CN111849900A - 一种自体脂肪MSCs来源的前体细胞智能培养方法 - Google Patents
一种自体脂肪MSCs来源的前体细胞智能培养方法 Download PDFInfo
- Publication number
- CN111849900A CN111849900A CN202010777919.7A CN202010777919A CN111849900A CN 111849900 A CN111849900 A CN 111849900A CN 202010777919 A CN202010777919 A CN 202010777919A CN 111849900 A CN111849900 A CN 111849900A
- Authority
- CN
- China
- Prior art keywords
- culture
- mesenchymal stem
- cells
- culture medium
- stem 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.)
- Pending
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 67
- 210000004027 cell Anatomy 0.000 title claims abstract description 54
- 239000002243 precursor Substances 0.000 title claims abstract description 24
- 238000012136 culture method Methods 0.000 title claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 48
- 230000006698 induction Effects 0.000 claims abstract description 26
- 230000001537 neural effect Effects 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 241001465754 Metazoa Species 0.000 claims abstract description 4
- 210000004369 blood Anatomy 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims abstract description 4
- 210000001124 body fluid Anatomy 0.000 claims abstract description 4
- 239000010839 body fluid Substances 0.000 claims abstract description 4
- 230000002093 peripheral effect Effects 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 4
- 210000002966 serum Anatomy 0.000 claims abstract description 4
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims abstract 4
- 238000000034 method Methods 0.000 claims description 26
- 210000001519 tissue Anatomy 0.000 claims description 10
- 230000001464 adherent effect Effects 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 9
- 239000006059 cover glass Substances 0.000 claims description 8
- 239000003102 growth factor Substances 0.000 claims description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 102000007072 Nerve Growth Factors Human genes 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 3
- 210000000577 adipose tissue Anatomy 0.000 claims description 3
- 210000001185 bone marrow Anatomy 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000003900 neurotrophic factor Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- 210000003954 umbilical cord Anatomy 0.000 claims description 3
- 102000018386 EGF Family of Proteins Human genes 0.000 claims description 2
- 108010066486 EGF Family of Proteins Proteins 0.000 claims description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 2
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 claims description 2
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims description 2
- 239000012752 auxiliary agent Substances 0.000 claims description 2
- 239000007640 basal medium Substances 0.000 claims description 2
- 239000012894 fetal calf serum Substances 0.000 claims description 2
- 239000012634 fragment Substances 0.000 claims description 2
- 210000005059 placental tissue Anatomy 0.000 claims description 2
- 230000012010 growth Effects 0.000 abstract description 7
- 230000003321 amplification Effects 0.000 abstract description 6
