JP2018078862A - Adipose tissue preservation method and method of isolating and culturing stem cells from frozen adipose tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide effective means of isolating and culturing stem cells such as MSC from preserved adipose tissue which is preserved for a long period by a simple method.SOLUTION: The adipose tissue preservation method comprises: i) a step of shredding adipose tissue collected from a living organism to a maximum diameter of 8 mm; ii) a step of soaking the shredded adipose tissue in cryoprotective liquid such that the adipose tissue weight/(adipose tissue weight + cryoprotective liquid weight) is 10% (w/w) or more and less than 50% (w/w); and iii) a step of putting the adipose tissue and cryoprotective liquid in a vessel and carrying out cryopreservation in an environment of -80°C or less.SELECTED DRAWING: None

Description

  The present invention relates to a method for efficiently culturing stem cells from frozen adipose tissue.

  Mesenchymal stem cells (MSC = Mesenchymal Stem Cell) contained in bone marrow fluid have high proliferative ability, such as bone, cartilage, muscle, fat, hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, etc. It is known to differentiate into cells, and has been reported to be useful as a cell source for regenerative medicine, particularly cell transplantation treatment. Some have already been put into practical use, and further clinical application development is underway.

  Conventionally, MSC has been prepared mainly by culturing from bone marrow fluid. However, in recent years, adipose tissue has attracted attention as a cell source of MSC. This is because not only adipose tissue is rich in stem cells, but adipose tissue can be collected relatively easily compared to bone marrow, and can be prepared in large quantities if a large amount of tissue is obtained. It depends on the reason.

It is reported that about 5 × 10 ³ MSCs can be collected from 1 gram of adipose tissue, which is 500 times more than the same amount of bone marrow tissue (Non-patent Document 1). Further, since MSC derived from adipose tissue generally has a higher growth rate than that derived from bone marrow, the necessary number of cells can be secured relatively easily.

  Adipose tissue is also collected around the world by liposuction for slimming purposes, and it can be considered that there is a potentially large source of cells, assuming that it is used for cells for transplantation . Thus, it can be said that effective use of adipose tissue is extremely important for the development of regenerative medicine in the future.

  When preparing stem cells such as MSC from adipose tissue, only fresh adipose tissue is used. For example, MSC separation culture includes washing of adipose tissue (removal of attached blood, etc.), purification (removing excess tissue, blood vessels, etc.), enzyme treatment with protease, filter filtration, centrifugation, cell seeding operation, etc. A series of steps are performed at once to move to a cell culturing step.

  Therefore, if the obtained fresh adipose tissue is abundant, or if there is not enough manpower or time, or if the culture is not ready, the obtained adipose tissue can be processed up to the cell culture process for various reasons. In many cases, it is impossible to complete the process, and the fresh tissue that has been obtained cannot be used sufficiently and must be discarded.

  For this reason, an effective means for storing adipose tissue for a long period of time by a simple method and separating and culturing stem cells such as MSC from the preserved adipose tissue is required.

Japan Journal of Transfusion and Cell Therapy, Vol. 59. No. 3 59 (3): 450-456, 2013

  An object of the present invention is to provide an effective means capable of preserving adipose tissue for a long period of time by a simple method and capable of separating and culturing stem cells such as MSC from the preserved adipose tissue.

  As a result of intensive studies to solve the above problems, the present inventor found that the above problems can be solved by the following method, and completed the present invention.

