WO2021220731A1 - 幹細胞用培地及び幹細胞の培養方法 - Google Patents
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Definitions
- the present invention relates to a medium for stem cells and a method for culturing stem cells.
- pluripotent stem cells are undifferentiated cells having self-renewal ability and capable of differentiating into various cells.
- regenerative medicine has been actively studied in which pluripotent stem cells and cells induced to differentiate from pluripotent stem cells are transplanted into damaged tissues of patients to regenerate their functions.
- stem cells such as pluripotent stem cells and mesenchymal stem cells and their differentiated cells. ing.
- Non-Patent Document 2 mTeSR1 medium
- Essential-8 medium Non-Patent Document 3
- DMEM Dulbecco's modified Eagle's medium
- F12 F12 medium
- Additives such as the substitute (for example, albumin) are used.
- Patent Document 1 discloses a medium in which seven non-essential amino acids used as the additive are reduced.
- Patent Document 2 discloses that albumin and polyvinyl alcohol are added to the medium.
- Patent Document 3 discloses that a serum-free medium containing phospholipids and fatty acids as well as a plurality of cytokines is used for culturing mesenchymal stem cells among stem cells.
- an object of the present invention is to provide a medium for stem cells, which has good proliferative performance while maintaining the differentiation potential of stem cells, and a method for culturing stem cells using the medium.
- the present inventors have made good results while maintaining the differentiation potential of stem cells by containing at least one of carboxymethyl cellulose and polyvinylpyrrolidone as a water-soluble polymer compound in the medium.
- the present invention has been completed by discovering that it has excellent proliferation performance.
- the present invention has been made based on the above findings, and provides the following medium for stem cells and a method for culturing stem cells using the medium.
- a medium for stem cells containing at least one of carboxymethyl cellulose and polyvinylpyrrolidone as a water-soluble polymer compound.
- the medium for stem cells according to the above [1] wherein the amount of polyvinylpyrrolidone added to the medium is a final concentration of 0.05 ⁇ g / mL to 2 mg / mL.
- [4] The medium for stem cells according to any one of [1] to [3] above, wherein the amount of recombinant albumin added to the medium is 0.05 to 1 mg / mL at a final concentration.
- [5] The medium for stem cells according to any one of [1] to [4] above, further containing ⁇ -nicotinamide mononucleotide.
- [6] A method for culturing stem cells using the medium for stem cells according to any one of [1] to [5] above.
- FIG. 1 is a graph showing the measurement results of the absorbance of the growth performance test I in Examples 1 to 7 and Comparative Example 1 by the crystal violet staining method.
- FIG. 2 is a graph showing the results of absorbance by the crystal violet staining method of the growth performance test I in Example 11 and Comparative Example 1.
- FIG. 3A is a graph showing the transition of the cumulative number of divisions of the growth performance test II in Example 8 and Comparative Example 1
- FIG. 3B is a growth performance test in Example 13 and Comparative Example 1. It is a graph which showed the transition of the cumulative number of divisions of II.
- FIG. 4 is a graph showing the results of flow cytometry of the differentiation potential test I in Example 8, Example 13, and Comparative Example 1.
- FIG. 5 is an image showing the state of the stem cells that have been induced to differentiate in the differentiation potential test II in Example 8, Example 13, and Comparative Example 1.
- the medium for stem cells of the present invention is used for culturing stem cells.
- a “stem cell” is an undifferentiated cell having a self-renewal ability and a differentiating ability capable of differentiating into various cell tumors.
- stem cells include pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells), as well as somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, nerve stem cells, and skin stem cells. Can be mentioned.
- stem cells to be cultured using the stem cell medium according to the present invention animal-derived stem cells are preferable, mammalian-derived stem cells are more preferable, and human-derived stem cells are even more preferable.
- stem cells cultured using the stem cell medium of the present invention are divided into either ectodermal, mesoderm, or endoderm cells.
- Stem cells that can be transformed are preferable, and stem cells that can differentiate into mesoderm cells are more preferable. Examples of such stem cells include mesenchymal stem cells.
- the medium for stem cells according to the present invention is characterized by containing at least one of carboxymethyl cellulose and polyvinylpyrrolidone as a water-soluble polymer compound.
- Carboxymethyl cellulose (hereinafter also referred to as "CMC"; CAS registration number 9000-11-7) is one of the cellulose derivatives, and is a cellulose in which a part of the hydroxy group of glucopyranose constituting the cellulose is replaced with a carboxymethyl group. It is ether.
- CMC Carboxymethyl cellulose
- those conventionally used as a medium for cell culture can be used without particular limitation.
- the CMC preferably has a degree of substitution (etherification) of the carboxymethyl ether group per anhydrous glucose unit of 0.6 to 0.8.
