WO2022045201A1 - Method for efficiently producing tissue from adhesive cells - Google Patents

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

How to Efficiently Produce Adhesive Cells from Tissue

The present invention relates to a method for efficiently producing adhesive cells such as mesenchymal stem cells from tissues.

Adhesive mesenchymal stem cells are somatic stem cells that have been reported to be present in bone marrow, adipose tissue, dental pulp, etc., and have the ability to differentiate into bone, cartilage, fat, etc., and are therefore promising in cell therapy. It is attracting attention as a cell source. Mesenchymal stem cells have not only differentiation ability but also immunosuppressive ability, and clinical application for acute graft-versus-host disease (GVHD) and Crohn's disease is advancing. In addition, skeletal myoblasts separated from muscle tissue are also known to have adhesiveness and secrete cytokines that promote tissue regeneration, and have been put into practical use as myocardial regeneration therapy by Telmo. In addition, fibroblasts that can be separated from skin tissue also have adhesiveness and are used in clinical practice for anti-aging such as cosmetology.

These adhesive mesenchymal stem cells, skeletal myoblasts, and fibroblasts are known to be contained in sheep membrane, fat, umbilical cord, placenta, skin, and muscle tissues, and these tissues are treated. It is attracting attention as a promising raw material for producing cells for use. In commercial practice, these tissue collection and processing facilities (facilities that produce cells from the tissue) are separated from each other, so that the tissue is safely stored and / or transported from the tissue collection facility to the processing facility. There is a need. When the tissue is stored and / or transported, it is often immersed in a solution for the purpose of preventing the tissue from drying, and a buffer solution may be used as the solution. In addition, an organ preservation solution has been developed and is commercially available as a solution for preserving organs.

An object of the present invention is to provide a method for safely storing and / or transporting a raw material tissue containing adhesive cells such as mesenchymal stem cells, and to efficiently obtain adhesive cells from the raw material tissue containing the adhesive cells after storage / transportation. Is to provide a method of manufacturing.

In the course of the study under the above-mentioned problems, the tissue was immersed in a buffer solution or an organ preservation solution, preserved and / or transported, and an attempt was made to produce adhesive cells from the tissue. The tissue was preserved and / or transported. It was confirmed that the cells were damaged and induced a decrease in adhesiveness and damage to the adhesive cells in the tissue, and therefore the number of acquired cells decreased. As a result of further diligent studies, the present inventors surprisingly suppressed the damage to the tissue by storing and / or transporting the tissue in a medium having an appropriate compressive stress. It was found that the adhesive cells could be efficiently produced.

Based on the above results, the following inventions will be provided.

A method for producing a cell population containing the cells from a tissue containing the adhesive cells.
Tissues containing adherent cells are stored and / or transported embedded in a medium with a compressive stress greater than 0 and 12 N or less.
A method for producing adhesive cells from a tissue, which comprises removing the tissue embedded in the medium from the medium and separating the adhesive cells.

This specification includes the disclosure content of Japanese Patent Application No. 202-143577, which is the basis of the priority of the present application.

According to the present invention, tissues containing adherent cells such as mesenchymal stem cells can be safely preserved / transported, and adherent cells can be efficiently produced after storage / transport.

Hereinafter, embodiments of the present invention will be specifically described, but the following description is for facilitating the understanding of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

[1] Explanation of terms The "adhesive cells" in the present invention refer to cells that adhere to a petri dish, plate, or flask of a glass or plastic substrate and exhibit a spindle-shaped morphology. Examples of adhesive cells include mesenchymal stem cells, skeletal myoblasts, fibroblasts, epithelial cells, myocardial stem cells, nerve stem cells, hepatic stem cells, myocardial cells, nerve cells, hepatocytes, etc. Is not limited to these cells.
Tissues containing adhesive cells include solid tissues such as fat, placenta, fetal membrane, amniotic membrane, villous membrane, skin, umbilical cord, heart, brain, lung, corneal membrane, intestine, muscle, and synovial cord, bone marrow, dental pulp, sheep's water, and umbilical cord. There are liquid tissues such as blood and blood, but the present invention is not limited to these tissues. In addition, those obtained by processing these structures are also included.

The "medium" in the present invention may have any state, properties, and structure as long as the compressive stress is greater than 0 and 12 N or less. For example, it may have any properties such as solid, liquid, and gas, and may be in a mixed state. Specific examples of the medium having a compressive stress greater than 0 and 12 N or less include gels, sol, and the like. Something similar to. The gel refers to a gel in which colloidal particles are dispersed in a liquid or a gas and lose their fluidity, and examples thereof include konjac, yokan, agar, and pudding. The sol refers to a sol in which colloidal particles are dispersed in a liquid or a gas and the fluidity is not lost, and examples thereof include milk, yogurt to drink, and oil. As the sol / gel in the present invention, a colloid using water as a dispersion medium is preferable, and a so-called hydrogel is more preferable.

The medium of the present invention preferably has an appropriate hardness, and as described above, compressive stress is adopted as an index of the hardness. As a method for measuring compressive stress in the present invention, a 2 mL medium is placed in a 24-well plate, the medium is compressed with a plunger having a diameter of 1 cm, and the stress (unit: N) when the medium is compressed by 1.5 mm is EZ. -It is obtained by measuring with TEST (Shimadzu Corporation, EZ-SX), but it is not limited to the above method when the same result can be obtained. In the present invention, the compressive stress of the medium must satisfy the above range of "greater than 0 and 12 N or less" at the temperature during storage and / or transportation. For example, compressive stress at 4 ° C. for storage and transport at 4 ° C., and compressive stress at 15 ° C. for storage and transport at 15 ° C. The compressive stress is not particularly limited as long as it is greater than 0 and 12 N or less, but is preferably 0.001 N or more, more preferably 0.005 N or more, further preferably 0.01 N or more, and most preferably 0.1 N or more. The compressive stress is preferably 11 N or less, more preferably 10 N or less, and even more preferably 9 N or less. 8N or less is more preferable, and 7N or less is most preferable. If the temperature during storage and transportation is not constant, any one of the temperatures may be within the above compressive stress range, but at least 40% of the entire storage and / or transportation process. The above period preferably satisfies the above compressive stress, more preferably 60% or more, further preferably 80% or more, and most preferably satisfying the above compressive stress under all temperature conditions. For example, when stored once at 4 ° C and transported in the range of 15 ° C to 25 ° C, any one of the compressive stresses at 4 ° C and 15 ° C to 25 ° C falls within the above compressive stress range. However, it is more preferable to satisfy all of them.