That is, this invention consists of the following structures.
1. i) Step of chopping adipose tissue collected from a living body so that the maximum diameter is 8 mm or less ii) Adipose tissue weight / (Adipose tissue weight + Cryoprotection solution weight) Iii) a step of immersing so as to be 10 wt% or more and less than 50 wt%. A method for preserving adipose tissue comprising a step of placing adipose tissue and a cryoprotective solution in a container and cryopreserving in an environment of −80 ° C. or less.
2. vi) a step of thawing the frozen adipose tissue obtained by the method described in (1) and then washing with a culture solution v) a step of washing with a protease solution vi) a step of digesting the adipose tissue in the protease solution A method for preparing stem cells derived from adipose tissue.
3. The method according to (2), wherein the protease comprises collagenase and / or dispase.
4). The method according to (2), wherein the stem cell is a mesenchymal stem cell.
5. A method for culturing stem cells derived from frozen adipose tissue, comprising seeding a cell suspension obtained by the method according to any one of (2) to (4) on a culture substrate and culturing the adhered stem cells. .
6). The culture method according to (5), wherein the culture substrate is a hollow fiber membrane.

  According to the present invention, adipose-derived stem cells can be separated and cultured with high efficiency from cryopreserved adipose tissue.

It is a schematic diagram which shows an example of a cell culture apparatus. It is a microscopic image of mesenchymal stem cells isolated and cultured from canine frozen adipose tissue.

  In the present invention, adipose tissue refers to loose connective tissue composed of adipocytes. In vivo, adipose tissue is present around the viscera subcutaneously or in the abdominal cavity, but any adipose tissue present in any site can be suitably used in the present invention.

  In the present invention, adipose tissue collected from a living body is washed with a phosphate buffer (PBS) or the like, and then blood vessels and connective tissue are removed. Thereafter, the adipose tissue is appropriately chopped. The size to be chopped is preferably adjusted so that the handleability and the cooling rate can be made uniform. In the present invention, the size is chopped by using a scissors or forceps so that the maximum diameter is 8 mm or less. To. More preferably, it is 3-6 mm.

  In the present invention, the preservation of adipose tissue is preferably carried out by cryopreserving a fragmented adipose tissue by adding a cryoprotective solution. As a cryoprotective solution, it is easy to use a commercially available solution, but dimethyl sulfoxide (DMSO), glycerin, serum, etc. can also be used. In the present invention, it is inexpensive and preferable to use a culture solution containing 5% or more and less than 20% DMSO. The amount of DMSO added may be adjusted in the above range depending on the cell type and the like. DMSO must be sterilized. Further, serum may be added to the cryoprotective solution. The adipose tissue and the cryoprotective solution are preferably mixed so that the adipose tissue weight / (adipose tissue weight + cryoprotective solution weight) is 10 wt% or more and less than 50 wt%. Within such a range, the intracellular concentration of the cryoprotectant is appropriate, and the cell viability after thawing is increased. Moreover, it is preferable that the cryoprotectant is cooled to around 4 ° C. before being added to the adipose tissue.

The prepared cryoprotective solution is added to the adipose tissue and lightly suspended, and then transferred to a container such as a vial and cooling is started. The cooling temperature (the storage temperature of the adipose tissue) is preferably low because the cell survival rate is high, but is preferably −80 ° C. to −196 ° C. in terms of equipment and cost. Although a cooling rate is not specifically limited, The following conditions are illustrated as an example. It is preferable to appropriately control the cooling rate because the viability of the cells after thawing is increased.
(1) If necessary, cool to 4 ° C. at a rate of −2 ° C./min.
(2) After holding at 4 ° C. for 5 to 15 minutes, cool to −30 ° C. at a rate of −1 ° C./min.
(3) After holding at −30 ° C. for 5 to 15 minutes, cool to −80 ° C. at a rate of −5 ° C./min.
(4) After cooling to −80 ° C., store in a −80 ° C. freezer or liquid nitrogen.

The frozen adipose tissue stored in this manner can be thawed as necessary, and cell culture can be performed for growth.
Thaw the container taken out of the freezer or the like in advance by immersing it in a constant temperature bath at around 37 ° C. When the frozen contents are melted, the contents are taken out from the thermostatic bath, and the contents are transferred to a container such as a petri dish containing a culture solution previously cooled to about 4 ° C. After rinsing the container lightly, remove the supernatant using an aspirator or the like. Again, the culture medium is added to the container, and the adipose tissue is washed by repeating the above operation. It should be noted that if the thawing operation is slow, the viability of adipocytes is reduced, so that it is important to perform the operation quickly.

