WO2013187077A1 - 幹細胞保存媒体、幹細胞保存方法および幹細胞保存システム - Google Patents
幹細胞保存媒体、幹細胞保存方法および幹細胞保存システム Download PDFInfo
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Definitions
- the present invention relates to a storage medium, storage method, and storage system for stem cells, particularly primate inducible pluripotent stem cells (hereinafter referred to as iPS cells) and embryonic stem cells (ES cells).
- iPS cells primate inducible pluripotent stem cells
- ES cells embryonic stem cells
- the present invention relates to a technique for simple and efficient storage by cryopreservation.
- human iPS / ES cells have a problem that the survival rate after cryopreservation is low unlike that of mice, and industrialization requires cryopreservation techniques.
- DAP213 is a vitrification solution developed for the preservation of fertilized eggs of mice. Since it has a high solute concentration of 2M, acetamide 1M, propylene glycol 3M, it is very toxic due to osmotic pressure, and when primate iPS / ES cells including humans are frozen, they are immersed in liquid nitrogen after starting suspension. It takes about 10 seconds to complete the process, and after removing it from liquid nitrogen during thawing, it is necessary to quickly add the medium pre-warmed to 37 ° C and rapidly thaw. It requires considerable skill, such as inability to defrost.
- solute cell freezing protection agent
- the solute concentration the more limited the molecular motion of water and the easier the vitrification, but the higher the osmotic pressure, the higher the toxicity to the cells.
- the problem is that cells are damaged by recrystallization during lysis rather than freezing.
- T'Joen et al. Detached a cell clamp to reduce the cell clamp by two-step treatment of Collagenase IV followed by Cell Dissolution Solution against multiple strains of human ES cells, and cryopreservation solution 5% HES +5 % DMSO + Freeze slowly using a program freezer with storage medium (80% DMEM / F12 + 20% KSR + 400 ⁇ L HEPES), then add special recovery medium containing 0.1 M sucrose at the time of thawing and incubate for 5 minutes When treated, it reports a survival rate of 80% (see Non-Patent Document 4).
- cryopreservation solution 5% HES + 5% or 10% EG + Freezing medium (80% DMEM / F12 + 20% KSR + 400 ⁇ L HEPES) reports that the survival rate is 0% when slowly frozen, and DMSO is effective, but ethylene glycol alone It has also been reported that there is no cryoprotective effect.
- the vitrification method requires complicated operations such as increasing the concentration of the cell frost protection agent and increasing the cooling rate.
- the slow freezing method tried instead of the vitrification method has a low cell viability and is not simple compared with the vitrification method.
- the present invention provides a storage medium, storage method, and storage system for stem cells that are used in a slow freezing method instead of the vitrification method, have high cell viability, and are simple and efficient. For the purpose.
- the inventors of the present invention have made extensive studies on a storage medium for slow cryopreservation of stem cells such as human iPS / ES cells.
- Hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene are used as cell cryoprotectants. It was found that a high cell viability can be obtained by combining with glycol (EG).
- EG glycol
- a high cell viability can be obtained by slow freezing by reducing the size of the cell clamp using a pronase solution.
- the stem cell storage medium of the present invention is a medium for storing stem cells, and contains hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). It is characterized by.
- the stem cell storage medium of the present invention preferably contains hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and contains serum, serum substitute component, protein component (eg, albumin), animal Does not contain any of the derived components.
- the stem cell storage medium of the present invention is a medium that can be stored at room temperature and is excellent in convenience.
- DMSO dimethyl sulfoxide
- CP-1 manufactured by Kyokuto Pharmaceutical Co., Ltd.
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- composition of CP-1 consists of 12 g of HES, 10 mL of DMSO and physiological saline, and human serum albumin solution and RPMI 1640 medium are added at the time of use.
- the characteristics of CP-1 are that it can be stored frozen at ⁇ 80 ° C. and program freezing is unnecessary.
- the stem cell storage medium of the present invention is obtained by further adding ethylene glycol (EG) to a mixture of HES and DMSO, and a cell survival rate higher than that of CP-1 was obtained.
- EG ethylene glycol
- HES and ethylene glycol alone have no cryoprotective effect, but the effect of the present invention is high when HES, DMSO, and EG coexist at appropriate concentrations. This is a new finding that shows a cytoprotective effect.
- a frozen tube that has been slowly frozen and stored using the stem cell storage medium of the present invention is added to a warm medium and then ultra-rapidly thawed or rapidly thawed in a water bath (37 ° C.
- the temperature of the warm medium is, for example, 20 ° C. or higher, preferably 25 ° C. or higher, more preferably 30 ° C. or higher, for example 40 ° C. or lower, preferably 39 ° C. or lower, more preferably 38 ° C. or lower, most preferably. Is about 37 ° C.
- the high survival rate by normal culture medium addition was obtained without using the special recovery
- ethylene glycol (EG) is preferably present at a concentration in the range of 2 to 15% (v / v), preferably 4 to 12.5% (v / v).
- the EG concentration is 2% or more, the cell viability exceeds 20% even when the EG is thawed in a water bath (37 ° C. hot water bath).
- the EG concentration is 15%, the cell viability exceeds 20%.
- the EG concentration is in the range of 4 to 12.5% (v / v)
- the same high cells as in rapid thawing by adding a warm medium Survival rate is obtained.
- the ethylene glycol (EG) concentration is more preferably 4 to 10% (v / v), and particularly preferably 5 to 6% (v / v). That is, when three components of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG) coexist at appropriate concentrations, an excellent cytoprotective effect is exhibited.
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- EG ethylene glycol
- hydroxyethyl starch is empirically preferably present at a concentration within the range of 4 to 8% (w / v), more preferably 5 to 6% (v / v).
- HES is a polysaccharide that does not permeate the cell membrane and has an effect of protecting the cell membrane from cell damage due to ice block formation on the cell surface, and is thought to contribute to the improvement of cell viability after thawing.
- dimethyl sulfoxide is more preferably 4 to 6% (v / v), still more preferably about 5% (v / v).
- DMSO is permeable to the cell membrane and has the effect of protecting the cell membrane from cell damage caused by the formation of ice blocks in the cell, while it is cytotoxic and induces cell differentiation. Therefore, the DMSO concentration is generally The concentration is about 1/2 of 4 to 6% of 10% of the concentration during typical cryopreservation.
- ethylene glycol (EG) can reduce DMSO cell differentiation-inducing action.
- DMSO and ethylene glycol (EG) act as frost damage inhibitors and suppress the formation of ice crystals during freezing.
- DMSO has been pointed out to have a cell differentiation action
- ethylene glycol (EG) antagonizes and reduces its action.
- EG ethylene glycol
- pluripotency is maintained by confirming stem cell markers and in vitro three germ layer differentiation.
- the stem cell storage method of the present invention is a storage method for slowly freezing stem cells, and includes a peeling step for peeling stem cells using a pronase solution, and a freezing step for slowly freezing the peeled stem cells in a stem cell storage medium.
- the stem cell storage medium is the above-described stem cell storage medium of the present invention, and is a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG).
- the stem cell storage medium is a medium containing hydroxyethyl starch (HES) and dimethyl sulfoxide (DMSO) and a culture medium or albumin solution, as in the existing CP-1.
- stem cells suspended in a buffer or medium are subdivided into cell colonies from which feeder cells have been removed by treatment with a pronase solution, and then stored as a cryopreservation agent. Mix with media and cool. Cooling can be performed by slow freezing with a program freezer or simple freezing with an ultra-low temperature freezer.
- the program freezer is a method of cooling at a planned speed of -1 ° C to -2 ° C per minute.
- the simple freezing method a freezing tube is placed in a foamed polystyrene box or a commercially available freezing container and placed in a freezer at ⁇ 80 ° C. and slowly frozen at the same rate as described above.
- the simple freezing method allows simple and efficient work.
- the clamp size (cell mass size) is preferably 200 to 10,000 ⁇ m 2 . It was obtained empirically by performing stem cell detachment treatment using a pronase solution.