From the viewpoint of biocompatibility, the medium described in the present invention preferably contains at least one selected from the group consisting of proteins, peptides, polysaccharides and synthetic polymers, and in the present invention, these are dispersed in water. It is more preferable to use a medium as a medium, but the present invention is not limited thereto. As the protein, gelatin, collagen, fibrin, soybean protein and the like can be used. Polysaccharides or substances containing polysaccharides include agarose, pectin, carrageenan, curdlan, chitin, chitosan, arginic acids, soybean polysaccharides, celluloses such as carboxymethyl cellulose, mannans, arabic gum, gellan gum, guar gum, xanthan gum, starch, agar. , Fucoidan, etc. can be used. As the synthetic polymer, synthetic peptides (self-assembled peptides such as Panacea gel and PuraMatrix), polyvinyl alcohol, propylene glycol, silicon, polyacrylamide and the like can be used. Further, these may be used alone or in combination of two or more.

The "culture solution" in the present specification is not particularly limited, and any liquid medium for cell culture is used as a basal medium, and other components (albumin, blood-derived components, growth factors, etc.) are appropriately added as needed. Can be prepared by.

As the basal medium, BME medium, BGJb medium, CMRL1066 medium, Glasgo MEM medium, Applied MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco's Medium), Medium 199 Medium, Eagle's Medium, Eagle's Medium, Eagle's Medium, Eagle's Medium, Eagle's Medium, Eagle's Medium, Eagle's Medium of Medium Essential Medium Eagle's medium, DMEM medium (Dulvecco's Modified Eagle's Medium), ham F10 medium, ham F12 medium, RPMI 1640 medium, Fisher's medium, and a mixed medium thereof (for example, DMEM / F12 medium). (Dulvecco's Modified Eagle's Medium / Nutient Mixture F-12 Ham)) and the like can be used, but the medium is not particularly limited. In addition, various commercially available serum-free media can also be used.

Examples of other components added to the basal medium include albumin, blood-derived components, growth factors, and the like. In the embodiment in which albumin is added to the basal medium, the concentration of albumin is preferably 0.05% by weight or more and 5% by weight or less. Further, as the blood-derived component, various sera (animal-derived serum such as bovine fetal serum (FBS or FCS), human serum, various animal and / or human blood-derived multiplatelet plasma and platelet lysate are prepared as raw materials. Serum, etc.), various animal and / or human blood-derived platelet lysates, plasma, etc. The human serum may be derived from the same individual as the individual from which the tissue containing the adhesive cells was obtained, or may be derived from a different individual. In the embodiment in which the blood-derived component is added to the basal medium, the concentration of the blood-derived component is preferably 2% by volume or more and 40% or less by volume. More preferably, it is 3% by volume or more and 30% or less by volume. In the embodiment in which the growth factor is added, a reagent for stabilizing the growth factor in the medium (anticoagulant such as heparin, gel, polysaccharide, etc.) may be further added in addition to the growth factor. Pre-stabilized growth factors may be added to the basal medium. Growth factors include, for example, fibroblast growth factor (FGF), epithelial cell growth factor (EGF), transforming growth factor (TGF), vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF), and theirs. Families can be used, but are not particularly limited.

[2] Method for Efficiently Separating Adhesive Cells such as Mesenchymal Stem Cells The step of collecting tissue containing adherent cells such as mesenchymal stem cells can be performed by, for example, the following procedure. can. In the case of adipose tissue, make an incision of about 0.5 cm to 1 cm in any part of the patient (for example, abdomen, waist, tweezers) with a sharp-edged scalpel, and use any surgical instrument (for example, mosquito forceps, tweezers) to make fat. Is removed and excised. The incision should be sutured with one needle or taped. Adipose tissue collected by such means is generally called excised fat. On the other hand, fat can be aspirated from any part of the patient (for example, abdomen, waist, thigh) using a cannula or the like. Adipose tissue collected by such means is generally called aspirated fat. In the case of amniotic tissue, fetal appendages (placenta, fetal membrane, etc.) are collected at birth, and then the amniotic membrane is detached from the stump of the fetal membrane. In the case of muscular tissue, the muscular tissue is collected from the thigh. In the case of skin tissue, it may be collected from the back of the ear using a surgical disposable knife, a skin biopsy punch, or the like, but the present invention is not limited thereto.

The collected tissue is stored and / or transported in a state of being embedded in a medium, and can be carried out by the following procedure, for example. The tissue is embedded, stored and / or transported by embedding the collected tissue in a container-filled medium or by adding the medium to the container containing the collected tissue. The step of taking out the tissue embedded in the medium can be performed, for example, by the following procedure. The tissue is removed from the medium in which the tissue is embedded using any instrument (eg, mosquito forceps, tweezers, etc.).

When a sol or gel having the property of repeating reversible solidification (including gelation) and liquefaction such as gelatin is used as a medium, the tissue is preferably preserved / transported as follows by utilizing the property. be able to. When gelatin is used as an example, gelatin is dissolved or dispersed in a dispersion medium such as an aqueous solution as described below to prepare a gelatin solution, and then the gelatin solution is heated to liquefy and the collected tissue is embedded. The gelatin solution in which the tissue is embedded can be cooled to solidify and stored and / or transported in a gel-embedded state. Then, by heating, the gelatin solution can be liquefied and the embedded tissue can be taken out.

The temperature at which the above gelatin solution is liquefied varies depending on the gelatin concentration, and is, for example, 25 ° C. or higher and 60 ° C. or lower. Is more preferable. The temperature at which the gelatin solution is solidified varies depending on the gelatin concentration, but is more preferably 1 ° C. or higher and 25 ° C. or lower, and 1 ° C. or higher and 10 ° C. or lower.

