Method for producing amniotic membrane-derived adherent stem cells

Abstract

To provide production methods in which the growth rate during culture of adherent stem cells obtained from an amnion is increased by devising a method of preserving the amnion before culturing, to reduce the manufacturing costs.SOLUTION: By preserving an amnion in a solution in which platelet lysate (PL) is added to the solution for preserving the amnion, the proliferation during culture of adherent stem cells collected from the amnion is enhanced.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing amniotic membrane-derived adherent stem cells. [Background technology]

[0002] Adherent stem cells, also known as mesenchymal stromal cells, are somatic stem cells that have been reported to exist in bone marrow, fat, and dental pulp, and have recently been found to be present in the amniotic membrane. Furthermore, these adherent stem cells possess immunosuppressive capabilities, and their practical application is progressing in the treatment of acute graft-versus-host disease (GVHD) and Crohn's disease, an inflammatory bowel disease.

[0003] In recent years, with rising medical costs, there has been a growing emphasis on the medical economics of expensive pharmaceuticals, such as biopharmaceuticals. Reducing the manufacturing cost of cell therapies is crucial for the realization and widespread adoption of cell-based therapies that use living cells as active ingredients. The manufacturing process for cell therapies includes a cell expansion culture process to amplify cells to the required number. This expansion culture process generally takes several days to several weeks, during which time the culture medium must be changed, necessary components added, and CO2 concentration and temperature adjusted to maintain an optimal culture environment. Therefore, by shortening the expansion culture period by increasing the cell proliferation rate, it is possible to increase the annual production volume and significantly reduce culture costs, thereby lowering the price of cell therapies. Patent Document 1 discloses a method using αMEM medium containing human platelet lysate in the culture process as an example of a method to increase the proliferation rate of adherent stem cells. In this way, attempts are being made to increase the proliferation rate of mesenchymal stem cells and reduce manufacturing costs by improving the culture method. However, there are limitations to increasing the proliferation rate solely through improvements in the culture method. On the other hand, a technique for preserving amniotic membrane has been disclosed, which involves preserving the amniotic membrane in a culture medium at 2-8°C for up to 48 hours (Patent Document 2). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International release WO2019 / 132025 [Patent Document 2] Japanese Patent Publication No. 2015-61520 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a manufacturing method that improves the proliferation rate of adherent stem cells obtained from amniotic membranes during culture and reduces manufacturing costs by improving the method of preserving the amniotic membrane before culture. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors of this invention have discovered a novel method of using platelet lysate (PL) by preserving the amniotic membrane in a solution to which PL is added, thereby increasing the proliferative capacity of adherent stem cells collected from the amniotic membrane during culture. This invention was completed based on this finding. In other words, the following inventions are provided according to this specification.

[0007] (1) A method for producing amniotic membrane-derived adherent stem cells or a cell preparation containing them, comprising the step of preserving the amniotic membrane in a solution containing platelet lysate. (2) The manufacturing method according to (1), wherein the concentration of platelet lysate in the solution is 2 to 20 v / v%. (3) The method for producing the amniotic membrane according to (1) or (2), wherein the solution for preserving the amniotic membrane contains a basal culture medium. (4) The manufacturing method according to any one of (1) to (3), wherein the time for storing the amniotic membrane in the solution is 1 to 130 hours. (5) The manufacturing method according to any one of (1) to (4), wherein the temperature at which the amniotic membrane is stored in the solution is 1 to 40°C. (6) After the step of preserving the amniotic membrane, (a) a step of isolating amniotic membrane-derived adherent stem cells from the preserved amniotic membrane, and (b) The process of culturing the isolated amniotic membrane-derived adherent stem cells. A manufacturing method according to any one of (1) to (5), further comprising the above. (7)(a) A step of separating amniotic membrane-derived adherent stem cells from amniotic membrane stored in a solution containing platelet lysate, and (b) The process of culturing the isolated amniotic membrane-derived adherent stem cells. A method for producing amniotic membrane-derived adherent stem cells or cell preparations containing them. (8) The manufacturing method according to (7), wherein the concentration of platelet lysate in the solution is 2 to 20 v / v%. (9) The method of production according to (7) or (8), wherein the solution for preserving the amniotic membrane contains a basal culture medium. (10) The manufacturing method according to any one of (7) to (9), wherein the amniotic membrane is stored in the solution for 1 to 130 hours. (11) The manufacturing method according to any one of (7) to (10), wherein the amniotic membrane is stored in the solution at a temperature of 1 to 40°C. [Effects of the Invention]

[0008] The method of the present invention can enhance the proliferative capacity of adherent stem cells obtained from amniotic membrane during culture. This enables the rapid and low-cost production of cell therapies using amniotic membrane-derived adherent stem cells. [Modes for carrying out the invention]

[0009] [1] Explanation of terms In this specification, "amnion" refers to the transparent, vascularly sparse membrane that forms the innermost layer of the egg membrane. The inner layer of the amnion (also called the epithelial cell layer) is covered with a single layer of epithelial cells that have secretory function and secrete amniotic fluid. The outer layer of the amnion (also called the extracellular matrix layer and corresponding to the stroma) contains adherent stem cells. In this specification, the amnion may be taken from any mammal. Examples of mammals include humans, dogs, monkeys, birds, cattle, mice, rats, cats, guinea pigs, rabbits, and rodents, but humans are preferred.

