JP2018113903A - Method for preserving umbilical cord tissue and method of culturing stem cells isolated from preserved umbilical cord tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple method for preserving umbilical cord tissue over a long period and an effective means of culturing isolated stem cells such as MSC from the preserved umbilical cord tissue.SOLUTION: The method of preserving umbilical cord tissue comprises: a step of cutting umbilical cord tissue collected from the body so that the maximum diameter becomes 6 mm or less; a step of immersing the cut umbilical cord tissue in a cryoprotective liquid so that the umbilical cord tissue weight/(umbilical cord tissue weight+cryoprotective liquid weight) is 10 wt.% or more and less than 50 wt.%; a step of putting the cut umbilical cord tissue and cryoprotective solution in a container and performing cryopreservation in an environment of -80°C or lower. A method of preparing stem cells from the preserved umbilical tissue is also provided in addition to a method for culturing stem cells derived from the prepared umbilical cord tissue.SELECTED DRAWING: None

Description

  The present invention relates to a method for preserving umbilical cord tissue and a method for efficiently separating and culturing stem cells from cryopreserved umbilical cord 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 clinical application development is underway.

  Conventionally, mesenchymal stem cells have been prepared mainly by culturing from bone marrow fluid. However, in recent years, tissues other than bone marrow have attracted attention as a source of mesenchymal stem cells. For example, it has been found that abundant stem cells are present in tissues such as placenta, amniotic membrane, and umbilical cord that are normally discarded after delivery, and application to regenerative medicine is expected.

  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.

  Amnion 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. Further, differentiation of human amnion epithelial-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).

  Umbilical cord tissue is an elastic cord-like tissue that plays an important role in mass transport between the fetus and the placenta, and its surface is covered with amniotic membrane. The fetus receives oxygen and nutrients from the mother's side through the placenta and passes waste products to the mother's side, but the umbilical cord connects the fetus and the placenta. Two umbilical arteries and one umbilical vein flow through the umbilical cord, and the umbilical cord structure is filled with Walton's jelly. Umbilical cord blood collected from the umbilical cord isolated immediately after delivery is rich in hematopoietic stem cells and is used for transplantation for the treatment of several intractable diseases such as leukemia.

  It is known that Walton's colloid is rich in mesenchymal stem cells and differentiates into chondrocytes, adipose tissue, and bone (Non-patent Document 1). Thus, good mesenchymal stem cells can be prepared from the umbilical cord tissue, and effective use as a stem cell source is expected.

  When preparing stem cells such as mesenchymal stem cells from the umbilical cord, fresh tissues are exclusively used. For example, isolation culture of mesenchymal stem cells is a series of steps such as washing of the umbilical cord (removal of attached blood, etc.), shredding, 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 fresh umbilical cord tissue is abundant, if there is not enough manpower or time, or if the culture is not ready, the obtained umbilical cord 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 umbilical cord tissue to be frozen and stored for a long period of time by a simple method, and that stem cells such as mesenchymal stem cells can be separated and cultured from the cryopreserved umbilical cord 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 storing umbilical cord tissue for a long period of time by a simple method and capable of separating and culturing stem cells such as MSC from the stored umbilical cord 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 umbilical cord tissue collected from a living body so that the maximum diameter is 6 mm or less ii) The umbilical cord tissue that has been minced in lyoprotective solution, umbilical cord tissue weight / (umbilical cord tissue weight + frozen) Step of immersing so that the weight of the protective liquid is 10 wt% or more and less than 50 wt% iii) The umbilical cord tissue including the step of putting the umbilical cord tissue and the cryoprotective liquid into a container and cryopreserving them in an environment of -80 ° C or lower How to save.
2. iv) a step of thawing the frozen umbilical cord tissue obtained by the method described in (1) and washing with a culture solution v) a step of washing with a protease solution vi) a step of digesting the umbilical cord tissue in a protease solution A method of preparing tissue-derived stem cells.
3. The method according to (2), wherein the protease is collagenase and / or dispase.
4). The method according to (2) or (3), wherein the stem cell is a mesenchymal stem cell.
5. A method for culturing stem cells derived from umbilical cord 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, umbilical cord tissue can be stably stored for a long time, and umbilical cord-derived stem cells can be separated and cultured with high efficiency from the stored umbilical cord tissue.

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

  In the present invention, the umbilical cord collected from a living body is washed and removed from blood adhering to the surroundings in a sterile environment using a sterilized phosphate buffer (PBS) or the like. Next, the umbilical cord is incised using a scissors or the like, and the Walton's colloid including the umbilical vein and the umbilical artery is separated from the perumbilical membrane. After that, the Walton colloid is chopped appropriately. The size of the walton colloid is preferably adjusted so that the handling and the cooling rate can be made uniform. In the present invention, the wrinkles and forceps are adjusted so that the maximum diameter is 6 mm or less. Use to shred. More preferably, it is 2-5 mm. Thus, the umbilical cord tissue in the present invention substantially refers to Walton's colloid.

  In the present invention, the umbilical cord tissue is preferably preserved by adding a cryoprotective solution to the subdivided umbilical cord tissue. 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. It is preferable to mix the umbilical cord tissue and the cryoprotective solution so that the umbilical cord tissue weight / (umbilical cord tissue weight + cryoprotective solution weight) is 10 wt% or more and less than 50 wt%. The cryoprotectant is preferably cooled to around 4 ° C. before being added to the umbilical cord tissue.