- CMCs are commercially available, such as Nacalai Tesque, Sodium Carboxymethyl Cellulose (cat.no.07326-95), Fujifilm Wako Pure Chemical Industries, Ltd., Carboxymethyl Cellulose (cat.no.11676-85), etc. Commercially available products can be used.
- Polyvinylpyrrolidone (hereinafter also referred to as “PVP”; CAS Registry Number 9003-39-8) is a water-soluble polymer compound in which N-vinyl-2-pyrrolidone is polymerized.
- PVP polyvinylpyrrolidone
- those conventionally used as a medium for cell culture can be used without particular limitation.
- the weight average molecular weight of PVP is preferably 40,000 to 1,200,000.
- Such PVPs are commercially available, for example, Nacalai Tesque, Polyvinylpyrrolidone K-30 (cat.no.06306-72) and Nacalai Tesque, Polyvinylpyrrolidone K-90 (cat.no.28315-72). Commercial products such as, etc. can be used.
- At least one of CMC and PVP is added to the medium for stem cells according to the present invention.
- Either one of CMC and PVP may be added to the stem cell medium, or both CMC and PVP may be added.
- the amount of carboxymethyl cellulose added to the stem cell medium according to the present invention is preferably a final concentration of 0.001 ⁇ g / mL to 1 mg / mL. More preferably, the concentration is 0.02 ⁇ g / mL to 0.5 mg / mL.
- the amount of polyvinylpyrrolidone added to the medium for stem cells according to the present invention is preferably a final concentration of 0.05 ⁇ g / mL to 2 mg / mL, and a final concentration of 0.1 ⁇ g / mL to 1 mg / mL. Is more preferable.
- the medium used for culturing stem cells contains albumin (albumin protein) such as serum albumin.
- albumin albumin protein
- the medium for stem cells of the present invention preferably contains albumin, more preferably contains albumin from mammals, and is the same type of albumin as the stem cells to be cultured. It is even more preferable to have it.
- the albumin is recombinant albumin.
- human-derived recombinant albumin for example, in order to culture human-derived stem cells, it is preferable to use human-derived recombinant albumin.
- the amount of albumin added to the stem cell medium according to the present invention can be in the range of 0.05 to 1 mg / mL final concentration, preferably 0.05 to 1 mg / mL final concentration, and more preferably 0. It can be in the range of 1 to 0.5 mg / mL.
- albumin preferably has a reduced amount of fatty acids supported.
- albumin albumin that has been subjected to defatty acid treatment such as activated carbon treatment can be appropriately used.
- the defatty acid treatment can be carried out, for example, by the method described in WO2014-192938.
- the medium for stem cells according to the present invention preferably further contains ⁇ -nicotinamide mononucleotide.
- Nicotinamide mononucleotide hereinafter, also referred to as "NMN”. Chemical formula: C 11 H 15 N 2 O 8 P
- ⁇ -NMN CAS number: 1094-61-7
- the structure of ⁇ -NMN is shown below.
- ⁇ -NMN those prepared by any method can be used.
- ⁇ -NMN artificially synthesized and purified by a chemical synthesis method, an enzymatic method, a fermentation method or the like can be used as an active ingredient.
- a chemical synthesis method for synthesizing ⁇ -NMN for example, ⁇ -NMN can be produced by reacting NAM with L-ribose tetraacetate and phosphorylating the obtained nicotinamide mononucleoside.
- ⁇ -NMN can be produced from NAM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) by nicotinamide phosphoribosyl transferase (NAMPT).
- PRPP 5'-phosphoribosyl-1'-pyrophosphate
- NAMPT nicotinamide phosphoribosyl transferase
- ⁇ -NMN can be produced from NAM by utilizing the metabolic system of a microorganism expressing NAMPT.
- ⁇ -NMN is a component widely present in a living body, ⁇ -NMN obtained by extracting and purifying from natural raw materials such as animals, plants and microorganisms can also be used as an active ingredient.
- a commercially available purified ⁇ -NMN may be used.
- the amount of ⁇ -NMN added to the stem cell medium according to the present invention is preferably 0.01 to 5 mM, and the final concentration is 0. It is more preferably 05 to 2 mM, and even more preferably a final concentration of 0.1 to 1 mM.
- the basal medium used for the stem cell medium As the basal medium used for the stem cell medium according to the present invention, a medium used for maintaining or proliferating the pluripotency (undifferentiated state) of stem cells, a medium used for culturing animal cells, and the like are generally used.
- Basic medium can be used.
- As such a basal medium commercially available culture media for various stem cells can also be used. Examples of the basal medium include Eagle's minimum essential medium (MEM), Dalveco's modified eagle medium (DMEM), ⁇ -Eagle's minimum essential medium ( ⁇ MEM), Iscove's modified Dalveco medium (IMDM), F-12 medium, and F-10 medium.