- 210000001178 neural stem cell Anatomy 0.000 abstract description 6
- 210000002569 neuron Anatomy 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 238000011476 stem cell transplantation Methods 0.000 abstract description 5
- 230000002159 abnormal effect Effects 0.000 abstract description 2
- 230000004936 stimulating effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 10
- 230000004069 differentiation Effects 0.000 description 7
- 238000012546 transfer Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- MRBKEAMVRSLQPH-UHFFFAOYSA-N 3-tert-butyl-4-hydroxyanisole Chemical compound COC1=CC=C(O)C(C(C)(C)C)=C1 MRBKEAMVRSLQPH-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- FINHMKGKINIASC-UHFFFAOYSA-N Tetramethylpyrazine Chemical compound CC1=NC(C)=C(C)N=C1C FINHMKGKINIASC-UHFFFAOYSA-N 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 210000004248 oligodendroglia Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 239000004255 Butylated hydroxyanisole Substances 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 241000305491 Gastrodia elata Species 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 208000001738 Nervous System Trauma Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- IPQKDIRUZHOIOM-UHFFFAOYSA-N Oroxin A Natural products OC1C(O)C(O)C(CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IPQKDIRUZHOIOM-UHFFFAOYSA-N 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 description 1
- 241000304195 Salvia miltiorrhiza Species 0.000 description 1
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- IKIIZLYTISPENI-ZFORQUDYSA-N baicalin Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IKIIZLYTISPENI-ZFORQUDYSA-N 0.000 description 1
- 229960003321 baicalin Drugs 0.000 description 1
- AQHDANHUMGXSJZ-UHFFFAOYSA-N baicalin Natural products OC1C(O)C(C(O)CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 AQHDANHUMGXSJZ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940043253 butylated hydroxyanisole Drugs 0.000 description 1
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000002304 esc Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002608 insulinlike Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 208000028412 nervous system injury Diseases 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
-
- 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
- C12N2501/105—Insulin-like growth factors [IGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (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)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Neurology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，自制培养基为无动物血清无异型蛋白的临床用培养液，且自制培养基还包括人外周静脉血或体液；培养基置于35‑36℃、饱和湿度的4‑6％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养5‑12天，即获得神经前体细胞；本发明同现有技术相比，能够大大缩短神经干细胞、神经元的生长周期，诱导细胞长势良好，符合临床干细胞移植的需要，解决了其他培养基和刺激因子所带来的诸多问题。
Description
[技术领域]
本发明涉及细胞培养技术领域，具体地说是一种自体脂肪MSCs来源的前体细胞智能培养方法。
[背景技术]
骨髓间充质干细胞(mesenchymal stem cells，MSCs)除了参与构成支持造血的微环境外，还具有强大自我复制能力以及多向分化潜能，且不同的体外诱导条件可决定其分化方向，这些诱导条件启动了决定分化方向的转录因子。而将MSCs通过静脉途径或局部注射移植至体内不同的组织时，即可在相应的组织内分化形成该类组织细胞。长期以来神经系统损伤后神经细胞的再生移植困扰着人们，胚胎神经干细胞移植可以促进神经损功能的恢复，但因伦理问题限制了其应用。