- the stem cells in the stem cell storage medium are preferably present in the range of 1 ⁇ 10 3 to 1 ⁇ 10 6 per milliliter of the storage medium.
- the stem cell storage medium is preferably about 0.2 to 1 milliliter per freezing tube.
- ethylene glycol (EG) is preferably present at a concentration in the range of 2 to 15% (v / v), preferably 4 to 12.5% (v / v), From the viewpoint of cell viability, the ethylene glycol (EG) concentration is more preferably 4 to 10% (v / v), still more preferably 5 to 6% (v / v).
- hydroxyethyl starch (HES) is preferably present at a concentration in the range of 4 to 8% (w / v), more preferably 5 to 6% (w / v).
- dimethyl sulfoxide (DMSO) is preferably present at a concentration in the range of 4 to 6% (v / v), more preferably about 5% (v / v).
- the medium preferably contains a medium selected from the group consisting of Dulbecco's modified Eagle (DMEM medium) and F12 medium, or a mixture thereof.
- DMEM medium Dulbecco's modified Eagle
- F12 medium F12 medium
- the albumin solution is preferably present at a concentration of about 4% (w / v).
- the stem cell preservation medium is a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), and is cryopreserved after the freezing step. Further, it includes a thawing step for rapid thawing, and the thawing step is preferably performed by adding a warm medium to a freezing tube and rapidly thawing.
- a cell that has been cryopreserved using the above-described stem cell storage medium of the present invention can obtain a high cell viability even when the cryotube is thawed in a water bath (37 ° C. hot water bath). Can do.
- cultivates with the warm culture medium which added the ROCK inhibitor is further included after the said thawing
- a higher cell survival rate can be obtained compared to the culture without the addition of the ROCK inhibitor.
- culture without adding a ROCK inhibitor is one of the important factors for obtaining high cell viability at the research reagent level.
- the ROCK inhibitor any substance that inhibits Rho-associated coiled-coil kinase (ROCK) can be used, and examples thereof include Y-27632, Fasudil, and H-1152.
- the stem cells are stem cells selected from the group consisting of tissue stem cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells, for example.
- Suitable cells for cryopreservation using the stem cell storage medium of the present invention include stem cells (particularly human and primate inducible pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)). These cells may be already established cell lines or newly established cell lines.
- the stem cell storage system of the present invention is a system for storing stem cells, and includes the following 1) to 3). 1) Pronase solution as exfoliation means for exfoliating stem cells 2) Stem cell storage medium of the present invention described above 3) Slow freezing means for slowly freeing exfoliated stem cells in cell storage medium
- stem cells can be slowly frozen and stored conveniently and efficiently. Thereby, cells can be stored semipermanently while maintaining pluripotency and proliferation ability, and high cell viability can be obtained after thawing.
- the cell viability is high, and destabilization of the cell viability associated with cryopreservation by the conventional vitrification method can be avoided.
- the viability of human iPS cells subjected to slow freezing using cryopreservation solutions A to E is shown.
- the result compared with the vitrification method using DAP213 is shown.
- the results of preparing cryopreservation solutions having a final concentration of ethylene glycol (EG) of 1 to 15% and comparing the survival rates of the respective solutions are shown.
- the influence which the combination of various protease and cryopreservation liquid A has on survival rate is shown.
- Shows the results of quantifying the area of the clamp after cell detachment treatment with various proteases from microscopic image data The comparison of the survival rate by the presence or absence of the addition of a ROCK inhibitor is shown.
- FIG. 3 shows the results of RT-PCR gene expression analysis and immunocytochemical analysis of main stem cell markers of human iPS / ES cells before and after freezing. The results of gene expression analysis and immunocytochemical analysis by RT-PCR of human iPS cells differentiated in vitro after embryoid body (EB) formation is shown.
- Flow chart of stem cell storage method of Example 2 It is a graph which shows that there is almost no difference in the survival rate after freeze-thaw of the human iPS cell 201B7 between the batches of the cryopreservation solution A. It is a graph which shows that there is no change in proliferative property before and after freezing and thawing of human iPS cell 201B7. It shows that the chromosome after freezing and thawing of human iPS cell 201B7 strain is maintained normally.
- a stem cell storage medium is produced by dissolving 6 g of hydroxyethyl starch (HES), 5 mL of dimethyl sulfoxide (DMSO), and 5 mL of ethylene glycol (EG) in 100 mL of physiological saline. be able to. Then, stem cells suspended in a buffer solution or medium are subdivided into cell colonies from which feeder cells have been removed by treatment with a pronase solution, and then the cell pellets after centrifugation of the stem cells and the above stem cell storage medium are used. Mix. After the stem cell storage medium and the stem cells are mixed, the stem cell storage medium is cooled to ⁇ 80 ° C.
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- EG ethylene glycol
- Cooling is performed by placing the cell suspension in a protective case into a foamed polystyrene box in a freezer at ⁇ 80 ° C. Further, it may be further cooled to the temperature of liquid nitrogen and stored in liquid nitrogen.
- Thawing can be performed by adding a medium preliminarily warmed to 37 ° C. to the ice block so as to be diluted 10 times or more and thawing rapidly, or by immersing in 37 ° C. warm water. Specifically, frozen cells are placed in a thermostatic layer at 37 to 40 ° C., and thawed rapidly with good shaking. For cell suspensions in 0.5 mL cryotubes, it is desirable to thaw within 2 to 3 minutes as a guide.
- the stem cell storage method of the present invention includes a peeling step of peeling stem cells using a pronase solution, and a freezing step of slowly freezing the peeled stem cells in a stem cell storage medium.
- a stem cell storage medium as a cryopreservation solution to be used is a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), or hydroxyethyl starch (HES) and dimethyl sulfoxide (DMSO).
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- EG ethylene glycol
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- DMSO dimethyl sulfoxide
- DMSO dimethyl sulfoxide
- DMSO dimethyl sulfoxide
- a pronase solution for cell dispersion manufactured by Kyokuto Pharmaceutical Co., Ltd.
- the following five types of cryopreservation solutions were prepared.
- the concentration notation below represents the final concentration.
- the above BSA is a 25% bovine serum albumin solution (albumin made by Sigma dissolved in physiological saline made by Otsuka Pharmaceutical Co., Ltd.).
- the DMEM / F12 is a medium in which Dulbecco's modified Eagle (DMEM medium) and F12 medium are mixed.
- Cryopreservation solutions A to E were prepared using cell frost damage protection solution CP-1 (551-27200-0, Kyokuto Pharmaceutical Co., Ltd., 68 mL of physiological saline containing 12 g of HES and 10 mL of DMSO).
- cryopreservation solution A contains 10% EG (manufactured by Wako Pure Chemical Industries, Ltd.) after adding 3.2 mL of physiological saline to 6.8 mL of CP-1 stock solution to make 10 mL and mixing well.
- the physiological saline was prepared by mixing gently at a low temperature with an equal volume of 10 mL.
- Cryopreservation solution B was prepared by adding 3.2 mL of 25% BSA to 6.8 mL of CP-1 stock solution to make 10 mL and mixing well, and then gently mixing DMEM / F12 medium in an equal volume of 10 mL at a low temperature. .
- Cryopreservation solution C was prepared by adding 3.2 mL of 25% BSA solution to 6.8 mL of CP-1 stock solution, mixing well, and then mixing physiological saline in an equal volume of 10 mL gently at low temperature. did.
- Cryopreservation solution D is prepared by adding 3.2 mL of physiological saline to 6.8 mL of CP-1 stock solution to make 10 mL and mixing well, then gently mixing DMEM / F12 medium in an equal volume of 10 mL at low temperature. did.
- the cryopreservation solution E was prepared by adding 3.2 mL of physiological saline to 6.8 mL of CP-1 stock solution to make 10 mL, and gently mixing at low temperature.