- MEM Eagle's minimum essential medium
- DMEM Dalveco's modified eagle medium
- ⁇ MEM ⁇ -Eagle's minimum essential medium
- IMDM Iscove's modified Dalveco medium
- F-12 medium F-12 medium
- F-10 medium F-10 medium.
- Examples thereof include DMEM / F12 medium, RPMI-1640 medium, Membrane cell basal medium (MSCBM), E8 (Essential 8) medium, TeSR-E8 medium, mTeSR1 medium, and a mixed medium thereof.
- the medium for stem cells according to the present invention may contain active ingredients such as amino acids, inorganic salts, vitamins and antibiotics, if necessary, in addition to the above-mentioned components, and only one of these active ingredients or Two or more types may be contained in combination.
- the active ingredient that can be contained in the medium for stem cells according to the present invention is a component that is known to enhance the survival efficiency and proliferation efficiency of stem cells, a component that is known to enhance the differentiation efficiency, and the like.
- various amino acids such as ascorbic acid and ⁇ -tocopherol, growth factors such as insulin and transferase, minerals such as sodium selenate, ethanolamine, Rock inhibitor, valproic acid, dimethylsulfoxide, dexamethasone, butyric acid
- physiologically active substances such as tricostatin A, GSK3 inhibitor, BMP inhibitor, Wnt inhibitor, and cytokines such as PDGF-BB, EGF, VEGF, TGF- ⁇ , FGF2 activin, and nogin.
- physiologically active substances such as tricostatin A, GSK3 inhibitor, BMP inhibitor, Wnt inhibitor, and cytokines such as PDGF-BB, EGF, VEGF, TGF- ⁇ , FGF2 activin, and nogin.
- cytokines such as PDGF-BB, EGF, VEGF, TGF- ⁇ , FGF2 activin, and nogin.
- the method for culturing stem cells according to the present invention is a method for efficiently proliferating the stem cells while maintaining the differentiation potential of the stem cells. That is, it is a culturing method for culturing stem cells stably and efficiently.
- the medium for stem cells according to the present invention can be preferably used.
- the method for culturing the stem cells can be carried out in the same manner as the conventional method for culturing stem cells, except that at least one of carboxymethyl cellulose and polyvinylpyrrolidone is contained as the water-soluble polymer compound.
- the culture conditions can generally be the culture conditions for culturing animal cells, and may be appropriately modified as necessary.
- the culture can be performed at a culture temperature of 30 to 40 ° C., a CO 2 concentration of 1 to 10% by volume, and an O 2 concentration of 0.1 to 25% by volume.
- a medium for stem cells was prepared.
- a medium having the same composition as the serum-free medium A described in Japanese Patent No. 5804385 was used except that CMC or PVP, which is a water-soluble compound, was added instead of polyvinyl alcohol (PVA).
- the serum-free medium A is a basal medium obtained by mixing DEME (Sigma, cat.no.D6046) and MCDB201 (Sigma, cat.no.M6770) at a ratio of 1: 1 with FGF, PDGF, TGF- ⁇ , and HGF. , EGF, phospholipids, fatty acids, PVA and other active ingredients are added (see Table 1 of Japanese Patent No. 5804385).
- the amount of albumin added to the serum-free medium A was 0.2 mg / mL at the final concentration.
- Example 1 in which CMC (Nacalai Tesque, sodium carboxymethyl cellulose, degree of etherification 0.59 to 0.85, cat.no.07326-95) was added instead of PVA in serum-free medium A.
- Example 11 was prepared by adding PVP (Nacalai Tesque, Polyvinylpyrrolidone K-90, weight average molecular weight 360,000, cat.no.28315-72) instead of PVA in serum-free medium A as a medium for stem cells of -10.
- the medium was used as a medium for stem cells of -14.
- ⁇ -NMN was further added to the stem cell media of Examples 8 to 10 and Examples 12 to 14.
- the medium consisting of only the serum-free medium A was used as the medium of Comparative Example 1.
- the amounts of CMC, PVP, and ⁇ -NMN added (final concentrations in the medium) in the stem cell media of each Example and Comparative Example are as shown in the table below.
- Proliferation performance test I A 96-well plate for cell culture was coated with 2.5 ⁇ g / cm 2 fibronectin (Corning, product number: 356008). Stem cells were seeded on this 96-well plate at 800 cells / well using the medium for stem cells of Examples 1 to 7 or Comparative Example 1 on a 100 ⁇ L scale, and allowed to stand under 37 ° C. and 5% CO 2 conditions. It was cultured.