[0010] As used herein, "adhesive stem cells" refer to stem cells that meet the following definitions i) and ii), and "mesenchymal stromal cells" and "mesenchymal stem cells (MSC)" are also included in the adhesive stem cells of the present invention.

[0011] Definition of Adhesive Stem Cells in this Specification i) Under culture conditions in a standard medium, it shows adhesion to a hydrophobic substrate, such as plastic. The standard medium referred to here is a medium obtained by adding serum, a serum replacement reagent, or a growth factor (e.g., human PL, which is a serum replacement reagent) to a basal medium (e.g., αMEM medium). ii) It is positive for surface antigens CD73 and CD90 and negative for CD45 and CD326.

[0012] As used herein, "amnion-derived adhesive stem cells" refer to amnion-derived stem cells that meet the above definitions i) and ii). Further, the amnion-derived adhesive stem cells of the present invention are not particularly limited with respect to the presence or absence of the ability to differentiate into bone, cartilage, fat, etc. For example, the amnion-derived adhesive stem cells include cells that have the ability to differentiate into bone, cartilage, and fat, like mesenchymal stem cells. Also, the amnion-derived adhesive stem cells include cells that meet the above definition but do not have the ability to differentiate into bone, cartilage, and fat. Further, the amnion-derived adhesive stem cells include cells that meet the above definition but can only differentiate into any one or two of bone, cartilage, and fat.

[0013] As used herein, "proliferation ability" refers to the ability of cells to increase by undergoing cell division. In this specification, "high proliferation ability" can be used interchangeably with "high proliferative ability". The proliferation ability of an amnion-derived adhesive stem cell population can be evaluated using the specific growth rate, doubling number, doubling time, and / or passage number. The method for measuring the specific growth rate is as described later in this specification.

[0014] In this specification, "platelet lysate (PL)" (often abbreviated as "PL") refers to a cell culture additive containing multiple growth factors and chemokines as components of platelets derived from blood. The animal species from which PL is derived is not particularly limited, but is preferably human. Examples of the growth factors mentioned above include platelet-derived growth factor isoforms (PDGF-AA, -AB, -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neutrophil factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and morphogenetic protein-2, -4, -6 (BMP-2, -4, -6). Examples of the chemokines mentioned above include interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), regulated on activation, normal T cell expressed and secreted (TANTES), monocyte chemotactic protein-1,-3 (MCP-1,3), macrophage inflammatory proteins-1 alpha (MIP-1α), and beta-thromboglobulin. Methods for producing PL include, but are not limited to, the buffy coat method, platelet-rich plasma method, and apheresis method.PLs that can be used include, but are not limited to, UltraGRO-Advanced (AvenataCell BioMedical), Stemulate (Cook Reagent), PLTMax / PLTGold (Mill Creek Life Sciences), PLUS (Compass Biomedical), Multi PL' (Macopharma), PL SOLUTION (PL BioScience GmbH), and CRUX RUFA GMP (Trinova Biochem).

[0015] In this specification, "preservation" refers to a state in which the amniotic membrane (for example, the amniotic membrane detached from the amniotic membrane collected from a pregnant woman) is in contact with a solution. Part of the amniotic membrane may be in contact with the solution, or the entire amniotic membrane may be in contact with the solution. "Preservation" is not limited to static preservation, but can also include preservation in a state involving movement such as transport, shaking, or inversion, but does not include "culture" which involves growing isolated cells.

[0016] [2] Method for producing adherent stem cells The present invention relates to a method for producing amniotic membrane-derived adherent stem cells, comprising the step of preserving the amniotic membrane in a solution containing platelet lysate. In the manufacturing method of the present invention, the step of preserving the amniotic membrane can be performed, for example, by immersing the amniotic membrane collected from a living organism in a solution containing PL. The amniotic membrane to be preserved may be in the state in which it was collected, or it may be cut into multiple pieces with scissors or the like after collection. The amniotic membrane to be preserved may also have the blood vessels and blood clots of the living organism removed, or it may have the blood vessels and blood clots of the living organism attached to it.

[0017] The manufacturing method of the present invention may include a step of washing the amniotic membrane before the step of preserving the amniotic membrane described above. The washing solution used to wash the amniotic membrane is not particularly limited, but examples include physiological saline, Dulbecco's phosphate-buffered saline (DPBS), Earl's equilibrium salt solution (EBSS), Hanks' equilibrium salt solution (HBSS), phosphate-buffered saline (PBS), etc.

