CN115216445B - Culture medium and application thereof in culturing retina progenitor cells - Google Patents
Culture medium and application thereof in culturing retina progenitor cells Download PDFInfo
- Publication number
- CN115216445B CN115216445B CN202210936477.5A CN202210936477A CN115216445B CN 115216445 B CN115216445 B CN 115216445B CN 202210936477 A CN202210936477 A CN 202210936477A CN 115216445 B CN115216445 B CN 115216445B
- Authority
- CN
- China
- Prior art keywords
- progenitor cells
- cells
- culture medium
- retina
- culture
- 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.)
- Active
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 51
- 238000012258 culturing Methods 0.000 title claims abstract description 21
- 210000000130 stem cell Anatomy 0.000 title abstract description 34
- 210000001525 retina Anatomy 0.000 title abstract description 33
- 210000004027 cell Anatomy 0.000 claims abstract description 67
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 34
- 239000001301 oxygen Substances 0.000 claims abstract description 28
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 23
- 210000001164 retinal progenitor cell Anatomy 0.000 claims description 52
- 239000002609 medium Substances 0.000 claims description 15
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 12
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 12
- BIZNHCWFGNKBBZ-JLHYYAGUSA-N (e)-2-cyano-3-(1-phenylindol-3-yl)prop-2-enoic acid Chemical compound C12=CC=CC=C2C(/C=C(C(=O)O)\C#N)=CN1C1=CC=CC=C1 BIZNHCWFGNKBBZ-JLHYYAGUSA-N 0.000 claims description 8
- LLJYFDRQFPQGNY-QINSGFPZSA-N (z)-5-(4-chlorophenyl)-3-phenylpent-2-enoic acid Chemical compound C=1C=CC=CC=1C(=C/C(=O)O)\CCC1=CC=C(Cl)C=C1 LLJYFDRQFPQGNY-QINSGFPZSA-N 0.000 claims description 8
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 claims description 7
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 claims description 7
- 229960002930 sirolimus Drugs 0.000 claims description 7
- XPYBSIWDXQFNMH-UHFFFAOYSA-N D-fructose 1,6-bisphosphate Natural products OP(=O)(O)OCC(O)C(O)C(O)C(=O)COP(O)(O)=O XPYBSIWDXQFNMH-UHFFFAOYSA-N 0.000 claims description 6
- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 claims description 6
- RNBGYGVWRKECFJ-UHFFFAOYSA-N fructose-1,6-phosphate Natural products OC1C(O)C(O)(COP(O)(O)=O)OC1COP(O)(O)=O RNBGYGVWRKECFJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000005715 Fructose Substances 0.000 claims description 4
- 239000001177 diphosphate Substances 0.000 claims description 4
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 4
- 235000011180 diphosphates Nutrition 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000007640 basal medium Substances 0.000 claims description 3
- 206010021143 Hypoxia Diseases 0.000 abstract description 20
- 230000007954 hypoxia Effects 0.000 abstract description 18
- 210000004498 neuroglial cell Anatomy 0.000 abstract description 8
- 230000000324 neuroprotective effect Effects 0.000 abstract description 7
- 230000036961 partial effect Effects 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 6
- 239000000470 constituent Substances 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 230000002207 retinal effect Effects 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 18
- 230000006907 apoptotic process Effects 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000004069 differentiation Effects 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 7
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 7
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 7
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 description 7
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 7
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 7
- 102000004374 Insulin-like growth factor binding protein 3 Human genes 0.000 description 7
- 108090000965 Insulin-like growth factor binding protein 3 Proteins 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 210000001178 neural stem cell Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 210000003986 cell retinal photoreceptor Anatomy 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000037149 energy metabolism Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 2
- 108010024986 Cyclin-Dependent Kinase 2 Proteins 0.000 description 2
- 102100036239 Cyclin-dependent kinase 2 Human genes 0.000 description 2
- 108010041834 Growth Differentiation Factor 15 Proteins 0.000 description 2
- 102000000597 Growth Differentiation Factor 15 Human genes 0.000 description 2
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 2
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 description 2
- 102000004369 Insulin-like growth factor-binding protein 4 Human genes 0.000 description 2
- 108090000969 Insulin-like growth factor-binding protein 4 Proteins 0.000 description 2
