CN106190964A - A kind of mesenchymal stem cell serum-free culture medium - Google Patents

A kind of mesenchymal stem cell serum-free culture medium Download PDF

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CN106190964A
CN106190964A CN201610560103.2A CN201610560103A CN106190964A CN 106190964 A CN106190964 A CN 106190964A CN 201610560103 A CN201610560103 A CN 201610560103A CN 106190964 A CN106190964 A CN 106190964A
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culture medium
stem cell
mesenchymal stem
vitamin
free culture
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CN106190964B (en
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李志远
程娜
王飞
林佐贤
叶威
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Guangzhou Institute of Biomedicine and Health of CAS
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Abstract

The invention discloses a kind of mesenchymal stem cell serum-free culture medium.The culture medium of the present invention is culture medium based on L DMEM, DMEM F12 or α MEM culture medium, also includes following adding ingredient: Fibronectin, laminin，LN, transferrins, trypsin, aprotinin, growth hormone, insulin, hydrocortisone, dexamethasone, bFGF, EGF, B27, IGF I, IGF II, choline, linoleic acid, sodium selenite, phosphoethanolamine, glutathion, vitamin C, vitamin E, vitamin B12 and biotin.This medium component determines, quality controllable, cultivates growth of mesenchymal stem cells with it normal；Still maintain " dryness " of mescenchymal stem cell and be divided into the ability of the cell types such as osteoblast, chondrocyte, adipose cell and neuron after repeatedly passing on time.

