WO2014208295A1 - 多能性幹細胞の増殖促進因子のスクリーニング法 - Google Patents
多能性幹細胞の増殖促進因子のスクリーニング法 Download PDFInfo
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Definitions
- the present invention relates to a highly efficient feeder-free serum-free culture technique for cell culture and a screening method based on the culture technique.
- pluripotent stem cells such as human ES cells (hESC) and human iPS cells (hiPSC) has increased the possibility of practical application of regenerative medicine. Since these cells have the ability to proliferate indefinitely and have the ability to differentiate into various cells, regenerative medicine using pluripotent stem cells is a treatment method for intractable diseases, lifestyle-related diseases, etc. Is expected to fundamentally change From pluripotent stem cells, it has already been possible to induce differentiation into various types of cells such as nerve cells, cardiomyocytes, blood cells, and retinal cells in vitro.
- human pluripotent stem cells such as hESC and hiPSC have been mainly cultured on feeder cell layers using mouse-derived fetal fibroblasts MEF (mouse embryonic fibroblast).
- Feeder cells have a function of supplying stem cells with growth factors useful for maintaining and culturing human pluripotent stem cells.
- the activity that enables maintenance culture of human pluripotent stem cells has been reported in various human cell types in addition to MEF (Non-Patent Documents 1 to 4).
- Non-Patent Documents 1 to 4 Non-Patent Documents 1 to 4).
- it takes time to prepare feeder cells at the time of culture and there is a risk that the feeder cells are mixed with stem cells, and therefore, a safer alternative method is required to be developed.
- Non-Patent Document 5 a method in which a medium supplemented with serum such as FBS or a serum substitute is previously conditioned with MEF (MEF-CM) or MEF is chemically immobilized.
- MEF-CM a medium supplemented with serum
- MEF-CM a serum substitute
- MEF chemically immobilized.
- Non-Patent Document 6 a method of using various human-derived cells (fibroblasts, placental cells, bone marrow cells, endometrial cells, etc.) as live feeder cells without using heterogeneous cells has been reported.
- bovine serum in order to culture human pluripotent stem cells, bovine serum, KNOCKOUT TM SR (Knockout Serum Replacement: an additive capable of culturing ES / iPS cells by being used instead of serum), etc.
- KNOCKOUT TM SR Knockout Serum Replacement: an additive capable of culturing ES / iPS cells by being used instead of serum
- bovine serum in order to culture human pluripotent stem cells, bovine serum, KNOCKOUT TM SR (Knockout Serum Replacement: an additive capable of culturing ES / iPS cells by being used instead of serum), etc.
- BSE bovine spongiform encephalopathy
- human-derived serum is also used in some cases, it is not suitable for practical use because it is limited in use and limited in quantity.
- Non-patent Documents 7 and 8 development of a completely synthetic medium for culturing without using MEF is also in progress.
- Analysis of functional proteins for MEF secretions has also been performed (Non-patent Document 9).
- An object of the present invention is to provide a highly efficient feeder-free and serum-free culture technique for cell culture. It is another object of the present invention to provide a method for screening a growth promoting factor applying the culture technique.
- the present inventors have conditioned a serum-free medium that can be used for culturing pluripotent stem cells in advance with feeder cells without co-culturing with feeder cells. Finding that a medium capable of stably culturing pluripotent stem cells and improving proliferation can be prepared, and screening the prepared medium enables efficient identification of growth promoting factors for pluripotent stem cells The inventors have found the technology and have completed the present invention.
- the present inventors have also found that the proliferation of pluripotent stem cells can be further enhanced by cultivating pluripotent stem cells in a serum-free medium containing a predetermined component, followed by feeder-free culture. .
- the present invention includes the following.
- a method for screening a growth promoting factor for pluripotent stem cells a) culturing feeder cells in a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate but not serum and serum replacement, and recovering the conditioned medium produced; and b ) Detecting a growth promoting factor for pluripotent stem cells contained in the collected conditioned medium, Including methods.
- the serum-free medium may be a DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.
- this screening method it is also preferable to culture feeder cells by adding a growth factor to the serum-free medium.
- the growth factor added in this screening method is preferably FGF2 and / or TGF- ⁇ 1.
- the feeder cells can be mouse embryonic fibroblasts.
- the pluripotent stem cell is preferably an ES cell or an iPS cell.
- a method for preparing a culture medium for pluripotent stem cell culture
- the serum-free medium may be a DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.
- the preparation method in one embodiment, it is also preferable to culture feeder cells by adding a growth factor to the serum-free medium.
- the growth factor added in this preparation method is preferably FGF2 and / or TGF- ⁇ 1.
- the feeder cells can be mouse embryonic fibroblasts.
- the pluripotent stem cell is preferably an ES cell or an iPS cell.
- Pluripotent in a conditioned medium produced by culturing feeder cells in a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate but not serum and serum replacement A method for proliferating pluripotent stem cells, comprising culturing stem cells without feeder.
- the serum-free medium may be a DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.
- feeder cells it is also preferable to culture feeder cells by adding a growth factor to the serum-free medium.
- feeder cells are cultured without adding a growth factor to the serum-free medium, and feeder-free culture of pluripotent stem cells is performed with a growth factor added to the conditioned medium.
- the growth factor added in this growth method is preferably FGF2 and / or TGF- ⁇ 1.
- the feeder cells can be mouse embryonic fibroblasts.
- the pluripotent stem cells are preferably ES cells or iPS cells.
- the serum-free medium preferably does not contain albumin.
- the proliferation of pluripotent stem cells in feeder-free culture can be improved.
- FIG. 1 is a photograph showing the effect of serum-free MEF-conditioned medium on iPS cell growth in feeder-free culture.
- A is proliferative (+++) in conditioned medium prepared from basal medium (serum-free medium) A + ITS + FGF2 + TGF- ⁇ 1
- B is proliferative (+) in basal medium (serum-free medium) A + ITS + FGF2 + TGF- ⁇ 1 (no acclimation)
- C is basal Proliferation (++) in conditioned medium prepared from medium (serum-free medium) A + ITS (after acclimation, FGF2 + TGF- ⁇ 1 added)
- D is conditioned medium prepared from basal medium (serum-free medium) A (after conditioned, ITS + FGF2 + TGF- ⁇ 1)
- E is basal medium (serum-free medium)
- B is conditioned medium prepared from B + ITS (after acclimation, FGF2 + TGF- ⁇ 1 added) ( ⁇ )
- FIG. 2 is a photograph showing the growth results of human iPS cells in a serum-free conditioned medium in plastic culture dishes coated with various culture substrates.
- a and B used Matrigel (registered trademark), C and D used vitronectin, and E and F used PCM-DM.
- A, C, and E were serum-free media that were not conditioned
- B, D, and F were MEF-conditioned media.
- FIG. 3 shows a table comparing the proliferation of human iPS cells in culture using a serum-free medium not conditioned with MEF or a serum-free medium not conditioned.
- FIG. 3 shows a table comparing the proliferation of human iPS cells in culture using a serum-free medium not conditioned with MEF or a serum-free medium not conditioned.
- FIG. 4 shows the results of analyzing the expression of undifferentiated markers by flow cytometry for human iPS cells cultured in a serum-free medium conditioned with or without MEF.
