CN112126626A - Limbal stem cell culture medium and culture method - Google Patents
Limbal stem cell culture medium and culture method Download PDFInfo
- Publication number
- CN112126626A CN112126626A CN202011064612.9A CN202011064612A CN112126626A CN 112126626 A CN112126626 A CN 112126626A CN 202011064612 A CN202011064612 A CN 202011064612A CN 112126626 A CN112126626 A CN 112126626A
- Authority
- CN
- China
- Prior art keywords
- limbal stem
- culture medium
- stem cell
- cell culture
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 111
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 46
- 238000012136 culture method Methods 0.000 title abstract description 6
- 210000004027 cell Anatomy 0.000 claims abstract description 25
- 102100029379 Follistatin-related protein 3 Human genes 0.000 claims abstract description 15
- 101001062529 Homo sapiens Follistatin-related protein 3 Proteins 0.000 claims abstract description 15
- BNMGUJRJUUDLHW-HCZMHFOYSA-N Madecassoside Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O)OC[C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)OC(=O)[C@]12CC[C@H]([C@@H]([C@H]1C=1[C@@]([C@@]3(C[C@@H](O)[C@H]4[C@](C)(CO)[C@@H](O)[C@H](O)C[C@]4(C)[C@H]3CC=1)C)(C)CC2)C)C)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O BNMGUJRJUUDLHW-HCZMHFOYSA-N 0.000 claims abstract description 12
- BNMGUJRJUUDLHW-HLUHVYOBSA-N Madecassoside Natural products C[C@@H]1CC[C@@]2(CC[C@]3(C)C(=CC[C@@H]4[C@@]5(C)C[C@@H](O)[C@H](O)[C@@](C)(CO)[C@@H]5[C@H](O)C[C@@]34C)[C@@H]2[C@H]1C)C(=O)O[C@@H]6O[C@H](CO[C@@H]7O[C@H](CO)[C@@H](O[C@@H]8O[C@H](C)[C@H](O)[C@@H](O)[C@H]8O)[C@H](O)[C@H]7O)[C@@H](O)[C@H](O)[C@H]6O BNMGUJRJUUDLHW-HLUHVYOBSA-N 0.000 claims abstract description 12
- QCYLIQBVLZBPNK-UHFFFAOYSA-N asiaticoside A Natural products O1C(C(=O)C(C)C)=CC(C)C(C2(C(OC(C)=O)CC34C5)C)C1CC2(C)C3CCC(C1(C)C)C45CCC1OC1OCC(O)C(O)C1O QCYLIQBVLZBPNK-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 229940090813 madecassoside Drugs 0.000 claims abstract description 12
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 7
- 102000009016 Cholera Toxin Human genes 0.000 claims abstract description 4
- 108010049048 Cholera Toxin Proteins 0.000 claims abstract description 4
- 102000004877 Insulin Human genes 0.000 claims abstract description 4
- 108090001061 Insulin Proteins 0.000 claims abstract description 4
- 102000004338 Transferrin Human genes 0.000 claims abstract description 4
- 108090000901 Transferrin Proteins 0.000 claims abstract description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229960000890 hydrocortisone Drugs 0.000 claims abstract description 4
- 229940125396 insulin Drugs 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 239000012581 transferrin Substances 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 claims description 11
- 230000029087 digestion Effects 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 229960001484 edetic acid Drugs 0.000 claims description 10
- 230000001079 digestive effect Effects 0.000 claims description 8
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 7
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 239000012091 fetal bovine serum Substances 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 9
- 230000003321 amplification Effects 0.000 abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 8
- 230000004663 cell proliferation Effects 0.000 abstract description 3
- 230000004069 differentiation Effects 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 3
- 239000012894 fetal calf serum Substances 0.000 abstract description 2
- 101150000157 ARHGEF1 gene Proteins 0.000 abstract 2
- 238000000506 liquid--solid chromatography Methods 0.000 abstract 2
- 101150055452 lsc gene Proteins 0.000 abstract 2
- 230000000052 comparative effect Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 5
- 230000003111 delayed effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 2
- 108010032788 PAX6 Transcription Factor Proteins 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000023715 Ocular surface disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000003560 epithelium corneal Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000003786 sclera Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Images
Classifications
-
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- 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
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a limbal stem cell culture medium and a culture method, wherein the culture medium comprises the following components: 8-10% of fetal calf serum, 10-15 ug/ml of madecassoside, 0.3-0.8 ug/ml of follistatin-related protein, 10-20 mg/ml of transferrin, 0.3-0.5 mg/ml of glutamine, 2-3 ml of 100 Xstreptomycin mixed solution, 5-8 ug/ml of insulin, 0.2-0.5 ug/ml of hydrocortisone, 10-15 ng/ml of cholera toxin, 3-5 ng/ml of platelet-derived factor and the balance of DMEM/F12(1: 1); the limbal stem cell basic culture medium can better promote the growth of LSCs stem cells, up-regulate the cell activity, improve the cell proliferation activity, has good stability, fully delay the exhaustion of the amplification capacity, inhibit the differentiation of the cells and maintain the characteristics of the LSCs stem cells.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a limbal stem cell culture medium and a culture method.