- the stem cells used were human adipose tissue-derived mesenchymal stem cells (Lonza, product number: PT-5006, 1 donor). The medium was changed 1 day and 4 days after sowing, and the culture was continued for 6 days from the start of the culture.
- the above-mentioned CMC-content medium for stem cells (medium for stem cells of Examples 1 to 7 or Comparative Example 1) was used.
- the proliferation performance of stem cells was evaluated by the crystal violet staining method.
- the culture supernatant in the 96-well plate was removed, 0.99% paraformaldehyde (PFA) was added to each well, and the mixture was allowed to stand at 4 ° C. for 1 hour or longer.
- PFA paraformaldehyde
- MilliQ water was used as the ultrapure water.
- MilliQ water is ultrapure water produced by MilliQ's ultrapure water production equipment MilliQ.
- the stem cells were set to 5 ⁇ 10 3 cells / cm 2 and the medium for stem cells of Example 11 or Comparative Example 1 was used.
- Stem cells were cultured in the same manner as in Examples 1 to 7 above except that they were seeded on a 3 mL scale, and the proliferation performance of the stem cells was evaluated. The measurement results are shown in FIG.
- the culture supernatant of the culture dish is removed, washed with phosphate buffered saline [1 x PBS (-)], and then 1 x Triple select (Thermo Fisher, product number: 12563011) is added. Then, the cells were allowed to stand for 4 minutes under the conditions of 37 ° C. and 5% CO 2. The cells were then suspended by pipetting and detached from the culture dish to single. Then, the medium for each stem cell was added and centrifuged (1500 rpm, 5 min) to remove the supernatant, and then each medium was added to the precipitated cells and suspended.
- phosphate buffered saline [1 x PBS (-)]
- 1 x Triple select Thermo Fisher, product number: 12563011
- the suspension was cell-counted and seeded in a culture dish (60 mm dish) previously coated with fibronectin at a rate of 5 ⁇ 10 3 cells / cm 2.
- the medium was exchanged once every 2 or 3 days using each stem cell medium. The above passage was repeated 8 times.
- the cumulative number of divisions of the stem cells obtained through the above passages was calculated and graphed.
- the cumulative number of divisions was calculated by accumulating the number of stem cell divisions based on the number of initial seeds of stem cells and the number of cells counted for each passage.
- the proliferative performance of the stem cells in each example was evaluated according to the following evaluation criteria by comparison with the cumulative number of divisions of Comparative Example 1. According to the following evaluation criteria, when the cumulative number of divisions is one or more times higher than that of Comparative Example 1, it is evaluated as "higher proliferation performance than Comparative Example 1", and the cumulative number of divisions is the same as that of Comparative Example 1 or-(minus). ) Once, it was evaluated as "proliferation performance equivalent to that of Comparative Example 1". +: Proliferation performance equivalent to that of Comparative Example 1 was shown. ++: Higher proliferation performance than Comparative Example 1 was shown.
- Table 2 shows the evaluation results of this growth performance test II using the stem cell media of Examples 8 to 10 and Comparative Example 1.
- the measurement results of the cumulative number of divisions of stem cells in Example 8 and Comparative Example 1 are shown in FIG. 3 (a).
- the above-mentioned growth performance test II was carried out using the stem cell media of Examples 12 to 14 and Comparative Example 1.
- the evaluation results are shown in Table 3.
- the measurement results of the cumulative number of divisions of stem cells in Example 13 and Comparative Example 1 are shown in FIG. 3 (b).
- the collected stem cells were washed with 1 ⁇ PBS, each antibody shown below was added, and the cells were reacted at 4 ° C. for 30 minutes or longer. Then, the cells were washed again with 1 ⁇ PBS and flow cytometric analysis was performed using a flow cytometer (Becton Dickinson, model: FACS Calibur) to confirm the expression of cell surface antigens in the recovered stem cells. The flow cytometry analysis was performed according to the instruction manual of the flow cytometer.
- fetal bovine serum has a final concentration of 10%
- dexamethasone has a final concentration of 100 nM
- ascorbic acid to a final concentration of 25 ⁇ g / mL
- pyruvate to a final concentration of 1 mM to the cartilage differentiation-inducing medium ⁇ -MEM medium.
- fetal bovine serum has a final concentration of 10%
- IBMX has a final concentration of 0.5 mM
- dexamethasone has a final concentration of 1 ⁇ M
- indomethacin has a final concentration of 200 ⁇ M
- penicillin-streptomycin has a final concentration of 0.
- the supernatant of the 48-well plate for cell culture was removed, 0.99% PFA was added, and the cells were allowed to stand for 30 minutes to fix the cells. Then, PFA was removed, the cells were washed with water, a cell staining solution was added, and the cells were allowed to stand for 20 minutes to perform cell staining. After cell staining, the cells were washed with MilliQ water three times or more, and the state of cell staining was observed with a microscope. The observation image is shown in FIG.