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

The dispersion medium for dissolving gelatin can be any water or an aqueous solution, and the aqueous solution can be any aqueous solution such as a buffer solution, an isotonic solution, a hypotonic solution, and a hypertonic solution. .. From the viewpoint of reducing damage to tissues, buffers and isotonic solutions are more preferable, for example, PBS, HBSS (-), Ringer's solution, Ringer's lactate, infusion solution, saline solution, culture solution, albumin solution, blood-derived components, and the like. Examples include a mixture of. Further, from the viewpoint of controlling bacteria, an antibiotic may be added to the above medium.

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

The storage and transportation temperatures are not particularly limited, but 37 ° C or lower is preferable from the viewpoint of reducing damage to tissues. 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 storage and transportation temperature is not particularly limited, but is, for example, −5 ° C. or higher and 0 ° C. or higher.

The step of separating the adhesive cells from the tissue taken out from the medium can be performed by, for example, the following procedure. The removed tissue is treated with an enzyme, then the adherent cells are separated by centrifugation, and washing and centrifugation are repeated multiple times with a washing solution. At that time, in order to improve the digestibility by the enzyme, it may be cut into small pieces with scissors before the enzyme treatment. Further, as the enzyme treatment solution, collagenase, dispase and the like can be used, but the present invention is not limited thereto.

[3] Method for Producing Cell Population Containing Adhesive Cells Separated from Tissue Containing Adhesive Cells The steps for producing a cell population containing adherent cells separated from tissue containing adhesive cells are, for example, as follows. It can be done by the procedure like. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Next, the cells are seeded in a culture vessel and cultured in a medium with a CO ₂ concentration of 3% or more and 5% or less and a confluence rate of 95% or less. As the above-mentioned medium, for example, the “culture solution” described in the explanation of the term [1] can be used, but the present invention is not limited thereto. In addition, as another method for separating the adherent cells from the tissue and producing a cell population containing the adherent cells, a method for producing the tissue without enzymatic treatment can be applied, for example, ceiling culture (property of floating on water). Tissue pieces are floated when the amount of medium is large. Therefore, the liquid amount can be manufactured by a method of minimizing the amount of the tissue to be immersed, a method of adhering the tissue to the dish by suppressing the floating of the tissue piece by a mesh, etc.), but the present invention is not limited to these. The cells obtained by the above-mentioned culture are cells that have been cultured once (cells of the 0th passage).

The culture period of the above-mentioned single culture can be, for example, 2 to 21 days, more preferably 3 to 19 days, and further preferably 4 to 17 days.

The above-mentioned single-cultured cells can be further subcultured and cultured as follows, for example. First, the cells once cultured are treated with a cell exfoliation means such as trypsin and exfoliated from the culture vessel. The resulting cell suspension is then centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in medium. Finally, the cells are seeded in a culture vessel and cultured in a medium with a CO ₂ concentration of 3% or more and 5% or less and a confluence rate of 95% or less. As the above-mentioned medium, the "culture medium" described in the explanation of [1] terminology can be used, but the present invention is not limited thereto. The culture period of the above culture may be, for example, 2 to 21 days, more preferably 3 to 19 days, still more preferably 4 to 17 days. For cells obtained by culturing, cells that have been passaged n times can be obtained by repeating passage and culture (n indicates an integer of 1 or more). The lower limit of the number of passages n is, for example, once or more, preferably 2 times or more, more preferably 3 times or more, still more preferably 4 times or more, still more preferably 5 times or more, from the viewpoint of mass production of cells. be. Further, the upper limit of the number of passages n is preferably 25 times or less, 20 times or less, 15 times or less, 10 times or less, for example, from the viewpoint of suppressing cell aging.

As the above-mentioned cell exfoliating means, for example, a cell exfoliating agent may be used. As the cell exfoliating agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) and the like can be used, but the cell stripping agent is not particularly limited. As the cell exfoliating agent, a commercially available cell exfoliating agent may be used. For example, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technologys), Acculex, etc. Further, as the cell detachment means, a physical cell detachment means may be used, and for example, a cell scraper (manufactured by Corning Inc.) can be used, but the present invention is not limited thereto. The cell ablation means may be used alone or in combination of two or more.

The means for cryopreserving a cell population containing adherent cells such as mesenchymal stem cells in the present invention is not particularly limited, and examples thereof include a program freezer, a deep freezer, and storage in liquid nitrogen. When a program freezer is used, 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 a program freezer is used, the preferred freezing speeds for freezing are, for example, -1 ° C / min or less, -2 ° C / min or less, -5 ° C / min or less, -9 ° C / min or less, -10 ° C / min or less. Minutes or less, -11 ° C / minute or less, or -15 ° C / minute or less. When a program freezer is used as the above freezing means, for example, at a freezing rate of -2 ° C./min or more and -1 ° C./min or less, up to a temperature between -50 ° C. and -30 ° C. (for example, -40 ° C.). Lower the temperature and further increase the temperature to a temperature of -100 ° C or higher and -80 ° C or lower (eg, -90 ° C) at a freezing rate of -11 ° C / min or higher and -9 ° C / min or lower (for example, -10 ° C / min). Can be lowered. When immersion in liquid nitrogen is used as the above-mentioned freezing means, for example, the temperature can be rapidly lowered to -196 ° C. for freezing, and then cryopreservation can be performed in liquid nitrogen (gas phase). It can also be stored in liquid nitrogen (liquid phase).

When frozen by the above freezing means, the above cell population may be frozen in an arbitrary storage container. Examples of the storage container include, but are not limited to, a cryotube, a cryovial, a freezing bag, and an infusion bag.

When frozen by the above freezing means, the above cell population may be frozen in any cryopreservation solution. As the above cryopreservation solution, a commercially available cryopreservation solution may be used. For example, CP-1 (registered trademark) (manufactured by Far East Pharmaceutical Co., Ltd.), BAMBANKER (manufactured by Lymphotech), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by Reprocell), CryoNovo (Acron Biotechnology). , MSC Freezing Solution (manufactured by Biomagnetic Industries), CryoStor (manufactured by HemaCare), etc., but are not limited thereto. The cryopreservation solution may be used alone or in combination of two or more.