Background
Limbal Stem Cells (LSCs) are the interface of the cornea, conjunctiva and sclera, and healing of the corneal epithelium is accomplished by migration and proliferation of limbal stem cells. The limbal stem cells play an important role in ocular surface reconstruction, and the limbal stem cells are obtained by an in vitro isolated culture method to carry out corneal transplantation to treat ocular surface diseases at present, so that the method overcomes and avoids the conditions of limited access to the limbal stem cells and potential immunological rejection of some patients.
At present, in the in vitro culture process of the limbal stem cells, the existing limbal stem cells have the problems of slow proliferation, low proliferation activity, high culture condition requirement, poor stability and the like due to the limitation of micro-environments created for the limbal stem cells by different culture media, so that the preparation of the limbal stem cell culture medium which can better maintain the characteristics of the limbal stem cells, delay the expansion capacity depletion and inhibit the differentiation process of the limbal stem cells in the in vitro culture process has important significance for realizing the efficient expansion culture of the number of the limbal stem cells and providing a rapid and stable cell source for the research and transplantation treatment of the specific mechanism of the limbal stem cells.
Disclosure of Invention
In view of the above, the present invention provides a limbal stem cell culture medium and a culture method thereof.
The technical scheme of the invention is realized as follows:
the invention provides a limbal stem cell culture medium, which comprises the following components:
more preferably, the concentration of the follistatin-related protein is 0.5-0.6 ug/ml follistatin-related protein. The depletion of the limbal stem cell amplification capacity can be effectively delayed by the follistatin-related protein with a certain concentration.
More preferably, the concentration of the madecassoside is 12-13 ug/ml. The madecassoside and the follistatin-related protein with certain concentration are preferably selected, so that the activity of the membrane edge stem cells is better adjusted on the ground, and the exhaustion of the amplification capacity is delayed.
More preferably, the limbal stem cell culture medium comprises the following components:
a method for culturing limbal stem cells using the above culture medium, comprising the steps of:
(1) adopting a limbal stem cell basic culture solution DMEM/F12(1:1), respectively adding fetal bovine serum, madecassoside, follistatin-related protein, transferrin, glutamine, 100 Xstreptomycin mixed solution, insulin, hydrocortisone, cholera toxin and platelet-derived factor according to the proportion, and mixing to prepare a limbal stem cell culture medium for later use;
(2) cutting the limbal stem cell tissue blocks into pieces, flushing the limbal stem cell tissue blocks for 2-3 times by using Hank liquid in an aseptic environment, adding 30-50 times of digestive juice by volume for digestion for 10min until the tissue blocks are loose, mixing 1.25g/l of trypsin and 0.2g/l of EDTA (ethylene diamine tetraacetic acid) 1:1, and removing the digestive juice; washing with Hank solution for 2-3 times, adding the limbal stem cell culture medium obtained in the step (1), sucking out the cell culture medium, adding an equivalent amount of new limbal stem cell culture medium, repeating for 1-2 times at 1000r/min, centrifuging for 5min, and removing the supernatant;
(3) adding the precipitated cells obtained in the step 2 into a limbal stem cell culture medium for resuspension, planting the cells into a culture plate with holes, and culturing the cells at 37 ℃ and 95% humidity and containing 5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
In step (2), the addition amount of the limbal stem cell culture medium is 5-6 times the volume of the limbal stem cells, and the use of a small amount of the limbal stem cell culture medium for a plurality of times can sufficiently disperse the cells and sufficiently eliminate the residual digestion effect by using the culture medium.