[0018] There is no particular lower limit to the amniotic membrane weight as a single unit for storage (the total amount if multiple cut amniotic membranes are used), but it must be a weight sufficient to recover the number of adherent stem cells necessary for culture. Therefore, the amniotic membrane weight as a single unit for storage is preferably 1 g or more, more preferably 2 g or more, and even more preferably 3 g or more. There is no particular upper limit to the amniotic membrane weight as a single unit for storage, but if the amniotic membrane weight is too large, the number of adherent stem cells recovered per unit weight of amniotic membrane will decrease, which is disadvantageous in terms of cost. Therefore, the amniotic membrane weight as a single unit for storage is preferably 80 g or less, more preferably 60 g or less, for example, 30 g or less, 25 g or less, 20 g or less, or 15 g or less. For example, the amniotic membrane weight as a single unit for storage is 1 to 80 g, 1 to 60 g, 1 to 30 g, 1 to 15 g, 2 to 15 g, or 3 to 15 g.

[0019] The PL concentration in the solution used to preserve the amniotic membrane is preferably 2 v / v% or higher, for example, 2.5 v / v% or higher, 3 v / v% or higher, 3.5 v / v% or higher, 4 v / v% or higher, 4.5 v / v% or higher, 4.6 v / v% or higher, 4.7 v / v% or higher, 4.8 v / v% or higher, or 4.9 v / v% or higher. If the PL concentration is less than 2 v / v%, the growth rate may decrease because the culture medium does not contain a sufficient amount of the components necessary for growth that are contained in the PL. There is no particular upper limit to the PL concentration in the solution used to preserve the amniotic membrane, but if it exceeds 20 v / v%, it becomes disadvantageous from a cost standpoint, so it is preferably 20 v / v% or less, for example, 19 v / v% or less, 18 v / v% or less, 17 v / v% or less, 16 v / v% or less, 15 v / v% or less, 14 v / v% or less, 13 v / v% or less, 12 v / v% or less, 11 v / v% or less, 10 v / v% or less, 9 v / v% or less, 8 v / v% or less, 7 v / v% or less, 6 v / v% or less, 5.5 v / v% or less, 5.4 v / v% or less, 5.3 v / v% or less, 5.2 v / v% or less, or 5.1 v / v% or less. The PL concentration in the amniotic membrane preservation solution may be, for example, 2-20 v / v%, 2-15 v / v%, 2-10 v / v%, 2-7 v / v%, 4-7 v / v%, or 4-6 v / v%. The PL concentration in the amniotic membrane preservation solution can be converted by the protein concentration. The PL concentration in the amniotic membrane preservation solution converted by the protein concentration (i.e., the PL-derived protein concentration in the amniotic membrane preservation solution) may be, for example, 1-10 mg / mL, 1-7.5 mg / mL, 1-5 mg / mL, 1-3.5 mg, 2-3.5 mg / mL, or 2-3 mg / mL.

[0020] The solution used to preserve the amniotic membrane is not particularly limited as long as it contains PL (polynitrate). The aqueous solution is preferably an isotonic solution or a basal culture medium to which PL has been added. The isotonic solution is not particularly limited, but examples include physiological saline, DPBS, EBSS, HBSS, and PBS. As basal media, you may use, but are not limited to, 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, RPMI (Roswell Park Memorial Institute) 1640 medium, Fischer's medium, and mixed media of these (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). In the present invention, the solution for preserving the amniotic membrane is preferably a solution containing a basal medium and PL, for example, a basal medium to which PL has been added. Examples of preferred basal media used in this case include αMEM medium or RPMI1640 medium.

[0021] The solution used to preserve the amniotic membrane may further contain antibiotics. Examples of antibiotics include, but are not limited to, actinomycin D, antefocillin B, ampicillin, antimycin A, bafilomycin A1, bleomycin, carbenicillin, chloramphenicol, concanamycin B, erythromycin, G418, gentamicin, hygromycin, kanamycin, mitomycin C, neomycin, oligomycin, penicillin, puromycin, rapamycin, streptomycin, tetracycline, tobranomycin, and valinomycin.

[0022] The volume of the solution used to preserve the amniotic membrane is not particularly limited as long as the entire amniotic membrane is immersed, but for example, the lower limit of the volume per gram of amniotic membrane can be 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, or 10 mL. Increasing the volume of solution relative to the amniotic membrane beyond what is necessary will only increase the cost of preserving the amniotic membrane, therefore, the upper limit of the volume per gram of amniotic membrane is preferably 1,000 mL, more preferably 750 mL, for example, 500 mL, 400 mL, 300 mL, 200 mL, 100 mL, 50 mL, 40 mL, or 30 mL. The volume of the solution used to preserve the amniotic membrane is, for example, 3 to 1,000 mL, 3 to 500 mL, 3 to 400 mL, 3 to 200 mL, 3 to 100 mL, 3 to 50 mL, 3 to 40 mL, 3 to 30 mL, 4 to 30 mL, 5 to 30 mL, 6 to 30 mL, or 7 to 30 mL per gram of amniotic membrane.

