CN113201500A - Non-nutritive layer culture medium for culturing iPS cells - Google Patents
Non-nutritive layer culture medium for culturing iPS cells Download PDFInfo
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- CN113201500A CN113201500A CN202110495652.7A CN202110495652A CN113201500A CN 113201500 A CN113201500 A CN 113201500A CN 202110495652 A CN202110495652 A CN 202110495652A CN 113201500 A CN113201500 A CN 113201500A
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- 238000012258 culturing Methods 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 title abstract description 34
- 108010059616 Activins Proteins 0.000 claims abstract description 23
- 239000000488 activin Substances 0.000 claims abstract description 23
- 102000005606 Activins Human genes 0.000 claims abstract 6
- 102100033438 Tyrosine-protein kinase JAK1 Human genes 0.000 claims description 20
- 101000997835 Homo sapiens Tyrosine-protein kinase JAK1 Proteins 0.000 claims description 19
- 102000042838 JAK family Human genes 0.000 claims description 7
- 108091082332 JAK family Proteins 0.000 claims description 7
- 230000006907 apoptotic process Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 3
- 230000000050 nutritive effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 60
- IYOZTVGMEWJPKR-VOMCLLRMSA-N 4-[(1R)-1-aminoethyl]-N-pyridin-4-yl-1-cyclohexanecarboxamide Chemical compound C1CC([C@H](N)C)CCC1C(=O)NC1=CC=NC=C1 IYOZTVGMEWJPKR-VOMCLLRMSA-N 0.000 description 19
- 102100026818 Inhibin beta E chain Human genes 0.000 description 17
- 230000012010 growth Effects 0.000 description 10
- 108010023082 activin A Proteins 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 210000001778 pluripotent stem cell Anatomy 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 239000012595 freezing medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001228 trophic effect Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DJMUYABFXCIYSC-UHFFFAOYSA-N 1H-phosphole Chemical compound C=1C=CPC=1 DJMUYABFXCIYSC-UHFFFAOYSA-N 0.000 description 1
- 108010000837 Janus Kinase 1 Proteins 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Abstract
The invention discloses a culture medium without a culture layer for culturing iPS cells, belonging to the technical field of biology. The culture medium for culturing the non-nutritive layer of the iPS cell comprises: y27632, Activin a, and JAK 1. The culture medium without the culture layer for culturing the iPS cells can promote the iPS cells to obviously grow, can quickly expand the cells and obtain a large number of cells in a short time, and has the advantages of good cell state, high cell survival rate and low apoptosis rate of the cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium without a culture layer for culturing iPS cells.
Background
iPS cells (Induced pluripotent stem cells) are stem cells Induced from somatic cells and have developmental multipotency. The iPS cell technology has huge potential application value, and the iPS cell technology can be used for obtaining the pluripotent stem cells specific to patients or diseases, so that the problem of immunological rejection in the transplanting process can be avoided, and the ethical problem brought by the research of human embryonic stem cells is avoided, so that the iPS cell technology has wide application prospect in the field of regenerative medicine. In the iPS cell technology, an iPS cell culture medium and a culture system are very important, and iPS cells cultured by the existing iPS cell culture medium and the culture system have the condition of massive apoptosis in the culture process, so that the cell survival rate is low, and the development of the iPS cell technology is not facilitated.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides an auxotrophy-free culture medium for culturing iPS cells. The technical scheme is as follows:
the invention provides an auxotrophy layer culture medium for culturing iPS cells, which is characterized by comprising the following components in parts by weight: y27632, Activin a, and JAK 1.
Specifically, the final concentration ratio of Y27632, Activin a and JAK1 is: 1 (1-5) and (1-7).
Specifically, the final concentration ratio of Y27632, Activin a and JAK1 is: 1 (1-3) and (1-5).
Specifically, the final concentration ratio of Y27632, Activin a and JAK1 is: 1:2:3.