- 108010088225 Nestin Proteins 0.000 description 2
- 102000008730 Nestin Human genes 0.000 description 2
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 2
- 101150081664 PAX6 gene Proteins 0.000 description 2
- 208000017442 Retinal disease Diseases 0.000 description 2
- 206010038923 Retinopathy Diseases 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 239000002771 cell marker Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 210000005055 nestin Anatomy 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- YPSXFMHXRZAGTG-UHFFFAOYSA-N 4-methoxy-2-[2-(5-methoxy-2-nitrosophenyl)ethyl]-1-nitrosobenzene Chemical compound COC1=CC=C(N=O)C(CCC=2C(=CC=C(OC)C=2)N=O)=C1 YPSXFMHXRZAGTG-UHFFFAOYSA-N 0.000 description 1
- 229940088872 Apoptosis inhibitor Drugs 0.000 description 1
- 101150012716 CDK1 gene Proteins 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 108090000567 Caspase 7 Proteins 0.000 description 1
- 102100029855 Caspase-3 Human genes 0.000 description 1
- 102100038902 Caspase-7 Human genes 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 101710106279 Cyclin-dependent kinase 1 Proteins 0.000 description 1
- 102100032857 Cyclin-dependent kinase 1 Human genes 0.000 description 1
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 101100351026 Drosophila melanogaster ey gene Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 101000596404 Homo sapiens Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 description 1
- 101000596402 Mus musculus Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 description 1
- 101000800539 Mus musculus Translationally-controlled tumor protein Proteins 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 1
- 201000007737 Retinal degeneration Diseases 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 101000781972 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Protein wos2 Proteins 0.000 description 1
- 101150037203 Sox2 gene Proteins 0.000 description 1
- 101001009610 Toxoplasma gondii Dense granule protein 5 Proteins 0.000 description 1
- 102100040250 Transcription elongation factor A protein-like 1 Human genes 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000158 apoptosis inhibitor Substances 0.000 description 1
- 208000034615 apoptosis-related disease Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 102000055102 bcl-2-Associated X Human genes 0.000 description 1
- 108700000707 bcl-2-Associated X Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000005157 neural retina Anatomy 0.000 description 1
- 230000009223 neuronal apoptosis Effects 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- -1 oxygen free radical Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004243 retinal function Effects 0.000 description 1
- 210000001116 retinal neuron Anatomy 0.000 description 1
- 210000000880 retinal rod photoreceptor cell Anatomy 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
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
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- 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
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- 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
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Ophthalmology & Optometry (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Neurosurgery (AREA)
- Zoology (AREA)
- Neurology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of cell biology, in particular to a culture medium and application thereof in culturing retina progenitor cells. The culture medium provided by the invention is suitable for the growth of retina progenitor cells under the condition of hypoxia, can maintain the culture of retina progenitor cells under the condition of hypoxia, and can continuously maintain the dryness of retina progenitor cells. The retina progenitor cells cultured by the culture medium still have the capability of differentiating into various retina constituent cells at the downstream, are less differentiated into glial cells, and can secrete a large amount of neuroprotective factors. Under the condition of low oxygen, the cell can be expanded to P20 generation by using the culture medium, the cell characteristics are maintained, and the yield is improved by more than 40 times compared with other commercial culture media. The method can reduce the frequency and the liquid exchange amount of liquid exchange, more effectively simulate the retina physiological oxygen partial pressure environment, reduce the influence of active oxygen free radicals on cells, reduce the instability caused by frequent operation, and can produce retina progenitor cells with stable and uniform batches, thereby being more beneficial to industrialization.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a culture medium and application thereof in culturing retina progenitor cells.