对于MSCs定向分化机制的研究仍处于探索阶段，筛选近年来神经向分化的所有文献，统计间充质干细胞分化为神经样细胞的方法。目前应用于间充质干细胞分化为神经样细胞的方法有:(1)细胞生长因子法：NGF、EGF、bFGF、低浓度TNF-α等；(2)化学诱导剂法：β-巯基乙醇、二甲基亚砜、丁化羟基苯甲醚、3-叔丁基-4-羟基茴香醚、维甲酸等；(3)生长因子与化学诱导剂联合；(4)其他：创伤性脑组织匀浆、脑组织上清液、脱细胞神经移植物、与神经细胞、神经干细胞或视网膜色素上皮细胞共培养或用接近生理状态的条件培养基、基因转染、传统中药黄芩苷、红景天苷、川芎嗪、丹参、天麻、人参等。
此外，MSCs具有来源丰富，不涉及伦理问题等优点，可能为神经系统疾病的治疗提供新途径。尽管目前已经具有相关报道MSCs具有向神经细胞分化的潜能，但诱导体系仍需要改善，系统重复性较差或者细胞诱导后活性不良，最终不能获得功能细胞。
针对上述问题，目前尚未提出有效的解决方案。
[发明内容]
本发明的目的就是要解决上述的不足而提供一种自体脂肪MSCs来源的前体细胞智能培养方法，能够大大缩短神经干细胞、神经元的生长周期，诱导细胞长势良好，符合临床干细胞移植的需要，解决了其他培养基和刺激因子所带来的诸多问题。
为实现上述目的设计一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于35-36℃、饱和湿度的4-6％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养5-12天，即获得神经前体细胞。
进一步地，所述间充质干细胞包括骨髓、脐带、胎盘、脂肪组织来源的间充质干细胞中的一种。
进一步地，所述自制培养基为无动物血清无异型蛋白的临床用培养液，且自制培养基还包括人外周静脉血或体液。
进一步地，所述诱导培养基为添加有生长因子的基础培养基，生长因子包括表皮生长因子、碱性成纤维生长因子、胰岛素样生长因子-1和神经营养因子3。
进一步地，所述传代培养为按常规贴壁细胞传代方法传代，所述常规贴壁细胞传代方法传代过程中的接种密度为1.5×104/cm2，其培养体系为含1％OPCs培养添加剂的OPCs无血清基础培养基，培养表面为经OPCs培养表面包被液包被的培养表面。
进一步地，待间充质干细胞传代至第3代时，采用胰蛋白酶消化贴壁细胞，离心，用MSC培养液重悬，即得到细胞悬液；再调整细胞悬液间充质干细胞的细胞浓度，将细胞悬液滴于盖玻片上，盖玻片周边滴加基础培养基覆盖，细胞即在盖破片中生长，得到第4代细胞；待细胞生长覆盖玻片表面62％～66％时，倾去MSC培养液，加入诱导培养基，在36℃、5％CO2条件下培养，培养8天，即获得神经前体细胞。
进一步地，所述基础培养基为含2％胎牛血清和1％N2辅剂的低糖DMEM液。
本发明同现有技术相比，完成了自体脂肪MSCs诱导分化为临床用神经前体细胞，可以大大缩短神经干细胞、神经元的生长周期，诱导细胞长势良好，其提供的微环境更利于MSCs化为神经样细胞,更符合临床干细胞移植的需要，解决了其他培养基和刺激因子所带来的诸多问题；此外，本发明避免了采用ESCs来源的少突胶质细胞细胞制剂而产生的的严重的伦理问题以及体内移植致瘤的严重临床风险，也避免了采用iPSCs来源的少突胶质细胞制剂产生的外源基因转染和更严重的体内移植致瘤性的严重临床风险，且操作简单，OPCs收率高。
[具体实施方式]
下面结合具体实施例对本发明作以下进一步说明：
实施例1
一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，自制培养基为无动物血清无异型蛋白的临床用培养液，且自制培养基还包括人外周静脉血或体液；培养基置于36℃、饱和湿度的4％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养5-12天，即获得神经前体细胞。
实施例2
一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于35℃、饱和湿度的4％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养10天，即获得神经前体细胞。其中，间充质干细胞包括骨髓、脐带、胎盘、脂肪组织来源的间充质干细胞中的一种。
实施例3
一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于36℃、饱和湿度的6％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养基为添加有生长因子的基础培养基，生长因子包括表皮生长因子、碱性成纤维生长因子、胰岛素样生长因子-1和神经营养因子3；诱导培养12天，即获得神经前体细胞。
实施例4
一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于35℃、饱和湿度的6％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养11天，即获得神经前体细胞；其中，传代培养为按常规贴壁细胞传代方法传代，常规贴壁细胞传代方法传代过程中的接种密度为1.5×104/cm2，其培养体系为含1％OPCs培养添加剂的OPCs无血清基础培养基，培养表面为经OPCs培养表面包被液包被的培养表面。
实施例5
一种自体脂肪MSCs来源的前体细胞智能培养方法，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于35.5℃、饱和湿度的5％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养10天，即获得神经前体细胞；其中，待间充质干细胞传代至第3代时，采用胰蛋白酶消化贴壁细胞，离心，用MSC培养液重悬，即得到细胞悬液；再调整细胞悬液间充质干细胞的细胞浓度，将细胞悬液滴于盖玻片上，盖玻片周边滴加基础培养基覆盖，基础培养基为含2％胎牛血清和1％N2辅剂的低糖DMEM液，细胞即在盖破片中生长，得到第4代细胞；待细胞生长覆盖玻片表面62％～66％时，倾去MSC培养液，加入诱导培养基，在36℃、5％CO2条件下培养，培养8天，即获得神经前体细胞。
本发明并不受上述实施方式的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (7)
1.一种自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于，包括以下步骤：先从人组织中分离出间充质干细胞,将间充质干细胞置于自制培养基内进行间充质干细胞的扩增，培养基置于35-36℃、饱和湿度的4-6％CO2培养箱培养，定期换液经传代培养，取稳定传代的自体脂肪充质干细胞转入诱导培养基，诱导培养5-12天，即获得神经前体细胞。
2.如权利要求1所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：所述间充质干细胞包括骨髓、脐带、胎盘、脂肪组织来源的间充质干细胞中的一种。
3.如权利要求1所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：所述自制培养基为无动物血清无异型蛋白的临床用培养液，且自制培养基还包括人外周静脉血或体液。