- FIG. 5 shows the growth after human iPS cells cultured on feeder using serum-replacement-containing medium (B to E) or serum-free medium (G to J) containing a predetermined component are transferred to feeder-free culture. It is a photograph which shows the result of having compared.
- the level of proliferation is indicated by the number of ⁇ (not proliferating) or +.
- the present invention relates to a method for preparing a medium suitable for growing pluripotent stem cells in feeder-free culture by acclimating a serum-free medium with feeder cells.
- a “pluripotent stem cell” is a cell having multipotency (pluripotency) that can be differentiated into all types of cells constituting a living body, and is cultured in vitro. Refers to cells that can continue to grow indefinitely while maintaining pluripotency.
- pluripotent stem cells to be proliferated in the present invention include, for example, embryonic stem cells (ES cells), EG cells that are pluripotent stem cells derived from fetal primordial germ cells (Shamblott MJ et al., Proc. Natl Acad. Sci. USA.
- iPS cells induced pluripotent stem cells
- the pluripotent stem cell grown in the present invention is particularly preferably an ES cell or an iPS cell.
- the ES cell is a cultured cell derived from an undifferentiated cell collected from an inner cell mass existing in an early embryo called a blastocyst.
- An iPS cell is a cultured cell to which a somatic cell is initialized to an undifferentiated state by introducing an reprogramming factor into the somatic cell, thereby imparting pluripotency.
- reprogramming factors include Oct family genes (eg, Oct3 / 4) and Klf family genes (eg, Klf4), and Myc family genes (eg, c-Myc) and / or Sox family genes (eg, Sox2) are used. be able to.
- Pluripotent stem cells may be derived from any animal, for example, rodents such as mice, rats, hamsters, primates such as humans, gorillas, chimpanzees, dogs, cats, rabbits, cows, horses Although it may be derived from livestock such as sheep and goats or mammals such as pets, human-derived pluripotent stem cells are particularly preferred.
- pluripotent stem cells including ES cells and iPS cells commercially available products or cells that have been distributed may be used, or newly prepared cells may be used.
- Stimulus-triggered acquisition cells Stimulus-triggered acquisition cells (Stimulus-Acquisition of Pluripotency cells: STAP cells) may also be used as pluripotent stem cells.
- STAP cells are cells in which animal cells are given strong stimulation (stress) from the outside to have differentiation pluripotency (Nature, 505, 641-647, (2014)).
- a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, and not serum and serum replacement is used for conditioning.
- serum refers to serum derived from any animal (eg, human, cow, horse, goat, etc.).
- a “serum replacement” is a reagent used for maintaining and culturing cells in an undifferentiated state as a substitute for serum (FBS, etc.) in the culture of ES cells and iPS cells.
- KNOCKOUT TM SR KnockOut TM Serum Replacement (KSR); GIBCO
- StemSure registered trademark Serum Replacement (SSR; Wako Pure Chemical Industries), N2 supplement (Wako Pure Chemical Industries), and the like.
- This serum-free medium can be prepared using any liquid medium for animal cell culture that does not contain serum and serum substitutes as a basal medium.
- BME medium BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199M medium, EMEM medium, EMag medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / A medium such as Nutrient Mixture F-12 Ham)) can be used, but is not particularly limited.
- L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate may be added to the basal media.
- a serum-free medium is prepared using a liquid medium that does not contain serum and serum replacement, to which at least one of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate is added in advance. You can also.
- a serum-free medium may be prepared by adding components not included in the medium among L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate to the medium.
- a serum-free medium may be prepared by adding L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate to the medium including the components contained therein.
- CHO-S-SFM II manufactured by GIBCO BRL
- Hybridoma-SFM manufactured by GIBCO BRL
- eRDF Dry Powdered Media manufactured by GIBCO BRL
- UltraCULTURE TM manufactured by BioWhittaker
- UltraDOMA TM manufactured by BioWhittaker
- UltraCHO TM manufactured by BioWhittaker
- UltraMDCK TM manufactured by BioWhittaker
- STEMPRO registered trademark
- hESC SFM manufactured by Life Technologies
- mTeSR1 manufactured by Veritas
- TeSR2 manufactured by Veritas
- Essential 8 TM medium manufactured by Life Technologies in which the protein component is limited to a small part can also be suitably used.
- a preferred example of the serum-free medium is a DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.
- the medium used for the preparation of the serum-free medium in the present invention may contain a fatty acid, a collagen precursor, a trace element, 2-mercaptoethanol, 3 ′ thiolglycerol, or an equivalent thereof.
- the content of the protein component is preferably as low as possible.
- the serum-free medium used in the present invention preferably does not contain albumin. This is because albumin is often added to a serum-free medium, but there are concerns about problems such as large variations in quality from lot to lot.
- the medium used for the preparation of a serum-free medium in the present invention preferably has a known composition. For example, when a growth promoting factor for pluripotent stem cells is screened from a conditioned medium, the medium composition is preferably known.
- L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate may be added to the animal cell culture medium in the form of a solution, derivative, salt, or mixed reagent. It can.
- L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-phosphate ascorbate.
- Selenium may be added to the medium in the form of selenite (such as sodium selenite).
- Insulin and transferrin may be of natural origin isolated from tissues or sera of animals (preferably, humans, mice, rats, cows, horses, goats, etc.) or recombinants prepared by genetic engineering. It may be a protein.
- Insulin, transferrin, and selenium may be added to the medium in the form of the reagent ITS (insulin-transferrin-selenium).
- ITS is an additive for promoting cell growth, including insulin, transferrin, and sodium selenite.
- the serum-free medium used for acclimation in the present invention contains fatty acids or lipids, amino acids (for example, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like. You may contain.
- 2-mercaptoethanol when 2-mercaptoethanol is included, its concentration is not limited as long as it is suitable for culturing stem cells, but may be, for example, about 0.05 to 1.0 mM, preferably about 0.1 to 0.5 mM.
- the medium is acclimated by culturing feeder cells in the serum-free medium as described above.
- the feeder cells are preferably used for acclimation after inactivating mitosis by mitomycin treatment, ⁇ -irradiation treatment or the like.
- the feeder cells used in the present invention are cells that can be used for culturing pluripotent stem cells on a feeder cell layer (on-feeder culture).
- the feeder cells may be cells such as fibroblasts, placental cells, bone marrow cells, endometrial cells derived from mammalian embryos or tissues such as humans, mice, rats, and cows.
- feeder cells include mouse embryonic fibroblast MEF or STO cell lines, STO cell derivatives (for example, SNL cells stably incorporating a neomycin resistance gene expression vector and an LIF expression vector), etc. However, it is not limited to these.
- the serum-free medium used for acclimation with feeder cells may contain a growth factor or may not contain a growth factor. Regardless of whether the serum-free medium contains or does not contain growth factors, the serum-free medium does not need to contain growth factors when acclimated, but the serum-free medium contains growth factors. If not, it is more preferable to add a growth factor for acclimation.