Specifically, the final concentrations of Y27632, Activin a and JAK1 are: 10 μ M, 20ng/mL and 30 ng/mL.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the embodiment of the invention provides an unstrained layer culture medium for culturing iPS cells, which can promote the iPS cells to obviously grow.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph showing the growth of hPSC cells on the first day according to one embodiment of the present invention;
FIG. 2 is a graph showing the growth of hPSC cells on the third day according to one embodiment of the present invention;
FIG. 3 is a graph showing the growth of hPSC cells at the fifth day according to the first embodiment of the present invention;
FIG. 4 is a graph showing the growth of hPSC cells at day seven according to an embodiment of the present invention;
FIG. 5 is a graph showing the growth of hPSC cells on the first day according to the comparative example of the present invention;
FIG. 6 is a graph showing the growth of hPSC cells on the third day according to the comparative example of the present invention;
FIG. 7 is a graph showing the growth of hPSC cells on the fifth day according to the comparative example of the present invention;
FIG. 8 is a graph showing the growth of hPSC cells at day seven according to the comparative example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The invention provides an auxotrophy layer-free culture medium for culturing iPS cells, which comprises the following components in parts by weight: y27632, Activin a, and JAK 1.
Specifically, the final concentration ratios of Y27632, Activin a and JAK1 are: 1 (1-5) and (1-7).
Specifically, the final concentration ratios of Y27632, Activin a and JAK1 are: 1 (1-3) and (1-5).
Specifically, the final concentration ratios of Y27632, Activin a and JAK1 are: 1:2:3.
Specifically, final concentrations of Y27632, Activin a and JAK1 were: 10 μ M, 20ng/mL and 30 ng/mL.
Y27632 provided in the examples herein is distinguished as (R) - (+) -trans-4- (1-aminoethyl) -N- (4-pyridyl) cyclohexanecarboxamide; activin A is Activin A; JAK1 is Janus kinase 1, purchased from midsea america biomedical corporation.
Example one
The embodiment of the invention provides an unstrained layer culture medium for culturing iPS cells, which comprises the following components in part by weight: y27632, Activin a, and JAK 1.
Specifically, the final concentration ratios of Y27632, Activin a and JAK1 are: 1:2:3.
Briefly introduced below, the culture medium without a culture layer provided in the embodiment of the present invention is used for culturing iPS cells, and the embodiment of the present invention selects Human Pluripotent Stem cells (hpscs) in iPS cells to perform experiments, and the specific method is as follows:
1. reviving human pluripotent stem cells
1) Coating a culture dish: mu.L of stock solution of human recombinant laminin LN521 (purchased from Shanghai Xiaopeng Biotech Co., Ltd.) was taken from six well plates per well, and 950. mu.L of Dulbecco's Phosphole Buffered Saline (DPBS) including Ca was added to the stock solution of human recombinant laminin LN5212+And Mg2+And mixing the two solutions uniformly, adding the mixture into a six-hole plate, and placing the six-hole plate in a refrigerator at the temperature of 2-8 ℃ overnight for at least 4 hours to obtain a coated culture dish.
2) Preparing a basic culture medium: 50mL of Stem-Partner ACF medium is taken, 100 μ L of b-FGF solution with the concentration of 40 μ g/mL and basic fibroblast growth factor (b-FGF) with the final concentration of 80ng/μ L are added and mixed evenly, and the mixture is stored at the temperature of 2-8 ℃ and is used up within 2 weeks.
3) Preparing a culture medium without a culture layer: taking 20mL of the basic culture medium obtained in the step 2), and adding Y27632, Activin A and JAK1 into the basic culture medium, wherein the final concentration of Y27632 is 10 muM; final concentration of Activin a was 20ng/mL and final concentration of JAK1 was 30 μ M.