The above cryopreservation solution can contain a predetermined concentration of polysaccharides. The preferable concentration of the polysaccharide is, for example, 1% by mass or more, 2% by mass or more, 4% by mass or more, or 6% by mass or more. The preferable concentration of the polysaccharide is, 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 the polysaccharide include, but are not limited to, hydroxylethyl starch (HES) and dextran (Dextran40, etc.). The polysaccharide may be used alone or in combination of two or more.

The above cryopreservation solution can contain a predetermined concentration of dimethyl sulfoxide (DMSO). The preferred concentration of DMSO is, 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. The preferable concentration of DMSO is, 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.

The above cryopreservation solution may contain albumin having a predetermined concentration higher than 0% by mass. The preferable concentration of albumin is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, or 4% by mass or more. The preferable concentration of albumin is, for example, 30% by mass or less, 20% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin (BSA), mouse albumin, human albumin and the like.

According to one aspect of the present invention, the cell population containing the mesenchymal stem cells provided by the present invention satisfies that the ratio of mesenchymal stem cells positive for CD73, CD90, and CD105 is 80% or more. You may.

CD73 means a differentiation cluster 73 and is a protein also known as 5-nucleotidase or Ecto-5'-nucleotidase.

CD90 means differentiation cluster 90 and is a protein also known as Thy-1.

CD105 means differentiation cluster 105 and is a protein also known as Endoglin.

The proportion of mesenchymal stem cells positive for CD73 in the cell population is 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%. It may be 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The proportion of mesenchymal stem cells positive for CD90 in the cell population is 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%. It may be 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The proportion of mesenchymal stem cells positive for CD105 in the cell population is 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%. It may be 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

According to one aspect of the present invention, even if the cell population containing the mesenchymal stem cells provided by the present invention satisfies that the ratio of mesenchymal stem cells exhibiting negative for CD45 and CD31 is 80% or more. good.

CD45 means a differentiation cluster 45, and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type, C) or LCA (Leukocyte common engine).

CD31 means a differentiation cluster 31, and is a protein also known as Hematopoietic progenitor cell antigen CD31.

The proportion of mesenchymal stem cells that are negative for CD45 in the cell population is 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%. It may be 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The proportion of mesenchymal stem cells that are negative for CD31 in the cell population is 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%. It may be 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

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

In this example, the compressive stress of the medium for transporting and storing the excised fat was as follows: 2 mL of the medium was placed in a 24-well plate, the medium was compressed with a plunger having a diameter of 1 cm at 4 ° C, and the medium was compressed by 1.5 mm. The stress at this time (unit is N) was measured by EZ-TEST (Shimadzu Corporation, EZ-SX).

<Comparative Example 1: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
The abdomen of the donor (donor A) who gave informed consent was incised with a scalpel with a sharp edge, and the fat was excised with mosquito forceps and tweezers. The weight of the excised fat collected was weighed, and 0.06 g of the excised fat was placed in a 1.5 mL microtube as it was, and transported and stored in a refrigerated (4 ° C.) environment for about 20 hours.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF (stromal vascular cell fraction))
The excised fat taken out from the 1.5 mL microtube is cut into small pieces with scissors, immersed in a Hanks balanced salt solution containing 0.1% (v / w) collagenase (without Ca / Mg), and immersed at 37 ° C. for 30 minutes at 200 rpm. The enzyme treatment was carried out by shaking and stirring under the conditions of. The solution after the enzyme treatment was centrifuged, the supernatant was removed, and then the solution was washed. The process of centrifugation and washing was repeated 5 times to obtain an SVF containing a fat-derived MSC.

(Process 3: Culture of fat-derived MSC)
The entire amount of SVF obtained in "Process 2: Enzymatic treatment of excised fat and acquisition of SVF" was inoculated into a 6-well plate (manufactured by Corning Inc.) in a culture vessel, and the final concentration was 5% of human platelet lysate. The cells were adherently cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing the above. These adherently cultured cells are called a cell population of the 0th passage. When the subconfluent or arbitrary culture period was reached, the cell population of the 0th passage was exfoliated using a TrypLE Select (manufactured by Thermo Fisher Scientific).

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The dead cell concentration and the total cell concentration were measured using a nucleocounter for the cell population of the 0th passage exfoliated in "Process 3: Culture of fat-derived MSC" described above.

The cell concentration was measured using a Nucleocounter (model: NC-100) manufactured by ChemoMetec. The concentration of dead cells in this measurement was measured by sucking the cell suspension into a cassette (model number: 941-0002) containing a PI solution for staining dead cells. The total cell concentration in this measurement is determined by mixing the cell suspension with the cell treatment reagent A100 (model number: 910-0003) and the cell treatment reagent B (model number: 910-0002) in equal amounts to control all cells. The PI solution was targeted for staining, and the cells were sucked into the above-mentioned cassette and measured.

From the numerical values of the cell concentration measurement, the cell number and the survival rate of the obtained fat-derived MSC were calculated. The calculation formula to be calculated was carried out as follows.
(1) Number of cells of fat-derived MSC (cells) = total cell concentration (cells / mL) x 3 (dilution ratio at the time of measurement) x cell suspension amount (mL)
(2) Survival rate of fat-derived MSC (%) = 100- (dead cell concentration (cells / mL) / (total cell concentration (cells / mL) x 3 (dilution ratio at the time of measurement)) x 100)
As a result, in the cell population of the 0th passage derived from the donor A of Comparative Example 1, no adherent cells to the culture vessel could be confirmed after culturing for 8 days, and the obtained cell suspension was detected by the above-mentioned measuring instrument. It was below the limit. From the above, the fat-derived MSC could not be produced by the method of Comparative Example 1.

In Comparative Example 2 and Example 1 below, the same donor B excised fat was used.