- Growth factors include, but are not limited to, FGF2 (Basic fibroblast growth factor), TGF- ⁇ 1 (Transforming growth factor- ⁇ 1), MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF and IGFBP Preferably one or more selected from the group consisting of -7, for example FGF2 and / or TGF- ⁇ 1. Particularly preferred growth factors are FGF2 and / or TGF- ⁇ 1.
- Conditioning of the medium by the feeder cells can be carried out by replacing the medium in the culture vessel with the above serum-free medium after culturing the feeder cells for proliferation.
- the culture of feeder cells can be performed by a conventional method. Culturing in a serum-free medium for conditioning can be performed at a temperature of 4 to 45 ° C., for example, 25 to 40 ° C., for 1 to 72 hours, for example, 8 to 36 hours. This culture is also preferably carried out at a CO 2 concentration of 4 to 10%, for example 5%.
- the culture vessel is not particularly limited as long as it is a vessel that can be used for cell culture.
- a flask a flask for tissue culture, a dish, a petri dish, a culture dish, a multi-dish, a microplate, a microwell plate, a multiplate
- Examples include multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, hollow fiber incubators.
- feeder cells can be cultured in a serum-free medium, and growth-promoting factors and the like can be secreted from the feeder cells into the medium to acclimate the serum-free medium.
- the conditioned medium thus produced can be separated from the feeder cells and recovered by a conventional method.
- the conditioned medium may be collected by filtering and / or centrifuging, for example, by centrifuging at 1000 rpm for 5 minutes to separate the liquid medium from the feeder cells and collecting it. After the conditioned medium is collected, the culture in a serum-free medium for acclimation can be further repeated (for example, 2 to 10 times).
- conditioned medium preparation procedure monolayer MEFs are cultured until confluent, treated with 10 ⁇ g / ml mitomycin C, then the cells are detached with a cell detachment solution such as Trypsin-EDTA, and the recovered MEF was seeded on a culture dish at a cell density of 3-5 ⁇ 10 5 cells / 60 mm dish and cultured for 1-2 days. After replacing the medium in the culture dish with the above serum-free medium, every 24 hours The conditioned medium can also be prepared by collecting the liquid medium.
- a cell detachment solution such as Trypsin-EDTA
- the obtained conditioned medium can be suitably used as a culture medium for pluripotent stem cells, particularly for feeder-free serum-free and serum-free substitute culture.
- “feeder-free serum-free serum-free culture” refers to culture without a feeder cell layer (feeder-free culture), which is performed in a medium that does not contain serum or serum substitutes. Means culture.
- the growth factor or medium component does not contain a heterogeneous component for pluripotent stem cells, it can be particularly preferably used as a heterogeneous component-free medium (xenofree medium).
- the present invention also relates to a method for preparing such a conditioned medium for pluripotent stem cell culture, and a conditioned medium for pluripotent stem cell culture obtained by this method.
- pluripotent stem cells can be cultured in a feeder-free manner using the conditioned medium produced as described above.
- the proliferation of pluripotent stem cells in feeder-free culture can be significantly improved.
- the present invention also relates to a method for proliferating pluripotent stem cells, comprising culturing pluripotent stem cells in a conditioned medium as described above in a feeder-free manner.
- Pluripotent stem cells cultured in a conditioned medium can be maintained and cultured in advance by a conventional method.
- the pluripotent stem cells that have been cultured for maintenance are preferably dissociated from the culture vessel with a dissociation solution such as a collagenase solution, and collected in a small mass of about several tens, for example, about 20 to 50.
- a dissociation solution such as a collagenase solution
- the recovered pluripotent stem cells are incubated in a gelatin-coated culture vessel to adhere the MEF to the culture vessel and float in the medium. The MEF can be removed by collecting the.
- the pluripotent stem cells thus prepared are preferably seeded in a culture vessel coated with a culture substrate that serves as a cell scaffold.
- the culture vessel is similar to that described for the preparation of the conditioned medium.
- the culture substrate is not particularly limited as long as it can be used for cell culture.
- Matrigel registered trademark
- laminin laminin-511, laminin-511, produced from gelatin
- Engelbreth-Holm-Swarm (EHS) mouse sarcoma Laminin-111, laminin-332, etc.
- fibronectin vitronectin
- collagen E-cadherin
- synthetic peptides synthetic polymers, etc.
- MEF extracellular matrix derived from human serum or decidual mesenchymal cells (PCM-CM) Etc.
- a hydrogel such as a temperature-sensitive hydrogel having 2- (diethylamino) ethyl acrylate as a basic skeleton can also be used as a culture substrate (Zhang (etZal., Nature Communications, (2013) 4). , Article number: 1335).
- the coating of culture vessels with these culture substrates is well known to those skilled in the art and can be performed by conventional methods.
- the culture container can be coated by placing a culture substrate solution (for example, vitronectin solution) in the culture container and incubating for a certain time (for example, 1 hour).
- the culture of pluripotent stem cells in the above-mentioned conditioned medium is not limited, but is preferably performed at 20 to 40 ° C., for example, 35 to 40 ° C., for 1 to 7 days, for example, 1 to 24 hours. Just do it. Cultivation of pluripotent stem cells in a conditioned medium is also preferably performed at a CO 2 concentration of 4 to 10%, for example 5%.
- the culture of pluripotent stem cells may involve passage.
- the proliferation of the pluripotent stem cells cultured in this manner is remarkably improved as compared with the case where an unconditioned medium is used.
- the number of pluripotent stem cells grown in a conditioned medium is preferably 10 times or more, more preferably 100 times or more, and even more preferably 200, as compared to the case where an unconditioned medium is used. More than double, for example, 250 to 300 times.
- the increase in the number of cells can be based on, for example, a value measured after 5 passages.
- pluripotent stem cells cultured in this conditioned medium can maintain an undifferentiated state.
- the undifferentiated state of pluripotent stem cells can be confirmed by the expression of undifferentiation markers (for example, genes or proteins such as SSEA3, SSEA4, Tra1-60, Tra1-81, Oct4, NANOG, and SOX2).
- the conditioned medium obtained as described above contains a substance (growth promoting factor for pluripotent stem cells) secreted from feeder cells such as MEF that can promote the proliferation of pluripotent stem cells in an undifferentiated state. Including. Therefore, the present invention can be further subjected to screening for the conditioned medium obtained as described above to identify such a growth promoting factor. In this screening, a growth promoting factor can be identified by detecting a growth promoting factor for pluripotent stem cells contained in the conditioned medium. That is, the present invention screens a growth promoting factor for pluripotent stem cells, which comprises collecting the conditioned medium produced as described above and detecting a growth promoting factor for pluripotent stem cells contained in the collected conditioned medium. A method is also provided.
- the growth promoting factor for pluripotent stem cells may be a protein or nucleic acid (RNA or the like), or may be an amino acid, a peptide, a sugar chain, or a low molecular compound such as a metabolite.
- the collected conditioned medium it is preferable to separate and / or purify the collected conditioned medium by any method to identify the growth promoting factor.
- any method to identify the growth promoting factor For example, two-dimensional electrophoresis, isoelectric focusing, electrophoresis such as SDS-PAGE, high performance liquid chromatography (HPLC), ion exchange chromatography, chromatography such as affinity chromatography, matrix-assisted laser desorption ionization / Separation, purification, and identification of growth-promoting factors using mass spectrometry such as time-of-flight mass spectrometry (MALDI / TOFMS), liquid chromatography / tandem mass spectrometry (LC-MS / MS), etc. it can.