Further, in the step (3), the addition amount of the limbal stem cell culture medium is 2-3 times of the volume of the precipitated cells, so that the cell resuspension effect is ensured.
Further, in step (3), the density of the seeded resuspended cells in the well plate is 1X 105Cells/cm2。
Compared with the prior art, the invention has the beneficial effects that: the invention adds a certain content of madecassoside and follistatin related protein on the basis of the limbal stem cell basic culture solution DMEM/F12(1:1), so that the growth of the limbal stem cells can be better promoted, the cell activity is up-regulated, the cell proliferation speed is increased, the stability is good, and simultaneously a platelet-derived factor with a certain concentration is combined, so that a high-quality culture microenvironment is created for the limbal stem cells by a low-content fetal calf serum culture medium, the limbal stem cells can maintain the undifferentiated state of the limbal stem cells as far as possible while being highly amplified, the process of effectively delaying the amplification capability and inhibiting the epithelial cells with mature differentiation is realized, the number of the limbal stem cells is more efficiently amplified, and the number of the limbal stem cells is greatly expanded, the characteristics of the LSCs stem cells are maintained, and a rapid and stable cell source basis is provided for the research of the specificity mechanism of the corneal limbal stem cells in the later stage and the transplantation treatment.
Drawings
FIG. 1 is a line graph showing the expansion results of the cells of the limbal stem cell culture method of the present invention at successive passages of 3 generations.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-a limbal stem cell culture medium comprising the following components:
example 2-a limbal stem cell culture medium comprising the following components:
example 3-a limbal stem cell culture medium comprising the following components:
example 4-a limbal stem cell culture medium comprising the following components:
example 5-a limbal stem cell culture medium comprising the following components:
comparative example 1-limbal stem cell culture medium according to example 3, except that madecassoside was not added.
Comparative example 2-limbal stem cell culture medium according to example 3, except that no follistatin-related protein was added and 15% fetal bovine serum.
Comparative example 3-limbal stem cell culture medium according to example 3, with the difference that the content of madecassoside is 20ug/ml and that of follistatin-related protein is 1.0 ug/ml.
Example 6-in vitro culture of limbal stem cells was performed according to the limbal stem cell culture media of examples 1-5 and comparative examples 1-3, comprising the steps of:
(1) adopting a limbal stem cell basic culture solution DMEM/F12(1:1), respectively adding fetal bovine serum, madecassoside, follistatin-related protein, transferrin, glutamine, 100 Xstreptomycin mixed solution, insulin, hydrocortisone, cholera toxin and platelet-derived factor according to the proportion, and mixing to prepare a limbal stem cell culture medium for later use;
(2) cutting the limbal stem cell tissue block into pieces, flushing the limbal stem cell tissue block for 2 times by using Hank liquid in an aseptic environment, adding 30 times of digestive juice with volume for digestion for 10min until the tissue block is loose, mixing 1.25g/l of trypsin and 0.2g/l of EDTA (ethylene diamine tetraacetic acid) 1:1, and removing the digestive juice; washing with Hank solution for 2 times to eliminate EDTA digestion, adding the limbal stem cell culture medium with 5 times volume in the step (1) for terminating trypsinase digestion, sucking out the cell culture medium, adding an equal amount of new limbal stem cell culture medium, repeating for 1 time to eliminate residual digestion, centrifuging for 5min at 1000r/min, and discarding the supernatant;
(3) adding the precipitated cells obtained in the step 2 into a limbal stem cell culture medium with 2 volumes for resuspension, and inoculating the cells at a density of 1 × 105Cells/cm2Planted in 10% matrigel coated 6-well plates at 37 deg.C, 95% humidity, 5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
1. The morphological structure of the limbal stem cells after 20 days of culture in examples 1 to 5 and comparative examples 1 to 3 were observed, and the cell expansion at 3 successive passages was counted, and the results are shown in the following table,
as can be seen from the above table, by using the limbal stem cell culture medium according to examples 1 to 5 of the present invention, a superior culture microenvironment for the limbal stem cells can be created, and the cell density at 3 successive passages can be maintained at 9.5X 105Above, the maximum of 10.4 x 10 in P3 generation5Indicating that the exhaustion of the amplification capacity is effectively delayed while the high-amount amplification is carried out,the number of limbal stem cells has expanded dramatically. Meanwhile, by comparison in examples 3-5, it can be seen that the activity of the membrane edge stem cells can be effectively adjusted and the exhaustion of the amplification capacity can be delayed under different concentrations of the follistatin-related protein and the madecassoside. Compared with the comparative examples 1 to 3, it can be seen that in the comparative examples 1 to 3, when the limbal stem cells P3 are in generation, the amplification capacity of the limbal stem cells is obviously depleted (as shown in fig. 1), which indicates that the growth of the limbal stem cells can be effectively promoted, the cell activity can be up-regulated, the cell proliferation speed can be increased, the stability is good, and the number of the limbal stem cells can be more efficiently amplified by matching the follistatin-related protein and the madecassoside with a certain concentration with low fetal bovine serum content.