Background
Retinal progenitor cells (Retinal Progenitor Cells, RPCs) are a class of tissue-specific stem cells that are located in retinal tissue and that can specifically differentiate into mature retinal function cells, which underlie retinal cell formation and function. In theory, adult retinal cells cannot regenerate after being damaged, so that in a patient with retinopathy, the retinal progenitor cells can be cultured in vitro and transplanted to a lesion site to interfere or delay the progress of retinopathy, improve the vision of the patient and improve the functions of the retinal cells. At present, a plurality of clinical test researches are carried out to transplant the retinal progenitor cells cultured in vitro into eyes of patients with retinal pigment degeneration, and all the researches report that the retinal progenitor cells are safe to transplant in eyes, and the vision of the patients can be obviously improved after the treatment, and the living condition of the patients can be improved. Thus, in vitro cultured retinal progenitor cells provide a new approach to the treatment of retinal degenerative diseases. At present, the culture systems of retina progenitor cells and nerve stem cells are mostly cultured under the normoxic condition, on one hand, the cells utilize sugar metabolism to generate energy, and on the other hand, oxidative phosphorylation is also applied to generate ATP energy, and in vitro normal oxygen partial pressure culture can generate a large number of active oxygen free radicals, so that the stem cells are damaged, and the stem cells are subjected to non-directional differentiation and apoptosis.
The retinal progenitor cell transplantation is one of the in-vitro therapies for treating retinal degenerative diseases, the retinal progenitor cells are isolated from fetal eye retinal tissues in a development stage, the content of the retinal progenitor cells is low, the in-vitro amplification capability is limited, so that in order to improve the amplification efficiency of the in-vitro retinal progenitor cells, a great deal of research is conducted on amplifying the retinal progenitor cells by adopting culture conditions which simulate the physiological oxygen partial pressure conditions of the retinal tissues in the development stage, the method can achieve the aim of amplifying the retinal progenitor cells in vitro, but has limited effect on improving the yield of the cells, and the reported culture medium still adopts a normal oxygen partial pressure scheme, does not consider the energy metabolism conversion problem of cells under the hypoxia condition, is not suitable for culturing the cells under the hypoxia condition, or needs to frequently replace the energy required by maintaining the growth of the cells by the fresh culture medium, and is unstable due to the fact that the cells are subjected to normal oxygen partial pressure in the process of liquid replacement, and stem cell damage is easy to be caused by frequent hypoxia and reoxygenation.
In the prior art, the normal oxygen culture medium is used for culturing the retinal progenitor cells and the neural stem cells under the condition of hypoxia, and the requirement of change of cell energy metabolism under the condition of hypoxia is not considered, so that the prior culture medium is not suitable for the growth requirement of the retinal progenitor cells under the condition of hypoxia, and the frequent reoxygenation-hypoxia liquid exchange operation can increase the damage condition of cells, thereby reducing the treatment effect. There is therefore a strong need to increase the expansion of retinal progenitor cells and neural stem cells and to maintain cell stem properties during expansion.
Disclosure of Invention
In view of the above, the technical problem underlying the present invention is to provide a culture medium and its use for culturing retinal progenitor cells. Aiming at the characteristics of cell energy metabolism under hypoxia, the invention innovatively invents a culture system which meets the requirement of hypoxia culture, and the culture system can be applied to culture of neural stem cells and retina progenitor cells under normoxic conditions, so that the influence of active oxygen free radicals on cells under normoxic conditions can be reduced, and the cells are protected from damage.
The present invention provides a composition comprising: EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and fructose-1, 6-bisphosphate. The influence of lack of trial factors on cell proliferation in the construction process of the culture medium is finally optimized, and the combination of the factors is found to be the most vigorous on cell proliferation.
The composition of the invention, wherein the mass ratio of EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and 1, 6-fructose diphosphate is 1:2:3000000:100:1000:500:50000000.
The invention provides a culture medium, which comprises a basal culture medium and the composition.