- Human iPS cells 201B7, 253G1, or human ES cells KhES1 and H1 are cultured according to the maintenance culture protocol for human iPS cells (RIKEN Center for Developmental Sciences, Human Stem Cell Research Support Office) It was. That is, the medium is 80% DMEM / F12 (without Glutamax-containing HEPES), 1% non-essential amino acid, 20% alternative serum KSR (above, manufactured by Invitrogen), 100 IU / mL penicillin / 100 ⁇ g / mL streptomycin (manufactured by Meiji Seika Co., Ltd.) and 0 immediately before use .1 mM 2-mercaptoethanol (Wako Pure Chemical Industries, Ltd.), 5 ng / mL bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) was added and used.
- the medium is 80% DMEM / F12 (without Glutamax-containing HEPES), 1% non-essential amino acid, 20% alternative serum K
- the feeder cells were stopped in advance by treatment of SNL cells (manufactured by DS Pharma Biomedical Co., Ltd.) with mitomycin C (manufactured by Kyowa Hakko Kirin Co., Ltd.) at a final concentration of 10 ⁇ g / mL in 3 ⁇ 10 5 / well / 6 well plates.
- the culture plate was gelatin-coated with 0.1% Gelatin Type A (Sigma) solution and seeded the day before.
- the medium was changed for 1 day and subcultured for 4 days for 1 passage.
- dissociation solution for CTK human ES cells (1 mg / mL Collagenase IV + 0.25% Trypsin + 20% KSR + 1 mM Peeling was performed using (CaCl 2 / PBS) (CaCl 2 manufactured by Nacalai Tesque, others manufactured by Invitrogen), and subculture was performed at a dilution of 1/6.
- the medium of confluent human iPS cells was removed, and the medium was washed with 2-3 mL of phosphate buffered saline PBS ( ⁇ ). To this was added 1 mL of the pronase solution for cell dispersion, and the treatment was carried out at 37 ° C. for about 1 minute, so that only the feeder cells were peeled and suspended first and removed by suction. Immediately after washing with PBS ( ⁇ ) and removing by suction, 2 mL of medium was added, peeled and collected by gentle pipetting with a P1000 pipette, and cell count was performed by trypan blue staining.
- Melting was performed by the following two protocols: melting method 1 and melting method 2.
- Melting method 2 Melting with a warm bath (water bath) Specifically, a tube is taken out from a deep freezer at -150 ° C, rapidly thawed in a 37 ° C hot water bath, 5 mL of medium is added, and then a 15 mL centrifuge tube (3 mL of medium is added in advance), centrifuged (300 g, 3 minutes, 4 ° C.), and the supernatant is removed by aspiration.
- a warm bath water bath
- a tube is taken out from a deep freezer at -150 ° C, rapidly thawed in a 37 ° C hot water bath, 5 mL of medium is added, and then a 15 mL centrifuge tube (3 mL of medium is added in advance), centrifuged (300 g, 3 minutes, 4 ° C.), and the supernatant is removed by aspiration.
- FIG. 10 shows the flow of the stem cell storage method in this example.
- the flow of FIG. 10 shows the stem cell preservation method when the melting method 1 (dilution and thawing by adding warm medium) is used. Specifically, as shown in FIG.
- stem cells are detached using a pronase solution (detachment step: step S01), and then the detached stem cells are slowly frozen in a stem cell storage medium (freezing step: step). S03) and stored frozen (step S05).
- a pronase solution detachment step: step S01
- the detached stem cells are slowly frozen in a stem cell storage medium (freezing step: step). S03) and stored frozen (step S05).
- Any of the above-described cryopreservation solutions A to E is used as the stem cell storage medium used in the freezing step.
- thawing cryopreserved stem cells add warm medium to a cryotube (ultra-rapid thawing) or rapidly thaw in a water bath at 37 ° C., and then quickly remove the freezing medium by centrifugation (thawing).
- Step S07 cultured in a medium supplemented with a ROCK inhibitor (maximum 48 hours), and then cultured in a normal medium (cultivation process: step S09). And it expands (step S11) or returns to a peeling process (step S01), and freeze-preserves again.
- step S11 rapid melting and ultra-rapid melting can be used.
- the rapid melting method in which melting is performed in a water bath at 37 ° C. is the same as the melting method of a normal cell line, and has the advantage of increasing robustness with few operation errors.
- DAP213 frozen solution (2M Add 0.2 mL of DMSO (manufactured by Calbiochem), 1M acetamide (manufactured by Sigma), 3M propylene glycol (manufactured by Nacalai Tesque)) and quickly freeze in liquid nitrogen within 10 seconds. It was transferred to a deep freezer at 0 ° C. and stored frozen for 7 days.
- alkaline phosphatase (ALP) staining was performed by removing the medium from the 6-well plate by suction, washing 3 times with PBS (-) (pH 7.4) (Invitrogen), and 4% paraformaldehyde (Nacalai Tesque).
- ALP staining solution 100 mM Tris-HCl (pH 9.5) to 2.5 mL, ALP Substrate Kit IV (Vector Laboratory, SK-5400) Reagent-1, Reagent-2 and Reagent-3 were added in a drop-by-drop order) and allowed to stand at room temperature to develop a black color.
- the number of colonies was counted based on a digital image (4x) obtained by an imaging system (manufactured by Keyence).
- FIG. 1 is a graph showing the survival rate of human iPS cells subjected to slow freezing using cryopreservation solutions A to E.
- survival rate of the cryopreservation solution A was 88.4%
- survival rate of the cryopreservation solution B was 96.4%.
- the survival rate of the one using the cryopreservation solution C was 94.3%
- the survival rate of the one using the cryopreservation solution D was 76.3%
- the survival rate of the one using the cryopreservation solution E was 88. 0.1% (all survival rates are mid-point values).
- the survival rate of the cryopreservation solution A was 78.7%
- that of the cryopreservation solution B was 44.5%.
- the survival rate of the sample using the cryopreservation solution C was 47.3%
- the survival rate of the sample using the cryopreservation solution D was 33.5%
- the survival rate of the sample using the cryopreservation solution E was 38.3%. 5% (all survival values are midpoint values).
- the slow freezing method using the above-described cryopreservation solutions A to E and thawing by thawing method 1 all have a survival rate of 75% or more. there were.
- the cryopreservation solutions A, D, and E diluted with physiological saline and medium containing no BSA can be cryopreserved. Further, as shown in FIG.
- cryopreservation solution A has a survival rate of 75 when the slow freezing method is performed using the above-described cryopreservation solutions A to E and thawed by the thawing method 2 (thaw in a warm bath). % Or more. It can be seen that the slow freezing method using the cryopreservation solution A showed a high survival rate regardless of the thawing method.
- FIG. 11 shows a comparative experiment between batches of the slow freezing method using the cryopreservation solution A. It can be seen that even in different batches, the survival rate was high regardless of the melting method.
- cryopreservation solution A (the stem cell preservation medium of the present invention) is an animal component-free cryopreservation solution that does not contain BSA and a medium, and is useful for clinical application of regenerative medicine using human iPS cells.
- Example 3 for the purpose of searching for the optimum concentration of ethylene glycol (EG), the final concentration of ethylene glycol (EG) in the cryopreservation solution A (stem cell storage medium of the present invention) of Example 2 is 1 to A 15% cryopreservation solution was prepared and the survival rate of each was compared. The result is shown in FIG. As shown in FIG. 3, as a result of examining cryopreservation solutions of various concentrations of ethylene glycol (EG), those having an ethylene glycol (EG) concentration of 5% are not affected by the melting method, and the survival rate is 90% or more. It was confirmed that.
- EG ethylene glycol
- Example 4 shows that the stem cell preservation method of the present invention in which stem cells are detached using a pronase solution has a higher survival rate by cryopreservation compared to cell separation by various proteases. Specifically, detachment is performed with the following five types of protease cell detachment solutions, cryopreserved with the cryopreservation solution A (stem cell storage medium of the present invention) of Example 2, and thawed by rapid thawing by adding a warm bath medium. Cultured and examined for optimal combination.