4.如权利要求1所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：所述诱导培养基为添加有生长因子的基础培养基，生长因子包括表皮生长因子、碱性成纤维生长因子、胰岛素样生长因子-1和神经营养因子3。
5.如权利要求1所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：所述传代培养为按常规贴壁细胞传代方法传代，所述常规贴壁细胞传代方法传代过程中的接种密度为1.5×104/cm2，其培养体系为含1％OPCs培养添加剂的OPCs无血清基础培养基，培养表面为经OPCs培养表面包被液包被的培养表面。
6.如权利要求1所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：待间充质干细胞传代至第3代时，采用胰蛋白酶消化贴壁细胞，离心，用MSC培养液重悬，即得到细胞悬液；再调整细胞悬液间充质干细胞的细胞浓度，将细胞悬液滴于盖玻片上，盖玻片周边滴加基础培养基覆盖，细胞即在盖破片中生长，得到第4代细胞；待细胞生长覆盖玻片表面62％～66％时，倾去MSC培养液，加入诱导培养基，在36℃、5％CO2条件下培养，培养8天，即获得神经前体细胞。
7.如权利要求6所述的自体脂肪MSCs来源的前体细胞智能培养方法，其特征在于：所述基础培养基为含2％胎牛血清和1％N2辅剂的低糖DMEM液。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010777919.7A CN111849900A (zh) | 2020-08-05 | 2020-08-05 | 一种自体脂肪MSCs来源的前体细胞智能培养方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010777919.7A CN111849900A (zh) | 2020-08-05 | 2020-08-05 | 一种自体脂肪MSCs来源的前体细胞智能培养方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111849900A true CN111849900A (zh) | 2020-10-30 |
Family
ID=72971364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010777919.7A Pending CN111849900A (zh) | 2020-08-05 | 2020-08-05 | 一种自体脂肪MSCs来源的前体细胞智能培养方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111849900A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852737A (zh) * | 2021-01-14 | 2021-05-28 | 香港再生医学有限公司 | 一种提高msc分化为神经前体细胞产量的方法 |
-
2020
- 2020-08-05 CN CN202010777919.7A patent/CN111849900A/zh active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852737A (zh) * | 2021-01-14 | 2021-05-28 | 香港再生医学有限公司 | 一种提高msc分化为神经前体细胞产量的方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100449141B1 (ko) | 간엽 간세포를 신경세포로 분화시키는 방법 | |
| KR102573180B1 (ko) | 분화 배지 및 희소돌기아교세포 전구체를 제조하는 방법 | |
| CN107254443B (zh) | 一种促进骨髓间充质干细胞向神经元分化的诱导培养基和诱导方法 | |
| CN111154718B (zh) | 一种人间充质干细胞体外快速扩增用添加剂及扩增方法 | |
| CN114480273A (zh) | 用于获得间充质干细胞及其外泌体的培养基及其制备方法 | |
| Yang et al. | Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo | |
| CN113444689B (zh) | 人源自体脂肪干细胞与脐带间充质干细胞共培养诱导分化成神经样干细胞的方法 | |
| CN113151165B (zh) | 一种人脐带间充质干细胞扩增用培养基及培养方法 | |
| CN107384864B (zh) | 将脐带间充质干细胞诱导成神经干细胞的细胞培养液及其使用方法 | |
| CN106282113B (zh) | 一种利用无血清培养基通过转分化获得神经细胞的方法 | |
| CN111849900A (zh) | 一种自体脂肪MSCs来源的前体细胞智能培养方法 | |
| US10760051B2 (en) | Process for preparing astrocytes | |
| US20130108589A1 (en) | Method of Producing Neurons from Stem Cells, the Neurons and Uses Thereof | |
| CN116694567A (zh) | 将人诱导性多能干细胞间接诱导为间充质干细胞的培养液和方法 | |
| CN107164325B (zh) | MSCs来源的少突胶质细胞的制备方法及试剂盒 | |
| CN110093309B (zh) | 一种诱导成纤维细胞转分化为脂肪细胞的方法 | |
| CN109385399A (zh) | 一种羊水间充质干细胞分化为神经干细胞的方法 | |
| CN112608904B (zh) | 一种高效快速重编程体细胞为神经干细胞的方法及其应用 | |
| CN111304167B (zh) | 人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用 | |
| CN112708598A (zh) | 无血清成分的神经前体细胞培养基及其配制方法和应用 | |
| US20150147409A1 (en) | Adipose stromal vascular fraction-conditioned medium | |
| CN104774807A (zh) | 将脐带间充质干细胞诱导分化成少突胶质细胞的方法 | |
| CN111235110A (zh) | 一种神经干细胞的体外培养方法 | |
| CN110885787A (zh) | 脐带间充质干细胞向多巴胺能神经元分化的方法 | |
| CN112852737A (zh) | 一种提高msc分化为神经前体细胞产量的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201030 |
|
| WD01 | Invention patent application deemed withdrawn after publication |