[0023] The upper limit of the time for storing the amniotic membrane in a solution containing PL is not particularly limited, but is preferably 130 hours, more preferably 75 hours. The lower limit of the time for storing the amniotic membrane in a solution containing PL is not particularly limited, but is preferably 1 hour, more preferably 8 hours, and even more preferably 15 hours. The time for storing the amniotic membrane in a solution containing PL may be, for example, 1 to 130 hours, 1 to 75 hours, 8 to 75 hours, or 15 to 75 hours.

[0024] The upper limit of the solution temperature when storing amniotic membrane in a solution containing PL is not particularly limited, but for example, it may be 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 30°C, 25°C, 20°C, 15°C, or 10°C, preferably 40°C, more preferably 25°C, and even more preferably 10°C. The lower limit of the solution temperature when storing amniotic membrane in a solution containing PL may be any temperature at which the solution does not freeze, for example 0°C, 1°C, 2°C, 3°C, or 4°C under atmospheric pressure. The solution temperature may be determined by the external temperature of the storage container or by the internal temperature of the container. The solution temperature may also be controlled by measuring the external temperature of the storage container or by directly measuring the temperature of the solution inside the storage container. The solution temperature when storing amniotic membrane in a solution containing PL may be, for example, 1-40°C, 2-39°C, 3-38°C, or 4-37°C.

[0025] The amniotic membrane is preferably preserved in a sealed, sterile container. Examples of such storage containers include, but are not limited to, square culture bottles (Thermo Fisher Scientific), PET storage bottles with caps (Corning), PRTG sterile culture bottles (TriForest), EOG sterile bottles (PE wide mouth) milky white (Sanplatec), and clear square PC bottles (NALGEN).

[0026] The present invention's method for producing adherent stem cells may preferably further include, after the step of preserving the amniotic membrane, a step of separating amniotic membrane-derived adherent stem cells from the preserved amniotic membrane by, for example, enzymatic treatment of the preserved amniotic membrane.

[0027] In the present invention's method for producing adherent stem cells, the enzyme used to separate adherent stem cells from the amniotic membrane is preferably an enzyme (or a combination thereof) that can release adherent stem cells contained in the extracellular matrix layer of the amniotic membrane without degrading the epithelial cell layer. Examples of preferred enzymes, though not particularly limited, include collagenase and / or metalloproteinase. 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.

[0028] The activity concentration of the collagenase that can be used in the present invention is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, and even more preferably 200 PU / ml or more. Furthermore, the activity concentration of the collagenase that can be used in the present invention is not particularly limited, but for example, it could be 1,000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, 500 PU / ml or less, or 300 PU / mL or less. For example, the activity concentration of the collagenase that can be used in the present invention is 50 PU / ml to 1,000 PU / ml, 50 PU / ml to 500 PU / ml, or 100 to 300 PU / mL. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 μg of FITC-collagen (fluorescein isothiocyanate-labeled collagen) in 1 minute at pH 7.5 and 30°C.

[0029] The activity concentration of the metal proteinase (e.g., thermolysin and / or dispase) that can be used in the present invention is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, even more preferably 150 PU / ml or more, and even more preferably 190 PU / ml or more. Furthermore, the activity concentration of the metal proteinase that can be used in the present invention 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, even more preferably 500 PU / ml or less, and even more preferably 300 PU / ml or less. For example, the activity concentration of the metal proteinase that can be used in the present invention is 50 PU / ml to 1000 PU / ml, 50 PU / ml to 500 PU / ml, or 100 to 300 PU / mL. In this embodiment, where dispase is used as the metalloproteinase, PU (Protease Unit) can be defined as the amount of enzyme that releases an amino acid equivalent to 1 μg of tyrosine per minute from lactate casein at pH 7.5 and 30°C. By enzymatically treating the amniotic membrane with the above enzyme within the above enzyme concentration range, mesenchymal cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of the epithelial cell layer of the amniotic membrane with epithelial cells. The preferred combination of collagenase and / or metalloproteinase concentrations can be determined by microscopic observation of the amniotic membrane after enzymatic treatment or by flow cytometry of cells separated by enzymatic treatment.

[0030] In the process of isolating the amniotic membrane-derived adherent stem cells described above, it is preferable to enzymatically treat the amniotic membrane using a combination of collagenase and a metalloproteinase (e.g., dispase) from the viewpoint of efficiently recovering viable cells. In this case, the collagenase and metalloproteinase can be used simultaneously or sequentially, but it is preferable to treat the amniotic membrane simultaneously and all at once using a combination of collagenase and a metalloproteinase. In this case, thermolysin and / or dispase can be used as the metalloproteinase, but are not limited to these. For example, by treating the amniotic membrane only once with an enzyme solution containing collagenase and a metalloproteinase, the amniotic membrane-derived adherent stem cells can be easily isolated. Furthermore, treating the amniotic membrane simultaneously and all at once as described above can reduce the risk of contamination by bacteria, viruses, etc.