- Cell detachment solution A Pronase solution, 37 ° C., 1 minute
- Cell detachment solution B 0.05% Trypsin / EDTA (manufactured by Invitrogen)
- 37 ° C., 1 minute cell detachment solution C
- Cell detachment solution D 2 mg / mL Dissease II (Roche, 10x stock diluted with DMEM / F12), 37 ° C., 15 minutes
- cell detachment solution D 1.5 mg / mL Collagenase IV (powder dissolved in DMEM / F12), 37 ° C., 40 minutes
- Cell detachment fluid E CTK, 37 ° C., 1 minute
- FIG. 4 shows that pronase is optimal. 4 and 5, Pronase indicates cell detachment solution A, Trypsin indicates cell detachment solution B, Disease indicates cell detachment solution C, Collagenase indicates cell detachment solution D, and CTK indicates cell detachment solution E.
- the size of the cell mass (clamp size) at the time of cell detachment using various proteases was quantified using the 201B7 strain. After cell detachment, the cells are collected with a conventional medium, the suspension is put into a 6-well plate, a digital image (4x) is taken with an imaging system (manufactured by Keyence), and the clamp size is set using the area measurement mode of the attached software. Quantified.
- the clamp size is 2089 ⁇ 1485. ⁇ m 2 .
- This size 25454 ⁇ 41002 ⁇ m 2 in the case of dispase treatment, as compared to 8600 ⁇ 13267 ⁇ m 2 at 8551 ⁇ 18884 ⁇ m 2, CTK process when the collagenase treatment, reduced significantly (P ⁇ 0.01), also variation in It was small.
- trypsin treatment 1936 ⁇ 1663 ⁇ m 2 was almost the same as in the case of pronase treatment, and there was no significant difference.
- a) and b) are presumed as the reason why the pronase treatment is superior to the trypsin treatment.
- the protease efficacy of trypsin is stronger than that of pronase, it is difficult to inactivate after the action, and after measuring the colony size, some iPS cells can be transferred to single cells by pipetting or moving between tubes. It fell apart.
- human iPS cells in the epistem state have a good survival rate if they are in a certain amount of cell mass, but when they become single cells, they are cells in the ground state derived from the inner cell mass such as mouse ES cells. I can no longer survive.
- Example 5 the difference in survival rate between the case of ROCK inhibitor (+) culture and the case of ROCK inhibitor ( ⁇ ) culture in the stem cell preservation method of the present invention will be described.
- Cultivation and freezing in the stem cell storage method were performed in the same manner as in Example 2. Thawing was carried out after rapid thawing using a warm medium, and the difference in survival rate with and without the addition of the ROCK inhibitor 10 ⁇ M Y-27632 was measured. When added, the cells were cultured for 2 days in the presence of Y-27632, and then further cultured for 3 days in a medium not containing Y-27632. On the other hand, when not added, the cells were cultured in a normal medium for 5 days. In both cases, the survival rate was measured according to the method of Example 2.
- ROCK inhibitor (+) culture is an important reagent that supports high survival rate in the stem cell preservation method.
- Example 6 stem cells containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), ethylene glycol (EG) for the established human iPS cells 201B7 strain, 253G1 strain and the established human ES cells KhES1 strain, H1 strain
- HES hydroxyethyl starch
- DMSO dimethyl sulfoxide
- EG ethylene glycol
- Example 7 with respect to the state of human iPS cells before and after freezing, the presence or absence of changes in gene expression was determined by RT-PCR (Reverse Transcriptase-Polymerase Chain). Results of semi-quantitative gene expression analysis by Reaction) are shown. Table 1 shows a primer list for RT-PCR gene expression analysis. Primers were synthesized from Invitrogen based on a paper (Takenaka et al. Experimental Hematology. 2010. 38 (2): 154-62.).
- RNA concentration was quantified by measuring the absorbance at 260 nm using Nanodrop (ThermoFisher).
- the reverse transcription reaction was carried out according to the attached protocol of River Tra Ace qPCR RT kit (manufactured by Toyobo Co., Ltd.), and after rapid heating at 65 ° C. for 5 minutes, 100 ng of denatured Total RNA was subjected to 5 ⁇ RT Buffer 2 ⁇ L, RT Enzyme.
- reverse transcription reaction was performed at 37 ° C. for 15 minutes, followed by heat treatment at 98 ° C. for 5 minutes to prepare cDNA. .
- the obtained cDNA was diluted 10-fold by adding 90 ⁇ L of RNase-free water to obtain a cDNA solution.
- the PCR reaction was performed using 10 ⁇ L of a 10 ⁇ L system using a total of 10 ⁇ L of a cDNA solution of 1 ⁇ L as a template. 1 ⁇ L, 2.5 mM dNTP 0.8 ⁇ L, ExTaq HS 0.05 ⁇ L (all manufactured by Takara), 10 ⁇ M PCR was performed with 1 ⁇ L of primer mix at 98 ° C. for 10 seconds, 55 ° C. for 20 seconds, 72 ° C. for 30 seconds, and 72 ° C. for 5 minutes.
- pluripotency markers OCT4, SSEA3, SSEA4, TRA-1-60, TRA-1-81
- human iPS cell 201B7 strain cultured in an on-feeder for 5 days in a 24-well plate was aspirated and removed, and then PBS ( ⁇ ) containing 4% paraformaldehyde was added at 500 ⁇ L / well, And allowed to stand for 15 minutes. The fixative was removed by suction and washed 3 times with about 2 mL (5 minutes) of PBS ( ⁇ ).
- anti-OCT4 mouse monoclonal antibody (Santa Cruz), anti-SSEA3 rat monoclonal antibody (Millipore), anti-SSEA4 mouse monoclonal antibody (CST), anti-TRA-1-60 mouse monoclonal antibody (primary antibody) CST) and anti-TRA-1-81 mouse monoclonal antibody (CST) were all diluted 200-fold with antibody diluent (PBS (-) containing 1% BSA, 0.3% Triton X-100). The reaction was allowed to stand overnight at 4 ° C.
- the plate was washed 3 times with about 2 mL of PBS ( ⁇ ) (5 minutes), and AlexaFluoro488-labeled anti-mouse IgG goat antibody, AlexaFluoro488-labeled anti-mouse IgM goat antibody, AlexaFluoro488-labeled anti-rat IgM goat antibody ( (All manufactured by Invitrogen) were similarly diluted 500-fold with an antibody diluent, added, and allowed to react at room temperature for 2 hours while being shielded from light with aluminum foil.
- AlexaFluoro488-labeled anti-mouse IgG goat antibody AlexaFluoro488-labeled anti-mouse IgM goat antibody
- AlexaFluoro488-labeled anti-rat IgM goat antibody (All manufactured by Invitrogen) were similarly diluted 500-fold with an antibody diluent, added, and allowed to react at room temperature for 2 hours while being shielde
- Example 8 shows the results of studying three germ layer differentiation in vitro.
- the stem cell preservation medium of the present invention containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)
- SNL cells were removed from strain 201B7 cultured on feeder for 7 days with CTK stripper. Thereafter, it was collected with human iPS cell culture medium (-bFGF) and cultured as it was on a low adhesion surface plate (manufactured by Corning) for 10 days to form embryoid bodies (EB). The medium was changed every other day.
- Half of the embryoid body (EB) was collected in a tube, and gene expression analysis of three germ layer-specific markers was performed by RT-PCR. Specifically, RLT RNA was extracted with Buffer (attached to RNeasy mini kit manufactured by Qiagen), and reverse transcription reaction and PCR were performed as usual. Table 2 shows a list of primers for RT-PCR gene expression analysis.
- EB embryoid body
- -bFGF human iPS cell medium
- FIG. 9 (1) human iPS cell 201B7 strain was transformed into in embryoid body (EB). Differentiated in vitro, major differentiation markers (ectodermal PAX6, mesoderm BRACHYRY, endoderm SOX17, GATA4) were confirmed by RT-PCR analysis, and the gene expression of the differentiation gene was confirmed. In addition, as shown in FIG. 9 (2), main differentiated cell markers (ectodermal ⁇ -tubulin, mesoderm ⁇ SMA, endoderm ⁇ Fetoprotein (AFP)) are stained in all cases by immunocytochemical analysis. Thus, it was confirmed that the ability to differentiate three germ layers was maintained.