Description

A kind of mesenchymal stem cell serum-free culture medium
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of mesenchymal stem cell serum-free culture medium.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) be mesoderma origin have height self renewal Ability and the pluripotent stem cell of multi-lineage potential, separate from the Various Tissues such as muscle, fat, periodontal matter, umbilical blood, umbilical cord Obtain, osteoblast, chondrocyte, adipose cell and neuron etc. can be divided under given conditions.Wide material sources and not depositing In problems such as ethics, it is careful that MSC is proved to be autologous and variant cell transplanting, treatment ideal kind in many-side Born of the same parents.Carry out multinomial MSC clinical application research both at home and abroad at present, relate to the MSC type in multiple source and multiple disease type.
The medium component used by stem cell medicine preparation of clinical practice should have enough purity and meet aseptic, without causing Sick microorganism and endotoxic quality standard, receptor should be had no adverse effects by the culture medium of residual；Normally give birth to meeting stem cell In the case of length, do not affect stem cell " dryness " and differentiation capability.Containing substantial amounts of aminoacid, nucleoside, albumen in serum The micro constitutents such as matter, hormone, lipid, the content of these compositions and specifically acting on still without determining completely.And serum often quilt Virus is polluted, possibly together with possible cytostatic materials such as PDGFs.Therefore, composition determines and quality controllable Serum-free medium be clinical practice MSC cultivate inevitable choice.
Summary of the invention
It is an object of the invention to provide a kind of mesenchymal stem cell serum-free culture medium, this culture medium does not contains serum, composition Determine, meet aseptic, without pathogenic microorganism and endotoxic quality standard, mescenchymal stem cell normal growth can be met, and Do not affect " dryness " and the differentiation capability of mescenchymal stem cell；Receptor should be had no adverse effects by the culture medium of residual.
The mesenchymal stem cell serum-free culture medium of the present invention, including basal medium and adding ingredient, adding ingredient and Its content is as follows:
Preferably, described basal medium is L-DMEM, DMEM-F12 or α-MEM culture medium.
The mesenchymal stem cell serum-free culture medium composition that the present invention provides determines, quality controllable, cultivates mesenchyme with it Stem cell growth is normal；Still maintain " dryness " of mescenchymal stem cell and be divided into osteoblast, cartilage after repeatedly passing on time The ability of the cell types such as cell, adipose cell and neuron.
Accompanying drawing explanation
Fig. 1 is umbilical cord mesenchymal stem cells Secondary Culture to the upgrowth situation in second day the 3rd generation.
Fig. 2 is umbilical cord mesenchymal stem cells Secondary Culture to the upgrowth situation in the 3rd day the 3rd generation.
Detailed description of the invention
Following example are to further illustrate the present invention rather than limitation of the present invention.
Fibronectin used in the present invention is purchased from Gene Operation, and laminin，LN is purchased from R&D, transferrins, Trypsin, aprotinin, growth hormone, insulin, hydrocortisone, dexamethasone, choline, linoleic acid, sodium selenite, phosphoric acid Ethanolamine, vitamin C, vitamin E, vitamin B12 and biotin be purchased from Sigma, bFGF, EGF, B27, IGF-I, IGF-II and Glutathion is purchased from Gibco.L-DMEM, DMEM-F12 or α-MEM culture medium used is purchased from Gibco.
Embodiment 1:
A kind of mesenchymal stem cell serum-free culture medium of the present embodiment, culture medium based on L-DMEM culture medium, also As follows including following adding ingredient, adding ingredient and content (final concentration) thereof: Fibronectin 25 μ g/mL, laminin，LN 25 μ g/ ML, transferrins 0.2ng/mL, trypsin 10 μ g/mL, aprotinin 0.1mg/mL, growth hormone 20ng/mL, insulin 1U/ ML, hydrocortisone 0.5 μ g/mL, dexamethasone 0.2nM, bFGF 20ng/mL, EGF 30ng/mL, B27 volume fraction 1%, IGF-I 10ng/mL, IGF-II 30ng/mL, choline 30nM, linoleic acid 10nM, sodium selenite 30nM, phosphoethanolamine 50nM, Glutathion 2.5 μ g/mL, vitamin C 50 μ g/mL, vitamin E 30 μ g/mL, vitamin B12 10 μMs, biotin 20 μMs.
Its concrete compound method is to add above-mentioned interpolation composition in L-DMEM culture medium, and makes it final concentration of above-mentioned dense Degree, thus obtains mesenchymal stem cell serum-free culture medium.
Embodiment 2:
A kind of mesenchymal stem cell serum-free culture medium of the present embodiment, culture medium based on DMEM-F12 culture medium, Also include that following adding ingredient, adding ingredient and content (final concentration) thereof are as follows: laminin，LN 40 μ g/mL, transferrins 2ng/mL, trypsin 20 μ g/mL, aprotinin 0.1mg/mL, growth hormone 30ng/mL, insulin 10U/mL, hydrocortisone 1 μ g/mL, dexamethasone 0.3nM, bFGF 40ng/mL, EGF 20ng/mL, B27 volume fraction 2%, IGF-I 10ng/mL, IGF-II 20ng/mL, choline 30nM, linoleic acid 20nM, sodium selenite 15nM, phosphoethanolamine 30nM, glutathion 5 μ g/ ML, vitamin C 50 μ g/mL, vitamin E 30 μ g/mL, vitamin B12 20 μMs, biotin 30 μMs.
Its concrete compound method is to add above-mentioned interpolation composition in DMEM-F12 culture medium, and makes it final concentration of above-mentioned Concentration, thus obtains mesenchymal stem cell serum-free culture medium.
Embodiment 3:
A kind of mesenchymal stem cell serum-free culture medium of the present embodiment, culture medium based on α-MEM culture medium, also wrap Include following adding ingredient, adding ingredient and content (final concentration) thereof as follows: laminin，LN 50 μ g/mL, transferrins 0.2ng/ ML, trypsin 30 μ g/mL, aprotinin 0.1mg/mL, growth hormone 30ng/mL, insulin 6U/mL, hydrocortisone 1 μ g/ ML, dexamethasone 0.3nM, bFGF 30ng/mL, EGF 30ng/mL, B27 volume fraction 3%, IGF-I 10ng/mL, IGF-II 10ng/mL, choline 20nM, linoleic acid 20nM, sodium selenite 20nM, phosphoethanolamine 20nM, glutathion 2.5 μ g/mL, dimension are raw Element C 50 μ g/mL, vitamin E 30 μ g/mL, vitamin B12 20 μMs, biotin 30 μMs.
Its concrete compound method is to add above-mentioned interpolation composition in α-MEM culture medium, and makes it final concentration of above-mentioned dense Degree, thus obtains mesenchymal stem cell serum-free culture medium.
Embodiment 4: the mesenchymal stem cell serum-free culture medium of embodiment 1 preparation cultivates MSC cell
(1) MSC method for resuscitation
From liquid nitrogen container, take out umbilical cord mesenchymal stem cells (UMSC cell) cryopreservation tube, be directly immersed in 37 DEG C of water-baths, And shake makes it melt as early as possible frequently.After dissolving, UMSC cell is transferred in centrifuge tube, and thin toward dropping UMSC in centrifuge tube The mesenchymal stem cell serum-free culture medium that 10 times of born of the same parents are prepared with the embodiment 1 of upper volume, mixing.Centrifugal, remove supernatant, add The mesenchymal stem cell serum-free culture medium of embodiment 1 preparation, counting, adjust cell density, be seeded to Tissue Culture Dish or training Support in bottle, 37 DEG C, CO2Incubator quiescent culture.
(2) MSC propagating method
MSC cell reaches 90% when converging, and sucking-off culture medium, with PBS, adds appropriate 0.25% trypsinization 2-5min, inhales the mesenchymal stem cell serum-free culture medium abandoning trypsin addition embodiment 1 preparation, is dispelled by cell, centrifugal, Remove supernatant, add the mesenchymal stem cell serum-free culture medium of embodiment 1 preparation, counting, adjust cell density, be seeded to cell In culture dish or culture bottle, 37 DEG C, CO2Incubator quiescent culture.Fig. 1 and Fig. 2 be umbilical cord mesenchymal stem cells Secondary Culture extremely 3rd second day generation and the upgrowth situation of the 3rd day.
After carrying out and so forth being passaged to obtain the 20th generation umbilical cord mesenchymal stem cells, dry thin to the 20th generation umbilical cord mesenchyma Born of the same parents carry out stem cell surface mark mensuration, to the detection of osteoblast differentiation potential, to the detection of Adipocyte Differentiation potential, to The detection of neuron differentiation potential.Concrete detection method includes: stem cell surface mark measures, to osteoblast differentiation potential Detection and detection to Adipocyte Differentiation potential according to document (Wang Juan, Lu Yan, He Dongmei. human umbilical cord mesenchymal stem cells In-vitro separation, purification and qualification. Ji'nan University's journal: medicine, 2009,30:367-372) in method carry out.Result table Bright, carrying out in the case of 20 generations passed on, the mescenchymal stem cell of separation and Culture also have stem cell specific surfaces mark, To osteoblast differentiation potential, to Adipocyte Differentiation potential.As can be seen here, can to perform well in mesenchyme dry thin for the present invention The In vitro culture of born of the same parents, and repeatedly pass on rear cell and do not lose differentiation potential yet.
Embodiment 5:
The present embodiment is substantially the same manner as Example 4, and mesenchymal stem cell serum-free culture medium the most used is embodiment 2 Or the mesenchymal stem cell serum-free culture medium of embodiment 3 preparation, find after testing according to the method for embodiment 4, implement It is normal that the mesenchymal stem cell serum-free culture medium of example 2 or embodiment 3 preparation also can cultivate growth of mesenchymal stem cells；Repeatedly pass Still maintain " dryness " of mescenchymal stem cell after generation and be divided into osteoblast, chondrocyte, adipose cell and neuron Ability Deng cell type.