2. The limbal stem cells of 3 generations after continuous passage in examples 1 to 5 and comparative examples 1 to 3 are subjected to immunochemical staining by antibodies of the limbal stem cell specificity markers PAX6, P63, K12 and Ki67 respectively, blue color is used as DAPI, green color is used as an antibody for color development, and the results show that the limbal stem cells of 3 generations after continuous passage in examples and comparative examples are positive, have the characteristics of the limbal stem cells, have the antibody positive rate of over 96 percent and the highest antibody positive rate of 100 percent, and show that the invention can effectively maintain the undifferentiated state of the limbal stem cells and inhibit the process of differentiating mature epithelial cells while expanding the limbal stem cells at high amount, effectively maintain the characteristics of LSCs stem cells, and provide a rapid and stable cell source for the mechanism research and transplantation treatment of the limbal stem cells in later period.
Example 7-in vitro culture of limbal stem cells was performed according to the culture medium from example 3, comprising the following steps:
(1) cutting the limbal stem cell tissue block into pieces, flushing the limbal stem cell tissue block for 3 times by using Hank liquid in an aseptic environment, adding 50 times of digestive juice by volume for digestion for 10min until the tissue block is loose, mixing 1.25g/l of trypsin and 0.2g/l of EDTA (ethylene diamine tetraacetic acid) 1:1, and removing the digestive juice; washing with Hank solution for 3 times to eliminate EDTA digestion, adding the limbal stem cell culture medium with 6 times volume in the step (1) for terminating trypsin digestion, sucking out the cell culture medium, adding an equal amount of new limbal stem cell culture medium, repeating for 2 times to eliminate residual digestion, centrifuging for 5min at 1000r/min, and discarding the supernatant;
(2) adding the precipitated cells obtained in the step 1 into a 3-volume limbal stem cell culture medium for re-suspension, and performing inoculation at a density of 1 × 105Cells/cm2Planted in 10% matrigel coated 6-well plates at 37 deg.C, 95% humidity, 5% CO2Culturing in incubator, and changing culture medium every other day to obtain limbal stem cells, wherein the cell density of 3 generations after continuous passage can be maintained at 9.5 × 105The specific markers PAX6, P63, K12 and Ki67 have the characteristics of the limbal stem cells, and the anti-positive rate is more than 97.5%.
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 (8)
1. A limbal stem cell culture medium comprising: the culture medium comprises the following components:
the balance being DMEM/F12(1: 1).
2. The limbal stem cell culture medium of claim 1, wherein: the concentration of the follistatin-related protein is 0.5-0.6 ug/ml follistatin-related protein.
3. The limbal stem cell culture medium of claim 1, wherein: the concentration of the madecassoside is 12-13 ug/ml.
4. The limbal stem cell culture medium according to claim 2 or 3, wherein: the culture medium comprises the following components:
the balance being DMEM/F12(1: 1).