<Comparative Example 2: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
0.06 g of excised fat collected from a donor (donor B) different from the donor in Comparative Example 1 containing 1 mL of a Hanks balanced salt solution (without Ca / Mg) (compressive stress of the medium is 0.00 N) 1 The excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1, except that it was housed in a 5.5 mL microtube.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 2 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, in the cell population of the 0th passage derived from the donor B of Comparative Example 2, adherent cells to the culture vessel could not be confirmed after culturing for 13 days, and the obtained cell suspension was the detection limit of the measuring instrument. It was as follows. From the above, the fat-derived MSC could not be produced by the method of Comparative Example 2.

<Example 1: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
0.06 g of excised fat collected from the same donor (donor B) as the donor in Comparative Example 2 was placed in a 1.5 mL microtube, and a gelatin-containing Hanks balanced salt solution (Ca. Excised fat was transported and stored by the same method as in Process 1 of Comparative Example 1 except that it was embedded in 1 mL (without Mg). For embedding of the excised fat, a method was used in which the prepared gelatin-containing Hanks balanced salt solution was liquefied at 37 ° C., the excised fat was placed in the solution, and then solidified at 4 ° C. Then, it was transported and stored in a refrigerator (4 ° C.) for 20 hours, heated to 37 ° C. to liquefy the gelatin-containing Hanks balanced salt solution, and the excised fat was taken out to carry out Process 2.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 1 were evaluated by the same method as in Process 4 of Comparative Example 1.

As a result, after culturing for 13 days, the number of cells in the 0th passage cell population derived from Donor B of Example ¹ was 3.0 × 105, and the survival rate was 95.9%. From the above, it was found that in the method of Example 1, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

(Process 5: Subculture of fat-derived MSC)
The cell population of the 0th passage obtained in the above-mentioned "Process 3: Culturing of fat-derived MSC" was seeded in a T-75 flask (manufactured by Corning Inc.) for subculture. This subcultured cell population is called the first subculture cell population.

The cell population of the first passage was exfoliated using TripLE Select when it reached the subconfluent, diluted with a medium, and recovered by centrifugation. The recovered cell population was suspended in a cryopreservation solution mixed at a ratio of CP-1 (registered trademark) (manufactured by Far East Pharmaceutical Co., Ltd.): 25% human serum albumin: physiological saline = 2: 1: 3, and -80 ° C. It was slowly frozen until then, and then cryopreserved at -80 ° C.

(Process 6: Surface antigen analysis)
With respect to the cell population of the first passage derived from the donor B of Example 1, each surface antigen (CD73 positive rate, CD90 positive rate, CD105 positive rate, CD45 positive rate and negative rate, using a flow cytometer, The positive rate and negative rate of CD31) were measured.

The surface antigen analysis was performed using a Guava easyCyte Single manufactured by MERCK, with the number of analyzed cells: 10,000 cells and the flow rate setting: Slow. In this measurement, as antibodies for isotype control, PE Mouse IgG1 k Isotype Control (BD company / model number: 555749), FITC Mouse IgG1 k Isotype Control (BD company / model number: 555748) and AlexaFluor647 (BD company / model number: 557714) was used as an antibody against the CD73 antigen, PE Mouse Anti-Human CD73 (manufactured by BD company / model number: 550257), and PE Mouse Anti-Human CD90 (manufactured by BD company) as an antibody against the CD90 antigen. / Model number: 555595), PE Mouse Anti-Human CD105 (manufactured by BD / model number: 560839) as an antibody against the CD105 antigen, and FITC Mouse Anti-Human CD45 (manufactured by BD / model number: 555482) as an antibody against the CD45 antigen. , AlexaFluor 647 Mouse Anti-Human CD31 (manufactured by BD / model number: 561654) was used as an antibody against the CD31 antigen.

As a result of surface antigen analysis, in the cell population of the first passage derived from donor B of Example 1, the positive rates of CD73, CD90, and CD105 were all 90% or more (specifically, CD73: 100%, The positive rate of CD90: 100%, CD105: 100%), CD45, and CD31 was less than 5% (negative rate was 95% or more) (specifically, the positive rate of CD45: 1% (negative rate: 99%). ), CD31 positive rate: 1% (negative rate: 99%)). From the above results, it was confirmed that the cell population of the first passage of Example 1 was a cell population containing high-purity mesenchymal stem cells.

In Example 2 and Comparative Example 3 below, the same donor C excised fat was used.

<Example 2: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
A gelatin-containing Hanks balanced salt having a compressive stress of 1.09 N was placed in a 1.5 mL microtube containing 0.15 g of agarose collected from a donor (donor C) different from the donors in Comparative Examples 1 and 2. It was embedded in a solution (without Ca / Mg) (donor C-gelatin), and the other was embedded with 1 mL of an agarose-containing Hanks balanced salt solution (without Ca / Mg) having a compressive stress of 1.71 N (without Ca / Mg). Except for donor C-agarose), the excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1. However, for embedding the excised fat of donor C-agarose, the prepared agarose-containing Hanks equilibrium salt solution was solidified at room temperature or lower, and the excised fat was embedded in a finely crushed gel. Then, it was transported and stored in a refrigerator (4 ° C.) for 20 hours, the excised fat was removed from the gel, and the process 2 was carried out.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 2 were evaluated by the same method as in Process 4 of Comparative Example 1.

As a result, after culturing for 17 days, the number of cells in the 0th passage cell population derived from the donor C-gelatin of Example 2 was 1.0 × ¹⁰⁶ , and the survival rate was 96.8%. In addition, the number of cells in the 0th passage cell population derived from the donor C-agarose of Example 2 was 1.5 × ¹⁰⁶ , and the survival rate was 98.6%. From the above, it was found that in the method of Example 2, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

<Comparative Example 3: Examination of Adipose Tissue Transport>
(Process 1: Collection, transport and storage of excised fat)
0.15 g of excised fat collected from the same donor (donor C) as the donor in Example 2 was collected into 1 mL of Astellas Pharma's UW solution (product name: Belzer UW cold storage solution) (compressive stress of the medium was 0.00N). The excised fat was transported and stored in the same manner as in Process 1 of Comparative Example 1, except that it was housed in a 1.5 mL microtube containing.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Comparative Example 3 were evaluated after culturing for 17 days by the same method as in Process 4 of Comparative Example 1. Table 1 shows the results of the cell number of each fat-derived MSC of Example 2 and Comparative Example 3 obtained from the adipose tissue of the same donor (donor C).