The culture medium consists of basic culture medium, EGF 1 ng/mL-50 ng/mL, bFGF 1 ng/mL-50 ng/mL, glutamax-1 mg/mL-30 mg/mL, rapamycin0.1 mug/mL-1 mug/mL, PS 48.5 mug/mL-20 mug/mL, UK5099 0.5 mug/mL-5 mug/mL and fructose 1, 6-bisphosphate 100 mg/mL-1 g/mL.
In some embodiments, the medium consists of basal medium and EGF 5 ng/mL-30 ng/mL, bFGF 5 ng/mL-30 ng/mL, glutamax-1 10 mg/mL-20 mg/mL, rapamycin 0.5 μg/mL-1 μg/mL, PS48 μg/mL-10 μg/mL, UK50991 μg/mL-5 μg/mL, and fructose 1, 6-bisphosphate 100 mg/mL-50 mg/mL.
In other embodiments, the medium consists of basal medium and EGF 10ng/mL, bFGF 20ng/mL, GIutaMAX-1 30mg/mL, rapamycin 1. Mu.g/mL, PS 48. Mu.g/mL, UK5099 5. Mu.g/mL, and fructose 1, 6-bisphosphate 500mg/mL.
The culture medium provided by the invention, wherein the basal culture medium is an Advanced DMFM/F12 culture medium.
In the culture medium provided by the invention, EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and 1, 6-fructose diphosphate are coordinated with each other, and each component is indispensable. Early experiments showed that replacement or reduction of either component significantly reduced the cell culture effect. In addition, the proportion of each component has a significant influence on the result. The culture medium of the invention is used for culturing the retina progenitor cells, so that the retina progenitor cells can be efficiently amplified until the P20 generation is not aged, and the cell stem property and the directional differentiation capability of the retina progenitor cells can be still maintained. Furthermore, the retinal progenitor cells cultured by the culture medium obviously reduce the differentiation towards GFAP (glial cellulose protein) cells, reduce the production of GFAP positive glial cells, more preferentially differentiate towards functional cells, and express at least one of neuroprotective factors GFBP-2, IGFBP-3, IGFBP-4, BDNF, GDF-15, FGF-4, FGF-7 and/or PDGF at high level. Wherein the improvement of IGFBP-3, BDNF, FGF-4, FGF-7, PDGF and other factors is more remarkable.
In addition, the retinal progenitor cells cultured by the culture medium of the invention highly express apoptosis inhibitor genes, and bcl2, p21, CDK2 and CDK1 prove that the culture medium has a protective effect on the retinal progenitor cells.
The invention provides a method for culturing retina progenitor cells, which comprises the steps of culturing cells and passaging by using the culture medium at an oxygen concentration of 1% -6.5%.
In some embodiments, the method comprises culturing the cells and passaging using the medium at an oxygen concentration of 2% to 6%.
In other embodiments, the method comprises culturing the cells and passaging using the medium at oxygen concentrations of 2%, 3%,4%, 5%, 6%, respectively.
In some embodiments, the method comprises culturing the cells and passaging using the medium at an oxygen concentration of 5%.
In the method for culturing retinal progenitor cells according to the embodiment of the invention, the seeding density is 9,000cells/cm 2 ～13,000cells/cm 2 The normoxic medium needs to be replaced every 2 days, the medium of the invention is replaced every 4 days, and CO 2 The concentration is controlled to be 5%, after 24 hours of inoculation, most cells can be seen to adhere to the wall, the cells are single when observed under a phase contrast microscope, the cell bodies are small and transparent, and few protrusions extend. And after the cells are more than 85% converged, starting the cell subculture expansion.
The culture medium can also be used for culturing retina progenitor cells with the oxygen concentration of 20%, can effectively reduce the generation of oxygen free radical ROS and protect the cells from damage.
The retinal progenitor cells cultured by the method still have the capability of differentiating into retinal component cells, and the glial cells with positive GFAP are expressed in a low level, so that the retinal photoreceptor cell markers and the retinal stem markers are expressed more preferentially, and the differentiation to the retinal photoreceptor cells is facilitated.