- mass spectrometry such as time-of-flight mass spectrometry (MALDI / TOFMS), liquid chromatography / tandem mass spectrometry (LC-MS / MS), etc.
- components separated or purified from the conditioned medium and / or identified in the culture system of pluripotent stem cells in the medium for pluripotent stem cell culture are added and cultured, and the proliferation of pluripotent stem cells (especially undifferentiated) is compared with a system in which the component is not added (control). It may be confirmed whether the proliferation has increased.
- the proliferative property the number of cells after proliferation
- the component can be confirmed to be a growth promoting factor for pluripotent stem cells.
- the screening method of the present invention may include such a step of detecting a proliferation promoting activity against pluripotent stem cells for components in the conditioned medium. Whether or not proliferation of pluripotent stem cells in an undifferentiated state (undifferentiated proliferation) can be promoted further depends on an undifferentiation marker (for example, SSEA3, SSEA4, Tra1-60, Tra1-81, Oct4, NANOG, and It can be determined by confirming that expression of a gene or protein such as SOX2 is maintained.
- an undifferentiation marker for example, SSEA3, SSEA4, Tra1-60, Tra1-81, Oct4, NANOG
- the growth promoting factor for pluripotent stem cells obtained by the screening method of the present invention can be used to promote undifferentiated proliferation of pluripotent stem cells by adding it to the culture system of pluripotent stem cells.
- the present invention also includes transferring pluripotent stem cells cultured on feeder cells in a serum-free medium usable for culturing pluripotent stem cells in the absence of feeder cells and culturing without feeders. Also provided are methods of expanding pluripotent stem cells. In a particularly preferred embodiment of this method for expanding pluripotent stem cells, feeder cells are used using serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, and no serum and serum replacement. The pluripotent stem cells cultured above are transferred to the absence of feeder cells and cultured without feeders.
- pluripotent stem cells are maintained and cultured while acclimating a serum-free medium with feeder cells, and then feeder-free culture is performed. According to this method, the proliferation of pluripotent stem cells in feeder-free culture can be further enhanced.
- the pluripotent stem cells, feeder cells, culture conditions and procedures used in this method are the same as described above.
- the serum-free medium the same serum-free medium as used for the preparation of the conditioned medium can be preferably used.
- pluripotent stem cells are cultured on feeder cells using a serum-free medium comprising L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, but not arginine, serum and serum replacement. Then, pluripotent stem cells can be proliferated with high efficiency by feeder-free culture.
- feeder-free culture can be particularly suitably performed using a polymer compound such as Matrigel (registered trademark) or a synthetic polymer as a culture substrate.
- synthetic polymers include hydrogels, for example, temperature sensitive hydrogels having 2- (diethylamino) ethyl acrylate as a basic skeleton.
- feeder-free culture can be performed using a culture vessel such as a culture dish coated on the inside with such a culture substrate.
- a culture vessel such as a culture dish coated on the inside with such a culture substrate.
- feeder-free culture is carried out using a conditioned medium of serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate as described above, but not serum and serum replacement.
- any other serum-free medium may be used instead of the conditioned medium.
- the proliferation (undifferentiated proliferation) of pluripotent stem cells in feeder-free culture can be remarkably enhanced.
- Example 1 undifferentiated human iPS cells maintained on mouse embryonic fibroblasts (MEF cells; feeder cells) inactivated by mitomycin treatment were coated with vitronectin (VTN-N) as follows. In the culture wells, the cells were transferred in the absence of feeder cells and cultured in the presence of MEF-conditioned nutrient medium.
- MEF cells mouse embryonic fibroblasts
- VTN-N vitronectin
- DMEM / F12 medium Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham; Sigma D6421
- KNOCKOUT TM SR KnockOut TM SerumGp
- NEAA non-essential amino acids; non-essential amino acids
- 2 mM L-glutamine 5 ng / ml human FGF2 (also called basic FGF or bFGF)
- 0.1 mM 2-mercaptoethanol Using the medium prepared by addition, the cells were cultured in a CO 2 incubator at 37 ° C. (5% CO 2 concentration). Passaging was performed every 6-7 days.
- human iPS cell colonies are dissociated from the feeder cell layer using a dissociation solution (collagenase solution), made into about 20-50 small clusters by pipetting, and then a new feeder cell layer is prepared. Sowing on top.
- a dissociation solution collagenase solution
- Human iPS cells maintained and cultured on feeder cells as described above were dissociated with a dissociation solution, made into about 20-50 small clusters by pipetting, and iPS cells were collected by centrifugation at 300 rpm for 5 minutes.
- the collected iPS cells were incubated on a gelatin-coated culture dish for 30 minutes to adhere the MEF cells to the dish, and the iPS cells floating in the medium were collected to remove the MEF cells. Subsequently, the collected iPS cells were divided into quarters (1/4 divisions) and seeded on plastic culture dishes coated with vitronectin (VTN-N; Gibco).
- the culture dish was coated with vitronectin (VTN-N) by incubating with a vitronectin solution at a concentration of 0.5 ⁇ g / cm 2 for 1 hour at room temperature.
- CM conditioned medium
- MEF cells mouse fetal fibroblasts
- DMEM medium supplemented with 10% FBS fetal fibroblasts
- the cells were cultured for at least 16 hours, then washed with PBS ( ⁇ ) and then with serum-free medium, and the medium was replaced with the same serum-free medium.
- the composition of the serum-free medium used is as follows.
- Serum-free medium A (DMEM / F12 medium, 64 mg / L 2-phosphate magnesium ascorbate, and 543 mg / L sodium bicarbonate) Serum-free medium A + ITS (DMEM / F12 medium, 64 mg / L 2-phosphate magnesium ascorbate, 543 mg / L sodium bicarbonate, 1% ITS (insulin-transferrin-selenium; Life technologies))
- FGF + TGF added 100 ⁇ g / L human FGF2 and 2 ⁇ g / L TGF- ⁇ 1 were added to the serum-free medium (FGF + TGF added).
- a conditioned medium was prepared using a serum-free medium to which no growth factor was added.
- the medium after 24 hours of culture was collected and centrifuged at 1000 rpm for 5 minutes, and the resulting liquid medium (supernatant) was used as the MEF-conditioned medium.
- iPS cells without using feeder cells
- iPS cells were cultured in MEF-conditioned medium for 5 days at 37 ° C. and 5% CO 2 concentration.
- the cultured cells were stained with alkaline phosphatase. Staining was performed by fixing the cells on the culture plate with 10% formalin, adding 1 ml of One-step NBT / BCIP solution (Pierce), and allowing to stand at room temperature for 30 minutes.
- FIG. 1 high proliferation of iPS cells was observed in MEF-conditioned medium prepared using serum-free medium A + ITS. It should be noted that iPS cells showed good growth even when growth factors were added after acclimation (FIG. 1C). However, using a conditioned medium prepared with a medium added with growth factors further improved the growth of iPS cells. Was observed (FIG. 1A).