- the stem cells cultured in the medium for each stem cell of Example 8 to which CMC was added and Example 13 to which PVP was added were compared with each other without CMC or PVP. Similar to the stem cells cultured in the medium of Example 1, the undifferentiated state was well maintained (see FIG. 4). Further, in the differentiation potential test II, the stem cells cultured in the stem cell media of Example 8 to which CMC was added and Example 13 to which PVP was added were the media of Comparative Example 1 to which neither CMC nor PVP was added. Similar to the stem cells cultured in, it was able to differentiate into osteoblasts, chondrocytes or fat cells (see FIG. 5).
- CMC or PVP did not change the differentiation potential of stem cells. From the results of the proliferation performance tests I and II and the differentiation potential tests I and II, it is shown that the medium for stem cells to which the CMC or PVP according to the present invention is added does not adversely affect the maintenance and proliferation of stem cells. It was shown that the proliferative potential can be improved while maintaining the differentiation potential of stem cells. As a result, stem cells can be cultured more stably and more efficiently.
- the medium for stem cells according to the present invention contains at least one of carboxymethyl cellulose and polyvinylpyrrolidone as a water-soluble polymer compound in the basal medium required for culturing stem cells, whereby good proliferation is maintained while maintaining the differentiation potential of stem cells. Has performance. Therefore, by using the stem cell medium, stem cells can be cultured more stably and more efficiently.
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Abstract
Description
特許文献1には、前記添加剤として用いられる7種の非必須アミノ酸が低減された培地が開示されている。特許文献2には、アルブミンとポリビニルアルコールとを培地に添加することが開示されている。また、特許文献3には、幹細胞のうち間葉系幹細胞の培養に、複数のサイトカインとともに、リン脂質や脂肪酸を含有する無血清培地を用いることが開示されている。
[1] 水溶性高分子化合物としてカルボキシメチルセルロース及びポリビニルピロリドンの少なくともいずれかを含有する、幹細胞用培地。
[2] 培地におけるカルボキシメチルセルロースの添加量が、終濃度0.001μg/mL~1mg/mLである、前記[1]に記載の幹細胞用培地。
[3] 培地におけるポリビニルピロリドンの添加量が、終濃度0.05μg/mL~2mg/mLである、前記[1]に記載の幹細胞用培地。
[4] 培地における組換えアルブミンの添加量が、終濃度0.05~1mg/mLで含有する、前記[1]~[3]のいずれかに記載の幹細胞用培地。
[5] さらにβ-ニコチンアミドモノヌクレオチドを含有する、前記[1]~[4]のいずれかに記載の幹細胞用培地。