The invention also provides the retinal progenitor cells obtained by culturing the method.
The retinal progenitor cells cultured by the method of the invention express neuroprotective factors at high levels. The neuroprotective factor comprises: at least one of GFBP-2, IGFBP-3, IGFBP-4, BDNF, GDF-15, FGF-4, FGF-7 and/or PDGF. Compared with the prior art, the neuroprotective factors in the retinal progenitor cells obtained by culture in the invention are obviously improved, wherein the improvement of IGFBP-3, BDNF, FGF-4, FGF-7 and PDGF is more obvious, and the concentration of each factor in the culture system of the invention can reach IGFBP-3:1206.03pg/ml, BDNF:728.1pg/ml, FGF-4:910.3pg/ml, FGF-7:758.4pg/ml, PDGF:961.967pg/ml. And the retinal progenitor cells cultured by the method of the invention have low expression of apoptosis promoting genes.
The invention provides application of the retinal progenitor cells in preparing products for treating and/or preventing diseases related to retinal nerve cell apoptosis.
In the use of the invention, the retinal neuronal apoptosis-related diseases include retinitis pigmentosa, diabetic retinopathy and/or age-related macular degeneration.
The invention also provides a product for treating and/or preventing diseases related to retinal nerve cell apoptosis, which comprises the retinal progenitor cells obtained by culturing the method.
The product of the invention is a cell preparation containing retinal progenitor cells. It is a cell suspension or cell lyophilized powder comprising retinal progenitor cells and agents that suspend cells, or comprises lyoprotectants. Other drugs having the ability to treat and/or prevent diseases associated with apoptosis of retinal nerve cells may also be included.
The invention also provides methods of treating and/or preventing diseases associated with apoptosis of retinal nerve cells comprising administering the products of the invention.
The invention provides a culture medium which is more suitable for the growth of retina progenitor cells under the condition of hypoxia, can maintain the long-term culture of the retina progenitor cells under the condition of hypoxia, can continuously maintain the stem property of the retina progenitor cells, and the retina progenitor cells cultured by the culture medium still have the capability of differentiating into various retina constituent cells at the downstream, are less differentiated into glial cells, and can secrete a larger amount of neuroprotective factors. Experiments prove that under the condition of hypoxia, cells can be amplified to P20 generation in vitro by using the culture medium, the characteristics of retina progenitor cells are still maintained, and the yield of the cells is improved by more than 40 times compared with other commercial culture media. The method can reduce the frequency and the liquid exchange amount of the liquid exchange, more effectively simulate the physiological oxygen partial pressure environment of retina, reduce the influence of active oxygen free radicals on cells, further improve the yield of retina progenitor cells, reduce the instability of cells caused by frequent operation, ensure that the retina progenitor cells obtained by the culture system can still keep the stem property and differentiation function of the retina progenitor cells, meet the requirement of establishing a three-level cell bank of cells, produce retina progenitor cells with stable and uniform batches, utilize industrialization more, obviously reduce the differentiation of cells under the system to GFAP (gliocellulose protein) cells, reduce the production of GFAP positive glial cells, differentiate to functional cells more advantageously and secrete nutritional factors required by tissues.
Drawings
For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:
FIG. 1 shows a comparison of the proliferative capacity of retinal progenitor cells at 5% oxygen concentration for different factor combinations;
FIG. 2 shows comparison of the doubling capacities of different generations of retinal progenitor cells at 5% oxygen concentration in different culture systems;
FIG. 3 shows comparative analysis of retinal progenitor cell cycle at 5% oxygen concentration in different culture systems;
FIG. 4 shows changes in retinal progenitor cell expression pro-apoptotic and apoptosis-inhibiting related genes at 5% oxygen concentration under different culture systems;
FIG. 5 shows the expression of the retinal progenitor stem markers at 5% oxygen concentration in different culture systems;
FIG. 6 shows a comparison of the differentiation capacity of retinal progenitor cells under differentiation induction conditions at 5% oxygen concentration;
figure 7 shows that at 5% oxygen concentration, retinal progenitor cells secrete neuroprotective factors under different culture systems.
