JP2018093782A - Preservation method of placental tissue, and method of culturing isolated stem cells from placental tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple long-term storage method for placental tissue and an effective means of culturing isolated stem cells such as MSC from stored placental tissue.SOLUTION: A method of preserving placental tissue comprises: a step of slicing placental tissue collected from a living organism so as to have a maximum diameter of 6 mm; a step of soaking the sliced placental tissue in cryoprotectant such that the weight of placental tissue/(weight of placental tissue+weight of cryoprotectant) is 10 to less than 50 wt.%; and a step of cryopreserving the placental tissue in a container with the cryoprotectant in an environment at -80°C or less. Further, a method for preparing stem cells derived from frozen placenta comprises: a step of washing the preserved placental tissue with a culture liquid after thawing; a step of washing with a protease solution; and a step of digesting the placental tissue in the protease solution. Further provided are a method for culturing stem cells from the prepared placental tissue, and a method in which the stem cells are mesenchymal cells.SELECTED DRAWING: None

Description

  The present invention relates to a method for preserving placental tissue and a method for efficiently preparing and culturing stem cells from cryopreserved placental 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 has been known to differentiate into cells, and many usefulness has been reported as a cell source for regenerative medicine, particularly cell transplantation treatment. Some of them have already been put into practical use and clinical applications are being promoted.

  Conventionally, MSC has been prepared mainly by culturing from bone marrow fluid. However, in recent years, tissues other than bone marrow have been spotlighted as cell sources of MSC. For example, amniotic membrane is an extraembryonic tissue composed of ectoderm-derived epithelial cells and mesoderm-derived mesenchymal cells, and has been confirmed to contain a group of cells having characteristics as pluripotent stem cells. For example, differentiation of human amniotic epithelium-derived stem cells into hepatocytes and insulin-producing cells, and differentiation of human amnion-derived mesenchymal stem cells into chondrocytes and cardiomyocytes are known (Patent Document 1). The umbilical cord is composed of an amnion surrounding the outer layer, a collagenous tissue called Walton's jelly, umbilical arteries, and umbilical veins, and mesenchymal stem cells are present in Walton's jelly, chondrocytes, adipose tissue It is known to differentiate into bone (Non-patent Document 1). Furthermore, the placenta includes a large number of cells such as syncytiotrophoblast cells, Langhans cells, fibroblasts, histiospheres, and decidual cells. Among them, cells showing CD59 positivity are promising as stem cells.

  Thus, tissues related to fetal growth such as the umbilical cord, placenta, and amniotic membrane were conventionally discarded after delivery, but are rich in stem cells and are expected to be applied to regenerative medicine.

  Among them, a large amount of placental MSCs can be prepared, and placenta-derived MSCs are considered to be more proliferative and immunomodulating than bone marrow-derived MSCs, and are expected to be used effectively as a high-quality stem cell source.

  When preparing stem cells such as MSCs from the placenta, fresh tissue is used exclusively. For example, MSC separation culture is a series of steps such as washing the placenta (removing attached blood, dew, etc.), chopping, enzyme treatment with protease, filter filtration, centrifugation, cell seeding, etc. It is necessary to proceed to the cell culture process promptly.

  Therefore, if the obtained placental tissue is abundant, if there is not enough manpower or time, or if the culture is not ready, the obtained placenta can be processed up to the cell culture process for various reasons. In many cases, it is impossible to finish the process, and the freshly obtained tissue must be discarded without being fully utilized.

  For this reason, there is a need for an effective means that allows the placenta to be frozen and stored for a long period of time by a simple method, and that stem cells such as MSC can be separated and cultured from the frozen placenta tissue.

JP 2003-231039 A

Cellular Reprogramming, 14 (5), 448-455 (2012)

  An object of the present invention is to provide an effective means capable of preserving placental 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 placental 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 slicing placenta tissue collected from a living body so that the maximum diameter is 6 mm or less ii) Placenta tissue weight / (placenta tissue weight + cryoprotection solution) into the cryoprotection solution A step of immersing so that the weight) is 10 wt% or more and less than 50 wt%.
2. iv) a step of thawing the frozen placental 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 placental tissue in the protease solution A method of preparing placental tissue-derived stem cells.
3. The method according to (2), wherein the protease comprises collagenase and / or dispase.
4). The method according to (2) or (3), wherein the stem cell is a mesenchymal stem cell.
5. (2) A method for culturing stem cells derived from frozen placental tissue, comprising seeding a cell suspension obtained by the method according to any one of (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, placental tissue can be stably stored for a long time, and placenta-derived stem cells can be separated and cultured with high efficiency from cryopreserved placental tissue.