- major differentiation markers ectodermal PAX6, mesoderm BRACHYRY, endoderm SOX17, GATA4
- AFP endoderm ⁇ Fetoprotein
- Example 9 demonstrates the effectiveness and stability of the stem cell storage method of the present invention using a stem cell storage medium without any change in proliferation before and after freezing and thawing for human iPS cells.
- the established human iPS cell strain 201B7 Prior to freezing, the established human iPS cell strain 201B7 was detached from the initial seeding concentration of about 25,000 / well / 6 well plate every 4 days with CTK and subcultured by 1/6 dilution. After thawing, 25,000 / vial was cultured in a 10 cm dish for 1 week, detached with CTK every 4 days, and subcultured by 1/6 dilution.
- the number of cells was sampled over time, dispersed to a single cell by 0.05% Trypsin / EDTA treatment (37 ° C., 10 minutes), stained with trypan blue, and measured under a microscope with a hemocytometer did. The result is shown in FIG. It was confirmed that the established human iPS cell 201B7 strain was almost constant before and after freeze-thawing.
- Example 10 the effectiveness and safety of the stem cell storage method of the present invention using a stem cell storage medium in which the chromosome after freezing and thawing is normally maintained for human iPS cells will be described.
- the result is shown in FIG. G-band analysis was performed on the established human iPS cell strain 201B7, which was passaged 3 to 5 after thawing, and it was confirmed that it was a normal karyotype.
- the present invention is useful for cell biological studies of stem cells (particularly human iPS cells / ES cells), and in the field of regenerative medicine such as organ transplantation using stem cell cell banks and cells differentiated from stem cells.
Abstract
Description
2M,アセトアミド 1M,プロピレングリコール 3Mという高い溶質濃度をもつため、浸透圧による毒性が非常に高く、ヒトを含む霊長類iPS/ES細胞を凍結する際には、懸濁を始めてから液体窒素に浸漬するまでに要する時間を10秒程度という短時間処理や解凍時に液体窒素から取り出した後、ただちに、37℃にあらかじめ温めておいた培地を迅速に添加し急速解凍が必要であり、インキュベータによるチューブごと解凍ができないなど手技的にかなりの熟練を要する。
また、凍結時よりもむしろ溶解時に再結晶化が起こることにより細胞がダメージを受けることがより大きな問題となっている。
WuCFらは、ヒトES細胞(hES1株)のトレハロースを含む凍結保存液により緩慢凍結後、急速解凍し、生存率48%であったことを報告している(非特許文献2を参照)。
+保存用培地 (80% DMEM/F12 + 20% KSR + 400μL HEPES)を用いてプログラムフリーザーを用いて緩慢凍結した後、融解時に0.1Mスクロースを含む特殊な回復培地を添加して5分間インキュベート処理すると生存率80%を報告している(非特許文献4を参照)。
なお、彼らは、凍結保存液5% HES+5%もしくは10% EG
+凍結用培地 (80% DMEM/F12 + 20% KSR + 400μL HEPES)を用い緩慢凍結した場合、生存率が0%であることも報告しており、DMSOは有効であるが、エチレングリコール単独では凍結保護効果がないことも報告している。
また、最近ではTrypLE(登録商標)Expressやtrypsin/EDTAを用いて細胞をシングルセルに細胞剥離した後、凍結保存し、ROCK阻害剤Y27632を10μM添加培養により生存率を向上させる方法も複数のグループから報告もある(非特許文献5~非特許文献9を参照)。
従って、もっとハンドリングが容易で効率が高く、毒性が低く、再現性が高く堅牢な新しい凍結保存方法の開発が望まれている。
上記状況に鑑みて、本発明は、ガラス化法に代わる緩慢凍結法に用いられ、高い細胞生存率を有し、かつ、簡便で効率良い、幹細胞の保存媒体、保存方法および保存システムを提供することを目的とする。
また、緩慢凍結保存の保存方法における細胞剥離操作において、プロナーゼ溶液を用いて細胞クランプのサイズを小さくして緩慢凍結することにより、高い細胞生存率が得られることの知見を得た。
本発明の幹細胞保存媒体は、好ましくは、ヒドロキシエチルスターチ(HES)、ジメチルスルホキシド(DMSO)、エチレングリコール(EG)を含有し、かつ、血清、血清の代替成分、蛋白成分(例えばアルブミン)、動物由来成分のいずれの成分も含まない。血清、血清の代替成分、アルブミンなどの蛋白成分、動物由来成分のいずれの成分も含まない、いわゆるアニマルコンポーネントフリーの幹細胞保存液の場合、ヒトiPS/ES細胞を用いた再生医療の臨床応用に有益である。
また、本発明の幹細胞保存媒体は、常温で保存可能な媒体であり、利便性に優れている。
また、本発明の幹細胞保存媒体を用いて緩慢凍結保存した凍結チューブを、温培地を添加して超急速解凍、もしくはウォーターバス(37℃湯浴)にて急速融解した場合でも、他の幹細胞保存媒体を用いた場合より、高い細胞生存率が得られた。ここで温培地の温度は、例えば20℃以上、好ましくは25℃以上、より好ましくは30℃以上であり、例えば40℃以下、好ましくは39℃以下、より好ましくは38℃以下であり、最も好ましくは約37℃である。また、独自の解凍液CELLOTIOM(登録商標)や0.1Mスクロースを含む特殊な回復培地を用いず、通常の培地添加による高い生存率が得られた。プログラムフリージング不要、特殊な回復培地が不要、通常の細胞株と同様に凍結融解が可能である点は、操作が簡便であり、堅牢性が向上し、ヒトiPS/ES細胞を用いた再生医療の臨床応用に有益である。
すなわち、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)とエチレングリコール(EG)の3成分が適当な濃度で共存すると、優れた細胞保護効果を示すのである。
HESは多糖類であり、細胞膜を透過せず、細胞表面において氷塊形成による細胞障害から細胞膜を保護する効果があり、融解後の細胞生存率の向上に寄与すると考えられている。
また、エチレングリコール(EG)添加により、DMSOの細胞分化の誘導作用を軽減できる。DMSOおよびエチレングリコール(EG)は、凍害防止剤として作用して、凍結の際の氷の結晶の形成を抑制する。DMSOは、細胞分化作用があることが指摘されているが、エチレングリコール(EG)がその作用と拮抗し低減するものと考えられる。