Detailed Description
The invention provides a culture medium and application thereof in culturing retina progenitor cells. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 establishment of human retinal progenitor cell hypoxia culture System
1. Establishment of isolated culture system of primary human retina progenitor cells
(1) Culturing the matrigel Cell start coating of the culture container row in advance: matrigel Cell start is prepared with Ca at a ratio of 1:50 2+ 、Mg 2+ After dilution of DPBS in culture flask, 2 ml/flask, 5% CO 2 Coating at 37 ℃ in an incubator for 1h or overnight at 4 ℃;
(2) Separating whole eye retina tissue under a dissecting microscope, transferring to a DMEM culture solution at 4 ℃ for rinsing for 2 times;
(3) Transfer retinal tissue to a medium containing 1ml TrypLE TM -Express digest, vortexed every 5 minutes at 37 ℃ until a single cell suspension;
(4) After digestion, adding 3ml of culture medium containing 5% KOSR to stop digestion, gently blowing the cell suspension, centrifuging for 1000 revolutions for 3 minutes, and discarding the supernatant;
(5) Adding 5ml of normal oxygen medium (ULTCULTURE) TM Culture medium, EGF 10ng/mL;20ng/mL bFGF;1% GlutaMAX-1) and 5mL of the culture medium of the invention of the patent (Advanced DMFM/F12 medium, EGF 10ng/mL;20ng/mL bFGF;1% GlutaMAX-1; rapamycin0.1 nM; PS48 50. Mu.M; 0.5mM UK5099;1, 6-Fructose Diphosphate (FDP) 50 mM), cell counts, and inoculated into culture flasks previously coated with celltar. The cell density was 9000cells/cm 2 ～13000cells/cm 2 Adding 5ml of culture medium containing 5% KOSR, placing in a hypoxia culture chamber, and adding CO 2 Concentration is controlled to 5%, O 2 The concentration is controlled to be 5 percent, and the temperature is 37 ℃; the culture medium of the invention has the influence of lack of try factors on cell proliferation in the construction process, and finally, the combination cell proliferation of the factors is found to be the most vigorous through optimization.
(6) After 24h inoculation, most cells can be seen to adhere to the wall, the cells are single when observed under a phase contrast microscope, the cell bodies are small and transparent, and few protrusions extend. The normoxic culture medium needs to be replaced every 2 days, the culture medium is replaced every 4 days, and the cell subculture expansion culture is started after more than 85% of cells are converged.
2. Hypoxia culture system promotes proliferation of human retinal progenitor cells
Human retinal progenitor cells under hypoxic conditions (5% O) 2 Concentration), the culture medium and the normoxic culture medium are cultured, cells with good growth state are taken to prepare a cell suspension with certain concentration, and 2 multiplied by 10 is added into each hole 3 Mu.l/100. Mu.l of cells per well was added to 96-well cell culture plates. Detecting proliferation capacity, doubling time, cell cycle and cell pro-apoptosis of cells and inhibiting expression change of apoptosis related genes. The results are shown in FIGS. 1 to 5.
As shown in fig. 1: in the process of constructing the culture medium, the proliferation capacity of the retinal progenitor cells is analyzed through comparison of different factor combinations, and as can be seen from fig. 1, after 72 hours of culture, the proliferation capacity of the retinal progenitor cells is obviously improved compared with that of a conventional culture medium (basic combination EGF+bFGF+Glu containing three factors), wherein the seven combination factor culture mediums provided by the invention have the strongest proliferation capacity of the cells. * Represents P < 0.05, P < 0.01, P < 0.001, P < 0.0001.
As shown in fig. 2: at 5% O 2 Retinal progenitor cells are cultured with the culture medium of the present patent at a concentrationCan be expanded to the P20 generation, whereas normoxic medium is expanded to the P10 generation only under this condition. The ordinate represents doubling time, and the abscissa represents passage times.