[6] 前記[1]~[5]のいずれかに記載の幹細胞用培地を使用する、幹細胞の培養方法。
本発明の幹細胞用培地は、幹細胞の培養に使用される。「幹細胞」とは、自己複製能を有し、且つ多様な細胞腫へ分化可能な分化能を備える未分化細胞である。幹細胞としては、例えばES細胞(胚性幹細胞)、iPS細胞(人工多能性幹細胞)等の多能性幹細胞の他、間葉系幹細胞、造血幹細胞、神経幹細胞、皮膚幹細胞等の体性幹細胞が挙げられる。
本発明に係る幹細胞用培地を使用して培養する幹細胞としては、動物由来の幹細胞が好ましく、哺乳類に由来する幹細胞がより好ましく、ヒトに由来する幹細胞がさらに好ましい。
分化能を維持しながらのより良好な増殖性能が得られる観点から、本発明の幹細胞用培地を使用して培養される幹細胞は、外胚葉、中胚葉、及び内胚葉のいずれかの細胞に分化しうる幹細胞であることが好ましく、中胚葉細胞に分化しうる幹細胞であることがより好ましい。斯かる幹細胞としては間葉系幹細胞等が挙げられる。
本発明に用いられるCMCは、細胞培養の培地に従来用いられているもの等を特に制限なく用いることができる。また、カルボキシメチルセルロース塩を用いてもよい。
増殖性能をより向上させる観点から、CMCは、無水グルコース単位当たりのカルボキシメチルエーテル基の置換度(エーテル化度)が0.6~0.8であることが好ましい。そのようなCMCは市販されており、例えばナカライテスク社製、カルボキシメチルセルロースナトリウム(cat.no.07326-95)や富士フィルム和光純薬社製、カルボキシメチルセルロース(cat.no.11676-85)等の市販品を使用することができる。
本発明に用いられるPVPは、細胞培養の培地に従来用いられているもの等を特に制限なく用いることができる。増殖性能をより向上させる観点から、PVPの重量平均分子量が40,000から1,200,000であることが好ましい。そのようなPVPは市販されており、例えばナカライテスク社製、ポリビニルピロリドンK-30(cat.no.06306-72)やナカライテスク社製、ポリビニルピロリドンK-90(cat.no.28315-72)等の市販品を使用することができる。
幹細胞の分化能を維持しながら増殖性能をより向上させる観点から、本発明に係る幹細胞用培地におけるカルボキシメチルセルロースの添加量は、終濃度0.001μg/mL~1mg/mLであることが好ましく、終濃度0.02μg/mL~0.5mg/mLであることがより好ましい。
上記と同様の観点から、本発明に係る幹細胞用培地におけるポリビニルピロリドンの添加量は、終濃度0.05μg/mL~2mg/mLであることが好ましく、終濃度0.1μg/mL~1mg/mLであることがより好ましい。
増殖性能をより向上させる観点から、本発明の幹細胞用培地は、アルブミンを含有していることが好ましく、哺乳動物のアルブミンを含有していることがより好ましく、培養する幹細胞と同じ種のアルブミンであるのがさらに好ましい。また、当該アルブミンは組換えアルブミンであることが好ましい。例えば、ヒト由来の幹細胞を培養するには、ヒト由来の組換えアルブミンを用いることが好適である。
本発明に係る幹細胞用培地は、前述のカルボキシメチルセルロース及びポリビニルピロリドンの少なくともいずれかを含有させることにより、コストが掛かるアルブミンの含有量を優位に低減することができる。このため、本発明に係る幹細胞用培地におけるアルブミンの添加量を、終濃度0.05~1mg/mLの範囲とすることができ、好ましくは終濃度0.05~1mg/mL、より好ましくは0.1~0.5mg/mLの範囲とすることができる。
増殖性能をより向上させる観点から、アルブミンは、担持する脂肪酸が低減されたものが好ましい。斯かるアルブミンとして、活性炭処理等の脱脂肪酸処理が施されたものを適宜用いることができる。脱脂肪酸処理は、例えば国際公開2014-192938号公報に記載の方法により行うことができる。
また、β-NMNは広く生体に存在する成分であるため、動物や、植物、微生物等の天然原料から抽出・精製することによって得られたβ-NMNを有効成分として用いることもできる。また、市販の精製されたβ-NMNを使用してもよい。
上記の有効成分は、幹細胞の生存効率や増殖効率を高めることや、幹細胞の未分化状態を維持する作用を高めることが知られている。
本発明に係る幹細胞の培養方法は、本発明に係る幹細胞用培地を好適に用いることができる。当該幹細胞の培養方法は、水溶性高分子化合物としてカルボキシメチルセルロース及びポリビニルピロリドンの少なくともいずれかを含有させる点以外は、常法の幹細胞の培養方法と同様の方法により行うことができる。
また、培養条件は、一般的に動物細胞を培養する培養条件とすることができ、必要に応じて適宜改変してもよい。例えば、培養温度が30~40℃、CO2濃度が1~10体積%、O2濃度が0.1~25体積%で培養できる。