- Serum-free medium A (DMEM / F12, 64 mg / L 2-phosphate magnesium ascorbate, 543 mg / L sodium bicarbonate) was used as the serum-free medium, and a MEF-conditioned medium was prepared without adding growth factors.
- DMEM / F12, 64 mg / L 2-phosphate magnesium ascorbate, 543 mg / L sodium bicarbonate was used as the serum-free medium, and a MEF-conditioned medium was prepared without adding growth factors.
- 1% ITS insulin-transferrin-selenium
- 100 ⁇ g / L human FGF2 and 2 ⁇ g / L TGF- ⁇ 1 were added to iPS cells and used for culture.
- IPS cells were cultured in serum conditioned medium.
- FIG. 1D The observation results after alkaline phosphatase staining are shown in FIG. 1D.
- MEF-conditioned medium prepared using serum-free medium A containing no ITS and growth factor even if ITS, human FGF2 and TGF- ⁇ 1 were added during iPS cell culture, almost no proliferation of iPS cells was observed. .
- IPS cells were prepared in the same manner as in Example 1, and serum-free medium A + ITS (DMEM / F12, 64 mg / L) not conditioned by MEF was applied to iPS cells seeded in a culture dish coated with vitronectin (VTN-N). 2-sodium ascorbate phosphate, 543 mg / L sodium bicarbonate, 1% ITS (insulin-transferrin-selenium; Life technologies)) was added. Further, 100 ⁇ g / L human FGF2 and 2 ⁇ g / L TGF- ⁇ 1 were added, and iPS cells were cultured in the same manner as in Example 1.
- FIG. 1B The observation results after alkaline phosphatase staining are shown in FIG. 1B.
- iPS cells were cultured using a serum-free medium A + ITS that had not been conditioned by MEF, the growth of iPS cells was low even when growth factors were added.
- serum-free medium B + ITS DMEM (Dulbecco's Modified Eagle's Medium), 2 mM L-glutamine, 0.1 mM NEAA (non-essential amino acids; non-essential amino acids), 1% ITS (insulin-transferrin) Example 1 except that selenium) and 0.1 mM ⁇ -mercaptoethanol
- ITS insulin-transferrin
- Example 2 undifferentiated human iPS cells maintained on mouse fetal fibroblasts MEF (feeder cells) inactivated by mitomycin treatment were treated with matrigel, vitronectin (VTN-N), or PCM as follows. In culture wells coated with DM, they were transferred in the absence of feeder cells and cultured in the presence of MEF cell conditioned nutrient medium.
- PCM-DM is an extracellular matrix of mesenchymal cells derived from human decidua (D. Kanematsu et al: Differentiation, 82, 77-88, 2011).
- Human iPS cells The 201B7 cell line obtained from iPS Academia Japan was used as human iPS cells (undifferentiated human iPS cells). Human iPS cells were maintained and cultured on plastic culture dishes seeded with mouse fetal fibroblasts MEF (feeder cells) inactivated by mitomycin treatment.
- mouse fetal fibroblasts MEF feeder cells
- DMEM / F12 medium Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham; Sigma D6421
- KNOCKOUT TM SR KnockOut TM SerumGp
- NEAA non-essential amino acids; non-essential amino acids
- 2 mM L-glutamine 5 ng / ml human FGF2 (also referred to as bFGF or FGF2)
- 0.1 mM 2-mercaptoethanol The medium prepared above was cultured in a CO 2 incubator at 37 ° C. (5% CO 2 concentration). Passaging was performed every 6-7 days.
- human iPS cell colonies are dissociated from the feeder cell layer using a dissociation solution (collagenase solution), made into about 20-50 small clusters by pipetting, and then a new feeder cell layer is prepared. Sowing on top.
- a dissociation solution collagenase solution
- the human iPS cells maintained on feeder cells as described above were dissociated with a dissociation solution, made into about 20 to 50 small clusters by pipetting, and centrifuged at 300 rpm for 5 minutes to collect iPS cells.
- the collected iPS cells were incubated on a gelatin-coated culture dish for 30 minutes to adhere the MEF to the dish, and the iPS cells floating in the medium were collected to remove the MEF.
- CM conditioned medium
- MEF cells mouse fetal fibroblasts
- FBS fetal fibroblasts
- serum-free medium A (DMEM / F12 medium, 64 mg / L 2-phosphorus) supplemented with PBS ( ⁇ ) and then with 1% ITS, 100 ⁇ g / L human FGF2 and 2 ⁇ g / L TGF- ⁇ 1 Washed with acid magnesium ascorbate and 543 mg / L sodium bicarbonate (also called basal medium A), the medium was replaced with the same serum-free medium.
- VTN-N vitronectin
- IPS cells prepared and recovered in step (b) and MEF removed were cultured in the same medium not conditioned with MEF.
- Example 3 IPS cells in medium conditioned with MEF (serum-free medium A + ITS + FGF + TGF) and medium not conditioned with MEF (serum-free medium A + ITS + FGF + TGF) using plastic culture dishes coated with Matrigel or PCM-DM, prepared according to Example 2 was continued for 5 passages. The result of comparing the cell growth rate during that time is shown in FIG. When iPS cells were cultured using the MEF-conditioned medium of serum-free medium A + ITS + FGF + TGF, the growth efficiency was about 300 times higher than when the unconditioned medium was used.
- Example 4 Culture of iPS cells in medium conditioned by MEF (serum-free medium A + ITS + FGF + TGF) and medium not conditioned by MEF (serum-free medium A + ITS + FGF + TGF) using plastic culture dishes coated with vitronectin prepared according to Example 2 Continued over 4 passages. As controls, culture of iPS cells using MEF as feeder cells (on-feeder culture) and iPS cells in MEF-CM were also performed.
- Control of MEF-CM is, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F12 Ham; Sigma D6421) to a final concentration of 20% KNOCKOUT TM SR (KnockOut TM Serum Replacement (KSR); GIBCO
- TM SR KnockOut TM Serum Replacement
- GIBCO TM Serum Replacement
- iPS cells in culture were positive for alkaline phosphatase (ALP) and maintained in an undifferentiated state regardless of the conditions.
- ALP alkaline phosphatase
- iPS cells were cultured in serum-free conditioned medium with improved proliferative properties, including culture in serum-free medium not conditioned, on-feeder culture, and culture in MEF-CM. In comparison, the expression state of the undifferentiated marker was not changed, and it was confirmed that there was no change in the cell properties.
- the serum-free medium conditioned with MEF can increase the proliferation efficiency of iPS cells while maintaining the undifferentiated state.
- Example 5 The conditioned medium prepared in the medium added with ITS and growth factor in Example 1 (FIG. 1A) and the conditioned medium prepared in the medium containing no ITS and growth factor in Comparative Example 1 (FIG. 1D) As a control, the protein secreted into each medium was analyzed by two-dimensional gel electrophoresis. First, proteins were collected from 1 ml of each medium by acetone precipitation. Each recovered protein is suspended in a swelling buffer, added to an immobilized pH gradient gel ReadyStrip IPG strip (pH 3-10, 11 cm; Bio-Rad), and an isoelectric focusing device Protean (registered trademark) IEF cell ( Bio-Rad) was swelled at 50 V and 20 ° C.