As shown in fig. 3: it can be seen that retinal progenitor cells cultured in the medium system of the present invention have significantly increased S phase and decreased G1 phase, indicating that most cells are in a proliferative state.
As shown in fig. 4: retinal progenitor cells at 5% O 2 The difference between the expression of pro-apoptosis genes (Caspase-3, caspase-9, caspase-7, bax) and the expression of apoptosis-inhibiting genes (Bcl 2, P21, CDK2, CDK 1) in the two culture mediums under the culture conditions can be seen that the culture mediums of the invention can obviously promote the cell to express the apoptosis-inhibiting genes, while the cell to express the apoptosis-promoting genes at low level. The culture medium of the invention can promote cell proliferation and inhibit cell apoptosis. * Represents P < 0.05, P < 0.01, P < 0.001.
As shown in fig. 5: for culture at 5% O 2 Concentration of retinal progenitor cells fluorescent staining of stem specific marker proteins, nestin: neural stem cell-specific marker protein, sox2: neural stem cell marker proteins; pax6: retinal progenitor cell-specific marker protein, ki67: cell proliferation marker proteins. From the statistical analysis results, the retinal progenitor cells cultured by the patent culture medium can highly express the retinal progenitor cell specific marker protein Pax6, and the proliferation marker Ki-67 expression is also remarkably improved. From the statistical figures, it can be seen that Nestin and Sox2 expression were slightly elevated, with no significant difference at that time.
3. Hypoxia culture system for maintaining stem property of human retina progenitor cells and related protein expression after induction
The result shows that the retinal progenitor cells cultured by the method still have the capability of differentiating into retinal component cells, and the glial cells with positive GFAP are expressed in a low level, so that retinal photoreceptor cell markers and retinal stem markers are expressed more preferentially. The results are shown in FIG. 6: as can be seen, at 5% O 2 Under the condition, the retinal progenitor cells still have the capacity of multi-directional differentiation, and under the culture system of the patent of the invention, the retinal progenitor cellsSignificantly reducing the differentiation to retinal glial cells and more to functional cells. GFAP: a retinal glial cell marker protein; map2: mature retinal neuron markers; NF: retinal neuropipe protein markers; recoverin: retinal photoreceptor cell markers; rhodopsin: retinal rhodopsin markers, how much of the rhodopsin marker reflects how much of the retinal rod cells.
4. Hypoxia culture system for promoting human retina progenitor cells to secrete neurotrophic factors
Investigation of the ability of retinal progenitor cells to secrete factors under different culture systems using ELISA kits, it can be seen from the results of the assays that cells secrete more IGFBP-3, BDNF, FGF-4, FGF-7, PDGF in the medium of the present invention. The results are shown in FIG. 7: the difference of cytokine expression under different culture medium culture conditions can be seen from the figure when retinal progenitor cells are cultured under physiological oxygen partial pressure: IGFBP-3, BDNF, FGF-4, FGF-7, PDGF expression was significantly increased.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (4)
1. A composition comprising EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and fructose-1, 6-bisphosphate;
wherein the mass ratio of EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and 1, 6-fructose diphosphate is 1:2:3000000:100:1000:500:50000000.
2. A culture medium is characterized by comprising a basal culture medium, EGF 1 ng/mL-50 ng/mL, bFGF 1 ng/mL-50 ng/mL, glutaMAX-1.3 mg/mL-30 mg/mL, rapamycin0.1 mu g/mL-1 mu g/mL, PS 48.5 mu g/mL-20 mu g/mL, UK 5099.5 mu g/mL-5 mu g/mL and fructose 1, 6-bisphosphate 100 mg/mL-1 g/mL.