[0031] The enzymatic treatment of the amniotic membrane is preferably carried out by directly immersing the amniotic membrane, which has been stored in a solution containing PL, in an enzyme solution containing the enzyme, either after washing with a washing solution such as physiological saline or HBSS, and then stirring with a stirring device. As such a stirring device, 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 stirring with a stirrer or shaker is not particularly limited, but if the stirring speed is too slow, the efficiency of the enzymatic treatment will decrease, so it is preferably 5 rpm, more preferably 10 rpm. The upper limit of the stirring speed is not particularly limited, but if the stirring speed is too fast, it may damage the cells, so it is preferably 100 rpm, more preferably 60 rpm, for example, 50 rpm, 40 rpm, or 30 rpm. The lower limit of the enzymatic 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. Furthermore, while there is no particular upper limit to the enzyme treatment time, 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. There is no particular lower limit to the enzyme treatment temperature, but in order to efficiently carry out the enzymatic reaction, it is preferably 15°C, more preferably 25°C, and even more preferably 35°C. Furthermore, while there is no particular upper limit to the enzyme treatment temperature, if the temperature is too high, it may cause the death of amniotic cells or the inactivation of the enzyme, so it is preferably 40°C.

[0032] In the manufacturing method of the present invention, if desired, the free adherent stem cells can be separated and / or recovered from the enzyme solution containing the adherent stem cells released by enzyme treatment, etc., by known methods such as filters, centrifugation, hollow fiber separation membranes, cell sorters, etc. 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 cannot pass through the filter and remains on the filter, so that the free adherent stem cells can be easily separated and / or recovered, and the risk of contamination by bacteria, viruses, etc. can also be reduced. The filter for filtering the enzyme solution is not particularly limited, but for example, a mesh filter can be used. The pore size (mesh size, opening) of the mesh filter is not particularly limited, but for example, it is 40 μm or more, 50 μm or more, 60 μm or more, 70 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, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, or 100 μm or less. The filtration speed is not particularly limited, but 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.

[0033] Nylon is preferred as the material for the mesh filter. For example, tubes containing nylon mesh filters with pore sizes of 40 μm, 70 μm, 95 μm, or 100 μm, such as the Falcon® cell strainer commonly used for research purposes, can be used as mesh filters. Medical mesh cloths (nylon and polyester) used in hemodialysis and other applications can also be used as mesh filters. Furthermore, arterial filters used during extracorporeal circulation (polyester mesh filters, pore size: 40 μm to 120 μm) can also be used as mesh filters. Other materials, such as stainless steel mesh filters, can also be used.

[0034] When filtering amniotic membrane-derived adherent stem cells, it is preferable to use gravity-assisted free fall. While forced filtering methods such as suction using a pump or pressurization are also possible, in such cases, it is desirable to use the lowest possible pressure to avoid damaging the cells.

[0035] Cell populations containing adherent stem cells, filtered through the medium, can be recovered by centrifugation after diluting the filtrate with culture medium or equilibrium salt buffer. Suitable equilibrium salt buffers include, but are not limited to, Dulbecco's phosphate-buffered saline (DPBS), Earl's equilibrium salt solution (EBSS), Hanks' equilibrium salt solution (HBSS), and phosphate-buffered saline (PBS).

[0036] The present invention's method for producing adherent stem cells may preferably include a step of culturing adherent stem cells (a cell population including adherent stem cells) obtained from the amniotic membrane. For example, the present invention's method for producing adherent stem cells may include a step of culturing the isolated amniotic membrane-derived adherent stem cells after the above-mentioned step of isolating the amniotic membrane-derived adherent stem cells.

[0037] The cell seeding density in the process of culturing adherent stem cells (cell populations including adherent stem cells) is not particularly limited, but for example, 500 to 10,000 cells / cm³. 2 Seeds can be sown at the following density. The lower limit of the seeding density is preferably, for example, 500 cells / cm³. 2 More preferably 750 cells / cm² 2 More preferably 1,000 cells / cm² 2 Furthermore, the upper limit of the seeding density is not particularly limited, but is preferably 10,000 cells / cm². 2 More preferably 7,000 cells / cm² 2 More preferably, 5,000 cells / cm² 2 That is the case.

[0038] The above-mentioned culture process may include a subculturing process, or it may include a process of repeating the culture multiple times under different culture conditions.

[0039] The culture period for one culture as described above can be, for example, 2 to 15 days, and more specifically, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0040] 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 in which 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 and then adding it, or the culture medium may be prepared by stabilizing the growth factor in advance with a gel or polysaccharide and then adding the stabilized growth factor to the base medium.