- the ReadyStrip IPG strip was shaken in SDS-PAGE equilibration buffer (containing 2% DTT) for 10 minutes and then in SDS-PAGE equilibration buffer (containing 2.5% iodoacetamide) for 10 minutes, and then the protein was analyzed by SDS-PAGE. Expanded. In the conditioned medium prepared in the medium added with ITS and growth factor in Example 1, 40 spots were found in the conditioned medium prepared in the medium not containing ITS and growth factor in Comparative Example 1, and 12 spots of MEF-derived secreted proteins were spotted. I was able to detect it. From this, it was shown that the conditioned medium (FIG. 1A) prepared in the medium added with ITS and growth factor in Example 1 contains a protein that contributes to growth promotion.
- SDS-PAGE equilibration buffer containing 2% DTT
- SDS-PAGE equilibration buffer containing 2.5% iodoacetamide
- Example 6 Feeder-free culture using MEF-conditioned medium Human iPS cells were cultured on mouse fetal fibroblasts MEF (feeder cells) inactivated by mitomycin treatment as described in “1. Preparation of human iPS cells” in Example 2. (On-feeder culture) and collected to remove MEF.
- the human iPS cells prepared in this way were transferred to the absence of feeder cells in a culture well coated with Matrigel, and cultured using various media (feeder-free culture). The following media were used.
- Serum-free medium A + ITS + FGF + TGF (DMEM, 2 mM L-glutamine, 0.1 mM NEAA, 1% ITS, 0.1 mM ⁇ -mercaptoethanol with 100 ⁇ g / L human FGF2 and 2 ⁇ g / L TGF- ⁇ 1) (FIG. 5) Middle, E8) -MEF-conditioned medium of serum-free medium A + ITS + FGF + TGF prepared according to the description in “2. Preparation of conditioned medium” in Example 2 (E8-CM in FIG.
- Example 5 -MEF-CM prepared as described in Example 4 -MTeSR TM 1 medium (modified Tenure Serum Replacer 1) (STEMCELL Technologies) As a control, it was prepared by adding 20% final concentration of KNOCKOUT TM SR, 0.1 mM NEAA, 2 mM L-glutamine, 5 ng / ml human FGF2 and 0.1 mM 2-mercaptoethanol to DMEM / F12 medium. A test in which only on-feeder culture in the medium was performed was also conducted. (KSR in FIG. 5) The culture dish was coated with Matrigel according to the description in “3. Preparation of substrate-coated culture dish” in Example 2.
- the culture and alkaline phosphatase staining were performed according to the procedure described in “4. Cultivation of iPS cells without using feeder cells” in Example 2 except that the culture period was 4 days.
- FIGS. 5A to 5E The results are shown in FIGS. 5A to 5E. After the transition from on-feeder culture to non-feeder culture, growth was observed in the serum-free medium A + ITS + FGF + TGF MEF-conditioned medium (E8-CM), but almost no growth was observed in other media.
- Non-feeder culture medium after transfer from on-feeder culture in a medium containing a predetermined component As final medium, DMEM / F12 medium with 20% final concentration of KNOCKOUT TM SR, 0.1 mM NEAA, 2 mM L-glutamine, 5 ng / According to the description in “1.
- Preparation of human iPS cells in Example 2, except that serum-free medium A + ITS + FGF + TGF was used instead of the medium prepared by adding ml human FGF2 and 0.1 mM 2-mercaptoethanol.
- Human iPS cells were cultured on mouse fetal fibroblasts MEF (feeder cells) inactivated by mitomycin treatment (on-feeder culture), collected, and MEF was removed.
- the human iPS cells prepared in this way were transferred to a Matrigel-coated culture well in the absence of feeder cells. In the same manner, no feeder culture was performed using the same various media as described above, and alkaline phosphatase staining was performed.
- pluripotent stem cells can be further enhanced by performing on-feeder culture using the above serum-free medium and then shifting to feeder-free culture.
- Example 7 A culture dish coated with a temperature-sensitive hydrogel based on 2- (diethylamino) ethyl acrylate was prepared (Zhang et al., Nature Communications, (2013) 4, Article number: 1335). Specifically, N-methyl-2-pyrrolidone contains N-acryloyl-N′-propylpiperazine, 2,2 ′-(ethylenedioxy) bis (ethylamine) monoacrylamide, a crosslinking agent and a photopolymerization initiator.
- the dissolved mixed solution was added to a plastic culture dish previously treated with 3- (trimethoxysilyl) propyl methacrylate, irradiated with 365 nm UV light for 30 minutes, and then allowed to stand at 50 ° C. overnight. Then, it wash
- the hydrogel coat culture dish prepared in this way was used in place of the Matrigel coat culture dish in Example 6-2.
- human iPS cells were subjected to on-feeder culture and subsequent feeder-free culture using various media. As a result, iPS cells showed high growth in any medium.
- the present invention can be suitably used for feeder-free serum-free culture of pluripotent stem cells.
- the present invention also provides a serum-free medium for culturing human pluripotent stem cells, such as a serum-free fully synthetic medium, containing a growth promoting factor that facilitates the proliferation of highly safe pluripotent stem cells. Can be used to By using such a medium, it becomes possible to stably and efficiently carry out feeder-free culture of human pluripotent stem cells.
- a medium that makes it possible to isolate a growth promoting factor secreted from feeder cells such as MEF.
- Such a medium can be used to screen for factors useful for the growth of human pluripotent stem cells.