  Subsequently, a protease solution is added to the washed adipose tissue, the container is lightly shaken and rinsed, and then the supernatant is removed using an aspirator or the like. After adding the protease solution to the washed adipose tissue, it is transferred to a centrifuge tube or the like, and shaken in a thermostatic bath to digest the adipose tissue. As digestion conditions, it is preferable to treat at 35 to 38 ° C. for 30 to 90 minutes.

  In the present invention, the protease (proteolytic enzyme) is preferably collagenase type II or dispase, or a mixed solution of collagenase and dispase, and the concentrations thereof are preferably 1-2 mg / mL and 3000-7000 units / mL, respectively. More preferably, they are 1.3-1.8 mg / mL and 4000-6000 units / mL. The treatment temperature with protease is preferably 35 to 37 ° C., and the treatment time is 1 to 3 hours.

  In the present invention, the culture medium (medium) is a so-called basal medium for growing and proliferating cells and containing amino acids, vitamins, inorganic salts, sugars and the like as nutrients. Examples of such a medium include Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Media199, Dulbecco's Modified Eagle Medium (DMEM), α-Minimum Essential Medium (α-MEM), and Ham's F-10 Nutrient Mixture. (Ham's F-10), Ham's F-12 Nutrient Mixture (Ham's F-12), RPMI 1640, L-15, Iscove's Modified Dulbecco's Medium (IMDM), ES medium, MCDB 131 Medium, CMRL 1066 Media, DM-160 Medium , Fisher's Medium, StemSpan Medium, StemPro Medium, Hybridoma Serum Free Medium (Hybridoma SFM), mTeSR1 (modified Tenneille Serum Replacer 1), Essential 8, Repro FF / FF2 / XF, StemSure (registered trademark), CELRENA, S-Medium, etc. Commercially available cell culture media and mixtures thereof can be used, but are not limited thereto.

  In addition, serum may be added to the culture solution. As the serum, serum such as bovine serum, fetal bovine serum (FBS), horse serum, human serum is preferably used. The amount added is preferably 1 vol% or more with respect to the basal medium. If the amount added is too small, the cells will not grow and proliferate, so it is more preferred to add 5 vol% or more, and even more preferred to add 10 vol% or more. Even if the addition amount is increased more than necessary, the culture cost only rises, and it is preferable that the upper limit is about 20 vol%.

  The digested adipose tissue suspension is passed through gauze filtration or a cell strainer (100 μm mesh) to remove contaminating components. This is transferred to a centrifuge tube and centrifuged at 100 to 3000 rpm for 1 to 10 minutes. After removing the supernatant (protease) using an aspirator or the like, the culture medium with FBS added to the residue is poured to float the adipocytes. The FBS concentration to be added is preferably 1 to 20 wt%. If the FBS concentration is too low, cell growth (proliferation) defects are likely to occur, so it is preferable to add 5 wt% or more. Although the FBS concentration is rarely a problem, excessive addition only leads to an increase in cost, and it is more preferably 15 wt% or less.

  This is seeded on a culture substrate such as a culture plate, a petri dish, or a hollow fiber membrane, and then the adherent adipose tissue-derived stem cells are cultured in a plane (two-dimensional) by a conventional method and proliferated. As a culture substrate, it is preferable to use a hollow fiber membrane in view of the trouble of exchanging the culture medium and the risk of contamination.

  In the present invention, the material of the hollow fiber membrane is not particularly limited as long as it can hold cells on the membrane surface and can pass through a solution or a low-molecular substance. For example, cellulose acetate, regenerated cellulose, polysulfone, polyethersulfone , Ethylene vinyl alcohol, polymethyl methacrylate, polyacrylonitrile and the like. In addition, in order to improve cell adhesion and familiarity with the culture solution, hydrophilic polymers such as polyvinylpyrrolidone, hydroxyalkyl cellulose, vinylpyrrolidone / vinyl acetate copolymer, and cell adhesion factors such as collagen and fibronectin are added. You may use together.

