10 ⁶ /mL 8.0×10 ⁶ /mL or more, 9.0×10 ⁶ /mL or more, 9.5×10 ⁶ /mL or more, or 1.0×10 ⁷ /mL or more. ^The upper limit of the cell concentration ^of the cell therapeutic agent of the present invention ^is not particularly limited, but is mL or less, 6.0×10 ⁸ cells/mL or less, 4.0×10 ⁸ cells/mL or less, 2.0×10 ⁸ cells/mL or less, or 1.0×10 ⁸ cells/mL or less.

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

Also, the liquid formulation may be a suspension of cells or a liquid preparation in which cells are dispersed in a liquid formulation. Furthermore, the form of the cells contained in the liquid preparation is not particularly limited, and may be, for example, single cells or cell aggregates. Needless to say, the liquid agent can be used for coating, sticking, and spraying.

Moreover, according to one aspect of the present invention, the cell therapeutic agent of the present invention may be a preparation for transplantation. The preparation for implantation is a solid or gel preparation. For example, the solid preparation for implantation includes a preparation for implantation having a sheet-like structure or a pellet structure. In addition, as a preparation for implantation with a gel-like structure, for example, in International Publication WO2017/126549, a preparation for implantation containing a gel obtained by adhering separated cells with an adhesive (e.g., fibrinogen) is known. It is Moreover, according to one aspect of the present invention, the cell therapeutic agent of the present invention may be a gel formulation in which cells and any gel are mixed. As a gel preparation, for example, a cell therapeutic agent composed of an adhesive stem cell-hydrogel composition is known in JP-A-2017-529362. The cell therapeutic agent of the present invention can also be made into a gel formulation, for example, by using the method described in the above literature.

In addition, as a preparation for transplantation with a sheet-like structure, for example, in International Publication WO2006/080434, Japanese Patent Application Laid-Open No. 2016-52272, etc., a temperature-responsive culture dish (for example, UpCell (registered trademark) (manufactured by Cellseed Co., Ltd.) ), a laminate of a sheet-like cell culture and fibrin gel, and a cell-coated sheet obtained by applying a cell suspension to a sheet-like substrate. The cell therapy agent of the present invention can also be made into various sheet-like structures for transplantation, for example, by using the methods described in the above documents.

The administration method of the cell therapy agent of the present invention is not particularly limited, but examples include subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrapleural injection, Local direct injection, direct application, or local direct transplantation may be mentioned. According to one aspect of the present invention, a liquid preparation for injection is filled in a syringe and injected intravenously, intraarterially, intramyocardially, intrasegmentally, intrahepatic artery, intramuscularly, epidurally, or gingivally through an injection needle or a catheter. , intracerebroventricularly, subcutaneously, intradermally, intraperitoneally, and intraportally. Methods for administering cell therapeutic agents are described, for example, in Japanese Unexamined Patent Application Publication No. 2015-61520, Onken JE, t al. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121. , Garcia-Olmo D, et al. Dis Colon Rectum 2005;48:1416-23. Intravenous injection, drip intravenous injection, local direct injection, local direct transplantation, and the like are known. The cell therapeutic agent of the present invention can also be administered by various methods described in the above documents.

The dose of the cell therapeutic agent of the present invention is the amount of cells that, when administered to a patient or subject, can produce a therapeutic effect on the disease compared to a patient or subject who has not been administered. A specific dosage can be appropriately determined depending on the dosage form, administration method, purpose of use, age, body weight and symptoms of the patient or subject. A single dose of adherent stem cells to humans is not particularly limited, but is, for example, 1×10 ⁴ cells/kg body weight or more, 1×10 ⁵ cells/kg body weight or more, 5×10 ⁵ cells/kg body weight or more, 1×10 ⁶ cells/kg body weight or more, 2×10 ⁶ cells/kg body weight or more, 4×10 ⁶ cells/kg body weight or more, 6×10 ⁶ cells/kg body weight or more, or 8×10 ⁶ cells/kg body weight or more is. In addition, the dose of adherent stem cells to humans is not particularly limited, but for example, 1×10 ¹² cells/kg body weight or less, 1×10 11 cells/kg body weight or less, 1×10 10 cells/kg body weight or less, 1 x 10 ⁹ cells/kg body weight or less, 5 x 10 ⁸ cells/kg body weight or less, 1 x 10 ⁸ cells/kg body weight or less, 8 x 10 ⁷ cells/kg body weight or less, 6 x 10 ⁷ cells/kg body weight or less, 4×10 ⁷ cells/kg body weight or less, or 2×10 ⁷ cells/kg body weight or less.