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Abstract
Description
a)L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含み、血清及び血清代替物を含まない無血清培地において、フィーダー細胞を培養し、生成した馴化培地を回収する工程、及び
b)回収した馴化培地に含まれる、多能性幹細胞に対する増殖促進因子を検出する工程、
を含む方法。
本実施例では、以下のように、マイトマイシン処理により不活性化したマウス胎児線維芽細胞(MEF細胞;フィーダー細胞)上で維持培養した未分化ヒトiPS細胞を、ビトロネクチン(VTN-N)でコーティングした培養ウェル中、フィーダー細胞の非存在下に移行し、MEF馴化栄養培地の存在下で培養した。
iPSアカデミアジャパン株式会社(日本、京都)より入手した201B7細胞株(Takahashi K., et al., Cell 131, 1-12 (2007))をヒトiPS細胞(未分化ヒトiPS細胞)として使用した。ヒトiPS細胞は、マイトマイシン処理により不活性化したマウス胎児線維芽細胞MEF(フィーダー細胞)を播いたプラスチック培養ディッシュの上で維持培養した。培養には、DMEM/F12培地(Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham; Sigma D6421)に終濃度20%のKNOCKOUTTM SR(KnockOutTM Serum Replacement(KSR); GIBCO社)、0.1 mM NEAA(non-essential amino acids; 非必須アミノ酸)、2 mM L-グルタミン、5 ng/ml ヒトFGF2(塩基性FGF又はbFGFとも称される)及び0.1 mM 2-メルカプトエタノールを添加して調製した培地を用い、37℃にてCO2インキュベーター内で培養した(5%CO2濃度)。継代は6~7日毎に行った。継代の際には、解離液(コラゲナーゼ溶液)を用いて、ヒトiPS細胞のコロニーをフィーダー細胞層から解離し、ピペット操作で20~50個程度の小塊にした後、新しいフィーダー細胞層の上に播いた。
馴化培地(CM)は、マイトマイシン処理により不活性化したマウス胎児線維芽細胞(MEF細胞)を用いて無血清培地から調製した。マイトマイシン処理により不活性化したマウス胎児線維芽細胞MEFを、MEF用培地(10%FBSを添加したDMEM培地)中に約500,000 細胞/直径60mmディッシュの細胞密度で播種した。細胞を少なくとも16時間培養した後、PBS(-)、次いで無血清培地で洗浄し、培地を同じ無血清培地に置換した。用いた無血清培地の組成は以下の通りである。
・無血清培地A+ITS(DMEM/F12培地、64mg/L 2-リン酸アスコルビン酸マグネシウム、543mg/L 炭酸水素ナトリウム、1% ITS(インスリン-トランスフェリン-セレン; Life technologies社))
無血清培地には、馴化前に、増殖因子として100μg/L ヒトFGF2及び2μg/L TGF-β1を添加した(FGF+TGF添加)。並行して、増殖因子を添加しない無血清培地を用いた馴化培地の調製も行った。
本実施例の1.でビトロネクチン(VTN-N)でコーティングされた培養ディッシュに播種したiPS細胞に、本実施例の2.で調製したMEF馴化培地を2ml加えた。増殖因子としてヒトFGF2及び2μg/L TGF-β1を添加せずに調製したMEF馴化培地には、この段階で100μg/L ヒトFGF2及び2μg/L TGF-β1を添加した。iPS細胞をMEF馴化培地で37℃、5%CO2濃度で5日間培養した。
無血清培地として、無血清培地A(DMEM/F12、64mg/L 2-リン酸アスコルビン酸マグネシウム、543mg/L 炭酸水素ナトリウム)を使用し、増殖因子を加えずにMEF馴化培地を調製し、それをiPS細胞に添加して培養に用いる際に1% ITS(インスリン-トランスフェリン-セレン)、100μg/L ヒトFGF2及び2μg/L TGF-β1を添加したこと以外は、実施例1と同様にして無血清馴化培地でiPS細胞を培養した。
実施例1と同様にしてiPS細胞を調製し、ビトロネクチン(VTN-N)でコーティングされた培養ディッシュに播種したiPS細胞に、MEFにより馴化していない無血清培地A+ITS(DMEM/F12、64mg/L 2-リン酸アスコルビン酸マグネシウム、543mg/L 炭酸水素ナトリウム、1% ITS(インスリン-トランスフェリン-セレン; Life technologies社))を添加した。さらに100μg/L ヒトFGF2及び2μg/L TGF-β1を添加し、実施例1と同様にしてiPS細胞を培養した。
無血清培地として、無血清培地B+ITS(DMEM(Dulbecco’s Modified Eagle’s Medium)、2mM L-グルタミン、0.1mM NEAA(non-essential amino acids; 非必須アミノ酸)、1% ITS(インスリン-トランスフェリン-セレン)、0.1mM β-メルカプトエタノール)を使用し、増殖因子として100μg/L ヒトFGF2及び2μg/L TGF-β1を添加して(FGF+TGF添加)馴化を行ったこと以外は、実施例1と同様にして無血清馴化培地でiPS細胞を培養した。また、MEFにより馴化していない無血清培地B+ITSを用いて、比較例2と同様に100μg/L ヒトFGF2及び2μg/L TGF-β1を添加してiPS細胞を培養した実験も行った。
本実施例では、以下のように、マイトマイシン処理により不活性化したマウス胎児線維芽細胞MEF(フィーダー細胞)上で維持培養した未分化ヒトiPS細胞を、マトリゲル、ビトロネクチン(VTN-N)、又はPCM-DMでコーティングした培養ウェル中、フィーダー細胞の非存在下に移行し、MEF細胞馴化栄養培地の存在下で培養した。PCM-DMは、ヒト脱落膜由来の間葉系細胞の細胞外マトリクス(D. Kanematsu et al: Differentiation, 82, 77-88, 2011)である。
iPSアカデミアジャパン株式会社より入手した201B7細胞株をヒトiPS細胞(未分化ヒトiPS細胞)として使用した。ヒトiPS細胞は、マイトマイシン処理により不活性化したマウス胎児線維芽細胞MEF(フィーダー細胞)を播いたプラスチック培養ディッシュの上で維持培養した。培養には、DMEM/F12培地(Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham; Sigma D6421)に終濃度20%のKNOCKOUTTM SR(KnockOutTM Serum Replacement (KSR); GIBCO社)、0.1 mM NEAA(non-essential amino acids; 非必須アミノ酸)、2 mM L-グルタミン、5 ng/ml ヒトFGF2(bFGF又はFGF2とも称される)及び0.1 mM 2-メルカプトエタノールを添加して調製した培地を用い、37℃にてCO2インキュベーター内で培養した(5%CO2濃度)。継代は6~7日毎に行った。継代の際には、解離液(コラゲナーゼ溶液)を用いて、ヒトiPS細胞のコロニーをフィーダー細胞層から解離し、ピペット操作で20~50個程度の小塊にした後、新しいフィーダー細胞層の上に播いた。
馴化培地(CM)は、マイトマイシン処理により不活性化したマウス胎児線維芽細胞(MEF細胞)を用いて無血清培地から調製した。マイトマイシン処理により不活性化したマウス胎児線維芽細胞(MEF細胞)を、MEF用培地(10%FBSを添加したDMEM培地)中に約500,000 細胞/直径60mmディッシュの細胞密度で播種した。細胞を少なくとも16時間培養した後、PBS(-)、次いで1% ITS、100μg/L ヒトFGF2及び2μg/L TGF-β1を添加した無血清培地A(DMEM/F12培地、64mg/L 2-リン酸アスコルビン酸マグネシウム、及び543mg/L 炭酸水素ナトリウム)(基本培地Aとも呼ぶ)で洗浄し、培地を同じ無血清培地に置換した。
マトリゲルでの培養ディッシュのコーティングは、Life technologies社のプロトコールに従い、マトリゲル(登録商標)(BD社)の、DMEM/F12での30倍希釈液で、室温にて1時間インキュベートすることにより実施した。