  Another advantage of using a hollow fiber membrane as a culture substrate is space saving of culture. Therefore, the inner diameter of the hollow fiber membrane is preferably 100 to 1000 μm, and the film thickness is preferably about 10 to 150 μm.

The pore diameter of the hollow fiber membrane does not allow cells to pass through, but it is sufficient that the culture solution components such as water, salts, and proteins pass therethrough. Is preferred. Specifically, the average pore size is preferably about 0.001 to 0.5 μm. The water permeability is preferably 10 to 5000 mL / m ² / hr / mmHg. In the present invention, the structure of the hollow fiber membrane is not particularly limited, and may be a homogeneous structure or a heterogeneous (asymmetric) structure.

  Hereinafter, the effectiveness of the present invention will be described with reference to examples, but the present invention is not limited thereto.

(Counting the number of cells)
The suspension containing the cells was finally suspended in 1 ml of culture solution by centrifugation. A solution obtained by mixing this suspension and trypan blue staining solution at a ratio of 1: 1 was added to a hemocytometer, and the number of cells was measured under a microscope.
1. The surface of the hemocytometer and cover glass is washed with 70% isopropanol, excess isopropanol is wiped off and air-dried.
2. Wet the side of the cover glass with Reagent grade water and attach it to the hemocytometer.
3. After thoroughly agitating the cell suspension with a Pasteur pipette, immediately pour it into a hemocytometer and fill the groove.
4. Perform steps 1 to 3 using a separate hemocytometer (measure twice and average).
5. Place a hemocytometer on the microscope and focus on the grid lines (10 × objective).
6). Using a counter, rapidly measure the number of cells in a 1 mm ² area.
* Since errors are likely to occur, at least 100-500 cells are counted for accurate counting.
Method of calculation:
C = N × 10 ⁴
C: Number of cells per ml N: Average number of measured cells 10 ⁴ : Conversion value of volume for 1 mm ² Total number = C × V
V = volume of the liquid in which the cells are suspended

[Example 1]
(Freezing of adipose tissue)
1. 3 g of dog adipose tissue from which blood and excess tissue had been removed was placed in a φ100 mm dish containing 10 ml of a cryoprotective solution (10 wt% DMSO + DMEM) previously cooled to 4 ° C.
2. Next, the adipose tissue was quickly chopped to a size of about 3 to 6 mm with scissors.
3. A cryoprotective solution in which adipose tissue was suspended was aspirated with a 25 ml disposable pipette and dispensed in approximately 1.5 ml portions into 10 cryopreservation tubes. At this stage, the adipose tissue weight / (adipose tissue weight + cryoprotective solution weight) was 22.9 wt%.
After holding at 4 ° C. for 4.5 to 15 minutes, it was cooled to −30 ° C. at a rate of −1 ° C./min.
After maintaining at −30 ° C. for 5.5 to 15 minutes, it was cooled to −80 ° C. at a rate of −5 ° C./min.
6). Placed in a deep freezer at −80 ° C. and stored frozen.

(Thawing and washing frozen adipose tissue)
1. The cryopreservation tube was removed from the -80 ° C deep freezer and quickly thawed in warm water warmed to 37 ° C.
2. The thawed adipose tissue and cryoprotective solution were placed in a φ100 mm dish containing 10 ml of DMEM medium that had been cooled to 4 ° C. in advance. After the dish was lightly shaken and rinsed, the medium was aspirated and removed with an aspirator, and the tissue was washed.
3. Once again, 10 ml of DMEM medium cooled to 4 ° C. was added and the dish was lightly shaken to rinse, and the medium was removed by suction with an aspirator to wash the tissue.