幹細胞用培地を用意した。斯かる培地は、ポリビニルアルコール(PVA)の代わりに水溶性化合物であるCMC又はPVPを添加した点以外は、特許第5804385号公報に記載の無血清培地Aと同じ組成のものを用いた。前記無血清培地Aは、DEME(Sigma社、cat.no.D6046)及びMCDB201(Sigma社、cat.no.M6770)を1:1で混合した基礎培地に、FGF、PDGF、TGF-β、HGF、EGF、リン脂質、脂肪酸、PVA等の有効成分を加えたものである(特許第5804385号公報の表1参照)。無血清培地Aにおけるアルブミンの添加量は、終濃度0.2mg/mLとした。詳細には、無血清培地AにおけるPVAの代わりにCMC(ナカライテスク社、カルボキシメチルセルロースナトリウム、エーテル化度0.59~0.85、cat.no.07326-95)を加えたものを実施例1~10の幹細胞用培地とし、無血清培地AにおけるPVAの代わりにPVP(ナカライテスク社、ポリビニルピロリドンK-90、重量平均分子量360,000、cat.no.28315-72)を加えたものを実施例11~14の幹細胞用培地とした。また、実施例8~10及び実施例12~14の幹細胞用培地には、さらにβ-NMNを添加した。前記無血清培地Aのみからなる培地を、比較例1の培地とした。
各実施例及び比較例の幹細胞用培地におけるCMC、PVP、及びβ-NMNの各添加量(培地中の終濃度)は下記表のとおりである。
細胞培養用96ウェルプレートを2.5μg/cm2のフィブロネクチン(コーニング社、製品番号:356008)でコーティングした。この96ウェルプレートに、幹細胞を800cells/ウェルとなるように且つ実施例1~7又は比較例1の幹細胞用培地を用いて100μLスケールで播種し、37℃、5%CO2条件下で静置培養した。使用した幹細胞は、ヒト脂肪組織由来間葉系幹細胞(Lonza社、品番号:PT-5006、1ドナー)であった。播種1日後及び4日後に培地交換し、培養開始から6日間培養を継続した。この培養には、上述したCMC含有量の幹細胞用培地(実施例1~7又は比較例1の幹細胞用培地)を用いた。
次いで、クリスタルバイオレット染色法により、幹細胞の増殖性能を評価した。先ず、96ウェルプレートにおける培養上清を除き、各ウェルに0.99%パラホルムアルデヒド(PFA)を添加して4℃に1時間以上静置した。その後、各ウェルについて、PFAを除き、超純水及び100%エタノールそれぞれで洗浄した。超純水としてはMilliQ水を用いた。MilliQ水は、ミリポア社の超純水製造装置MilliQで製造した超純水である。洗浄後、2.5%クリスタルバイオレットを各ウェルに添加し、室温で10分間静置した。次いで、各ウェルをさらに3回ずつ水道水で洗浄した後、ウェルを乾燥させ、マイクロプレートリーダーにて595nm波長の吸光度を測定した。以上の測定を独立した6ウェルで行い、その有意性をT検定により解析した。測定結果を図1に示す。
2.5μg/cm2のフィブロネクチンでコーティングした培養用ディッシュ(60mmディッシュ)に、増殖性能試験Iで用いた幹細胞と同じものを5×103cells/cm2となるように且つ実施例8~10又は比較例1の幹細胞用培地を用いて3mLスケールで播種し37℃、5%CO2条件下で静置培養した。播種1日後に培地交換し、以降2日又は3日に一度、各幹細胞用培地を用いて培地交換を実施した。この培養用ディッシュ内の細胞密度が約80~90%のサブコンフルエントになった時点で、継代を行った。より具体的には、培養用ディッシュの培養上清を除き、リン酸緩衝食塩水〔1×PBS(-)〕で洗浄した後、1×Tryple select(Thermo Fisher社、製品番号:12563011)を加えて、37℃、5%CO2の条件下で4分静置させた。次いで、ピペッティングによって懸濁し、培養用ディッシュから剥離した細胞をシングル化した。次いで、各幹細胞用培地を加えて遠心し(1500rpm,5min)、上清を除いた後、沈殿した細胞に前記各培地を加えて懸濁した。この懸濁液についてセルカウントを行い、予め前記フィブロネクチンでコーティングした培養用ディッシュ(60mmディッシュ)に5×103cells/cm2となるように播種した。このように継代を行った培養用ディッシュに対し、各幹細胞用培地を用いて、2日又は3日に一度の頻度で培地交換を実施した。上記の継代を8回繰り返した。
+ :比較例1と同等の増殖性能が示された。
++:比較例1よりも高い増殖性能が示された。
実施例12~14及び比較例1の各幹細胞用培地を用いて上述した本増殖性能試験IIを実施した。評価結果を表3に示す。また、実施例13及び比較例1における幹細胞の累積分裂回数の測定結果を図3(b)に示す。
2.5μg/cm2のフィブロネクチンでコーティングした培養用ディッシュ(60mmディッシュ)に、増殖性能試験Iで用いた幹細胞と同じものを5×103cells/cm2となるように且つ実施例8、実施例13又は比較例1の幹細胞用培地を用いて3mLスケールで播種し37℃、5%CO2条件下で静置培養した。培養用ディッシュ内の細胞密度が約80~90%のサブコンフルエントになった時点で、前記継代と同様の操作を行い、幹細胞を培養用ディッシュから剥離して、1×105cellsずつ1.5mLチューブに回収した。回収した幹細胞を1×PBSで洗浄し、下記に示す各抗体を添加して4℃で30分以上反応させた。その後、1×PBSで再度洗浄し、フローサイトメーター(ベクトン ディッキンソン社、機種:FACS Calibur)を用いてフローサイトメトリー分析を行い、回収した幹細胞の細胞表面抗原の発現を確認した。フローサイトメトリー分析は、前記フローサイトメーターの取扱説明書に従って行った。