As shown in Table 1, the number of cells in the 0th passage cell population derived from Donor C of Comparative Example 3 was 0.1 × ¹⁰⁶ , the survival rate was 90.8%, and the donor C of Example 2 was used. -Only 1/10 or less of the cell population of the 0th passage derived from gelatin and donor C-agarose could be obtained, and the survival rate was also slightly low. From the above, it was found that the method of Comparative Example 3 was not suitable for producing a fat-derived MSC.

The following Examples 3, 4, and 5 and Comparative Examples 4, 5, and 6 used the same donor D suction fat.

<Example 3: Examination of adipose tissue transport>
(Process 1: Collection, transportation and storage of sucked fat)
1 mL of suction fat (donor D-suction) collected by liposuction from a donor (donor D) different from the donors in Examples 1 and 2 was placed in a 1.5 mL microtube, and each gelatin having a compressive stress shown in Table 2 was placed. It was embedded in 1 mL of a Hanks equilibrium salt solution (without Ca / Mg) and transported and stored in a refrigerated (4 ° C.) environment for about 72 hours.

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
Except for the fact that each sucked fat taken out from the 1.5 mL microtube was directly immersed in a Hanks balanced salt solution containing 0.1% (v / w) collagenase (without Ca / Mg), it was compared with Process 2 of Comparative Example 1. By the same method, SVF containing fat-derived MSC was obtained.

(Process 3: Culture of fat-derived MSC)
Cells of the 0th passage were seeded in the same manner as in Process 3 of Comparative Example 1 except that each SVF obtained from donor D-suction was seeded in a T-25 flask (manufactured by Corning Inc.) in a culture vessel. Populations were obtained and adherent cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 3 were evaluated after culturing for 7 days by the same method as in Process 4 of Comparative Example 1.

As a result, the number of cells in the 0th passage cell population derived from the donor D-suction (1) of Example 3 was 1.0 × ¹⁰⁶ , and the survival rate was 97.4%, and the donor of Example 3 was obtained. The number of cells in the 0th passage cell population derived from D-suction (2) was 1.6 × ¹⁰⁶ , and the survival rate was 98.1%, which was derived from the donor D-suction (3) of Example 3. The number of cells in the 0th passage cell population was 1.9 × 10 ⁶ , and the survival rate was 98.8%. The number of cells was 0.8 × ¹⁰⁶ , and the survival rate was 97.1%. From the above, it was found that in the method of Example 3, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

<Comparative Example 4: Examination of Adipose Tissue Transport>
(Process 1: Collection, transportation and storage of sucked fat)
1 mL of suction fat (donor D-suction) collected by liposuction from the same donor (donor D) as the donor in Example 3 was mixed with a Hanks balanced salt solution (without Ca / Mg) (the compressive stress of the medium was 0. 00N) The sucked fat was transported and stored in the same manner as in Process 1 of Example 3 except that it was housed in a 1.5 mL microtube containing 1 mL.

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Example 3.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 3, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 4 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, after culturing for 7 days, the number of cells in the 0th passage cell population derived from the donor D-suction of Comparative Example 4 was 0.1 × ¹⁰⁶ , and the survival rate was 93.3%. Only 1/5 or less of the cell population of each 0th passage derived from the donor D-suction of Example 3 could be obtained. From the above, it was found that the method of Comparative Example 3 was not suitable for producing a fat-derived MSC.

<Example 4: Examination of adipose tissue transport>
(Process 1: Collection, transportation and storage of sucked fat)
1 mL of suction fat (donor D-suction) collected by suction fat from the same donor (donor D) as the donor in Example 3 is housed in a 1.5 mL microtube, and the fibrin having a compressive stress of 6.86 N is contained in the medium. (Product name: Borheel) The aspirated fat was transported and stored by the same method as in Process 1 of Example 3 except that it was embedded in 1 mL.

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Example 3.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 3, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 4 were evaluated after 7 days of culturing in the same manner as in Process 4 of Comparative Example 1. As a result, the number of cells in the 0th passage cell population derived from the donor D-suction of Example 4 was 0.8 × ¹⁰⁶ , and the survival rate was 97.1%. From the above, it was found that in the method of Example 4, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

<Comparative Example 5: Examination of adipose tissue transport>
(Process 1: Collection, transportation and storage of sucked fat)
1 mL of suction fat (donor D-suction) collected by liposuction from the same donor (donor D) as the donor in Example 3 is housed in a 1.5 mL microtube, and the compressive stress of the medium is 12.27 N. The sucked fat was transported and stored by the same method as in Process 1 of Example 3 except that it was embedded in 1 mL of a Hanks balanced salt solution (without Ca / Mg).

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Example 3.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 3, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Comparative Example 5 were evaluated after culturing for 7 days by the same method as in Process 4 of Comparative Example 1. As a result, in the cell population of the 0th passage derived from the donor D-suction of Comparative Example 5, the number of cells adhered to the culture vessel was very small, and the number of cells that could be obtained was below the detection limit of the measuring instrument. From the above, the fat-derived MSC could not be produced by the method of Comparative Example 5.

<Example 5: Examination of adipose tissue transport>
(Process 1: Collection, transportation and storage of sucked fat)
The aspirated fat is transported by the same method as in Process 1 of Example 3 except that the conditions for transporting and storing the collected aspirated fat (donor D-suction) are about 192 hours in a refrigerated (4 ° C.) environment. And saved.

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Example 3.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 3, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 5 were evaluated by the same method as in Process 4 of Comparative Example 1.

As a result, after culturing for 7 days, the number of cells in the 0th passage cell population derived from the donor D-suction of Example ⁵ was 1.5 × 105, and the survival rate was 96.3%. From the above, it was found that in the method of Example 5, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

<Comparative Example 6: Examination of adipose tissue transport>
(Process 1: Collection, transportation and storage of sucked fat)
The aspirated fat is transported by the same method as in Process 1 of Comparative Example 4, except that the conditions for transporting and storing the collected aspirated fat (donor D-suction) are about 192 hours in a refrigerated (4 ° C.) environment. And saved.