(Digestion of frozen adipose tissue)
1. Following the above washing operation, 5 ml of DMEM containing collagenase type II at a concentration of 1.5 mg / ml was added to the dish.
2. After the dish was lightly shaken and rinsed, the medium was removed by suction with an aspirator.
3. Once again, 5 ml of DMEM containing collagenase type II at a concentration of 1.5 mg / ml was added to the dish.
4). The obtained adipose tissue suspension was transferred to a 50 ml centrifuge tube and shaken in a 37 ° C. warm bath for 60 minutes to digest the adipose tissue.

(Separation culture of mesenchymal stem cells)
1. The digested adipose tissue was passed through a 100 μm mesh, and the large tissue was removed and placed in a 50 ml tube.
2. Centrifuged at 1000 rpm for 5 minutes.
3. The supernatant was removed by aspiration, and the sediment was suspended in DMEM supplemented with 10% FBS, seeded on a collagen-coated φ60 mm dish, and cultured for 7 days.

[Example 2]
(Production of hollow fiber membrane)
Polyethersulfone (4800P, manufactured by Sumitomo Chemical Co., Ltd.) 20% by mass, N methylpyrrolidone (Mitsubishi Chemical Co., Ltd.) 36% by mass, and triethylene glycol (Mitsui Chemicals Co., Ltd.) 44% by mass are uniformly dissolved. Produced. This stock solution is simultaneously discharged from a cylindrical double tube nozzle heated to 70 ° C. together with an aqueous solution of 13.5% by mass of N-methylpyrrolidone, 16.5% by mass of triethylene glycol, and 70% by mass of water as a hollow forming agent. After passing through a 300 mm dry part, it was immersed in 75 ° C. water and solidified, and after sufficient washing with water, it was wound into a bundle. After cutting, the yarn bundle was immersed in a 50% aqueous glycerin solution at 50 ° C., and then excess glycerin solution was removed and dried by ventilation at 60 ° C. The discharge amount was adjusted so that the hollow fiber membrane had an inner diameter of 250 μm, an outer diameter of 340 μm, and a film thickness of 45 μm.

(Production of hollow fiber membrane module)
A hollow fiber membrane module for testing was produced as follows. After inserting 10 of the produced polyethersulfone hollow fiber membranes into a cylindrical acrylic module case with an inner diameter of 4 mm and a length of 10 cm, a polyurethane potting agent is used so as not to block the hollow portions of the hollow fiber membranes. Both ends were fixed to a module case to produce a hollow fiber membrane module.

(Preparation for cell culture)
Using the obtained hollow fiber membrane module as a cell culture container, a cell culture apparatus as shown in FIG. 1 was constructed. First, in order to remove glycerin adhering to the hollow fiber membrane, sterilized distilled water for injection is put into the culture solution storage containers 5 and 6, the pumps 8 and 9 are started, and a sufficient amount is placed in the hollow fiber membrane module. Rinse and wash. Thereafter, the culture medium storage container 5 was replaced with a medium containing DMEM medium to which FBS was added at 10 vol%. In addition, the culture medium storage container 6 was replaced with a medium containing Dulbecco's modified Eagle medium supplemented with 0.5 μM insulin, 10 nM selenium, and 250 μM glutamic acid without serum. Thereafter, the pumps 8 and 9 were started to perform priming treatment, and the water in the hollow fiber membrane module was replaced with the culture solution.

(Cell culture experiment)
The cell suspension obtained in the same manner as in Example 1 was filled in the hollow part side of the hollow fiber membrane and allowed to stand overnight to adhere the cells to the inner surface of the hollow fiber membrane. After the culture solution storage container 5 was replaced with a culture solution-only container, the liquid feeding was started at a flow rate of 0.33 mm / min on the hollow portion side of the hollow fiber membrane and at a flow rate of 3.46 mm / min on the outside of the hollow fiber membrane. At this time, the culture solution to be passed through the hollow part of the hollow fiber membrane is a DMEM medium to which FBS is added at 10 vol%, and the culture solution to be passed outside the hollow fiber membrane is free of serum. Dulbecco's modified Eagle medium supplemented with 0.5 μM insulin, 10 nM selenium and 250 μM glutamic acid was used. The cell culture experiment was performed at 37 ° C. for 7 days in a CO ₂ incubator.