[0041] As the basal medium, the basal medium exemplified in the amniotic membrane preservation solution described above can be used, but is not particularly limited. Preferred basal media include αMEM medium and M199.

[0042] Other components that can be added to the basal medium include, for example, albumin, bovine serum, serum replacement reagents, or growth factors, etc. Among them, serum replacement reagents are preferred, and particularly PL is preferred. In particular, it is preferable to perform the above-mentioned culture in a basal medium containing a serum replacement reagent and not containing albumin, bovine serum, and growth factors. As the lower limit of the concentration of PL in the medium used for the above-mentioned culture, for example, the final concentration can be 1 v / v% or more, 2 v / v% or more, 3 v / v% or more. Also, as the upper limit of the concentration of PL in the medium used for the above-mentioned culture, for example, 20 v / v% or less, 10 v / v% or less, 7 v / v% or less is preferred.

[0043] Also, for the medium used in the above-mentioned culture, a commercially available serum-free medium may generally be used. For example, STK R 1 or STK R 2 (DS Pharma Biomedical Co., Ltd.), EXPREP MSC Medium (Bionmimetics Sympathies Co., Ltd.), Corning R stemgro R human mesenchymal stem cell medium (Corning Co., Ltd.), etc. can be mentioned, but it is not particularly limited.

[0044] The culture of adherent stem cells (cell population containing adherent stem cells) can be carried out, for example, in the following steps. First, the cell suspension containing adherent stem cells is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture container and cultured using the medium in an environment with a CO2 concentration of 3% or more and 5% or less and at 37°C. The cells obtained by the above-mentioned culture are cells cultured once.

[0045] 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 to achieve a confluence rate of 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 or more, preferably 2 or more. From the viewpoint of suppressing cell senescence, the upper limit of the number of subculturings n is, for example, preferably 20 or less, or 10 or less. As the cell detachment method described above, for example, a cell detachment agent may be used. Examples of cell detachment agents include trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc., but are not particularly limited. Commercially available cell detachment agents may also be used. Examples of commercially available cell detachment agents include trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE TM Select (manufactured by Thermo Fisher Scientific), Accutase TM (manufactured by Stemcell Technologies), Accumax TM Examples include, but are not limited to, those manufactured by Stemcell Technologies. Furthermore, physical cell detachment methods may be used as cell detachment methods; for example, a cell scraper (manufactured by Corning) can be used, but is not limited to this. Cell detachment methods may be used individually or in combination.

[0046] The present invention also provides a method for producing a cell preparation containing amniotic membrane-derived adherent stem cells. The method for producing a cell preparation containing amniotic membrane-derived adherent stem cells according to the present invention may include the same steps as the method for producing amniotic membrane-derived adherent stem cells described above.

[0047] The present invention also, (a) A step of isolating amniotic membrane-derived adherent stem cells from amniotic membrane stored in a solution containing platelet lysate, and (b) The process of culturing the isolated amniotic membrane-derived adherent stem cells. A method for producing amniotic membrane-derived adherent stem cells or cell preparations containing them. We also offer it. This manufacturing method is the same as the manufacturing method described above, except that in step (a) above, amniotic membrane is obtained and used in a state that has been previously stored in a solution containing platelet lysate. There are no particular limitations on how the amniotic membrane in the aforementioned state of storage is obtained; it may be purchased from a commercially available product in such a form, or it may be prepared by a medical institution, supplier, company, etc., through a request or outsourcing. In this manufacturing method, the "amnion preserved in a solution containing platelet lysate" is amnion preserved by the same process as the amnion preservation process described above. [Examples]

[0048] <Comparative Example 1> (Step 1: Amniotic membrane harvesting) The amniotic membrane, including the amnion, and placenta were aseptically collected from a pregnant woman (donor #1) 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 separated 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.

[0049] (Step 2: Preservation of the amniotic membrane) 11.6 g of amniotic membrane was immersed in 120 mL of αMEM (Alpha Modification of Minimum Essential Medium Eagle, Life Technologies) and stored at 37°C for 6 hours.

[0050] (Step 3: Enzymatic treatment of amniotic membrane and recovery of adherent stem cells) The amniotic membrane was enzymatically treated by immersing it in Hanks equilibrium salt solution (containing Ca·Mg) containing 240 PU / mL collagenase and 200 PU / mL dispase I, and shaking it at 37°C for 60 minutes at 10 rpm. After enzymatic treatment, the solution was filtered through a 95 μm nylon mesh to remove undigested amniotic material, and a cell suspension containing adherent stem cells was recovered. This cell suspension was diluted with 2 v / v% human PL-containing Hanks equilibrium salt solution (Ca·Mg-free), centrifuged, and the supernatant was discarded to recover the cell population containing adherent stem cells.

[0051] (Step 4: Culture of adherent stem cells) The cell population containing adherent stem cells obtained in "Step 3: Enzymatic treatment of amniotic membrane and recovery of adherent stem cells" described above was placed in a 75 cm³ culture vessel. 2 Cells were seeded in a U-shaped canted-neck cell culture flask (vented cap) (Corning). 1,000 cells / cm² 2 The cells were seeded at a density and cultured in αMEM containing human PL at a final concentration of 5 v / v%, at a CO2 concentration of 5% and 37°C. Cell seeding and culture were performed with n=3.

[0052] (Step 5-1: Recovery of adherent stem cells after culture) After culturing the cells until they reached subconfluence, they were detached from the flask using trypsin-EDTA solution. The detached cells were washed with physiological saline and then suspended in Hanks equilibrium salt solution (Ca·Mg-free).

[0053] (Step 5-2: Measurement of the number of recovered cells) After measuring the amount of cell suspension obtained in step 5-1, a portion of this cell suspension was sampled and mixed with an equal volume of cell treatment reagent Reagent A 100. An equal volume of cell treatment reagent Reagent B was then added and mixed. This mixture was subjected to a cell counter (NucleoCounter NC-100®) to measure the total cell concentration. The total number of cells recovered was determined by multiplying the measured cell suspension volume by the total cell concentration.

[0054] (Example 1) In Example 1, amniotic membrane was collected from the same donor (#1) as in Comparative Example 1. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 1, as shown below. All other steps were carried out using the same method as in Comparative Example 1.

[0055] (Step 2: Preservation of the amniotic membrane) 4.9 g of amniotic membrane was immersed in 120 mL of αMEM containing 5 v / v% human PL (median protein concentration: approximately 2.5 mg / mL, the same applies to Examples 2 to 7-3 below) and stored at 37°C for 6 hours.

[0056] <Comparative Example 2> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #2) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0057] (Step 2: Preservation of the amniotic membrane) 8.1 g of amniotic membrane was immersed in 100 mL of Hanks equilibrium salt solution (Ca·Mg-free) (Thermo Fisher Scientific) and stored at 4°C for 3 hours. Subsequently, steps 3 to 5-2 described in Comparative Example 1 were carried out.

[0058] (Example 2) In Example 2, the amniotic membrane was collected from the same donor (#2) as in Comparative Example 2. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 2, as shown below. All other steps were carried out using the same method as in Comparative Example 2.

[0059] (Step 2: Preservation of the amniotic membrane) 4.2 g of amniotic membrane was immersed in 100 mL of Hanks equilibrium salt solution (Ca·Mg-free) containing 5 v / v% human PL, and stored at 4°C for 3 hours.

[0060] <Comparative Example 3> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #2) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0061] (Step 2: Preservation of the amniotic membrane) 5.3 g of amniotic membrane was immersed in 100 mL of RPMI1640 medium (Thermo Fisher Scientific) and stored at 4°C for 3 hours. Subsequently, steps 3 to 5-2 described in Comparative Example 1 were carried out.

[0062] (Example 3) In Example 3, amniotic membrane was collected from the same donor (#2) as in Comparative Example 3. Only step 2 (preservation of amniotic membrane) was carried out using a different method than in Comparative Example 3, as shown below. All other steps were carried out using the same method as in Comparative Example 3.

[0063] (Step 2: Preservation of the amniotic membrane) 5.0 g of amniotic membrane was immersed in 100 mL of RPMI1640 medium containing 5 v / v% human PL and stored at 4°C for 3 hours.

[0064] <Comparative Example 4> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #3) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0065] (Step 2: Preservation of the amniotic membrane) 11.8 g of amniotic membrane was immersed in 100 mL of αMEM medium and stored at 37°C for 4 hours. Subsequently, steps 3 to 5-2 described in Comparative Example 1 were carried out.

[0066] (Example 4) In Example 4, the amniotic membrane was collected from the same donor (#3) as in Comparative Example 4. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 4, as shown below. All other steps were carried out using the same method as in Comparative Example 4.

[0067] (Step 2: Preservation of the amniotic membrane) 4.1 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 37°C for 4 hours.

[0068] <Comparative Example 5> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #3) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0069] (Step 2: Preservation of the amniotic membrane) 12.5 g of amniotic membrane was immersed in 100 mL of αMEM medium and stored at 37°C for 73 hours. Subsequently, steps 3 to 5-2 described in Comparative Example 1 were carried out.

[0070] (Example 5) In Example 5, amniotic membrane was collected from the same donor (#3) as in Comparative Example 5. Only step 2 (preservation of amniotic membrane) was performed using a different method than in Comparative Example 5, as shown below. All other steps were performed using the same method as in Comparative Example 5.

[0071] (Step 2: Preservation of the amniotic membrane) 6.7 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 37°C for 73 hours.

[0072] <Comparative Example 6> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #3) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0073] (Step 2: Preservation of the amniotic membrane) 13.3 g of amniotic membrane was immersed in 100 mL of αMEM medium and stored at 37°C for 124 hours. Subsequently, steps 3 to 5-2 described in Comparative Example 1 were carried out.

[0074] (Example 6) In Example 6, amniotic membrane was collected from the same donor (#3) as in Comparative Example 6. Only step 2 (preservation of the amniotic membrane) was performed using a different method than in Comparative Example 6, as shown below. All other steps were performed using the same method as in Comparative Example 6.

[0075] (Step 2: Preservation of the amniotic membrane) 11.0 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 37°C for 124 hours.

[0076] <Comparative Example 7> (Step 1: Amniotic membrane harvesting) Amniotic membrane was collected from a pregnant woman (donor #4) undergoing elective cesarean section who had given informed consent, using the same method as in Comparative Example 1.

[0077] (Step 2: Preservation of the amniotic membrane) 4.7 g of amniotic membrane was immersed in 100 mL of Hanks equilibrium salt solution (Ca·Mg-free) and stored at 4°C for 1 hour. Subsequently, steps 3 to 5-2 of Comparative Example 1 were carried out.

[0078] (Example 7-1) In Example 7-1, the amniotic membrane was collected from the same donor (#4) as in Comparative Example 7. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 7, as shown below. All other steps were carried out using the same method as in Comparative Example 7.

[0079] (Step 2: Preservation of the amniotic membrane) 6.1 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 4°C for 1 hour.

[0080] (Example 7-2) In Example 7-2, the amniotic membrane was collected from the same donor (#4) as in Comparative Example 7. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 7, as shown below. All other steps were carried out using the same method as in Comparative Example 7.

[0081] (Step 2: Preservation of the amniotic membrane) 6.4 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 25°C for 1 hour.

[0082] (Example 7-3) In Example 7-3, the amniotic membrane was collected from the same donor (#4) as in Comparative Example 7. Only step 2 (preservation of the amniotic membrane) was carried out using a different method than in Comparative Example 7, as shown below. All other steps were carried out using the same method as in Comparative Example 7.

[0083] (Step 2: Preservation of the amniotic membrane) 5.6 g of amniotic membrane was immersed in 100 mL of αMEM medium containing 5 v / v% human PL and stored at 37°C for 1 hour.

[0084] <Comparison of specific growth rates> Based on the total number of cells recovered in step 5-2 of Comparative Examples 1 to 7 and Examples 1 to 7-3, the specific growth rate was determined using the following formula. Specific growth rate = ln(total number of cells recovered / 75,000) / total culture time

[0085] The mean relative growth rates and standard deviations for each species are summarized in Tables 1-7. In Tables 1-7, the asterisk (*) indicates a statistically significant difference (significance level 5%) compared to the corresponding comparative example, as determined by a t-test. Tables 1-7 show that when amniotic membranes were preserved in a solution containing human PL, the proliferative capacity of adherent stem cells collected from those membranes was enhanced compared to when they were preserved in a solution without human PL.

[0086] [Table 1] [Table 2] Table 3 Table 4 Table 5 Table 6 Table 7

Claims

1. A step of preserving the amniotic membrane in a solution containing platelet lysate, A step of separating amniotic membrane-derived adherent stem cells from the amniotic membrane, and The process of culturing isolated amniotic membrane-derived adherent stem cells. A method for producing amniotic membrane-derived adherent stem cells or cell preparations containing them.

2. The manufacturing method according to claim 1, wherein the concentration of platelet lysate in the solution is 2 to 20 v / v%.

3. The manufacturing method according to claim 1 or 2, wherein the solution for preserving the amniotic membrane includes a basal culture medium.

4. The manufacturing method according to any one of claims 1 to 3, wherein the time for storing the amniotic membrane in the solution is 1 to 130 hours.

5. The manufacturing method according to any one of claims 1 to 4, wherein the temperature at which the amniotic membrane is stored in the solution is 1 to 40°C.

6. The manufacturing method according to claim 5, wherein the temperature at which the amniotic membrane is stored in the solution is 1 to 25°C.

7. (a) A step of separating amniotic membrane-derived adherent stem cells from amniotic membrane stored in a solution containing platelet lysate, and (b) A step of culturing the isolated amniotic membrane-derived adherent stem cells. A method for producing amniotic membrane-derived adherent stem cells or cell preparations containing them.

8. The manufacturing method according to claim 7, wherein the concentration of platelet lysate in the solution is 2 to 20 v / v%.

9. The manufacturing method according to claim 7 or 8, wherein the solution for preserving the amniotic membrane includes a basal culture medium.

10. The manufacturing method according to any one of claims 7 to 9, wherein the amniotic membrane is stored in the solution for 1 to 130 hours.

11. The manufacturing method according to any one of claims 7 to 10, wherein the amniotic membrane is stored in the solution at a temperature of 1 to 40°C.

12. The manufacturing method according to claim 11, wherein the amniotic membrane is stored in the solution at a temperature of 1 to 25°C.

Images