・FITC Mouse IgG1, κ isotype Ctrl(BIOLEGEND社、製品番号:400108)
・PE Mouse IgG1, κ isotype Ctrl(BIOLEGEND社、製品番号:400112)
・PerCP/Cyanine5.5 Mouse IgG1, κ isotype Ctrl(BIOLEGEND社、製品番号:400150)
・FITC anti-human CD90(Thy1)(BIOLEGEND社、製品番号:328108)
・PE anti-human CD105(BIOLEGEND社、製品番号:323206)
・PerCP/Cyanine5.5 anti-human CD73(Ecto-5’-nucleotidase)(BIOLEGEND社、製品番号:344014)
・FITC anti-human CD235a(BIOLEGEND社、製品番号:349105)
・PE anti-human CD45(BIOLEGEND社、製品番号:304008)
・PerCP/Cyanine5.5 anti-human CD31(BIOLEGEND社、製品番号:303131)
上記の抗体が結合するタンパク質のうち、細胞表面に発現するCD90、CD105、及びCD73が間葉系幹細胞のマーカータンパク質であり、CD235a、CD45、及びCD31が間葉系幹細胞のネガティブマーカータンパク質である。
実施例8、実施例13又は比較例1の幹細胞用培地を用いて増殖性能試験IIの継代を4回繰り返した幹細胞について、細胞培養用48ウェルプレートに2×104cells/ウェルで播種し、細胞密度が約100%コンフルエントになるまで培養させた。次いで、骨芽細胞、軟骨細胞及び脂肪細胞の各細胞に分化させるための分化培地を用いて培地交換し、約3週間の分化誘導を行った。分化誘導下の幹細胞は、37℃、5%CO2条件下で静置培養した。
・骨分化誘導用培地
α-MEM培地に、ウシ胎児血清を終濃度10%、デキサメタゾンを終濃度10nM、βグリセロリン酸を終濃度10mM、アスコルビン酸を終濃度50μg/mL、及びペニシリン-ストレプトマイシンを終濃度0.1%となるように添加。
・軟骨分化誘導用培地
α-MEM培地に、ウシ胎児血清を終濃度10%、デキサメタゾンを終濃度100nM、アスコルビン酸を終濃度25μg/mL、及びピルビン酸を終濃度1mMとなるように添加。
・脂肪分化誘導用培地
DMEM-High Glucose培地に、ウシ胎児血清を終濃度10%、IBMXを終濃度0.5mM、デキサメタゾンを終濃度1μM、インドメタシンを終濃度200μM、及びペニシリン-ストレプトマイシンを終濃度0.1%となるように添加。
・アリザリンS染色液(メルクミリポア社、製品番号:TMS_008_C)
アリザリンS染色液は、カルシウムと結合する色素を含んでいるので、当該染色液の染色の程度に基づき、骨芽細胞への分化を評価することができる。
・アルシアンブルー(ナカライテスク社、製品番号:37154-44)
アルシアンブルーは、アグリカンやグリコサミノグリカン等の軟骨細胞の細胞外マトリクスを染色することができるので、その染色の程度に基づき、軟骨細胞への分化を評価することができる。
・オイルレッドO(ナカライテスク社、製品番号:25633-92)
オイルレッドOは、脂肪滴を染色することができるので、その染色の程度に基づき、脂肪細胞への分化を評価することができる。
また、分化能試験IIでは、CMCが添加された実施例8及びPVPが添加された実施例13の各幹細胞用培地で培養された幹細胞は、CMCもPVPも添加されていない比較例1の培地で培養された幹細胞と同様に、骨芽細胞、軟骨細胞又は脂肪細胞に分化することができた(図5参照)。即ち、CMC又はPVPにより、幹細胞が有する分化能は変化しないことが示された。
前記増殖性能試験I及びII並びに前記分化能試験I及びIIの結果より、本発明に係るCMC又はPVPが添加された幹細胞用培地は、幹細胞の維持や増殖に悪影響を与えることがないことが示されたとともに、幹細胞の分化能を維持しながら増殖性を向上させることができることが示された。これにより、幹細胞をより安定的、より効率的に培養することができる。
Claims (6)
- 水溶性高分子化合物としてカルボキシメチルセルロース及びポリビニルピロリドンの少なくともいずれかを含有する、幹細胞用培地。
- 培地におけるカルボキシメチルセルロースの添加量が、終濃度0.001μg/mL~1mg/mLである、請求項1に記載の幹細胞用培地。
- 培地におけるポリビニルピロリドンの添加量が、終濃度0.05μg/mL~2mg/mLである、請求項1に記載の幹細胞用培地。
- 培地における組換えアルブミンの添加量が、終濃度0.05~1mg/mLである、請求項1~3のいずれか1項に記載の幹細胞用培地。
- さらにβ-ニコチンアミドモノヌクレオチドを含有する、請求項1~4のいずれか1項に記載の幹細胞用培地。
- 請求項1~5のいずれか1項に記載の幹細胞用培地を使用する、幹細胞の培養方法。
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