It is a schematic diagram which shows an example of a cell culture apparatus.

  In the present invention, placental tissue refers to a placenta that is a composite tissue of a fetus and a mother. The placenta is a complex tissue in which embryonic and fetal tissues and maternal tissues are closely adhered, and plays a physiologically important role such as exchange of substances and interaction at the cellular level between them.

  In the present invention, placental 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 placental tissue is appropriately minced. 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 6 mm or less. To. More preferably, it is 2-5 mm.

  In the present invention, the placental tissue is preferably preserved by cryopreserving a segmented placental 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 DMSO of 5 wt% or more and less than 20 wt%. 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 placental tissue and the cryoprotective solution are preferably mixed so that the weight of placental tissue / (weight of placental tissue + weight of cryoprotective solution) is 10 wt% or more and less than 50 wt%. The cryoprotectant is preferably cooled to around 4 ° C. before being added to the placental tissue.

The prepared cryoprotective solution is added to the placental tissue and suspended briefly, and then transferred to a container such as a vial, and cooling is started. The cooling temperature (preservation temperature of placental 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 placental tissue thus preserved can be thawed as necessary and cultured for cell culture to proliferate. That is, the container taken out from the freezer or the like is thawed by immersing it in a constant temperature bath at around 37 ° C. in advance. 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 placenta tissue is washed by repeating the above operation. It should be noted that if the thawing operation is slow, the viability of the cells decreases, so it is important to perform the operation quickly.

  Subsequently, a protease solution is added to the placental tissue after washing, and the container is gently shaken to rinse, and then the supernatant is removed using an aspirator or the like. After adding a protease solution to the washed placental tissue, the protease solution is transferred to a centrifuge tube or the like and shaken in a thermostatic bath to digest the placental 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.

  The tissue suspension obtained by digestion is passed through gauze filtration or a cell strainer (100 μm mesh) to remove contaminant 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 added with fetal bovine serum (FBS) is poured into the residue to float the cells. The FBS concentration to be added is preferably 1 to 20 vol%. If the FBS concentration is too low, cell growth (proliferation) defects are likely to occur, so it is preferable to add 5 vol% or more. Further, although the FBS concentration is too high, there is little problem, but excessive addition leads to cost increase, so 15 vol% or less is more preferable.

  This is seeded on a culture substrate such as a culture plate, a petri dish or a hollow fiber membrane, and then the adherent placental tissue-derived stem cells are cultured in a planar (two-dimensional) manner by a conventional method to proliferate. 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 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%.

  In the present invention, the material of the hollow fiber membrane used as a culture substrate 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 can be mentioned. In order to improve cell adhesion and familiarity with the culture solution, hydrophilic polymers such as polyvinylpyrrolidone and hydroxyalkylcellulose, and cell adhesion factors such as collagen and fibronectin may be used in combination.

  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). The above operations 1 to 3 are performed using another hemocytometer (measured twice and averaged).
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, prepare a suspension so that there are 100-500 cells 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

(Measurement of cell growth rate)
The cell proliferation rate was calculated by the following formula using the number of viable cells collected from the cell culture container (hollow fiber membrane module) after completion of the culture and the initial number of seeded cells (1.05 × 10 ⁵ ).
Cell proliferation rate (%) = (number of viable cells recovered−number of seeded cells) / number of seeded cells × 100

[Example 1]
(Freezing placental tissue)
1. 3 g of placental 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 placenta tissue was quickly chopped to a size of about 3 to 5 mm with scissors.
3. The cryoprotectant in which the placenta tissue was suspended was aspirated with a 25 ml disposable pipette, and about 1.5 ml was dispensed into 10 cryopreservation tubes. At this time, the placental tissue weight / (placental tissue weight + cryoprotective solution weight) was 22.4 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 placental 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 placental tissue and cryoprotective solution were placed in a φ100 mm dish containing 10 ml of DMEM previously cooled to 4 ° C. 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 cooled to 4 ° C. was added, the dish was lightly shaken and rinsed, and then the medium was aspirated and removed with an aspirator, and the tissue was washed.

(Digestion of frozen placental tissue)
1. Following the 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 placental tissue suspension was transferred to a 50 ml centrifuge tube and shaken in a 37 ° C. warm bath for 60 minutes to digest the placental tissue.

(Separation culture of mesenchymal stem cells)
1. The digested placental tissue fluid was filtered through a 100 μm mesh to remove large undigested tissue and placed in a 50 ml tube.
2. Centrifuged at 1000 rpm for 5 minutes.
3. The supernatant was removed by aspiration, the precipitate was suspended in DMEM to which FBS was added to 10 vol%, and seeded on a collagen-coated φ60 mm dish, and stem cells were cultured for 7 days.