(Cell recovery from hollow fiber membrane module)
After culturing for 7 days, the perfusion of the culture solution was stopped, and the cells grown in the hollow fiber membrane module were collected. That is, after stopping the perfusion of the culture solution, in order to remove the culture solution existing on the hollow part side and the outside of the hollow fiber membrane, phosphate buffered saline (PBS) was perfused for a certain period of time, and the culture solution was sufficiently PBS Replaced with Next, PBS was removed, and a 0.25% trypsin solution (manufactured by Life Technologies) was gently filled into the hollow portion side and the outside of the hollow fiber membrane, and incubated at room temperature for 15 minutes. Then, the culture solution was poured into the hollow part side of the hollow fiber membrane, and the cells that had flowed out were collected.

(Differentiation into adipocytes)
Differentiation into adipocytes was examined by the following method with reference to the description of Gimble JM., Et al., J Cell Biochem. 58. 393-402 (1995).

MSCs separated and cultured from frozen adipose tissue were seeded in a 24-well culture plate at a density of 5000 to 10000 cells / cm ² in DMEM (10% FBS added) medium and cultured overnight, and the cells were allowed to adhere to the plate. The cells were divided into two groups, and one group of media was changed to indomethacin (final concentration 60 μM), IBMX (3-Isobutyl 1-methylxanthine, final concentration 0.5 mM), hydrocortisone (final concentration) in basal medium for adipocyte induction (Lonza). The medium was replaced with an adipocyte differentiation induction medium supplemented with a concentration of 0.5 μM, and cultured for 5 weeks while changing the medium every 3 to 4 days. This group was designated as an adipocyte induction group. In the other group, the medium was replaced with fresh DMEM (10% FBS added) medium, and cultured for 5 weeks while changing the medium every 3 to 4 days. This group was used as a control group.

  Lipid droplets are observed in cells differentiated into fat cells. Therefore, after culturing, the cells of both groups were stained with Oil Red O (Repit Assay Kit, Primary Cell) to confirm the presence or absence of neutral fat. However, neutral fat staining was not observed in any group. Furthermore, after staining, oil red O dye was extracted using an extract (Rippit Assay Kit, Primary Cell), and the absorbance at 540 nm of the extract was compared between the two groups. I was not able to admit.

  Next, changes in the expression level of Lipo Protein Lipase, one of the adipocyte differentiation markers, were examined. Total RNA was extracted from each group of cells using RNeasy Plus Mini Kit (QIAGEN) to prepare a total RNA extract, and then the RNA concentration contained in the total RNA extract using a multimode plate reader (Molecular Device) Was measured. After measuring the RNA concentration, a PCR reaction solution was prepared using TaqMan ™ RNA-to-CTTM 1-Step kit (Life Technologies), and 25 ng of total RNA of each group was used as a template, [(48 ° C./15 min) × 1 cycle, Real-time PCR was performed under PCR conditions of (95 ° C./10 min) × 1 cycle and (95 ° C./15 sec, 60 ° C./1 min) × 40 cycles] to amplify the Lipo Protein Lipase gene and the β-actin gene. Lipo Protein Lipase probe (Applied Biosystems / Assay ID: Hs00173425_m1) and β-actin probe (Applied Biosystems / Assay ID: Hs99999993_m1) were used as PCR primers, respectively.

  As a result, the Ct value (Threhold cycle) of Lipo Protein Lipase was 39.3 in the control group and 36.0 in the fat differentiation induction group, and the Ct value of LPL was slightly higher in the fat differentiation induction group than in the control group. It turned out to be low. These results indicate that the expression level of Lipo Protein Lipase, one of the adipocyte differentiation markers, was increased in the fat differentiation induction group, although the presence of fat globules that accumulated neutral fat in the cells could not be confirmed. This shows that adipose tissue-derived stem cells (MSCs) have a weak ability to differentiate into adipocytes.