さらに、解凍時に温培地添加による急速解凍と希釈と遠心除去によりにDMSOとの接触時間を極力短時間にすることにより分化誘導遺伝子の遺伝子発現を極力おさえることが可能である。実際、幹細胞のマーカーやin vitro三胚葉分化の確認により多能性が維持されていることを確認している。
ここで、幹細胞保存媒体は、上述の本発明の幹細胞保存媒体であり、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)とエチレングリコール(EG)を含有する媒体である。
或いは、幹細胞保存媒体は、既存のCP-1と同様、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)と、培地(culture medium)もしくはアルブミン溶液とを含む媒体である。
本発明の幹細胞保存方法においては、簡易凍結法により簡便で効率良く作業を行うことができる。
また、ヒドロキシエチルスターチ(HES)は、4~8%(w/v)の範囲内の濃度で存在することが好ましく、5~6%(w/v)が更に好ましい。一方、ジメチルスルホキシド(DMSO)は、4~6%(v/v)の範囲内の濃度で存在することが好ましく、略5%(v/v)が更に好ましい。
また、本発明の幹細胞保存方法において、アルブミン溶液は、略4%(w/v)の濃度で存在することが好ましい。
また、上記解凍工程の後、ROCK阻害剤を添加した温培地で培養する培養工程を更に含むことが好ましい。ROCK阻害剤を添加した培養を行うことにより、ROCK阻害剤を添加しない培養に比べて高い細胞生存率を得ることができる。効率よりも安全性やコストや重視する臨床応用の場合、ROCK阻害剤を添加しないで培養させるのが好ましい。ROCK阻害剤の添加による培養は、研究試薬レベルにおいては、高い細胞生存率を得る重要なファクターのひとつである。ROCK阻害剤としては、Rho-associated coiled-coilキナーゼ(ROCK)を阻害する任意の物質を使用することができ、例えば、Y-27632、FasudilおよびH-1152が例示される。
1)幹細胞を剥離する剥離手段としてのプロナーゼ溶液
2)上述の本発明の幹細胞保存媒体
3)剥離した幹細胞を細胞保存媒体中で緩慢凍結させる緩慢凍結手段
また、本発明によれば、高い細胞生存率であり、従来のガラス化法による凍結保存に伴う細胞生存率の不安定化を避けることができる。
そして、緩衝液や培地に懸濁した幹細胞を、プロナーゼ溶液を用いた処理によりフィーダー細胞を除去した細胞コロニーを一定サイズに細分化した後、幹細胞の遠心後の細胞ペレットと上記の幹細胞保存媒体を混合する。幹細胞保存媒体と幹細胞を混合した後、毎分1~2℃の速度で-80℃まで冷却し凍結保存する。冷却は、保護ケースに入れた細胞浮遊液を-80℃の冷凍庫内の発砲スチロール箱に入れて行う。なお、さらに液体窒素の温度まで冷却して、液体窒素中で保存してもよい。
上述の如く、本発明の幹細胞保存方法は、プロナーゼ溶液を用いて幹細胞を剥離する剥離工程と、剥離した幹細胞を幹細胞保存媒体中で緩慢凍結させる凍結工程を備える。
また、使用する凍結保存液としての幹細胞保存媒体は、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)とエチレングリコール(EG)を含有する媒体、或いは、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)と、培地(culture medium)もしくはアルブミン溶液とを含む媒体である。
また、下記の5種の凍結保存液を調製した。以下の濃度表記は最終濃度を表している。
(凍結保存液A)6%
HES + 5% DMSO + 5% EG+ 0.9% NaCl
(凍結保存液B)6%
HES + 5% DMSO + 4% BSA+50% DMEM/F12+ 0.9% NaCl
(凍結保存液C)6%
HES + 5% DMSO + 4% BSA+ 0.9% NaCl
(凍結保存液D)6%
HES + 5% DMSO+50% DMEM/F12+ 0.9% NaCl
(凍結保存液E)6%
HES + 5% DMSO+ 0.9% NaCl
具体的には、凍結保存液Aは、CP-1原液6.8mLに、生理食塩水3.2mLを添加し10mLとし、よく混合した後、10%のEG(和光純薬社製)を含む生理食塩水を等量10mLにおだやかに低温で混合し調製した。
また、凍結保存液Bは、CP-1原液6.8mLに、25%BSA3.2mLを添加し10mLとし、よく混合した後、DMEM/F12培地を等量10mLにおだやかに低温で混合し調製した。
また、凍結保存液Dは、CP-1原液6.8mLに、生理食塩水3.2mLを添加し10mLとし、よく混合した後、DMEM/F12培地を等量10mLにおだやかに低温で混合し調製した。
また、凍結保存液Eは、CP-1原液6.8mLに、生理食塩水3.2mLを添加し10mLとし、おだやかに低温で混合し調製した。
DMEM/F12(Glutamax含HEPES不含) 、1% 非必須アミノ酸、20%代替血清KSR(以上、Invitrogen社製)、100IU/mL ペニシリン/100μg/mL ストレプトマイシン(明治製菓社製)と使用直前に0.1mM 2-メルカプトエタノール(和光純薬社製)、5ng/mL
bFGF(和光純薬社製)を添加し使用した。
CaCl2/PBS)(CaCl2はナカライテスク社製、その他はInvitrogen社製)を用いて剥離し、6分の1希釈にて継代培養を行った。
a)融解法1:プレインキュベーションした温培地を直接バイアルに添加する希釈による急速融解(温培地添加による希釈融解)
具体的には、-150℃のディープフリーザーからチューブを取り出し、あらかじめ37℃にてプレインキュベーションしておいた培地5mLにて急速解凍し、15mLの遠心チューブ(培地3mLをあらかじめ添加)に移し、遠心し(300g,3分間,4℃)、上清を吸引除去する。
b)融解法2:温浴(ウォーターバス)による融解
具体的には、-150℃のディープフリーザーからチューブを取り出し、37℃湯浴にて急速融解し、培地を5mL添加した後、15mLの遠心チューブ(培地3mLをあらかじめ添加)に移し、遠心し(300g,3分間,4℃)、上清を吸引除去する。
本実施例における幹細胞保存方法のフローを図10に示す。図10のフローは、融解法1(温培地添加による希釈融解)を用いた場合の幹細胞保存方法を示している。具体的には、図10に示すように、プロナーゼ溶液を用いて幹細胞を剥離し(剥離工程:ステップS01)、次に、剥離した幹細胞を幹細胞保存媒体中で緩慢凍結して(凍結工程:ステップS03)、凍結保存する(ステップS05)。凍結工程で用いる幹細胞保存媒体は、上述の凍結保存液A~Eのいずれかを用いる。
凍結保存された幹細胞を解凍する時は、凍結チューブに温培地を添加するか(超急速融解)、若しくは37℃のウオーターバスにて急速融解した後、遠心により凍結媒体を迅速に除去し(解凍工程:ステップS07)、ROCK阻害剤を添加した培地で培養(最大48時間)し、その後は通常培地にて培養する(培養工程:ステップS09)。そして、拡大培養(ステップS11)するか、或いは、剥離工程(ステップS01)に戻り、再び凍結保存する。
なお、融解の際、急速融解と超急速融解のどちらも使用可能である。特に37℃のウオーターバスにて融解する急速融解法は、通常の細胞株の融解法と同じであり、操作ミスが少なく堅牢性が高まるというメリットがある。
1mL、37℃で約1分間処理により、フィーダー細胞のみを先に剥離浮遊させ、吸引除去した。ただちにPBS(-)にて洗浄し吸引除去し、培地5mLを添加し、P1000ピペットにより穏やかにピペッティングし、クランプを崩さないように500 μL(約2.5×105個/チューブ)を1.5mLチューブに分注した。遠心(300g,3分間,4℃)後、上清をできるだけ取り除き、細胞ペレットにDAP213凍結液(2M
DMSO(カルビオケム社製)、1M アセトアミド(シグマ社製)、3M プロピレングリコール(ナカライテスク社製))0.2mLを加え、10秒以内に迅速に液体窒素にて急速凍結し、液体窒素または-150℃のディープフリーザーに移し、7日間凍結保存した。
生存率(%)=
凍結融解後5日間培養後のコロニー数/継代後5日間培養後のコロニー数×100で算出した。
その結果を図1に示す。図1は、凍結保存液A~Eを用いて緩慢凍結法を行ったヒトiPS細胞の生存率を示すグラフである。