5. A method for culturing limbal stem cells using the culture medium according to any one of claims 1 to 4, wherein the culture medium comprises: the method comprises the following steps:
(1) adopting a limbal stem cell basic culture solution DMEM/F12(1:1), respectively adding fetal bovine serum, madecassoside, follistatin-related protein, transferrin, glutamine, 100 Xstreptomycin mixed solution, insulin, hydrocortisone, cholera toxin and platelet-derived factor according to the proportion, and mixing to prepare a limbal stem cell culture medium for later use;
(2) cutting the limbal stem cell tissue blocks into pieces, flushing the limbal stem cell tissue blocks for 2-3 times by using Hank liquid in an aseptic environment, adding 30-50 times of digestive juice by volume for digestion for 10min until the tissue blocks are loose, mixing 1.25g/l of trypsin and 0.2g/l of EDTA (ethylene diamine tetraacetic acid) 1:1, and removing the digestive juice; washing with Hank solution for 2-3 times, adding the limbal stem cell culture medium obtained in the step (1), sucking out the cell culture medium, adding an equivalent amount of new limbal stem cell culture medium, repeating for 1-2 times at 1000r/min, centrifuging for 5min, and removing the supernatant;
(3) adding the precipitated cells obtained in the step 2 into a limbal stem cell culture medium for resuspension, planting the cells into a culture plate with holes, and culturing the cells at 37 ℃ and 95% humidity and containing 5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
6. The method for culturing limbal stem cells according to claim 5, wherein: in the step (2), the addition amount of the limbal stem cell culture medium is 5-6 times of the volume of the limbal stem cells.
7. The method for culturing limbal stem cells according to claim 5, wherein: in the step (3), the addition amount of the limbal stem cell culture medium is 2-3 times of the volume of the precipitated cells.
8. The method for culturing limbal stem cells according to claim 5, wherein: in step (3), the density of the inoculation of the resuspended cells in the well-containing culture plate is 1X 105Cells/cm2。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011064612.9A CN112126626B (en) | 2020-09-30 | 2020-09-30 | Limbal stem cell culture medium and culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011064612.9A CN112126626B (en) | 2020-09-30 | 2020-09-30 | Limbal stem cell culture medium and culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112126626A true CN112126626A (en) | 2020-12-25 |
| CN112126626B CN112126626B (en) | 2023-04-07 |
Family
ID=73843884
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011064612.9A Active CN112126626B (en) | 2020-09-30 | 2020-09-30 | Limbal stem cell culture medium and culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112126626B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635115A (en) * | 2004-05-27 | 2005-07-06 | 天津医科大学眼科中心 | Human corneal limbal stem cell tissue engineering product nourished by fibroblast and preparing process thereof |
| US20070122906A1 (en) * | 2003-12-29 | 2007-05-31 | Allan Mishra | Method of culturing cells |
| CN106916787A (en) * | 2017-02-15 | 2017-07-04 | 中山大学中山眼科中心 | A kind of limbal stem cell culture medium and its cultural method |
| CN109415680A (en) * | 2016-05-18 | 2019-03-01 | 学校法人庆应义塾 | Organoid culture cell culture medium, cultural method and organoid |
| CN109439628A (en) * | 2018-10-10 | 2019-03-08 | 中国海洋大学 | Limbal stem cell primary culture method |
-
2020
- 2020-09-30 CN CN202011064612.9A patent/CN112126626B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070122906A1 (en) * | 2003-12-29 | 2007-05-31 | Allan Mishra | Method of culturing cells |
| CN1635115A (en) * | 2004-05-27 | 2005-07-06 | 天津医科大学眼科中心 | Human corneal limbal stem cell tissue engineering product nourished by fibroblast and preparing process thereof |
| CN109415680A (en) * | 2016-05-18 | 2019-03-01 | 学校法人庆应义塾 | Organoid culture cell culture medium, cultural method and organoid |
| CN106916787A (en) * | 2017-02-15 | 2017-07-04 | 中山大学中山眼科中心 | A kind of limbal stem cell culture medium and its cultural method |
| CN109439628A (en) * | 2018-10-10 | 2019-03-08 | 中国海洋大学 | Limbal stem cell primary culture method |
Non-Patent Citations (4)
| Title |
|---|