Claims (2)

1. a mesenchymal stem cell serum-free culture medium, it is characterised in that include basal medium and adding ingredient, add into Divide and content be as follows:
Mesenchymal stem cell serum-free culture medium the most according to claim 1, it is characterised in that described basal medium For L-DMEM, DMEM-F12 or α-MEM culture medium.
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CN106916784A (en) * 2017-05-04 2017-07-04 刘力伟 A kind of serum free medium and its application for human mesenchymal stem cell
CN107557331A (en) * 2017-09-15 2018-01-09 上海莱馥医疗科技有限公司 A kind of method for separating and cultivating human adipose-derived stem cell
CN107663514A (en) * 2017-06-29 2018-02-06 刘劼 A kind of serum free medium of human umbilical cord mesenchymal stem cells
CN110343660A (en) * 2018-04-08 2019-10-18 生物角（厦门）科技有限公司 A kind of mesenchymal stem cell serum-free culture medium composition
CN111621473A (en) * 2019-04-03 2020-09-04 成熙(上海)生物科技有限公司 Preparation method of novel human adipose-derived stem cell preparation
CN112029717A (en) * 2020-09-17 2020-12-04 英科博雅基因科技（天津）有限公司 Serum-free in vitro domestication culture of human mesenchymal stem cells
CN112063580A (en) * 2019-06-10 2020-12-11 西安臻瑞生物科技有限公司 Culture inducing liquid for umbilical cord and placenta mesenchymal stem cells
CN112391340A (en) * 2020-11-04 2021-02-23 深圳盛皓生物科技有限公司 Mesenchymal stem cell culture medium
CN112725269A (en) * 2021-02-26 2021-04-30 江苏普瑞康生物医药科技有限公司 Serum-free medium additive and preparation method and application thereof
CN112852719A (en) * 2019-11-12 2021-05-28 武汉济源高科技有限公司 Clinical-grade stem cell serum-free culture medium
CN113832099A (en) * 2021-10-13 2021-12-24 浙江领蔚生物技术有限公司 Mesenchymal stem cell preparation for preparing medicine for treating rheumatoid arthritis
CN114045258A (en) * 2021-10-21 2022-02-15 辽宁盛京干细胞科技有限公司 Serum-free medium for mesenchymal stem cell culture and application thereof
CN115433712A (en) * 2022-10-25 2022-12-06 成都旨蓄科技有限公司 Umbilical cord mesenchymal stem cell proliferation culture medium and culture method

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Cited By (18)

* Cited by examiner, † Cited by third party
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CN106916784A (en) * 2017-05-04 2017-07-04 刘力伟 A kind of serum free medium and its application for human mesenchymal stem cell
CN107663514A (en) * 2017-06-29 2018-02-06 刘劼 A kind of serum free medium of human umbilical cord mesenchymal stem cells
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