4) Reviving human pluripotent stem cells: and taking out the human pluripotent stem cells in the liquid nitrogen tank, thawing in a 37 ℃ rapid water bath, putting the human pluripotent stem cells into a safety cabinet after thawing, transferring the human pluripotent stem cells into a 15mL centrifuge tube, adding DPBS into the centrifuge tube to supplement the volume to 10mL, uniformly mixing, and centrifuging at the centrifuge speed of 400g for 5 min.
5) Inoculating cells: discarding supernatant in the centrifuge tube, retaining precipitate, adding 2mL culture medium without nourishing layer into the precipitate for resuspension to obtain resuspension solution, transferring the resuspension solution into coated culture dish, mixing, placing the coated culture dish in a container at 37 deg.C and filled with 5% CO2In an incubator.
2. hPSC cell liquid changing (18-24 h after recovery)
The culture medium was changed every day and was a basal medium.
3. Passage of hPSC cells
1) Coating a culture dish: the method is the same as that of step 1.1, and is not described herein again.
2) Preparing a basic culture medium: the method is the same as that of step 1.2, and is not described herein again.
3) Preparing a culture medium without a culture layer: the method is the same as that of step 1.3, and is not described herein again.
4) The coated petri dish, the non-trophoblast medium, ReLeSR and DPBS buffer (the DPBS buffer does not contain Ca) were taken out separately2+And Mg2+) And the temperature is recovered to room temperature.
5) Removing hPSC cells from the incubator, discarding the medium from the old dish, and applying DPBS buffer (the DPBS buffer does not contain Ca)2+And Mg2+) Washing once, respectively adding 0.5mL of ReLeSR into each culture dish, digesting for 5min at 37 ℃, then adding 1mL of basal medium to terminate, resuspending and collecting cells, wherein the method for collecting the cells is centrifugation, the centrifugation speed is 400g, and the centrifugation time is 5 min.
6) Removing the supernatant, retaining the precipitate, adding a culture medium without a culture layer into the precipitate for resuspension to obtain a cell suspension, wherein the weight-volume ratio of the precipitate to the culture medium without the culture layer is 1:100, blowing the cell suspension to blow the cells into single cells, and carrying out passage according to the ratio of 1 (80-100). Meanwhile, the cell growth of the hPSC cells was observed and photographed at day 3, day 5 and day 7, respectively, as shown in FIGS. 1 to 4.
4. hPSC cell cryopreservation
1) Preparing a cell freezing medium: the culture medium without the culture medium of the culture layer is mixed with dimethyl sulfoxide DMSO according to the volume ratio of 9: 1.
2) Removing hPSC cells from the incubator, discarding the old medium, and applying DPBS buffer (the DPBS buffer does not contain Ca)2+And Mg2+) Washing once, adding 0.5mL of ReLeSR into each culture dish, digesting for 5min at 37 ℃, then adding 1mL of basal medium for termination, resuspending and collecting cells, wherein the method for collecting the cells is centrifugation, the centrifugation speed is 400g, and the centrifugation time is 5 min.
3) Discarding the supernatant, retaining the precipitate, adding cell freezing medium into the precipitate to resuspend the cells, blowing and beating for no more than ten times to make the cells present small lumps, and subpackaging the blown and beaten resuspension freezing medium into a plurality of freezing tubes, wherein each volume is 1 mL.
The date, cell type, cell number, cell generation and operator are marked on each cryopreserved tube. And (3) placing the freezing tube into a programmed cooling box, temporarily storing at-80 ℃, or transferring to a biological sample library (liquid nitrogen) for long-term storage.
As can be seen from fig. 1 to fig. 4, the cells grow significantly, and have a significant growth trend on days 3, 5, and 7, and after 7 days, the cell clones have fused with each other, and can be passaged, which indicates that the culture medium containing the non-nutritive layer provided by the embodiment of the present invention can rapidly expand cells and obtain a large number of cells in a short time, and the cells have good states, high cell survival rates, and low apoptosis rates.