3. The culture medium of claim 2, wherein the basal medium is Advanced DMFM/F12 medium.
4. A method for culturing retinal progenitor cells, comprising culturing cells and passaging the cells in a medium according to claim 2 or 3 at an oxygen concentration of 1% to 6.5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210936477.5A CN115216445B (en) | 2022-08-04 | 2022-08-04 | Culture medium and application thereof in culturing retina progenitor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210936477.5A CN115216445B (en) | 2022-08-04 | 2022-08-04 | Culture medium and application thereof in culturing retina progenitor cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115216445A CN115216445A (en) | 2022-10-21 |
| CN115216445B true CN115216445B (en) | 2023-11-14 |
Family
ID=83615069
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210936477.5A Active CN115216445B (en) | 2022-08-04 | 2022-08-04 | Culture medium and application thereof in culturing retina progenitor cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115216445B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747029A (en) * | 2012-07-30 | 2012-10-24 | 何伟 | Culture method for retina progenitor cells and culture medium thereof |
| CN106318909A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Retinal progenitor cell and preparation thereof having function of treating degenerative retinal diseases |
| CN106323845A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change |
| CN113348243A (en) * | 2018-09-27 | 2021-09-03 | 加利福尼亚大学董事会 | Method for separating and culturing human retinal progenitor cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9107897B2 (en) * | 2011-05-18 | 2015-08-18 | The Regents Of The University Of California | Methods for isolating mammalian retinal progenitor cells |
-
2022
- 2022-08-04 CN CN202210936477.5A patent/CN115216445B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747029A (en) * | 2012-07-30 | 2012-10-24 | 何伟 | Culture method for retina progenitor cells and culture medium thereof |
| CN106318909A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Retinal progenitor cell and preparation thereof having function of treating degenerative retinal diseases |
| CN106323845A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change |
| CN113348243A (en) * | 2018-09-27 | 2021-09-03 | 加利福尼亚大学董事会 | Method for separating and culturing human retinal progenitor cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115216445A (en) | 2022-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10724000B2 (en) | Small molecule based conversion of somatic cells into neural crest cells | |
| JP2009542247A (en) | Cell growth medium | |
| CN113699097B (en) | Method for preparing retinal pigment epithelial cells | |
| US11781110B2 (en) | Induction of corneal endothelial cells | |
| KR20150009441A (en) | Composition for prevention and treatment of liver fibrosis or liver cirrhosis comprising of mesenchymal stem cells derived from human embryonic stem cells as an active ingredient | |
| CN115916240A (en) | Amplification culture medium and culture method of neural cells | |
| KR101982801B1 (en) | Composition for inducing differentiation to retinal pigment epithelium | |
| JP2013123436A (en) | Medium for inducing differentiation to nerve cell and method for inducing differentiation to nerve cell | |
| JP2023551851A (en) | Generation of neural progenitor cells from embryonic stem cells or induced pluripotent stem cells | |
| CN110872576A (en) | Method for transdifferentiation of mouse fibroblasts into dopaminergic neurons | |
| Bains et al. | Recovering vision in corneal epithelial stem cell deficient eyes | |
| CN108795864B (en) | Method for obtaining retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells | |
| CN115216445B (en) | Culture medium and application thereof in culturing retina progenitor cells | |
| RU2409663C1 (en) | Method for producing dedifferentiated cells of adult retinal pigment eye epithelium | |
| JP2024120064A (en) | Generation of neural stem cell lines derived from human pluripotent stem cells | |
| KR101793722B1 (en) | Process for preparing astrocyte | |
| CN110199985B (en) | Preparation method of neuron cryopreservation liquid | |
| CN110628696A (en) | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells | |
| Wu et al. | In vitro culture and characterization of oligodendrocyte precursor cells derived from neonatal rats | |
| JPWO2020154533A5 (en) | ||
| JP2022540579A (en) | A composition for promoting stem cell proliferation containing cP1P or a pharmaceutically acceptable salt thereof as an active ingredient | |
| Lorenzo et al. | Trypsin for dissociation of limbal cells for engineering of grafts may induce DNA strand breaks in the harvested cells | |
| CN112481199B (en) | Method for preparing photosensitive cells | |
| TWI745906B (en) | Poly(allylguanidine) and the manufacture method and the use thereof | |
| Li et al. | Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |