A method for efficiently producing adherent cells from tissue.

Abstract

The present invention provides a method for safely saving and / or transporting raw material tissue for producing therapeutic cells.　This method produces a cell mass including adhesive cells, from tissue including the adhesive cells. The method produces adhesive cells from tissue and includes: (1) saving and / or transporting tissue that includes adhesive cells, in a state in which the tissue is embedded in a medium having a compressive stress of greater than 0N and less than 12 N; and (2) retrieving from the medium the tissue embedded in the medium, and separating the adhesive cells.

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently producing adherent cells, such as mesenchymal stem cells, from tissue. [Background technology]

[0002] Adherent mesenchymal stem cells are somatic stem cells that have been reported to exist in bone marrow, adipose tissue, and dental pulp. Because they have the ability to differentiate into bone, cartilage, and fat, they are attracting attention as a promising cell source in cell therapy. Mesenchymal stem cells possess not only differentiation potential but also immunosuppressive capabilities, and their clinical application is progressing for acute graft-versus-host disease (GVHD) and Crohn's disease. Skeletal myoblasts isolated from muscle tissue also possess adhesive properties and are known to secrete cytokines that promote tissue regeneration; they have been commercialized by Terumo Corporation for myocardial regeneration therapy. Furthermore, fibroblasts that can be isolated from skin tissue also possess adhesive properties and are used clinically for anti-aging purposes, including cosmetic applications.

[0003] These adhesive mesenchymal stem cells, skeletal myoblasts, and fibroblasts are known to be found in amniotic membrane, fat, umbilical cord, placenta, skin, and muscle tissue, and these tissues are attracting attention as promising raw materials for producing therapeutic cells. In commercial practice, the tissue collection facilities and processing facilities (facilities that produce cells from the tissues) are located far apart, so it is necessary to safely store and / or transport the tissues from the collection facility to the processing facility. When storing and / or transporting tissues, they are often immersed in a solution to prevent drying, and buffer solutions are considered for this purpose. In addition, organ preservation solutions have been developed and are commercially available for preserving organs. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The object of the present invention is to provide a method for safely storing and / or transporting raw tissue containing adherent cells such as mesenchymal stem cells, and a method for efficiently producing adherent cells from raw tissue containing adherent cells after storage / transportation. [Means for solving the problem]

[0005] In our investigations under the above-mentioned challenges, we attempted to produce adherent cells from tissue by immersing it in a buffer or organ preservation solution, preserving and / or transporting it. We found that the tissue was damaged during preservation and / or transport, leading to a decrease in adhesion and damage to adherent cells within the tissue, resulting in a reduction in the number of cells obtained. The inventors then conducted further intensive investigations and, surprisingly, discovered that preserving and / or transporting the tissue while embedded in a medium with appropriate compressive stress could suppress tissue damage and efficiently produce adherent cells.

[0006] Based on the above results, the following invention is provided.

[0007] A method for producing a cell population containing adherent cells from tissue containing said cells, Tissues containing adherent cells are stored and / or transported embedded in a medium with a compressive stress greater than 0 and less than or equal to 12 N. The tissue embedded in the above medium is removed from the medium, and the adherent cells are separated. A method for producing adherent cells from tissue including the following.

[0008] This specification includes the disclosures of Japanese Patent Application No. 2020-143577, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0009] According to the present invention, tissues containing adherent cells such as mesenchymal stem cells can be safely stored and transported, and adherent cells can be efficiently produced after storage and transport. [Modes for carrying out the invention]

[0010] The following describes specific embodiments of the present invention. However, the following description is intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which those skilled in the art appropriately substitute the configurations of the embodiments described below are also included in the scope of the present invention.

[0011] [1] Explanation of terms In this invention, "adherent cells" refer to cells that adhere to glass or plastic substrates such as petri dishes, plates, and flasks, and exhibit a spindle-shaped form. Examples of adherent cells include mesenchymal stem cells, skeletal muscle blasts, fibroblasts, epithelial cells, cardiomyocytes, neural stem cells, hepatocytes, cardiomyocytes, nerve cells, and hepatocytes, but this invention is not limited to these cells. Tissues containing adhesive cells include solid tissues such as fat, placenta, amniotic membrane, chorionic membrane, skin, umbilical cord, heart, brain, lungs, cornea, intestines, muscle, and synovial membrane, as well as liquid tissues such as bone marrow, dental pulp, amniotic fluid, umbilical cord blood, and blood. However, the present invention is not limited to these tissues. Processed forms of these tissues are also included.

[0012] In this invention, the "medium" can be any state, property, or structure as long as its compressive stress is greater than 0 and 12N or less. For example, it can be a solid, liquid, gas, or any other state, or a mixture thereof. Specific examples of a medium with a compressive stress greater than 0 and 12N or less include gels, sols, and similar substances. A gel refers to a substance in which colloidal particles are dispersed in a liquid or gas and have lost their fluidity; examples include konjac, yokan, agar, and pudding. A sol refers to a substance in which colloidal particles are dispersed in a liquid or gas and have not lost their fluidity; examples include milk, drinking yogurt, and oil. In this invention, colloids using water as the dispersion medium are preferred as sols / gels, and so-called hydrogels are more preferred.

[0013] The medium of the present invention preferably has moderate hardness, and as described above, compressive stress is used as an indicator of that hardness. The method for measuring compressive stress in the present invention is to place 2 mL of the medium in a 24-well plate, compress the medium with a plunger with a diameter of 1 cm, and measure the stress (in N) when the medium is compressed by 1.5 mm using EZ-TEST (Shimadzu Corporation, EZ-SX). However, the present invention is not limited to the above method if equivalent results can be obtained. In the present invention, the compressive stress of the medium must satisfy the range of "greater than 0 and 12 N or less" at the temperature during storage and / or transport. For example, if stored and transported at 4°C, the compressive stress is at 4°C, and if stored and transported at 15°C, the compressive stress is at 15°C. The compressive stress is not particularly limited as long as it is greater than 0 and 12 N or less, but it is preferably 0.001 N or more, more preferably 0.005 N or more, even more preferably 0.01 N or more, and most preferably 0.1 N or more. Furthermore, the compressive stress is preferably 11N or less, more preferably 10N or less, and even more preferably 9N or less. It is even more preferably 8N or less, and most preferably 7N or less. If the temperature during storage and transport is not constant, it is sufficient if at least one point at any of the temperatures falls within the above compressive stress range, but it is preferable that the above compressive stress is satisfied for at least 40% of the entire storage and / or transport process, more preferably 60% or more, even more preferably 80% or more, and most preferably that the above compressive stress is satisfied under all temperature conditions. For example, if the product is stored at 4°C once and transported in the range of 15°C to 25°C, it is sufficient if at least one point at either 4°C or 15°C to 25°C falls within the above compressive stress range, but it is more preferable that both are satisfied.

[0014] The medium described in the present invention preferably contains at least one selected from the group consisting of proteins, peptides, polysaccharides, and synthetic polymers from the viewpoint of biocompatibility. In the present invention, it is more preferable to use a medium in which these are dispersed in water, but the present invention is not limited to these. Examples of proteins include gelatin, collagen, fibrin, and soy protein. Examples of polysaccharides or substances containing polysaccharides include agarose, pectin, carrageenan, curdlan, chitin, chitosan, alginic acid derivatives, soy polysaccharides, cellulose derivatives such as carboxymethylcellulose, mannans, gum arabic, gellan gum, guar gum, xanthan gum, starch, agar, and fucoidan. Examples of synthetic polymers include synthetic peptides (self-assembling peptides such as Panacea gel and PuraMatrix), polyvinyl alcohol, propylene glycol, silicone, and polyacrylamide. These may be used individually or in combination of two or more.

[0015] The term "culture medium" as used herein is not particularly limited and can be prepared by using any liquid cell culture medium as a base medium and adding other components (such as albumin, blood-derived components, growth factors, etc.) as appropriate.

[0016] The above-mentioned basal media can be, 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 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)). Various commercially available serum-free media can also be used.

[0017] Examples of other components to be added to the basal medium include albumin, blood-derived components, growth factors, and the like. In the mode of adding albumin to the basal medium, the concentration of albumin is preferably 0.05% by weight or more and 5% by weight or less. Examples of blood-derived components include various sera (animal-derived sera such as fetal bovine serum (FBS or FCS), human serum, sera prepared from various animals and / or human blood-derived platelet-rich plasma or platelet lysates), platelet lysates, plasma, and the like, derived from various animals and / or human blood. The human serum may be derived from the same individual or a different individual from whom the tissue containing adherent cells was obtained. In the mode of adding a blood-derived component to the basal medium, the concentration of the blood-derived component is preferably 2% by volume or more and 40% by volume or less, more preferably 3% by volume or more and 30% by volume or less. In the mode of adding a growth factor, a reagent (such as an anticoagulant like heparin, a gel, a polysaccharide, etc.) for stabilizing the growth factor in the medium may be further added in addition to the growth factor, or a growth factor that has been previously stabilized may be added to the basal medium. Growth factors that can be used include, for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their families, but are not particularly limited.

[0018] [2] Method for efficiently separating adherent cells such as mesenchymal stem cells from tissue The process of collecting tissue containing adherent cells such as mesenchymal stem cells can be carried out, for example, by the following procedure. In the case of adipose tissue, an incision of about 0.5 cm to 1 cm is made with a sharp scalpel at any location on the patient (e.g., abdomen, waist, thigh), and the fat is extracted and removed with any surgical instrument (e.g., mosquito forceps, tweezers). The incision is sutured with one stitch or fixed with tape. Adipose tissue collected by this means is generally called excised fat. Alternatively, fat can also be aspirated from any location on the patient (e.g., abdomen, waist, thigh) using a cannula or the like. Adipose tissue collected by this means is generally called aspirated fat. In the case of amniotic tissue, after collecting fetal appendages (placenta, amniotic membrane, etc.) at the time of birth, the amniotic membrane is separated from the cut end of the amniotic membrane. In the case of muscle tissue, muscle tissue is collected from the thigh. In the case of skin tissue, it can be collected from behind the ear, etc., using a disposable surgical knife or a skin biopsy punch, but the present invention is not limited to these.

[0019] The collected tissue is stored and / or transported embedded in a medium, which can be done, for example, by the following procedure: embedding the collected tissue in a medium filled in a container, or adding the medium to a container containing the collected tissue, and then storing and / or transporting the tissue. The process of removing the tissue embedded in the medium can be done, for example, by the following procedure: removing the tissue from the medium containing the tissue using any instrument (e.g., mosquito forceps, tweezers, etc.).

[0020] When a sol or gel with the property of reversibly solidifying (including gelling) and liquefying repeatedly, such as gelatin, is used as a medium, this property can be utilized to preferably preserve / transport tissue as follows. For example, when using gelatin, a gelatin solution is prepared by dissolving or dispersing gelatin in a dispersion medium such as an aqueous solution as described below. Then, the gelatin solution is heated to liquefy it, and the collected tissue is embedded in it. The gelatin solution with the embedded tissue is cooled to solidify, and can be preserved and / or transported in the state of being embedded in the gel. After that, the gelatin solution can be liquefied by heating, and the embedded tissue can be removed.

[0021] The temperature for liquefying the above gelatin solution varies depending on the gelatin concentration. For example, it is 25°C or higher and 60°C or lower. From the perspective of damage to tissue, 30°C or higher and 55°C or lower is preferable, and 35°C or higher and 45°C or lower is more preferable. Also, the temperature for solidifying the above gelatin solution varies depending on the gelatin concentration, but it is 1°C or higher and 25°C or lower, and more preferably 1°C or higher and 10°C or lower.

[0022] Gelatin is extracted from pig skin, pig bones, fish, cows, humans, etc., and any gelatin extracted from any animal can be used. Also, it may be hydrolyzed with an acid or an alkali, but the type of gelatin is not limited in the present invention.

[0023] Any dispersion medium that can dissolve gelatin can be used as long as it is water or an aqueous solution. As the aqueous solution, any aqueous solution such as a buffer solution, isotonic solution, hypotonic solution, hypertonic solution, etc. can be used. From the perspective of reducing damage to tissue, a buffer solution or an isotonic solution is more preferable. For example, PBS, HBSS(-), Ringer's solution, lactated Ringer's solution, infusion solution, physiological saline solution, culture solution, albumin solution, blood-derived components, mixtures thereof, etc. can be mentioned. Also, from the perspective of suppressing bacteria, an antibiotic may be added to the above medium.

[0024] The time for storage and transportation is not particularly limited, but from the perspective of reducing damage to tissue, within 10 days is preferable. More specifically, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day can be mentioned. The lower limit of the time for storage and transportation is not particularly limited, but for example, it is 30 minutes or longer, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer.

[0025] The temperature for storage and transportation is not particularly limited, but from the perspective of reducing damage to tissue, 37°C or lower is preferable. More specifically, it is 30°C or lower, 25°C or lower, 20°C or lower, 15°C or lower, 10°C or lower, or 5°C or lower. The lower limit of the temperature for storage and transportation is not particularly limited, but for example, it is -5°C or higher, 0°C or higher.

[0026] The process of separating adherent cells from tissue extracted from a medium can be carried out, for example, by the following procedure: The extracted tissue is treated with an enzyme, then the adherent cells are separated by centrifugation, and the washing and centrifugation are repeated multiple times using a washing solution. In this case, the tissue may be finely chopped with scissors before enzyme treatment to increase the efficiency of enzyme digestion. Collagenase, dispase, etc., can be used as the enzyme treatment solution, but the present invention is not limited to these.

[0027] [3] Method for producing a cell population containing adherent cells isolated from tissue containing adherent cells. The process of producing a cell population containing adherent cells, isolated from tissue containing adherent cells, can be carried out, for example, by the following procedure: First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a culture medium. Next, the cells are seeded in a culture vessel and cultured in a culture medium at a CO2 concentration of 3% to 5% and a temperature of 37°C until the confluence rate is 95% or less. As the culture medium, for example, the "culture medium" described in the explanation of terms in [1] can be used, but the present invention is not limited thereto. In addition, as other methods for separating adherent cells from tissue and producing a cell population containing adherent cells, methods that do not involve enzymatic treatment of the tissue can also be applied, for example, ceiling culture (a method of culturing tissue that has the property of floating in water by attaching it to the ceiling side of a culture vessel filled with culture medium) or tissue piece culture (a method of culturing small pieces of tissue by submerging them in a culture medium, where if the amount of culture medium is too large the tissue piece will float, methods such as using the minimum amount of liquid necessary to immerse the tissue or using a mesh to suppress the floating of the tissue piece and make it adhere to the dish) can be used, but the present invention is not limited thereto. The cells obtained through the culture method described above are cells that have been cultured once (passage 0 cells).

[0028] The culture period for one culture as described above can be, for example, 2 to 21 days, more preferably 3 to 19 days, and even more preferably 4 to 17 days.

[0029] The cells cultured once as described above can be further subcultured as follows: First, the cells cultured once are treated with a cell detachment method such as trypsin to detach them from the culture vessel. 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 culture vessel and cultured using 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. The culture medium described above can be the "culture medium" described in the explanation of terms in [1], but the present invention is not limited thereto. The culture period for the above culture can be, for example, 2 to 21 days, more preferably 3 to 19 days, and even more preferably 4 to 17 days. Cells obtained by culture can be subcultured and cultured repeatedly to obtain cells that have been subcultured n times (n is an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of subcultures n is, for example, 1 or more, preferably 2 or more, more preferably 3 or more, even more preferably 4 or more, and even more preferably 5 or more. Furthermore, from the viewpoint of suppressing cellular aging, the upper limit of the number of passages n is preferably, for example, 25 or less, 20 or less, 15 or less, or 10 or less.

[0030] As the cell detachment means described above, for example, a cell detachment agent may be used. Examples of cell detachment agents that can be used include, but are not limited to, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc. Commercially available cell detachment agents may also be used. Examples include, but are not limited to, trypsin-EDTA solution (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), Accutase (Stemcell Technologies), and Accumax (Stemcell Technologies). Furthermore, physical cell detachment means may also be used as the cell detachment means; for example, a cell scraper (Corning) may be used, but is not limited to this. The cell detachment means may be used individually or in combination.

[0031] The means for cryopreserving cell populations including adherent cells such as mesenchymal stem cells in the present invention are not particularly limited, but examples include a programmable freezer, a deep freezer, and storage in liquid nitrogen. When using a programmable freezer, the freezing temperature is preferably -30°C or lower, -40°C or lower, -50°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -150°C or lower, -180°C or lower, or -196°C (liquid nitrogen temperature) or lower. When using a programmable freezer, the preferred freezing rate is, for example, -1°C / min or lower, -2°C / min or lower, -5°C / min or lower, -9°C / min or lower, -10°C / min or lower, -11°C / min or lower, or -15°C / min or lower. When a programmable freezer is used as the freezing method described above, for example, the temperature can be lowered to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of -2°C / min to -1°C / min, and then further lowered to a temperature between -100°C and -80°C (e.g., -90°C) at a freezing rate of -11°C / min to -9°C / min (e.g., -10°C / min). Alternatively, when immersion in liquid nitrogen is used as the freezing method described above, the temperature can be rapidly lowered to -196°C to freeze the product, and then it can be stored frozen in liquid nitrogen (gas phase). It can also be stored in liquid nitrogen (liquid phase).

[0032] When freezing using the freezing method described above, the cell population may be frozen in any storage container. Examples of such storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, and infusion bags.

[0033] When freezing by the freezing method described above, the cell population may be frozen in any cryopreservation solution. Commercially available cryopreservation solutions may be used as the cryopreservation solution. Examples include, but are not limited to, CP-1® (manufactured by Kyokuto Pharmaceutical Co., Ltd.), BAMBANKER (manufactured by Lymphotec), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by Reprocell), CryoNovo (manufactured by Akron Biotechnology), MSC Freezing Solution (manufactured by Biological Industries), and CryoStor (manufactured by HemaCare). Cryopreservation solutions may be used individually or in combination.

[0034] The above cryopreservation solution may contain a predetermined concentration of polysaccharides. Preferred concentrations of polysaccharides are, for example, 1% by mass or more, 2% by mass or more, 4% by mass or more, or 6% by mass or more. Alternatively, preferred concentrations of polysaccharides are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, or 13% by mass or less. Examples of polysaccharides include, but are not limited to, hydroxyethyl starch (HES) and dextran (Dextran 40, etc.). Polysaccharides may be used individually or in combination.

[0035] The above cryopreservation solution may contain a predetermined concentration of dimethyl sulfoxide (DMSO). Preferred concentrations of DMSO are, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, or 5% by mass or more. Alternatively, preferred concentrations of DMSO are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, 12% by mass or less, or 10% by mass or less.

[0036] The above cryopreservation solution may contain albumin at a predetermined concentration greater than 0% by mass. Preferred albumin concentrations are, for example, 1% or more by mass, 2% or more by mass, 3% or more by mass, or 4% or more by mass. Alternatively, preferred albumin concentrations are, for example, 30% or less by mass, 20% or less by mass, 10% or less by mass, or 9% or less by mass. Examples of albumin include, but are not limited to, bovine serum albumin (BSA), mouse albumin, and human albumin.

[0037] According to one aspect of the present invention, the cell population provided by the present invention, including mesenchymal stem cells, may satisfy the requirement that the proportion of mesenchymal stem cells positive for CD73, CD90, and CD105 is 80% or more.

[0038] CD73 stands for differentiation cluster 73 and is a protein also known as 5-nucleotidase or ecto-5'-nucleotidase.

[0039] CD90 stands for Differentiation Cluster 90 and is a protein also known as Thy-1.

[0040] CD105 stands for Differentiation Cluster 105 and is a protein also known as Endoglin.

[0041] The proportion of CD73-positive mesenchymal stem cells in a cell population may be 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%.

[0042] The proportion of CD90-positive mesenchymal stem cells in a cell population may be 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%.

[0043] The proportion of mesenchymal stem cells that are CD105-positive in the cell population may be 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0044] According to one aspect of the present invention, the cell population provided by the present invention, including mesenchymal stem cells, may satisfy the requirement that the proportion of CD45 and CD31-negative mesenchymal stem cells is 80% or more.

[0045] CD45 stands for differentiation cluster 45 and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type C) or LCA (Leukocyte common antigen).

[0046] CD31 stands for differentiation cluster 31 and is a protein also known as hematopoietic progenitor cell antigen CD31.

[0047] The proportion of CD45-negative mesenchymal stem cells in a cell population may be 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%.

[0048] The proportion of CD31-negative mesenchymal stem cells in a cell population may be 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0049] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples. [Examples]

[0050] In this embodiment, the compressive stress of the medium used to transport and store the excised fat was measured using EZ-TEST (Shimadzu Corporation, EZ-SX) when 2 mL of the medium was placed in a 24-well plate, compressed with a 1 cm diameter plunger at 4°C, and the stress (in N) was measured when the medium was compressed by 1.5 mm.

[0051] <Comparative Example 1: Examination of Adipose Tissue Transport> (Process 1: Collection, transport, and storage of excised fat) An incision was made in the abdomen of a donor (Donor A) who had given informed consent, and fat was removed using mosquito forceps and tweezers. The weight of the removed fat was measured, and 0.06 g of the removed fat was placed directly into a 1.5 mL microtube and transported and stored in a refrigerated environment (4°C) for approximately 20 hours.

[0052] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF (interstitial vascular cell fraction)) The excised fat, removed from a 1.5 mL microtube, was finely chopped with scissors and immersed in a Hanks equilibrium salt solution (Ca·Mg-free) containing 0.1% (v / w) collagenase. The solution was then enzymatically treated by shaking at 200 rpm at 37°C for 30 minutes. After enzymatic treatment, the solution was centrifuged, the supernatant was removed, and the mixture was washed. The centrifugation and washing process was repeated five times to obtain SVF containing fat-derived MSCs.

[0053] (Process 3: Culture of adipose-derived MSCs) The SVF obtained in "Process 2: Enzymatic treatment of excised fat and acquisition of SVF" described above was entirely seeded into a 6-well culture plate (Corning) and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 5% human platelet lysate at a final concentration. These adherently cultured cells are referred to as the 0th passage cell population. Upon reaching subconfluence or a desired culture period, the 0th passage cell population was detached using TrypLE Select (Thermo Fisher Scientific).

[0054] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) In the cell population of passage 0 detached in "Process 3: Culture of Adipose-Derived MSCs" described above, the dead cell concentration and total cell concentration were measured using a nucleo counter.

[0055] Cell concentration was measured using a Nucleocounter (model: NC-100) from ChemoMetec. Dead cell concentration was measured by aspirating the cell suspension into a cassette (model: 941-0002) containing a PI solution for staining dead cells. Total cell concentration was measured by mixing equal volumes of cell suspension with cell treatment reagent A100 (model: 910-0003) and cell treatment reagent B (model: 910-0002) to stain all cells with the PI solution, aspirating the mixture into the aforementioned cassette, and measuring the total cell concentration.

[0056] The cell count and viability of the obtained adipose-derived MSCs were calculated from the cell concentration measurements. The calculation formula used was as follows: (1) Number of adipocyte-derived MSCs (cells) = Total cell concentration (cells / mL) × 3 (dilution ratio at the time of measurement) × Volume of cell suspension (mL) (2) Survival rate of adipose-derived MSCs (%) = 100 - (dead cell concentration (cells / mL) / (total cell concentration (cells / mL) × 3 (dilution ratio at the time of measurement)) × 100) As a result, in Comparative Example 1, no adherent cells were observed in the cell population of passage 0 derived from donor A after 8 days of culture, and the resulting cell suspension was below the detection limit of the above measuring instrument. Therefore, the method of Comparative Example 1 could not produce adipose-derived MSCs.

[0057] Comparative Example 2 and Example 1 below used excised fat from the same donor B.

[0058] <Comparative Example 2: Examination of Adipose Tissue Transport> (Process 1: Collection, transport, and storage of excised fat) Except for 0.06 g of excised fat taken from a different donor (Donor B) than the donor in Comparative Example 1, the excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1, except that it was placed in a 1.5 mL microtube containing 1 mL of Hanks equilibrium salt solution (Ca·Mg-free) (compressive stress of the medium was 0.00 N).

[0059] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0060] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0061] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Comparative Example 2 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0062] As a result, in Comparative Example 2, no adherent cells were observed in the cell population of passage 0 derived from donor B after 13 days of culture, and the resulting cell suspension was below the detection limit of the measuring instrument. Therefore, the method of Comparative Example 2 could not produce adipose-derived MSCs.

[0063] <Example 1: Investigation of adipose tissue transport> (Process 1: Collection, transport, and storage of excised fat) Except for 0.06 g of excised fat taken from the same donor (Donor B) as in Comparative Example 2, which was placed in a 1.5 mL microtube and embedded in 1 mL of gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.09 N, the excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1. Embedding of the excised fat was performed by liquefying the prepared gelatin-containing Hanks equilibrium salt solution at 37°C, placing the excised fat in the solution, and then solidifying it at 4°C. Subsequently, it was transported and stored under refrigeration (4°C) for 20 hours, then heated to 37°C to liquefy the gelatin-containing Hanks equilibrium salt solution, the excised fat was removed, and Process 2 was performed.

[0064] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0065] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0066] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Example 1 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0067] As a result, after 13 days of culture, the cell count of the 0th passage cell population derived from donor B in Example 1 was 3.0 × 10⁶. 5 The survival rate was 95.9%. Therefore, it was found that the method of Example 1 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0068] (Process 5: Passaging of adipose-derived MSCs) The cell population obtained in "Process 3: Culture of Adipose-Derived MSCs" described above (passage 0) was seeded into a T-75 flask (Corning) and then subcultured. This subcultured cell population is called the cell population of passage 1.

[0069] The cell population from the first passage was detached using TrypLE Select when it reached subconfluence, diluted with culture medium, and recovered by centrifugation. The recovered cell population was suspended in a cryopreservation solution consisting of CP-1 (registered trademark) (manufactured by Kyokuto Pharmaceutical Co., Ltd.):25% human serum albumin:physiological saline in a ratio of 2:1:3, slowly frozen to -80°C, and then cryopreserved at -80°C.

[0070] (Process 6: Surface antigen analysis) For the first passage cell population derived from donor B in Example 1, the positive rates of each surface antigen (CD73, CD90, CD105, CD45, and CD31) were measured using a flow cytometer.

[0071] Surface antigen analysis was performed using Merck's Guava easyCyte Single, with a cell count of 10,000 cells and a flow rate setting of Slow. In this measurement, the following antibodies were used as isotype control antibodies: PE Mouse IgG1 k Isotype Control (BD / model number: 555749), FITC Mouse IgG1 k Isotype Control (BD / model number: 555748), and Alexa Fluor 647 Mouse IgG1 k Isotype Control (BD / model number: 557714). PE Mouse Anti-Human CD73 (BD / model number: 550257) was used as the antibody against the CD73 antigen, PE Mouse Anti-Human CD90 (BD / model number: 555595) as the antibody against the CD90 antigen, PE Mouse Anti-Human CD105 (BD / model number: 560839) as the antibody against the CD105 antigen, FITC Mouse Anti-Human CD45 (BD / model number: 555482) as the antibody against the CD45 antigen, and Alexa Fluor as the antibody against the CD31 antigen. I used the 647 Mouse Anti-Human CD31 (manufactured by BD Corporation / model number: 561654).

[0072] Surface antigen analysis revealed that in the first-passage cell population derived from donor B in Example 1, the positive rates for CD73, CD90, and CD105 were all over 90% (specifically, CD73: 100%, CD90: 100%, CD105: 100%), while the positive rates for CD45 and CD31 were less than 5% (negative rates were over 95%) (specifically, CD45 positive rate: 1% (negative rate: 99%), CD31 positive rate: 1% (negative rate: 99%)). From these results, it was confirmed that the first-passage cell population in Example 1 contained a cell population with high purity mesenchymal stem cells.

[0073] Example 2 and Comparative Example 3 below used excised fat from the same donor C.

[0074] <Example 2: Investigation of adipose tissue transport> (Process 1: Collection, transport, and storage of excised fat) 0.15 g of excised fat, collected from a donor (Donor C) different from the donors used in Comparative Examples 1 and 2, was placed in 1.5 mL microcentrifuge tubes. One tube was embedded in a gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.09 N (Donor C-Gelatin), and the other was embedded in 1 mL of agarose-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.71 N (Donor C-Agarose). Except for this, the excised fat was transported and stored using the same method as in Process 1 of Comparative Example 1. However, for the Donor C-Agarose embedding of the excised fat, the prepared agarose-containing Hanks equilibrium salt solution was solidified at room temperature or below, and the excised fat was embedded in the finely crushed gel. After that, the tubes were transported and stored under refrigeration (4°C) for 20 hours, the excised fat was removed from the gel, and Process 2 was performed.

[0075] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0076] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0077] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Example 2 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0078] As a result, after 17 days of culture, the cell count of the 0th passage cell population derived from the donor C-gelatin in Example 2 was 1.0 × 10⁶. 6 The survival rate was 96.8%. In addition, the cell count of the 0th passage cell population derived from the donor C-agarose in Example 2 was 1.5 × 10⁶. 6The survival rate was 98.6%. Therefore, it was found that the method of Example 2 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0079] <Comparative Example 3: Examination of Adipose Tissue Transport> (Process 1: Collection, transport, and storage of excised fat) Except for 0.15 g of excised fat taken from the same donor (Donor C) as in Example 2, which was placed in a 1.5 mL microtube containing 1 mL of Astellas Pharma's UW solution (product name: Belzer UW Refrigerated Storage Solution) (compressive stress of the medium is 0.00 N), the excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1.

[0080] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0081] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0082] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) Following a 17-day culture period, the cell count and viability of the 0th passage cell population of Comparative Example 3 were evaluated using the same method as in Process 4 of Comparative Example 1. Table 1 shows the cell count results for each adipose-derived MSC obtained from the adipose tissue of the same donor (Donor C) in Example 2 and Comparative Example 3.

[0083] [Table 1]

[0084] As shown in Table 1, the number of cells in the 0th passage cell population derived from donor C in Comparative Example 3 was 0.1 × 10⁶. 6The number of cells obtained was 90.8%, which is less than 1 / 10 of the number of cells obtained in the 0th passage cell population derived from donor C-gelatin and donor C-agarose in Example 2, and the survival rate was also slightly lower. Therefore, it was found that the method of Comparative Example 3 is not suitable for the production of adipose-derived MSCs.

[0085] Examples 3, 4, and 5 and Comparative Examples 4, 5, and 6 below used aspirated fat from the same donor D.

[0086] <Example 3: Investigation of adipose tissue transport> (Process 1: Fat harvesting, transport, and storage) One mL of aspirated fat (Donor D - aspirated) collected by liposuction from a donor (Donor D) different from the donors used in Examples 1 and 2 was placed in a 1.5 mL microtube, embedded in one mL of each gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with the compressive stresses shown in Table 2, and transported and stored under refrigerated conditions (4°C) for approximately 72 hours.

[0087] [Table 2]

[0088] (Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF) SVF containing fat-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1, except that each aspirated fat sample taken from a 1.5 mL microtube was directly immersed in a Hanks equilibrium salt solution (Ca·Mg-free) containing 0.1% (v / w) collagenase.

[0089] (Process 3: Culture of adipose-derived MSCs) Except for seeding the entire volume of each SVF obtained from donor D-aspiration into a T-25 flask (Corning), a cell population of passage 0 was obtained and adherent cells were collected using the same method as in process 3 of Comparative Example 1.

[0090] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the 0th passage cell population of Example 3 were evaluated after 7 days of culture using the same method as in Process 4 of Comparative Example 1.

[0091] As a result, the number of cells in the 0th passage cell population derived from donor D-aspiration (1) in Example 3 was 1.0 × 10⁶. 6 The survival rate was 97.4%, and the cell count of the 0th passage cell population derived from donor D-aspiration (2) in Example 3 was 1.6 × 10⁶. 6 The survival rate was 98.1%, and the cell count of the 0th passage cell population derived from donor D-aspiration (3) in Example 3 was 1.9 × 10⁶. 6 The survival rate was 98.8%, and the number of cells in the 0th passage cell population derived from donor D-aspiration (4) in Example 3 was 0.8 × 10⁶. 6 The survival rate was 97.1%. Therefore, it was found that the method of Example 3 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0092] <Comparative Example 4: Examination of Adipose Tissue Transport> (Process 1: Fat harvesting, transport, and storage) The aspirated fat (Donor D-Aspiration), collected by liposuction from the same donor as in Example 3 (Donor D), was transported and stored in the same manner as in Process 1 of Example 3, except that it was placed in a 1.5 mL microtube containing 1 mL of Hanks equilibrium salt solution (Ca·Mg-free) (compressive stress of the medium was 0.00 N).

[0093] (Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Example 3.

[0094] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Example 3, a cell population of passage 0 was obtained, and adherent cells were collected.

[0095] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) Using the same method as in Process 4 of Comparative Example 1, the cell count and viability of the passage 0 cell population of Comparative Example 4 were evaluated.

[0096] As a result, after 7 days of culture, the cell count of the passage 0 cell population derived from Donor D - aspiration of Comparative Example 4 was 0.1×10 6 cells, and the viability was 93.3%. The cell count obtained was less than 1 / 5 of that of each passage 0 cell population derived from Donor D - aspiration of Example 3. From the above, it was found that the method of Comparative Example 3 was not suitable for the production of adipose - derived MSCs.

[0097] <Example 4: Examination of adipose tissue transportation> (Process 1: Collection, transportation, and preservation of aspirated adipose tissue) 1 mL of aspirated adipose tissue (Donor D - aspiration) collected by aspirating adipose tissue from the same donor (Donor D) as in Example 3 was placed in a 1.5 mL microtube. Except that it was embedded with 1 mL of fibrin (product name: Bolheal) with a medium compression stress of 6.86 N, the aspirated adipose tissue was transported and preserved using the same method as in Process 1 of Example 3.

[0098] (Process 2: Enzymatic treatment of aspirated adipose tissue and acquisition of SVF) Using the same method as in Process 2 of Example 3, SVF containing adipose - derived MSCs was obtained.

[0099] (Process 3: Culture of adipose - derived MSCs) Using the same method as in Process 3 of Example 3, a passage 0 cell population was obtained, and adherent cells were collected.

[0100] (Process 4: Evaluation of cell count and viability of cultured adipose - derived MSCs) Using the same method as in Process 4 of Comparative Example 1, after 7 days of culture, the cell count and viability of the passage 0 cell population of Example 4 were evaluated. As a result, the cell count of the passage 0 cell population derived from Donor D - aspiration of Example 4 was 0.8×10 6The survival rate was 97.1%. Therefore, it was found that the method of Example 4 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0101] <Comparative Example 5: Examination of Adipose Tissue Transport> (Process 1: Fat harvesting, transport, and storage) The aspirated fat (Donor D-Aspiration), collected by liposuction from the same donor (Donor D) as in Example 3, was placed in a 1.5 mL microtube and embedded in 1 mL of gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 12.27 N. The aspirated fat was transported and stored using the same method as in Process 1 of Example 3.

[0102] (Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Example 3.

[0103] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Example 3, a cell population of passage 0 was obtained, and adherent cells were collected.

[0104] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) Following a 7-day culture, the cell count and viability of the passage 0 cell population of Comparative Example 5 were evaluated using the same method as in Process 4 of Comparative Example 1. As a result, only a very small number of cells adhered to the culture vessel in the passage 0 cell population derived from donor D-aspiration in Comparative Example 5, and the number of cells obtained was below the detection limit of the measuring instrument. Therefore, the method of Comparative Example 5 could not produce adipose-derived MSCs.

[0105] <Example 5: Investigation of adipose tissue transport> (Process 1: Fat harvesting, transport, and storage) The harvested aspirated fat (donor D-aspirated) was transported and stored in the same manner as in Process 1 of Example 3, except that the transport and storage conditions were kept in a refrigerated (4°C) environment for approximately 192 hours.

[0106] (Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Example 3.

[0107] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Example 3, a cell population of passage 0 was obtained, and adherent cells were collected.

[0108] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Example 5 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0109] As a result, after 7 days of culture, the cell count of the 0th passage cell population derived from donor D-aspiration in Example 5 was 1.5 × 10⁶. 5 The survival rate was 96.3%. Therefore, it was found that the method of Example 5 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0110] <Comparative Example 6: Examination of Adipose Tissue Transport> (Process 1: Fat harvesting, transport, and storage) The harvested aspirated fat (donor D-aspirated) was transported and stored using the same method as in Process 1 of Comparative Example 4, except that the transport and storage conditions were kept in a refrigerated (4°C) environment for approximately 192 hours.

[0111] (Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Example 3.

[0112] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Example 3, a cell population of passage 0 was obtained, and adherent cells were collected.

[0113] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Comparative Example 6 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0114] As a result, after 7 days of culture, the cell count of the 0th passage cell population derived from donor D-aspiration in Comparative Example 6 was 0.5 × 10⁶. 5 The survival rate was 93.3%, and only about one-third the number of cells obtained from the 0th passage cell population derived from donor D-aspiration in Example 5 was found. Therefore, it was concluded that the method of Comparative Example 6 is not suitable for the production of adipose-derived MSCs.

[0115] Table 3 below shows the cell number and viability of the cell population at passage 0 for Examples 3, 4, and 5 and Comparative Examples 4, 5, and 6.

[0116] [Table 3]

[0117] As shown in Table 3, when aspirated adipose tissue is embedded in a medium with a compressive stress greater than 0 N and 12 N or less, and stored and / or transported in this state, many adherent cells can be observed in the culture vessel, and it was found that a large number of adipose-derived MSCs with high viability can be produced.

[0118] Example 6 and Comparative Example 7 used excised fat from the same donor D.

[0119] <Example 6: Investigation of adipose tissue transport> (Process 1: Collection, transport, and storage of excised fat) Except for 0.1 g of excised fat (Donor D-excision) taken from a donor different from the donor in Comparative Example 1 (Donor D), which was placed in a 1.5 mL microtube and embedded in 1 mL of gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.09 N, the excised fat was transported and stored using the same method as in Process 1 of Comparative Example 1.

[0120] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0121] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0122] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Example 6 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0123] As a result, after 16 days of culture, the cell count of the 0th passage cell population derived from donor D- excision in Example 6 was 5.0 × 10⁶. 5 The survival rate was 98.3%. Therefore, it was found that the method of Example 6 allows for the observation of many adherent cells in the culture vessel and enables the production of many high-viability adipose-derived MSCs.

[0124] <Comparative Example 7: Examination of Adipose Tissue Transport> (Process 1: Collection, transport, and storage of excised fat) The excised fat was transported and stored using the same method as in Process 1 of Comparative Example 1, except that 0.1 g of excised fat (Donor D-excision) was used, which was collected from a different donor (Donor D) than the donor used in Comparative Example 1.

[0125] (Process 2: Enzymatic treatment of excised fat and acquisition of SVF) SVF containing adipose-derived MSCs was obtained using the same method as in Process 2 of Comparative Example 1.

[0126] (Process 3: Culture of adipose-derived MSCs) Using the same method as in Process 3 of Comparative Example 1, a cell population of passage 0 was obtained, and adherent cells were collected.

[0127] (Process 4: Evaluation of cell number and viability of cultured adipose-derived MSCs) The cell count and viability of the cell population at passage 0 of Comparative Example 7 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0128] As a result, after 16 days of culture, the cell count of the 0th passage cell population derived from donor D- excision in Comparative Example 7 was 0.2 × 10⁶. 5 Because the number of cells obtained was small and low, the survival rate was below the detection limit of the measuring instrument. Furthermore, only about 1 / 25th the number of cells obtained from each passage 0 cell population derived from donor D- excision in Example 6 could be obtained. From the above, it was found that the method of Comparative Example 7 is not suitable for the production of adipose-derived MSCs.

[0129] Table 4 below shows the cell number and viability of the cell population at passage 0 for Example 6 and Comparative Example 7.

[0130] [Table 4]

[0131] As shown in Table 4, when excised adipose tissue is embedded in a medium with a compressive stress greater than 0 N and less than or equal to 12 N and stored and / or transported, many adherent cells can be observed in the culture vessel, and it was found that a large number of adipose-derived MSCs with high viability can be produced.

[0132] Examples 7 and 8 and Comparative Example 8 used amniotic tissue from the same donor E.

[0133] <Example 7: Investigation of amniotic tissue transport> (Process 1: Amniotic membrane harvesting, transport, and storage) The amniotic membrane and placenta, which are fetal appendages, were aseptically collected from a pregnant woman (donor E) who underwent elective cesarean section and gave informed consent. The collected amniotic membrane and placenta were placed in a container with physiological saline, and the amnion was detached from the cut end of the amniotic membrane. The amnion was washed with Hanks equilibrium salt solution (Ca·Mg-free). The weight of the collected amnion was measured, and 1 g of amnion was embedded in 1 mL of gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.09 N and placed in a 1.5 mL microtube. The amnion was embedded by liquefying the prepared gelatin-containing Hanks equilibrium salt solution at 37°C, placing the amnion in the solution, and then solidifying it at 4°C. After that, it was transported and stored under refrigeration (4°C) for approximately 216 hours, then the gelatin-containing Hanks equilibrium salt solution was liquefied by warming to 37°C, the amnion was removed, and process 2 was performed.

[0134] (Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSCs) 1 g of amniotic membrane, removed from a 1.5 mL microcentrifuge tube, was immersed in Hanks equilibrium salt solution (containing Ca·Mg) containing 240 PU / mL collagenase and 200 PU / mL dispase I, and the amniotic membrane was enzymatically treated by shaking and stirring at 37°C for 90 minutes. The solution after enzymatic treatment was filtered through a strainer to remove undigested amniotic membrane material, and a cell population containing amniotic membrane-derived MSCs was obtained.

[0135] (Process 3: Culture of amniotic membrane-derived MSCs) The cell population containing amniotic membrane-derived MSCs obtained in "Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSCs" described above was seeded at 1 / 5 volume into a T-25 flask (Corning Corporation) and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 5% human platelet lysate at a final concentration. These adherently cultured cells are referred to as the 0th passage cell population. Upon reaching subconfluence or a desired culture period, the 0th passage cell population was detached using TrypLE Select (Thermo Fisher Scientific).