融解法1(温培地添加による希釈融解)で解凍した場合、凍結保存液Aを用いたものの生存率は88.4%であり、凍結保存液Bを用いたものの生存率は96.4%であり、凍結保存液Cを用いたものの生存率は94.3%であり、凍結保存液Dを用いたものの生存率は76.3%であり、凍結保存液Eを用いたものの生存率は88.1%である(いずれの生存率も中間点の値)。
一方、融解法2(温浴による融解)で解凍した場合、凍結保存液Aを用いたものの生存率は78.7%であり、凍結保存液Bを用いたものの生存率は44.5%であり、凍結保存液Cを用いたものの生存率は47.3%であり、凍結保存液Dを用いたものの生存率は33.5%であり、凍結保存液Eを用いたものの生存率は38.5%である(いずれの生存率も中間点の値)。
また、図1に示すように、上述の凍結保存液A~Eを用いて緩慢凍結法を行い、融解法2(温浴による融解)で解凍したものは、凍結保存液Aのみが生存率が75%以上であった。凍結保存液Aを用いて緩慢凍結法を行ったものは、融解法に左右されず、高い生存率を示したことがわかる。
また、図11は、凍結保存液Aの用いて緩慢凍結法をバッチ間で比較実験をおこなったものである。異なるバッチにおいても融解法に左右されず、高い生存率を示したことがわかる。
特に、凍結保存液A(本発明の幹細胞保存媒体)を用いて緩慢凍結法を行ったものは、融解法に左右されず、生存率が80%以上であることがわかった。凍結保存液A(本発明の幹細胞保存媒体)は、BSAおよび培地を含まないアニマルコンポーネントフリーの凍結保存液であり、ヒトiPS細胞を用いた再生医療の臨床応用に有益である。
その結果を図3に示す。図3に示すように、様々な濃度のエチレングリコール(EG)の凍結保存液の検討結果、エチレングリコール(EG)濃度が5%のものが、融解法に左右されず、90%以上の生存率であることが確認できた。
実施例2の融解法2では、エチレングリコール(EG)が無添加、エチレングリコール(EG)濃度が2~5%で、80%以上の生存率であった。エチレングリコール(EG)濃度が5%で102%の生存率であり改善が最大になった。エチレングリコール(EG)濃度が4~10%では、融解法に左右されず安定して回復した。
具体的には、以下の5種類のプロテアーゼの細胞剥離液にて剥離を行い、実施例2の凍結保存液A(本発明の幹細胞保存媒体)により凍結保存し、温浴培地添加による急速解凍により融解し培養し、最適な組合せを検討した。
(細胞剥離液B)0.05% Trypsin/EDTA(インビトロジェン社製)、37℃、1分間
(細胞剥離液C)2mg/mL Disease II(ロシュ社製、10xストックをDMEM/F12で希釈)、37℃、15分間
(細胞剥離液D)1.5mg/mL Collagenase IV(粉末をDMEM/F12で溶解)、37℃、40分間
(細胞剥離液E)CTK、37℃、1分間
なお、図4および図5において、Pronaseは細胞剥離液A、Trypsinは細胞剥離液B、Diseaseは細胞剥離液C、Collagenaseは細胞剥離液D、CTKは細胞剥離液Eを示している。
また、様々なプロテアーゼを用いた場合の細胞剥離時の細胞塊の大きさ(クランプサイズ)を、201B7株を用いて、その後のクランプサイズの定量化した。
細胞剥離後、細胞をコンベンショナル培地で回収し、6wellプレートに懸濁液を入れてイメージングシステム(キーエンス社製)によりデジタル画像(4x)を撮影し、付属ソフトウェアのエリア計測モードを用いてクランプサイズを定量した。
μm2であった。このサイズは、ディスパーゼ処理の場合の25454±41002μm2、コラゲナーゼ処理の場合の8551±18884μm2、CTK処理で8600±13267μm2と比較して、有意(P<0.01)に小さく、またばらつきも小さかった。一方、トリプシン処理の場合は、1936±1663μm2とプロナーゼ処理の場合とほぼ同等であり、有意差がなかった。
また、それぞれの剥離液での剥離後の凍結保存において、クランプサイズの細かったプロナーゼおよびトリプシンに関しては、保存することが可能であった。
しかしながら、プロナーゼ処理とトリプシン処理の2つの生存率を比較すると、プロナーゼが88%、トリプシンが30%であり、プロナーゼが有意に高かった。
a)細胞表面のiPS細胞の生存に必要な未知の蛋白を分解や、細胞死を誘導する蛋白質が影響した。
b)トリプシンのプロテアーゼ効力は、プロナーゼと比べ強く、作用後の不活化も難しく、コロニーサイズを測定した後も残存活性により、ピペット操作やチューブ間移動により、一部のiPS細胞がシングルセルまでにばらばらになった。特に、エピステム状態にあるヒトiPS細胞はある程度の細胞塊の状態であれば生存率はよいが、シングルセルになった場合、マウスES細胞のような内部細胞塊由来のグランドステート状態にある細胞と違って生存できなくなった。
クランプサイズの微小化が凍結保存の成功には不可欠であり、本発明の幹細胞保存方法のプロナーゼ溶液を用いた細胞剥離によれば、短時間にフィーダー細胞との分離、コロニーの細分化が行え、また生存率が高く、さらに動物由来成分を含まないといった利点がある。
幹細胞保存方法における培養および凍結は、実施例2と同様の方法により行った。融解は、温培地を用いた急速融解をおこなった後、ROCK阻害剤10μM Y-27632の添加の有無による生存率の違いについて測定した。添加する場合は、Y-27632存在下で2日培養し、その後は、Y-27632を含まない培地でさらに3日間培養した。一方、添加しない場合は、通常培地で5日間培養した。双方とも、実施例2の方法に従い生存率を測定した。
図6に示すように、測定の結果、ROCK阻害剤(+)培養の場合は約120%の生存率であり、ROCK阻害剤(-)培養の場合の約30%の生存率よりも高い生存率を示した。以上から、ROCK阻害剤(+)培養が、幹細胞保存方法において高い生存率を支持する重要な試薬であることがわかる。
その結果を図7に示す。ヒトiPS細胞201B7株、253G1株および株化ヒトES細胞KhES1株、H1株において、80~100%の高い生存率が確認できた。すなわち、HES,DMSO,EGを含有する幹細胞保存媒体を用いた本発明の幹細胞保存方法が、複数の幹細胞株の凍結保存において有効であることを示された。
Reaction)により半定量的に遺伝子発現解析を行った結果を示す。表1に、RT-PCR遺伝子発現解析用プライマーリストを示す。プライマーは、論文(Takenaka et al. Experimental Hematology. 2010. 38(2):154-62.)に基づき、Invitrogen社に依頼して合成した。
RNA濃度は、Nanodrop(ThermoFisher社製)を用いて260nmの吸光度を測定し定量した。逆転写反応は、Rever Tra Ace qPCR RT kit(東洋紡社製)の添付プロトコールに従い、65℃、5分間の加温処理後に急冷し、変性させたTotal RNA 100ngに対し、5x RT Buffer 2μL、RT Enzyme Mix 0.5μL、Primer Mix 0.5μLを添加した10μLの系で、37℃、15分間逆転写反応、続いて98℃5分間の加熱処理にて酵素の失活処理を行い、cDNAを調製した。
1μL、2.5mM dNTP 0.8μL、ExTaq HS 0.05μL(すべてTakara社製)、10μM
primer mix 1μLにて98℃で10秒間、55℃で20秒間、72℃で30秒間を30サイクルおこない、さらに72℃で5分間のPCRを行った。反応液全量に10x Loading Bufferを1μLずつ添加し、全量を2%アガロースゲルにて定電圧120V、10分間電気泳動した。エチジウムブロマイド染色後UVにより検出した。
その結果を図8に示す。図8(1)に示すように、ヒトiPS/ES細胞(ヒトiPS細胞201B7,ヒトES細胞KhES-1)の主要な幹細胞マーカー(OCT4,KLF4,SOX2,C-MYC,NANOG)をRT-PCRによる解析によりバンドが確認でき遺伝子発現が凍結前後において顕著な差がないことが確認できた。また、図8(2)に示すように、主要な幹細胞マーカー(OCT4,SSEA3,SSEA4,TRA-1-60,TRA-1-81)が、免疫細胞化学的解析により全ての場合で染色されることから、凍結前後において顕著な差がないことが確認できた。
ヒドロキシエチルスターチ(HES)、ジメチルスルホキシド(DMSO)、エチレングリコール(EG)を含有する本発明の幹細胞保存媒体による凍結融解後、7日間オンフィーダー培養した201B7株をCTK剥離液により、SNL細胞を除去後、ヒトiPS細胞培地(-bFGF)にて回収し、そのまま低接着表面プレート(コーニング社製)にて10日間培養し胚葉体(EB)を形成させた。培地交換は1日置きに行った。