(Process 2: Enzymatic treatment of aspirated fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Example 3.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 3, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 6 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, after culturing for 7 days, the number of cells in the 0th passage cell population derived from the donor D-suction of Comparative Example 6 was ^0.5 × 105, and the survival rate was 93.3%. Only about 1/3 of the cell population of the 0th passage derived from the donor D-suction of Example 5 could be obtained. From the above, it was found that the method of Comparative Example 6 was not suitable for producing a fat-derived MSC.

Table 3 below shows the cell numbers and viability of the 0th passage cell populations of Examples 3, 4, and 5 and Comparative Examples 4, 5, and 6.

As shown in Table 3, when aspirated adipose tissue was stored and / or transported in a medium having a compressive stress greater than 0 N and 12 N or less, many adherent cells could be confirmed in the culture vessel, and the survival rate was increased. It has been found that many high fat-derived MSCs can be produced.

In Example 6 and Comparative Example 7, the same donor D excised fat was used.

<Example 6: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
A gelatin-containing Hanks having a compressive stress of 1.09 N in a 1.5 mL microtube containing 0.1 g of excised fat (donor D-excision) collected from a donor (donor D) different from that of the donor in Comparative Example 1. The excised fat was transported and stored by the same method as in Process 1 of Comparative Example 1 except that it was embedded in 1 mL of a balanced salt solution (without Ca / Mg).

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and viability of the 0th passage cell population of Example 6 were evaluated by the same method as in Process 4 of Comparative Example 1.

As a result, after culturing for 16 days, the number of cells in the 0th passage cell population derived from the donor D-resect of Example 6 was ^5.0 × 105, and the survival rate was 98.3%. From the above, it was found that in the method of Example 6, many adhesive cells could be confirmed in the culture vessel, and many fat-derived MSCs having a high survival rate could be produced.

<Comparative Example 7: Examination of adipose tissue transport>
(Process 1: Collection, transport and storage of excised fat)
Excised fat was removed by the same method as in Process 1 of Comparative Example 1 except that 0.1 g of excised fat (donor D-excision) collected from a donor (donor D) different from that of the donor in Comparative Example 1 was used. Transported and stored.

(Process 2: Enzymatic treatment of excised fat and acquisition of SVF)
An SVF containing a fat-derived MSC was obtained by the same method as in Process 2 of Comparative Example 1.

(Process 3: Culture of fat-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Comparative Example 1, and the adhesive cells were collected.

(Process 4: Evaluation of cell count and survival rate of cultured fat-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 7 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, after culturing for 16 days, the number of cells in the 0th passage cell population derived from the donor D-resect of Comparative Example ⁷ was as small as 0.2 × 105, and the number of obtained cells was low. Therefore, the survival rate was below the detection limit of the measuring instrument. Furthermore, only about 1/25 of the cell population of each 0th passage derived from the donor D-resect of Example 6 could be obtained. From the above, it was found that the method of Comparative Example 7 was not suitable for producing a fat-derived MSC.

Table 4 below shows the number of cells and the survival rate of the 0th passage cell populations of Example 6 and Comparative Example 7.

As shown in Table 4, when the excised adipose tissue was stored and / or transported in a medium having a compressive stress greater than 0 N and 12 N or less, many adherent cells could be confirmed in the culture vessel, and the survival rate was increased. It has been found that many high fat-derived MSCs can be produced.

Examples 7 and 8 and Comparative Example 8 used the same donor E amnion tissue.

<Example 7: Examination of amniotic membrane tissue transport>
(Process 1: Amniotic membrane collection, transportation and storage)
Fetal membranes and placenta, which are fetal appendages, were aseptically collected from a pregnant woman (donor E) who gave informed consent to a case of selective caesarean section. The obtained fetal membrane and placenta were placed in a container containing physiological saline, and the amniotic membrane was detached from the stump of the fetal membrane. The amniotic membrane was washed with a Hanks balanced salt solution (without Ca / Mg). The weight of the collected amniotic membrane was measured, and 1 g of amniotic membrane was embedded in 1 mL of a gelatin-containing Hanks balanced salt solution (without Ca / Mg) having a compressive stress of 1.09 N and contained in a 1.5 mL microtube. For the embedding of the amniotic membrane, a method was used in which the prepared gelatin-containing Hanks balanced salt solution was liquefied at 37 ° C., the amniotic membrane was placed in the solution, and then the amniotic membrane was solidified at 4 ° C. Then, after transporting and storing in a refrigerator (4 ° C.) for about 216 hours, the mixture was heated to 37 ° C. to liquefy the gelatin-containing Hanks balanced salt solution, and the amniotic membrane was taken out to carry out Process 2.

(Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSC)
1 g of amniotic membrane removed from a 1.5 mL microtube is immersed in a Hanks balanced salt solution (containing Ca / Mg) containing 240 PU / mL collagenase and 200 PU / mL dispase I, and stirred at 37 ° C. for 90 minutes with shaking. The amniotic membrane was treated with an enzyme. The enzyme-treated solution was filtered with a strainer to remove undigested amniotic membrane, and a cell population containing amniotic membrane-derived MSC was obtained.

(Process 3: Culture of amniotic membrane-derived MSC)
A 1/5 amount of the cell population containing the amniotic membrane-derived MSC obtained in "Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSC" was seeded in a T-25 flask (manufactured by Corning) of the culture vessel. Adhesion culture was performed on αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 5% human platelet lysate at a final concentration. These adherently cultured cells are called a cell population of the 0th passage. When the subconfluent or arbitrary culture period was reached, the cell population of the 0th passage was exfoliated using a TrypLE Select (manufactured by Thermo Fisher Scientific).

(Process 4: Evaluation of cell number and survival rate of cultured amniotic membrane-derived MSC)
The cell number and viability of the 0th passage cell population of Example 7 were evaluated by the same method as in Process 4 of Comparative Example 1.

As a result, after culturing for 12 days, the number of cells in the 0th passage cell population derived from Donor E of Example ⁷ was 9.5 × 105, and the survival rate was 98.6%. From the above, it was found that in the method of Example 7, many adhesive cells could be confirmed in the culture vessel, and many amniotic membrane-derived MSCs having a high survival rate could be produced.