[Example 2]
(Production of hollow fiber membrane)
Polyethersulfone (4800P, manufactured by Sumitomo Chemical Co., Ltd.) 20 wt%, N methylpyrrolidone (Mitsubishi Chemical Co., Ltd.) 36 wt%, and triethylene glycol (Mitsui Chemicals Co., Ltd.) 44 wt% were uniformly dissolved to prepare a membrane forming stock solution. This stock solution was simultaneously discharged from a cylindrical double tube nozzle heated to 70 ° C. together with an aqueous solution of 13.5 wt% N-methylpyrrolidone, 16.5 wt% triethylene glycol and 70 wt% water as a hollow forming agent. After passing through the part, it was immersed in 75 ° C. water and solidified, and after sufficient water washing, 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 of the membrane forming solution and the hollow forming agent was adjusted so that the hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 300 μm, and a film thickness of 50 μm.

(Production of hollow fiber membrane module)
A hollow fiber membrane module for testing was produced as follows. After inserting 10 prepared polyethersulfone hollow fiber membranes (culture substrate) into a cylindrical acrylic module case with an inner diameter of 4 mm and a length of 10 cm, do not block the hollow portion of the hollow fiber membrane. Both ends were fixed to the module case with a polyurethane potting agent to prepare a hollow fiber membrane module (culture vessel).

(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 4 and 5, the pumps 6 and 7 are started, and a sufficient amount is put in the hollow fiber membrane module. Rinse and wash. Thereafter, the culture medium storage container 4 was replaced with one containing DMEM to which FBS was added at 10 vol%. In addition, the culture medium storage container 5 was replaced with a serum-free DMEM containing 0.5 μM insulin, 10 nM selenium, and 250 μM glutamic acid. Thereafter, the pumps 6 and 7 were activated to perform priming, and the water in the hollow fiber membrane module 3 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 exchanging the culture solution storage container 4 with a vessel containing only the culture solution, liquid feeding was started at a flow rate of 0.33 mm / min on the hollow part 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 portion side of the hollow fiber membrane was DMEM to which FBS was added at 10 vol%, and the culture solution to be passed to the outside of the hollow fiber membrane was insulin-free without addition of serum. DMEM to which 0.5 μM, selenium 10 nM and glutamic acid 250 μM were added was used. This cell culture experiment was conducted 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).

Mesenchymal stem cells separated and cultured from frozen placental tissue are seeded in DMEM (10 vol% FBS added) at a cell density (5000 to 10,000 cells / cm ² ) in a 24-well culture plate and cultured overnight, and the cells adhere to the plate I let you. 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 vol% FBS added), 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. In addition, 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 placental tissue-derived stem cells (MSC) 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 separated and cultured from frozen placental tissue prepared by the same technique as used in the above-described measurement of cartilage differentiation ability were added to DMEM (10 vol% FBS added) 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. In the other group, the medium was replaced with fresh DMEM (10 vol% FBS added), 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 placental tissue are differentiated into osteoblasts by culturing in an osteoblast differentiation-inducing medium, and calcium accumulates in the cells. It shows that mesenchymal stem cells separated and cultured from frozen placental tissue have the ability to differentiate into osteoblasts.

  According to the present invention, placental 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 placental tissue. This makes it possible to effectively use placental tissue as a cell source used in regenerative medicine.

1a, 1b Entrance / exit (hollow part side)
2a, 2b Entrance / exit (external side)
3 Cell culture vessel (hollow fiber membrane module)
4, 5 Culture solution storage container 6, 7 Liquid feed pump 8, 9 Waste liquid collection container (cell collection container)

Claims (6)

i) Step of slicing placenta tissue collected from living body so that maximum diameter is 6 mm or less ii) Placenta tissue weight / (placenta tissue weight + cryoprotection solution weight) Iii) A placental tissue storage method comprising a step of placing a placental tissue and a cryoprotective solution in a container and cryopreserving in an environment of −80 ° C. or lower. iv) a step of thawing the frozen placental tissue obtained by the method according to claim 1 and then washing with a culture solution v) a step of washing with a protease solution vi) a step of digesting the placental tissue in the protease solution A method of preparing placental tissue-derived stem cells.   The method according to claim 2, wherein the protease comprises collagenase and / or dispase.   The method according to claim 2 or 3, wherein the stem cell is a mesenchymal stem cell.   A method for culturing frozen placental 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.