When the cell therapeutic agent of the present invention is a solution for injection, a single dose of the adhesive stem cells in the solution for injection to humans is 1×10 ⁵ cells/kg body weight or more, from the viewpoint of enhancing therapeutic effects on diseases. 5×10 ⁵ cells/kg body weight or more, 1×10 ⁶ cells/kg body weight or more, 2×10 ⁶ cells/kg body weight or more, 4×10 ⁶ cells/kg body weight or more, 6×10 ⁶ cells/kg body weight or more, Alternatively, it is preferably 8×10 ⁶ cells/kg body weight or more. In addition, from the viewpoint of facilitating the preparation and administration of the injectable solution, the dose of the adhesive stem cells for injection into humans is 1×10 ⁹ cells/kg body weight or less, 5×10 ⁸ cells/kg or less. Body weight or less, 1 x 10 ⁸ cells/kg body weight or less, 8 x 10 ⁷ cells/kg body weight or less, 6 x 10 ⁷ cells/kg body weight or less, 4 x 10 ⁷ cells/kg body weight or less, or 2 x 10 ⁷ cells/kg body weight It is preferably less than kg body weight.

The frequency of administration of the cell therapy agent of the present invention is such that a therapeutic effect can be obtained for a disease when administered to a patient or subject. The specific administration frequency can be appropriately determined depending on the dosage form, administration method, purpose of use, and age, body weight and symptoms of the patient or subject. 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 administration period of the cell therapeutic agent of the present invention is a period during which a therapeutic effect can be obtained for a disease when administered to a patient or subject. The specific administration period can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, body weight and symptoms of the patient or subject. 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

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

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

(Comparative example 1)
<Step 1: Enzymatic treatment of amniotic membrane and recovery of adherent stem cells>
Egg membranes including amniotic membranes and placentas were aseptically collected from pregnant women undergoing elective caesarean section cases with informed consent. The resulting egg membrane and placenta were placed in a sterile vat containing physiological saline, and the amniotic membrane was manually stripped from the egg membrane stump. After washing the amniotic membrane with Hank's balanced salt solution (not containing Ca/Mg) to remove the attached blood and clots, Hank's balanced salt solution containing 240 PU/mL collagenase and 200 PU/mL dispase I (Ca/Mg containing), and the amniotic membrane was enzymatically treated by shaking and stirring at 37° C. for 60 minutes at 10 rpm. The enzyme-treated solution was filtered through a nylon mesh with an opening of 95 μm to remove undigested amniotic membrane, and a cell suspension containing adherent stem cells was recovered.

<Step 2: Culture and collection of adherent stem cells>
The cell population containing adherent stem cells obtained in step 1 is seeded in a 75 cm ² U-shaped Kantneck cell culture flask (vent cap) (manufactured by Corning) in a culture vessel at a density of 1,000 cells/cm ² , Culture was performed 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 a trypsin-EDTA solution. Detached cells were washed with 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 2×10 ⁷ cells/mL. A solution (A) containing 10% by volume of dimethylsulfoxide, 12% by mass of hydroxyethyl starch and 8% by mass of human serum albumin was separately prepared and mixed with the cell suspension at a ratio of 1:1.

<Step 4: Freezing>
A Nunc cryotube 1.8 mL inner cap (manufactured by Thermo Fisher Scientific) was filled with 1 mL of the mixture of the cell suspension prepared in step 4 and the solution (A). Immediately after that, the cryovial filled with the mixed solution was placed in a CoolCell (manufactured by Corning) and allowed to stand in a deep freezer at −80° C. for 24 hours to freeze. After freezing, the cells were transferred into a liquid nitrogen storage container and stored in the liquid nitrogen liquid phase for 10 days.

(Comparative example 2)
In step 3, the adherent stem cells were suspended in physiological saline containing 2 mM L-ascorbic acid sodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a cell suspension with a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for the preparation.

(Comparative Example 3)
In step 3, the adherent stem cells were suspended in physiological saline containing 8 mM L-ascorbic acid sodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a cell suspension with a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for the preparation.