Example two
The invention provides an auxotrophy layer-free culture medium for culturing iPS cells, which comprises the following components in parts by weight: y27632, Activin a, and JAK 1. Specifically, the difference between the second embodiment and the first embodiment is that when the culture medium without a trophic layer is prepared, the final concentration ratio of Y27632, Activin A and JAK1 is as follows: 1:1:1.
EXAMPLE III
The invention provides an auxotrophy layer-free culture medium for culturing iPS cells, which comprises the following components in parts by weight: y27632, Activin a, and JAK 1. Specifically, the difference between the third embodiment and the first embodiment is that when the culture medium without a trophic layer is prepared, the final concentration ratio of Y27632, Activin a and JAK1 is as follows: 1:3:4.
Example four
The invention provides an auxotrophy layer-free culture medium for culturing iPS cells, which comprises the following components in parts by weight: y27632, Activin a, and JAK 1. Specifically, the difference between the fourth embodiment and the first embodiment is that when the culture medium without a trophic layer is prepared, the final concentration ratio of Y27632, Activin A and JAK1 is as follows: 1:5:7.
Comparative example
The present comparative example provides a complete medium, which is different from example one in that the complete medium provided by the comparative example is entirely substituted for the non-feeder medium provided by example one, and specifically, the complete medium includes: y27632 and Activin A, wherein the final concentration ratio of Y27632 to Activin A is 1:2, specifically, preparing a culture medium without a nutrient layer: taking 20mL of the basic culture medium obtained in the step 2), and adding Y27632 and Activin A into the basic culture medium, wherein the final concentration of Y27632 is 10 mu M; final concentration of Activin A was 20 ng/mL.
In particular, the method for recovering the human pluripotent stem cells provided in the first embodiment,
and the cell growth of the hPSC cells was observed after the passage of the hPSC cells in step 3, and the hPSC cells were photographed at day 3, day 5 and day 7, respectively, and their growth was shown in FIGS. 5 to 8, respectively. Comparing fig. 5 to 8 with fig. 1 to 4, respectively, it can be seen that the cells cultured in the complete medium provided in the comparative example were not significantly grown in the examples, and the cell status and the expansion rate were inferior to those of the examples of the present invention.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. An auxotroph-free medium for culturing iPS cells, said auxotroph-free medium comprising: y27632, Activin a, and JAK 1.
2. The auxotrophic layer medium of claim 1, wherein the final concentration ratio of Y27632, Activin a, and JAK1 is: 1 (1-5) and (1-7).
3. The auxotrophic layer medium of claim 1, wherein the final concentration ratio of Y27632, Activin a, and JAK1 is: 1 (1-3) and (1-5).
4. The auxotrophic layer medium of claim 1, wherein the final concentration ratio of Y27632, Activin a, and JAK1 is: 1:2:3.
5. The auxotrophic layer medium of claim 1, wherein the final concentrations of Y27632, Activin a, and JAK1 are respectively: 10 μ M, 20ng/mL and 30 ng/mL.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110495652.7A CN113201500A (en) | 2021-05-07 | 2021-05-07 | Non-nutritive layer culture medium for culturing iPS cells |
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| CN202110495652.7A CN113201500A (en) | 2021-05-07 | 2021-05-07 | Non-nutritive layer culture medium for culturing iPS cells |
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| US20160060593A1 (en) * | 2013-04-16 | 2016-03-03 | Universiteit Maastricht | Blastoid, cell line based artificial blastocyst |
| JPWO2015125926A1 (en) * | 2014-02-21 | 2017-03-30 | 国立研究開発法人理化学研究所 | Method for establishing and maintaining trophoblast stem cells |
| US20190330597A1 (en) * | 2017-01-11 | 2019-10-31 | The Francis Crick Institute Limited | Composition for culture of pluripotent stem cells |
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2021
- 2021-05-07 CN CN202110495652.7A patent/CN113201500A/en active Pending
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