The prepared cryoprotective solution is added to the umbilical cord tissue and lightly suspended, then transferred to a container such as a vial and cooling is started. The cooling temperature (the storage temperature of the umbilical cord 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 umbilical cord tissue thus cryopreserved can be thawed as necessary, and can be proliferated by cell culture. 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 contents are melted, the contents are removed 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 umbilical cord 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 washed umbilical cord 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 umbilical cord tissue, the protease solution is transferred to a centrifuge tube or the like and shaken in a thermostatic bath to digest the umbilical cord 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 3,000-7, respectively. 000 units / mL, more preferably 1.3 to 1.8 mg / mL and 4,000 to 6,000 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 3,000 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 umbilical cord tissue-derived stem cells are cultured in a flat (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 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 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 1,000 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 5,000 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) Wash the surface of the hemocytometer and cover glass with 70% isopropanol, wipe off excess isopropanol and air dry.
(2) Wet the side surface of the cover glass with Reagent grade water and attach it to the hemocytometer.
(3) After thoroughly stirring the cell suspension with a Pasteur pipette or the like, immediately pour it into a hemocytometer and fill it up to the top of 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 lens).
(6) Using a counter, quickly measure the number of cells in 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]
(Freeze storage of tissue)
(1) Use sterilized phosphate buffer (PBS) to wash and remove blood adhering to the umbilical cord, then open the umbilical cord and remove Walton's glue (= umbilical cord tissue) including the umbilical vein and umbilical artery. Cut off from surrounding membrane.
(2) About 10 g of the umbilical cord tissue was placed in a φ100 mm dish containing 10 ml of a cryoprotective solution (10 wt% DMSO + DMEM) previously cooled to 4 ° C.
(3) Next, the umbilical cord tissue was quickly chopped to a size of about 3 to 5 mm with scissors.
(4) The cryoprotection solution in which the umbilical cord tissue was suspended was sucked with a 25 ml disposable pipette, and about 1.5 ml each was dispensed into 10 cryopreservation tubes. At this time, the umbilical cord tissue weight / (umbilical cord tissue weight + cryoprotective solution weight) was 22.4 wt%.
(5) After holding at 4 ° C. for 5 to 15 minutes, it was cooled to −30 ° C. at a rate of −1 ° C./min.
(6) After being held at −30 ° C. for 5 to 15 minutes, it was cooled to −80 ° C. at a rate of −5 ° C./min.
(7) Placed in a deep freezer at −80 ° C. and stored frozen.

(Thaw and wash frozen umbilical cord tissue)
(1) The cryopreservation tube was taken out from the deep freezer at -80 ° C and quickly thawed in warm water warmed to 37 ° C.
(2) The thawed umbilical cord 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) 10 ml of DMEM cooled to 4 ° C. was added once more, and the dish was lightly shaken to rinse, and then the medium was sucked and removed with an aspirator to wash the tissue.

(Digestion of umbilical cord 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 umbilical cord tissue suspension was transferred to a 50 ml centrifuge tube and shaken in a 37 ° C. warm bath for 60 minutes to digest the umbilical cord tissue.

(Separation culture of mesenchymal stem cells)
(1) The digested umbilical cord tissue fluid was filtered through a 100 μm mesh to remove a large undigested tissue and put into a 50 ml tube.
(2) Centrifugation was performed at 1,000 rpm for 5 minutes.
(3) The supernatant was removed by aspiration, and the sediment was suspended in DMEM to which 10% by volume of FBS had been added, seeded in 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 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. DMEM supplemented with 0.5 μM insulin, 10 nM selenium and 250 μM glutamic acid 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 isolated and cultured from frozen umbilical cord tissue are seeded in a 24-well culture plate in DMEM (10 vol% FBS added) at a cell density (5,000 to 10,000 cells / cm ² ) and cultured overnight. Was adhered 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 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. It shows that stem cells derived from umbilical cord tissue (MSC) have a weak ability to differentiate into adipocytes.

(Differentiation into osteoblasts)
The ability to differentiate into osteoblasts 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 umbilical cord tissue prepared by the same method as used for the measurement of differentiation into adipocytes were added to DMEM (10 vol% FBS added) at a cell density (5,000 to 10,000 cells). / Cm ² ) in a 48-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 concentration of released calcium was quantified using Calcium E-Test Wako (Wako Pure Chemical Industries, Ltd.). 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 umbilical cord tissue are differentiated into osteoblasts by culturing in an osteoblast differentiation-inducing medium, and calcium accumulates in the cells. This shows that mesenchymal stem cells separated and cultured from frozen umbilical cord tissue have the ability to differentiate into osteoblasts.

  According to the present invention, umbilical cord 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 umbilical cord tissue. This makes it possible to effectively use the umbilical cord tissue as a cell source used for 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 umbilical cord tissue collected from a living body so that the maximum diameter is 6 mm or less ii) The umbilical cord tissue that has been minced in lyoprotective solution, umbilical cord tissue weight / (umbilical cord tissue weight + frozen) Step of immersing so that the weight of the protective liquid is 10 wt% or more and less than 50 wt% iii) The umbilical cord including the step of putting the umbilical cord tissue and cryoprotective liquid into the container and cryopreserving them in an environment of -80 ° C or lower How to save the organization. iv) a step of thawing the frozen umbilical cord tissue obtained by the method of claim 1 and washing with a culture solution v) a step of washing with a protease solution vi) a step of digesting the umbilical cord tissue in a protease solution A method of preparing tissue-derived stem cells.   The method according to claim 2, wherein the protease is collagenase and / or dispase.   The method according to claim 2 or 3, wherein the stem cells are mesenchymal stem cells.   A method for culturing stem cells derived from umbilical cord tissue, 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.