(Comparative Example 4)
In step 3, the adherent stem cells were suspended in physiological saline containing 10 mM L-ascorbic acid sodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a cell suspension with a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for the preparation.

(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 Fuji Film Wako Pure Chemical Industries, Ltd.) to give a cell concentration of 2×10 ⁷ cells/cell. The same operation as in Comparative Example 1 was performed except for preparing mL of cell suspension.

(Example 1)
In step 3, the 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 give a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for preparing a cell suspension.

(Example 2)
In step 3, the 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 give a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for preparing a cell suspension.

(Example 3)
In step 3, the 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 give a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for preparing a cell suspension.

(Example 4)
In step 3, the 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 give a cell concentration of 2×10 ⁷ cells/mL. The same operation as in Comparative Example 1 was performed except for preparing a cell suspension.

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

(Evaluation of survival rate of cryopreservation solution)
The adherent stem cells after cryopreservation in Examples 1 to 3 and Comparative Examples 1 to 4 above were thawed in a 37°C hot water bath, left to stand under 22°C temperature conditions after thawing, and 0, 3, The survival rate of each adherent stem cell at 20 and 26 hours was measured. Table 2 and FIG. 1 show the relative viability at 3, 20, and 26 hours after thawing when the viability at 0 hours is defined as 100%. As shown in Table 2 and FIG. 1, in Examples 1 to 3, the decrease in survival rate was suppressed to a range of 1 to 4% even after 26 hours, but in Comparative Examples 1 to 4, it was 9% or more. Decrease in survival rate was observed. These results indicate that the cryopreservation solutions of Examples are less toxic than those of Comparative Examples.

(Proliferative evaluation)
The specific proliferation rate of adherent stem cells frozen using the cryopreservation solutions of Example 4 and Comparative Examples 1 and 5 was evaluated after thawing. Specifically, first, frozen adherent stem cells were thawed in a 37°C water bath, and placed in a 75 cm ² U-shaped flask with a cant neck vent cap (manufactured by Corning) at a density of 1000 viable cells/cm ² using a cell culture medium. and sown. After culturing for 148 hours, the adherent stem cells adhering to the flask were collected, the number of collected cells was measured, and the specific growth rate was determined using the following formula (n=3).

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

The results obtained are shown in FIG. * in FIG. 2 indicates that there is a significant difference at the significance level of 0.05 with respect to Comparative Example 1. From the results of FIG. It was shown to be higher than the proliferation of adherent stem cells frozen in the cryopreservation solution of Example 1.

(Adhesion evaluation)
Adhesion rates of adherent stem cells frozen using the cryopreservation solutions of Example 4 and Comparative Examples 1 and 5 were evaluated after thawing. Specifically, first, frozen adherent stem cells were thawed in a 37° C. water bath and seeded in a 15 cm dish at 4×10 ⁶ viable cells/dish using a cell culture medium. After culturing for 22 hours, the adherent stem cells adhering to the dish were recovered, the number of recovered cells was measured, and the adhesion rate was calculated using the following formula. The results obtained are shown in Table 3 and FIG.

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

As shown in Table 3 and FIG. 3, the adhesion rate of the adherent stem cells frozen in the cryopreservation medium of Example 4 is higher than that of the adherent stem cells frozen in the cryopreservation medium of Comparative Examples 1 and 5. It has been shown.

Claims (8)

A method for producing an adherent stem cell formulation, comprising the step of cryopreserving adherent stem cells in a solution containing 0.5 to 7 mM of an ascorbic acid derivative. 2. The production method according to claim 1, wherein the ascorbic acid derivative is an ascorbic acid phosphate or a salt thereof. 3. The manufacturing method according to claim 1 or 2, further comprising a cryoprotectant in said solution. 4. The production method according to claim 3, wherein 2 to 10% by volume of dimethyl sulfoxide is contained in the solution as the cryoprotectant. The production method according to claim 4, further comprising 4 to 10% by mass of hydroxyethyl starch in the solution as the cryoprotectant. The production method according to claim 4 or 5, further comprising 0.1 to 5% by mass of human serum albumin in the solution as the cryoprotectant. The production method according to any one of claims 1 to 6, wherein the adherent stem cells are amnion-derived cells. A cell therapy agent comprising, as an active ingredient, an adhesive stem cell preparation obtained by the method according to any one of claims 1 to 7.

Images