(Differentiation into osteoblasts)
Osteoblast differentiation ability was determined by Pittenger MF. , Et al. , Science. 284, 143-7 (1999), Colter Dc. , Et al. , Proc Natl Acad Sci USA. 98, 7841-5 (2001), was examined by the following method.

Mesenchymal stem cells isolated and cultured from frozen adipose tissue prepared by the same method as used in the above-described measurement of cartilage differentiation ability were placed in DMEM (10% FBS) medium at a cell density (5000 to 10,000 cells / cm ² ). The cells were seeded in a well culture plate and cultured overnight. The cells are divided into two groups, and one group of media is a bone medium containing dexamethasone, L-glutamine, ascorbate, penicillin / streptomycin, MCGS, β-glycerophosphate in a basic medium for osteoblast differentiation (Lonza). It was replaced with an osteoblast differentiation induction medium supplemented with an additive factor set for blast differentiation (Lonza), and differentiation culture was performed for 3 weeks while changing the medium every 3 to 4 days. This group was designated as an osteoblast induction group. For the other group, the medium was replaced with fresh DMEM (10% FBS added) medium, and cultured for 3 weeks while changing the medium every 3 to 4 days. This group was used as a control group. After culturing, the cells were washed once with PBS, 0.2 mL of PBS and 0.2 mL of 2M hydrochloric acid were added to each well, and left at 37 ° C. for 1 hour to release calcium accumulated in the cells from the cells. I let you. The released calcium concentration was quantified using Calcium E-Test Wako (Wako Pure Chemical Industries). As a result, the free calcium concentration was 1.53 mg / dL in the control group, whereas it was as high as 24.88 mg / dL in the osteoblast differentiation induction group.

  This result shows that mesenchymal stem cells separated and cultured from frozen adipose tissue are differentiated into osteoblasts by culturing in an osteoblast differentiation induction medium, and calcium accumulates in the cells. This shows that mesenchymal stem cells separated and cultured from frozen adipose tissue have the ability to differentiate into osteoblasts.

  According to the present invention, adipose tissue can be stored for a long time by a simple method, and stem cells such as mesenchymal stem cells can be efficiently separated and cultured from the stored adipose tissue. This makes it possible to effectively use adipose tissue as a cell source used for regenerative medicine.

1a, 1b Entrance / exit (hollow part side)
2a, 2b Entrance / exit (external side)
3 Module Case 4 Hollow Fiber Membrane 5, 6 Culture Medium Storage Container 7 Cell Culture Container (Hollow Fiber Membrane Module)
8, 9 Liquid feed pump 10, 11 Waste liquid collection container (cell collection container)

Claims (6)

i) Step of chopping adipose tissue collected from a living body so that the maximum diameter is 8 mm or less ii) Adipose tissue weight / (Adipose tissue weight + Cryoprotection solution weight) ) Step of immersing so as to be 10% (w / w) or more and less than 50% (w / w) iii) Step of placing adipose tissue and cryoprotective solution in a container and cryopreserving in an environment of −80 ° C. or lower A method for preserving adipose tissue. vi) a step of thawing the frozen adipose tissue obtained by the method of claim 1 and then washing with a culture solution v) a step of washing with a protease solution vi) a step of digesting adipose tissue in the protease solution A method for preparing stem cells derived from adipose tissue.   The method according to claim 2, wherein the protease comprises collagenase and / or dispase.   The method according to claim 2, wherein the stem cell is a mesenchymal stem cell.   A method for culturing frozen adipose tissue-derived stem cells, comprising seeding a cell suspension obtained by the method according to any one of claims 2 to 4 on a culture substrate and culturing the adhered stem cells.   The culture method according to claim 5, wherein the culture substrate is a hollow fiber membrane.