胚葉体(EB)の半分量をチューブに回収し、RT-PCRにより三胚葉特異的マーカーの遺伝子発現解析を行った。具体的には、RLT
Buffer(キアゲン社製RNeasy mini kit付属)によりRNA抽出し、常法どおり、逆転写反応とPCRを行った。表2にRT-PCR遺伝子発現解析用プライマーリストを示す。
vitroで分化させ、主要な分化マーカー(外胚葉
PAX6、中胚葉BRACHYURY、内胚葉SOX17、GATA4)をRT-PCRによる解析によりバンドが確認でき分化遺伝子の遺伝子発現を確認した。また、図9(2)に示すように、主要な分化細胞マーカー(外胚葉β-tubulin、中胚葉αSMA、内胚葉αFetoprotein(AFP))が、免疫細胞化学的解析により全ての場合で染色されることから、三胚葉分化能が維持されていることが確認できた。
凍結前では、株化ヒトiPS細胞201B7株を初期播種濃度約 25,000/well/6ウエルプレートから4日おきにCTKにて剥離し、1/6希釈により継代培養した。融解後は、25,000/vialを、10cmディッシュにて1週間培養し、4日おきにCTKにて剥離し、1/6希釈により継代培養した。細胞数は、経時的にサンプリングし、0.05%Trypsin/EDTA処理(37℃、10分間)にてシングルセルまでに分散した後、トリパンブルー染色し、血球算定盤にて顕微鏡下にて測定した。
その結果を図12に示す。株化ヒトiPS細胞201B7株について凍結融解前後でほぼ一定であることを確認できた。
その結果を図13に示す。融解後3~5継代した株化ヒトiPS細胞201B7株についてG-バンド分析を行い、正常核型であることを確認できた。
なお、2012年6月15日に出願された日本国特許出願第2012-136422号をそのまま参照により本明細書に取り込むものとする。また、本明細書中で引用する全ての刊行物、特許及び特許出願をそのまま参照により本明細書に取り込むものとする。
Claims (18)
- 幹細胞を保存するための媒体であって、ヒドロキシエチルスターチ(HES)、ジメチルスルホキシド(DMSO)、エチレングリコール(EG)を含有する幹細胞保存媒体。
- エチレングリコール(EG)が、2~15%(v/v)の範囲内の濃度で存在する、請求項1に記載の幹細胞保存媒体。
- ヒドロキシエチルスターチ(HES)が、4~8%(w/v)の範囲内の濃度で存在する、請求項1又は2に記載の幹細胞保存媒体。
- ジメチルスルホキシド(DMSO)が、4~6%(v/v)の範囲内の濃度で存在する、請求項1~3のいずれかに記載の幹細胞保存媒体。
- 血清、血清の代替成分、蛋白成分、動物由来成分のいずれの成分も含有していない、請求項1~4のいずれかに記載の幹細胞保存媒体。
- 幹細胞を緩慢凍結させる保存方法であって、
プロナーゼ溶液を用いて幹細胞を剥離する剥離工程と、
剥離した幹細胞を幹細胞保存媒体中で緩慢凍結させる凍結工程と、
を備え、
前記幹細胞保存媒体は、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)とエチレングリコール(EG)を含有する媒体、
或いは、
ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)と、培地(culture medium)もしくはアルブミン溶液とを含む媒体、
である、幹細胞保存方法。 - 上記の剥離工程において、クランプサイズが200~10000μm2である、請求項6に記載の幹細胞保存方法。
- 上記の凍結工程において、前記幹細胞保存媒体中の幹細胞は、前記保存媒体1ミリリットル当り1×103~1×106個の範囲で存在する、請求項6に記載の幹細胞保存方法。
- 上記の凍結工程において、前記幹細胞保存媒体は、凍結チューブ1本当り略0.2~1ミリリットルとする、請求項8に記載の幹細胞保存方法。
- エチレングリコール(EG)が、2~15%(v/v)の範囲内の濃度で存在する、請求項6に記載の幹細胞保存方法。
- ヒドロキシエチルスターチ(HES)が、4~8%(w/v)の範囲内の濃度で存在する、請求項6に記載の幹細胞保存方法。
- ジメチルスルホキシド(DMSO)が、4~6%(v/v)の範囲内の濃度で存在する、請求項6に記載の幹細胞保存方法。
- 前記培地は、ダルベッコ改変Eagle(DMEM培地)およびF12培地からなる群より選択される培地またはそれらの混合物を含む、請求項6に記載の幹細胞保存方法。
- 前記アルブミン溶液は、略4%(w/v)の濃度で存在する、請求項6に記載の幹細胞保存方法。
- 前記幹細胞保存媒体は、ヒドロキシエチルスターチ(HES)とジメチルスルホキシド(DMSO)とエチレングリコール(EG)を含有する媒体であり、
上記の凍結工程の後、急速解凍を行う解凍工程を更に含み、
該解凍工程は、凍結チューブに温培地を添加して急速解凍、もしくはウォーターバス(37℃湯浴)にて融解するものである、請求項6~14のいずれかに記載の幹細胞保存方法。 - 上記解凍工程の後、ROCK阻害剤を添加した温培地で培養する培養工程を更に含む、請求項15に記載の幹細胞保存方法。
- 前記幹細胞が、組織幹細胞および胚性幹(ES)細胞、誘導多能性幹(iPS)細胞からなる群より選択される幹細胞である、請求項6に記載の幹細胞保存方法。
- 幹細胞を保存するためのシステムであって、
幹細胞を剥離する剥離手段としてのプロナーゼ溶液、
請求項1~5のいずれかの幹細胞保存媒体、
剥離した幹細胞を上記の幹細胞保存媒体中で緩慢凍結させる緩慢凍結手段、
を備える幹細胞保存システム。
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WO2015159950A1 (ja) * | 2014-04-17 | 2015-10-22 | 東京エレクトロン株式会社 | 多能性幹細胞の細胞集塊製造方法および細胞集塊製造システム |
JP2019110799A (ja) * | 2017-12-22 | 2019-07-11 | 三菱製紙株式会社 | 動物細胞のガラス化凍結保存方法 |
JP2019154329A (ja) * | 2018-03-14 | 2019-09-19 | 旭化成株式会社 | 幹細胞の凍結保存液 |
JP2020506682A (ja) * | 2017-02-01 | 2020-03-05 | ザ ユナイテッド ステイツ オブ アメリカ, アズ リプレゼンテッド バイ ザ セクレタリー, デパートメント オブ ヘルス アンド ヒューマン サービシーズ | 組織凍結保存および回復のためのデバイス |
JP2020039326A (ja) * | 2018-09-13 | 2020-03-19 | 極東製薬工業株式会社 | 間葉系幹細胞の凍害保護液とその利用 |
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KR101794544B1 (ko) | 2016-06-30 | 2017-11-07 | 주식회사 셀루메드 | BrdU 어세이를 개선하기 위한 방법 |
JP6626225B2 (ja) * | 2017-01-31 | 2019-12-25 | サラヤ株式会社 | 細胞の凍結保存組成物および凍結保存方法 |
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JP2019110799A (ja) * | 2017-12-22 | 2019-07-11 | 三菱製紙株式会社 | 動物細胞のガラス化凍結保存方法 |
JP2019154329A (ja) * | 2018-03-14 | 2019-09-19 | 旭化成株式会社 | 幹細胞の凍結保存液 |
JP7000212B2 (ja) | 2018-03-14 | 2022-01-19 | 旭化成株式会社 | 幹細胞の凍結保存液 |
JP2020039326A (ja) * | 2018-09-13 | 2020-03-19 | 極東製薬工業株式会社 | 間葉系幹細胞の凍害保護液とその利用 |
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JPWO2013187077A1 (ja) | 2016-02-04 |
EP2883951A1 (en) | 2015-06-17 |
JP5804437B2 (ja) | 2015-11-04 |
EP2883951A4 (en) | 2016-03-02 |
EP2883951B1 (en) | 2018-01-17 |
US20160021873A1 (en) | 2016-01-28 |
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