<Comparative Example 8: Examination of amniotic tissue transport>
(Process 1: Amniotic membrane collection, transportation and storage)
1 g of amniotic membrane collected from the same donor (donor E) as the donor in Example 7 is contained in a 1.5 mL microtube containing 1 mL of a Hanks balanced salt solution (without Ca / Mg) (compressive stress of the medium is 0.00 N). The amniotic membrane was transported and stored in the same manner as in Process 1 of Example 7, except that it was housed in.

(Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSC)
A cell population containing amnion-derived MSC was obtained by the same method as in Process 2 of Example 7.

(Process 3: Culture of amniotic membrane-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 7, and the adhesive cells were collected.

(Process 4: Evaluation of cell number and survival rate of cultured amniotic membrane-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 8 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, after culturing for 12 days, the number of cells in the 0th passage cell population derived from Donor E of Comparative Example 8 was ^5.6 × 105, and the survival rate was 94.6%, and Example 7 Only about 3/5 of the cell population of the 0th passage derived from Donor E could be obtained. From the above, it was found that the method of Comparative Example 8 was not suitable for producing amniotic membrane-derived MSC.

<Example 8: Examination of amniotic tissue transport>
(Process 1: Amniotic membrane collection, transportation and storage)
1 g of amniotic membrane collected from the same donor (donor E) as the donor in Example 7 by the same method as in Process 1 of Example 7, was obtained from a gelatin-containing Hanks balanced salt solution having a compressive stress of 1.09 N as a medium. It was embedded in 1 mL (without Ca / Mg) and contained in a 1.5 mL microtube. For the embedding of the amniotic membrane, a method was used in which the prepared gelatin-containing Hanks balanced salt solution was liquefied at 37 ° C., the amniotic membrane was placed in the solution, and then the amniotic membrane was solidified at 4 ° C. Then, it was transported and stored at room temperature (about 20 ° C.) for about 110 hours, heated to 37 ° C. to liquefy the gelatin-containing Hanks balanced salt solution, and the amniotic membrane was taken out to carry out Process 2.

(Process 2: Enzymatic treatment of amniotic membrane and acquisition of amniotic membrane-derived MSC)
A cell population containing amnion-derived MSC was obtained by the same method as in Process 2 of Example 7.

(Process 3: Culture of amniotic membrane-derived MSC)
The cell population of the 0th passage was obtained by the same method as in Process 3 of Example 7, and the adhesive cells were collected.

(Process 4: Evaluation of cell number and survival rate of cultured amniotic membrane-derived MSC)
The cell number and survival rate of the 0th passage cell population of Comparative Example 8 were evaluated by the same method as that of Process 4 of Comparative Example 1.

As a result, after culturing for 11 days, the number of cells in the 0th passage cell population derived from Donor E of Example 8 was 1.3 × ¹⁰⁶ , and the survival rate was 98.3%. From the above, it was found that in the method of Example 6, many adhesive cells could be confirmed in the culture vessel, and many amniotic membrane-derived MSCs having a high survival rate could be produced.

Table 5 below shows the cell numbers and viability of the 0th passage cell populations of Examples 7 and 8 and Comparative Example 8.

As shown in Table 5, it was found that many amniotic membrane MSCs with high survival rate can be produced by storing and / or transporting the amniotic membrane tissue in a medium having a compressive stress greater than 0 N and 12 N or less in a state of being embedded.

From the above, it was found that in the method of the example satisfying the following conditions (1) and (2), many adhesive cells could be confirmed in the culture vessel, and many fats and amniotic membrane-derived MSCs having high survival rates could be produced. ..
(1) Tissues containing adhesive cells are stored and / or transported in a state of being embedded in a medium having a compressive stress greater than 0 and 12 N or less.
(2) The tissue embedded in the medium is taken out from the medium and the adhesive cells are separated. That is, according to the present invention, the tissue containing the adhesive cells such as mesenchymal stem cells is safely stored / transported. This makes it possible to efficiently produce adherent cells from raw material tissues containing adherent cells after storage / transport. This can be expected to expand opportunities for providing treatment to patients, reduce the burden on cell cultures, and reduce manufacturing costs and medical costs.

All publications, patents and patent applications cited herein shall be incorporated herein by reference as is.

Claims (11)

1. A method for producing a cell population containing the cells from a tissue containing the adhesive cells.
   (1) Tissues containing adhesive cells are stored and / or transported in a state of being embedded in a medium having a compressive stress greater than 0 and 12 N or less.
   (2) A method for producing adhesive cells from a tissue, which comprises removing the tissue embedded in the medium from the medium and separating the adhesive cells.
2. The production method according to claim 1, wherein the medium is a gel or a sol.
3. The production method according to claim 2, wherein the gel or sol comprises at least one selected from the group consisting of proteins, peptides, polysaccharides and synthetic polymers.
4. The production method according to claim 2 or 3, wherein the gel or sol comprises at least one of gelatin, collagen, agarose, alginic acid, pectin, fibrin, silicon, and polyvinyl alcohol.
5. The production method according to any one of claims 1 to 4, wherein the gel or sol reversibly repeats liquefaction and solidification at least once during storage and / or transportation.
6. The production method according to claim 5, wherein the gel or sol is repeatedly liquefied and solidified by changing the temperature.
7. The invention according to any one of claims 2 to 6, wherein the tissue containing the adhesive cells is stored and / or transported in the gel, and then the gel in which the tissue containing the adhesive cells is embedded is liquefied to remove the tissue. Production method.
8. The production method according to any one of claims 1 to 7, wherein the adhesive cells are mesenchymal stem cells.
9. The production method according to any one of claims 1 to 8, wherein the tissue containing the adhesive cells is at least one selected from the group consisting of amniotic membrane, fat, umbilical cord, placenta, skin and muscle.
10. The production method according to any one of claims 1 to 9, wherein the storage and transportation time is 240 hours or less.
11. The production method according to any one of claims 1 to 10, further comprising a process of removing a tissue containing adhesive cells from a medium and then treating and culturing the tissue.