| JING ZHU ET AL: "SPARC promotes self-renewal of limbal epithelial stem cells and ocular surface restoration through JNK and p38-MAPK signaling pathways", 《STEM CELL》 * |
| RUSZYMAH BT HJ IDRUS ET AL: "Aqueous extract of Centella asiatica promotes corneal epithelium wound healing in vitro", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
| 余跃主编: "《干细胞基础与临床》", 29 February 2008, 中国科学技术大学出版社 * |
| 黄益麒等: "积雪草苷对人成纤维细胞增殖及其分泌胶原蛋白的影响", 《广西中医药大学学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112126626B (en) | 2023-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112522191B (en) | Culture method of mesenchymal stem cells | |
| CN109749997B (en) | Limbal stem cell serum-free medium and culture method thereof | |
| EP2177602A1 (en) | Method for isolation of cell, serum-free culture medium for cell, and method for culture of cell | |
| CN114540298A (en) | Stem cell serum-free medium and preparation method thereof | |
| CN109609461A (en) | A kind of primary tumor cell isolated culture method | |
| Robinson et al. | Culture of conducting airway epithelial cells in serum-free medium | |
| CN113881625A (en) | Cell slice culture additive and application thereof | |
| CN112126626B (en) | Limbal stem cell culture medium and culture method | |
| WO2023143006A1 (en) | Kit for inducing ips cells into nk cells and use method thereof | |
| CN108753681B (en) | Nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation culture medium | |
| Ginsburg et al. | Long-term cultivation in tissue culture of leukemic cells from mouse leukemia induced by Moloney virus or by X rays | |
| CN105820996A (en) | Human primary airway epithelial cell culture method | |
| CN110982783A (en) | Method for culturing spermatogonial stem cells and application thereof | |
| CN112126625B (en) | In-vitro amplification method of limbal stem cells | |
| CN110564688B (en) | Method for culturing limbal stromal stem cells without serum and inducing balling and differentiation in vitro | |
| CN111793605A (en) | Separation and extraction method of primary tumor cells of skin squamous carcinoma | |
| CN104928233A (en) | Culture solution and method of islet-like cells | |
| CN110923205A (en) | Lymphatic endothelial cell culture medium and preparation method and application thereof | |
| CN104818245A (en) | Liver stem cell culture medium and culture method | |
| CN107043742A (en) | A kind of serum free medium of culture hepatocyte and preparation method thereof | |
| CN112359011B (en) | Medium supplement, medium supplement composition, medium and culture method | |
| CN110904035B (en) | Culture method for promoting spermatogonial stem cell proliferation and application thereof | |
| CN116836933B (en) | Liver and gall cancer organoid culture solution, culture reagent combination and culture method | |
| CN112779218B (en) | Culture medium and culture method for primary culture of tumor infiltrating mast cells | |
| CN111808799B (en) | Multifunctional stem cell culture method and culture medium used by same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210419 Address after: 2806, foreign trade group building, 239 Zhongxing Road, Chengdong community, Dongmen street, Luohu District, Shenzhen, Guangdong 518000 Applicant after: Guangdong kangdun Innovation Industry Group Co.,Ltd. Address before: 511500 the third card shop on the first floor, No. 251, xibailing village, Shiling village committee, Longtang Town, Qingyuan City, Guangdong Province Applicant before: Guangdong kangdun Bioengineering Technology Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: No. 4, Cowboy City Heng Road, Xintang Town, Zengcheng District, Guangzhou City, Guangdong Province, 510000 Patentee after: Guangdong Kangdun Hi tech Industry Group Co.,Ltd. Address before: 2806, foreign trade group building, 239 Zhongxing Road, Chengdong community, Dongmen street, Luohu District, Shenzhen, Guangdong 518000 Patentee before: Guangdong kangdun Innovation Industry Group Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240316 Address after: No. 4, Cowboy City Heng Road, Xintang Town, Zengcheng District, Guangzhou City, Guangdong Province, 510000 Patentee after: Guangdong Zhikang Intelligent Biotechnology Co.,Ltd. Country or region after: China Address before: No. 4, Cowboy City Heng Road, Xintang Town, Zengcheng District, Guangzhou City, Guangdong Province, 510000 Patentee before: Guangdong Kangdun Hi tech Industry Group Co.,Ltd. Country or region before: China |