[0136] (Process 4: Evaluation of cell number and viability of cultured amniotic membrane-derived MSCs) The cell count and viability of the cell population at passage 0 of Example 7 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0137] As a result, after 12 days of culture, the cell count of the 0th passage cell population derived from donor E in Example 7 was 9.5 × 10⁶. 5 The survival rate was 98.6%. Therefore, it was found that the method of Example 7 allows for the observation of many adherent cells in the culture vessel and enables the production of many amniotic membrane-derived MSCs with high viability.

[0138] <Comparative Example 8: Examination of Amniotic Tissue Transport> (Process 1: Amniotic membrane harvesting, transport, and storage) The amniotic membrane was transported and stored in the same manner as in Process 1 of Example 7, except that 1 g of amniotic membrane collected from the same donor (Donor E) as in Example 7 was placed in a 1.5 mL microtube containing 1 mL of Hanks equilibrium salt solution (Ca·Mg-free) (compressive stress of the medium was 0.00 N).

[0139] (Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSCs) A cell population containing amniotic membrane-derived MSCs was obtained using the same method as in Process 2 of Example 7.

[0140] (Process 3: Culture of amniotic membrane-derived MSCs) A cell population of passage 0 was obtained and adherent cells were collected using the same method as in Process 3 of Example 7.

[0141] (Process 4: Evaluation of cell number and viability of cultured amniotic membrane-derived MSCs) The cell count and viability of the cell population at passage 0 of Comparative Example 8 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0142] As a result, after 12 days of culture, the cell count of the 0th passage cell population derived from donor E in Comparative Example 8 was 5.6 × 10⁶. 5 The survival rate was 94.6%, and only about 3 / 5 the number of cells obtained from the 0th passage cell population derived from donor E in Example 7 was found. Therefore, it was concluded that the method of Comparative Example 8 is not suitable for the production of amniotic membrane-derived MSCs.

[0143] <Example 8: Investigation of amniotic tissue transport> (Process 1: Amniotic membrane harvesting, transport, and storage) From the same donor (Donor E) as in Example 7, 1 g of amniotic membrane was collected using the same method as in Process 1 of Example 7. This membrane was embedded in 1 mL of a gelatin-containing Hanks equilibrium salt solution (Ca·Mg-free) with a compressive stress of 1.09 N and placed in a 1.5 mL microtube. The amniotic membrane was embedded by liquefying the prepared gelatin-containing Hanks equilibrium salt solution at 37°C, placing the amniotic membrane in the solution, and then allowing it to solidify at 4°C. Subsequently, the solution was transported and stored at room temperature (approximately 20°C) for approximately 110 hours. The solution was then heated to 37°C to liquefy the gelatin-containing Hanks equilibrium salt solution, the amniotic membrane was removed, and Process 2 was carried out.

[0144] (Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSCs) A cell population containing amniotic membrane-derived MSCs was obtained using the same method as in Process 2 of Example 7.

[0145] (Process 3: Culture of amniotic membrane-derived MSCs) A cell population of passage 0 was obtained and adherent cells were collected using the same method as in Process 3 of Example 7.

[0146] (Process 4: Evaluation of cell number and viability of cultured amniotic membrane-derived MSCs) The cell count and viability of the cell population at passage 0 of Comparative Example 8 were evaluated using the same method as in Process 4 of Comparative Example 1.

[0147] As a result, after 11 days of culture, the cell count of the 0th passage cell population derived from donor E in Example 8 was 1.3 × 10⁶. 6The survival rate was 98.3%. Therefore, it was found that the method of Example 6 allows for the observation of many adherent cells in the culture vessel and enables the production of many amniotic membrane-derived MSCs with high viability.

[0148] Table 5 below shows the cell number and viability of the cell population at passage 0 for Examples 7 and 8 and Comparative Example 8.

[0149] [Table 5]

[0150] As shown in Table 5, it was found that storing and / or transporting amniotic tissue embedded in a medium with a compressive stress greater than 0 N and 12 N or less can produce a large number of amniotic mesenchymal stem cells (MSCs) with high viability.

[0151] Based on the above, it was found that in the example method that satisfies the following conditions (1) and (2), many adherent cells can be observed in the culture vessel, and a large number of fat and amniotic membrane-derived MSCs with high viability can be produced. (1) Preserve and / or transport tissues containing adherent cells embedded in a medium having a compressive stress greater than 0 and 12 N or less. (2) Remove the tissue embedded in the above medium from the medium and separate the adherent cells. In other words, according to the present invention, tissues containing adherent cells such as mesenchymal stem cells can be safely stored and transported, and adherent cells can be efficiently produced from the raw tissue containing adherent cells after storage and transport. This is expected to expand opportunities for providing treatment to patients, reduce the burden on cell culture operators, and lower manufacturing and medical costs.

[0152] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for producing a cell population containing adherent cells from tissue containing said cells, (1) Tissue containing adhesive cells is stored and / or transported embedded in a biocompatible gel or sol having a compressive stress of 0.01 N or more and 7 N or less at the storage and / or transport temperature. (2) The procedure includes removing the tissue embedded in the biocompatible gel or sol from the gel or sol and separating the adherent cells, The biocompatible gel or sol has the property of liquefying upon heating, solidifying upon cooling, and reversibly liquefying and solidifying upon temperature changes. A method for producing adherent cells from tissue.

2. The method for producing a product according to claim 1, wherein the gel or sol comprises at least one selected from the group consisting of proteins, peptides, polysaccharides, and synthetic polymers.

3. The method for producing a product according to claim 1 or 2, wherein the gel or sol comprises at least one of gelatin, agarose, and fibrin.

4. A method for producing a tissue containing adhesive cells, comprising storing and / or transporting the tissue in a gel, and then liquefying the gel in which the tissue containing adhesive cells is embedded by heating to remove the tissue, according to any one of claims 1 to 3.

5. A method for producing a product according to any one of claims 1 to 4, characterized in that the adherent cells are mesenchymal stem cells.

6. The method for producing a product according to any one of claims 1 to 5, wherein the tissue containing adhesive cells is at least one selected from the group consisting of amniotic membrane, fat, umbilical cord, placenta, skin, and muscle.

7. A manufacturing method according to any one of claims 1 to 6, wherein the storage and transport time is 240 hours or less.

8. The method for producing a product according to any one of claims 1 to 7, further comprising the step of removing tissue containing adherent cells from a gel or sol, enzymatically treating the tissue, and culturing the separated adherent cells.

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