本実施例の1.において調製及び回収しMEF細胞を除去したiPS細胞を、本実施例の3.で調製したマトリゲル、ビトロネクチン、又はPCM-DMのそれぞれでコーティングされたプラスチック培養ディッシュに、4分の1に分け(1/4分割)、播種した。培地として、本実施例の2.で調製した無血清培地A+ITS+FGF+TGFのMEF馴化培地を用いて、培養を行った(5%CO2濃度、37℃、5日間)。
実施例2に従って調製した、マトリゲル又はPCM-DMでコーティングしたプラスチック培養ディッシュを用いた、MEFにより馴化した培地(無血清培地A+ITS+FGF+TGF)及びMEFにより馴化していない培地(無血清培地A+ITS+FGF+TGF)におけるiPS細胞の培養を、5継代にわたって継続した。その間の細胞増殖率を比較した結果を図3に示す。無血清培地A+ITS+FGF+TGFのMEF馴化培地を用いてiPS細胞を培養した場合、馴化していない培地を用いた場合と比べて、約300倍高い増殖効率が示された。
実施例2に従って調製した、ビトロネクチンでコーティングしたプラスチック培養ディッシュを用いた、MEFにより馴化した培地(無血清培地A+ITS+FGF+TGF)及びMEFにより馴化していない培地(無血清培地A+ITS+FGF+TGF)におけるiPS細胞の培養を、4継代にわたって継続した。対照として、MEFをフィーダー細胞として用いたiPS細胞の培養(オンフィーダー培養)、及びMEF-CMにおけるiPS細胞の培養も行った。対照のMEF-CMは、DMEM/F12培地(Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham; Sigma D6421)に終濃度20%のKNOCKOUTTM SR(KnockOutTM Serum Replacement(KSR); GIBCO社)を添加して調製した培地において、MEFを37℃にて5%CO2濃度で培養した後、培地を回収することにより、調製した。
実施例1においてITS及び増殖因子を加えた培地で調製した馴化培地(図1A)と、比較例1においてITS及び増殖因子を含まない培地で調製した馴化培地(図1D)について、馴化しない培地をコントロールとしてそれぞれの培地中に分泌されたタンパク質の解析を二次元ゲル電気泳動により行った。まずそれぞれの培地1mlからアセトン沈殿によりタンパク質を回収した。それぞれの回収したタンパク質を、膨潤バッファーに懸濁し、固定化pH勾配ゲルReadyStrip IPGストリップ(pH3-10、11cm; Bio-Rad)に添加して等電点電気泳動装置Protean(登録商標) IEF cell (Bio-Rad)により50V、20℃で12時間膨潤後、等電点電気泳動を行った。その後、ReadyStrip IPGストリップをSDS-PAGE平衡化バッファー(2% DTT含有)中で10分間、次いでSDS-PAGE平衡化バッファー(2.5% ヨードアセトアミド含有)で10分間振盪後、SDS-PAGEによりタンパク質を展開した。実施例1においてITS及び増殖因子を加えた培地で調製した馴化培地では40個、比較例1においてITS及び増殖因子を含まない培地で調製した馴化培地では12個のMEF由来の分泌タンパク質のスポットを検出することができた。このことから、実施例1においてITS及び増殖因子を加えた培地で調製した馴化培地(図1A)には、増殖促進に寄与するタンパク質が含まれることが示された。
1.MEF馴化培地を用いた無フィーダー培養
実施例2の「1.ヒトiPS細胞の調製」の記載に従って、マイトマイシン処理により不活性化したマウス胎児線維芽細胞MEF(フィーダー細胞)上でヒトiPS細胞を培養し(オンフィーダー培養)、回収し、MEFを除去した。
・実施例2の「2.馴化培地の調製」の記載に従って調製した、無血清培地A+ITS+FGF+TGFのMEF馴化培地(図5中、E8-CM)
・実施例4の記載に従って調製したMEF-CM
・mTeSRTM1培地(modified Tenneille Serum Replacer 1)(STEMCELL Technologies)
対照として、DMEM/F12培地に終濃度20%のKNOCKOUTTM SR、0.1 mM NEAA、2 mM L-グルタミン、5 ng/ml ヒトFGF2及び0.1 mM 2-メルカプトエタノールを添加して調製した培地中でのオンフィーダー培養のみを行う試験も実施した。(図5中、KSR) なおマトリゲルでの培養ディッシュのコーティングは、実施例2の「3.基質コーティング培養ディッシュの調製」の記載に従って行った。
培地として、DMEM/F12培地に終濃度20%のKNOCKOUTTM SR、0.1 mM NEAA、2 mM L-グルタミン、5 ng/ml ヒトFGF2及び0.1 mM 2-メルカプトエタノールを添加して調製した培地に代えて、無血清培地A+ITS+FGF+TGFを使用したこと以外は、実施例2の「1.ヒトiPS細胞の調製」の記載に従ってマイトマイシン処理により不活性化したマウス胎児線維芽細胞MEF(フィーダー細胞)上でヒトiPS細胞を培養し(オンフィーダー培養)、回収し、MEFを除去した。
アクリル酸2-(ジエチルアミノ)エチルを基本骨格とする温度感受性ハイドロゲルでコートした培養ディッシュを作製した(Zhang et al., Nature Communications, (2013) 4, Article number: 1335)。具体的には、N-メチル-2-ピロリドン中に、N-アクリロイル-N’-プロピルピペラジン、2,2’-(エチレンジオキシ)ビス(エチルアミン)モノアクリルアミド、架橋剤及び光重合開始剤を溶解した混合溶液を、3-(トリメトキシシリル)プロピルメタクリレートで予め処理したプラスチック培養ディッシュに添加し、365nmのUV光を30分照射した後、50℃で一晩放置した。その後、エタノール、アセトンで順次洗浄し、風乾した。
Claims (15)
- 多能性幹細胞に対する増殖促進因子をスクリーニングする方法であって、
a)L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含み、血清及び血清代替物を含まない無血清培地において、フィーダー細胞を培養し、生成した馴化培地を回収する工程、及び
b)回収した馴化培地に含まれる、多能性幹細胞に対する増殖促進因子を検出する工程、
を含む方法。 - 前記無血清培地が、L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含む、DMEM/F12培地である、請求項1に記載の方法。
- フィーダー細胞の培養を、前記無血清培地に増殖因子を添加して行う、請求項1に記載の方法。
- 増殖因子がFGF2及び/又はTGF-β1である、請求項3に記載の方法。
- フィーダー細胞が、マウス胎児線維芽細胞である、請求項1~4のいずれか1項に記載の方法。
- 多能性幹細胞がES細胞又はiPS細胞である、請求項1~5のいずれか1項に記載の方法。
- L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含み、血清及び血清代替物を含まない無血清培地でフィーダー細胞を培養することにより生成された馴化培地において、多能性幹細胞を無フィーダー培養することを含む、多能性幹細胞の増殖方法。
- 前記無血清培地が、L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含む、DMEM/F12培地である、請求項7に記載の方法。
- フィーダー細胞の培養を、前記無血清培地に増殖因子を添加して行う、請求項7又は8に記載の方法。
- フィーダー細胞の培養を、前記無血清培地に増殖因子を添加せずに行い、かつ、多能性幹細胞の無フィーダー培養を、前記馴化培地に増殖因子を添加して行う、請求項7又は8に記載の方法。
- 増殖因子がFGF2及び/又はTGF-β1である、請求項9又は10に記載の方法。
- フィーダー細胞が、マウス胎児線維芽細胞である、請求項7~11のいずれか1項に記載の方法。
- 多能性幹細胞がES細胞又はiPS細胞である、請求項7~12のいずれか1項に記載の方法。
- L-アスコルビン酸、インスリン、トランスフェリン、セレン、及び炭酸水素ナトリウムを含み、血清及び血清代替物を含まない無血清培地を用いてフィーダー細胞上で培養した多能性幹細胞を、フィーダー細胞非存在下に移行して無フィーダー培養することを含む、多能性幹細胞の増殖方法。
- 前記無血清培地がアルブミンを含まない、請求項14に記載の方法。
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