Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof
Active Publication Date: 2010-12-15
辽宁艾米奥干细胞与再生医学研究院有限公司
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[0004] The present invention aims at the problem that the source of existing stem cells is either limited by ethics or limited in quantity, and provides a kind of human amniotic mesenchymal stem cell which is allogeneic, has a wide range of sources, turns waste into wealth, is not subject to ethical restrictions and has no immunogenicity problems Serum-free medium and culture method thereof
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Embodiment 1
[0032] A serum-free medium for human amniotic mesenchymal stem cells of the present invention, comprising:
[0033] Basal medium DMEM / F12 15.6g / L
[0034] (According to the volume of 1:1, take 15.6g and dissolve in 1000ml water, supplier GIBCO)
[0035] Human serum albumin 8.0×10 -1 %
[0036] Human transferrin 5.0×10 -1 g / L
[0037] Human insulin 8.0×10 -1 g / L
[0038] Sodium selenite 6.0×10 -6 g / L
[0039] made into an aqueous solution.
Embodiment 2
[0041] A serum-free culture method for human amniotic mesenchymal stem cells of the present invention comprises the following steps:
[0042] 1. Isolation of human amniotic mesenchymal stem cells and preparation of single cell suspension
[0043] Take the human placenta of normal full-term caesarean section fetus (male) under sterile conditions; detect hepatitis A antibody, hepatitis B virus surface antigen, hepatitis B virus surface antibody, hepatitis B virus e antigen, hepatitis B virus e Antibody, hepatitis B core antibody IgM, hepatitis C antibody, hepatitis E antibody, HIV antibody, Treponema pallidum antibody and other related infectious indicators were all negative; Amniotic membrane 5×5cm 2 , fully rinsed with PH7.2 phosphate buffer solution (PBS), put the amniotic membrane in physiological saline containing 0.1 million U / ml gentamicin and 2.5ug / ml amphotericin B, and soak for 20 minutes; Cut into pieces, add 5ml of trypsin at a final concentration of 2.5g / L per gra...
Embodiment 3
[0054] A method for the separation and serum-free culture of human amniotic mesenchymal stem cells of the present invention, comprising the following steps:
[0055] The 1st step of the present invention can also be: trypsin final concentration is 2.5g / L, and digestion time is 30-60 minutes, and number of times is 2-4 times; Score can be 5.0×10 -1 %; When digested with collagenase IV and deoxyribonuclease I, V DMEM :V F12 =The concentration of collagenase IV in the DMEM / F12 culture fluid of 1:1 adopts 0.5-1.5g / L, deoxyribonuclease 0.05-0.15g / L, and digestion time is 1-2 hour; All the other are with embodiment 1.
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Abstract
The invention relates to a human amnion mesenchymal stem cell serum-free culture medium and a culture method thereof. The culture medium is formed by adding human serum albumin, human transferrin, human insulin and sodium selenite into a DMEM/F12 basic culture medium. The culture method for the culture medium comprises the following steps of: digesting human amnion by using trypsin, then digesting the human amnion by using collagenase IV and deoxyribonuclease I, and filtering the mixture to obtain single cell suspension; and adding the human serum albumin, the transferrin, the insulin and the sodium selenite into the DMEM/F12 basic culture medium in a ratio of VDMEM to VF12 of 1:1, and putting human amnion mesenchymal stem cells in a 37 DEG C CO2 incubator with saturated humidity and volume fraction of 5 percent under the serum-free condition, wherein culture in vitro and amplification are realized by solution change and transfer of culture, potentiality of multi-direction differentiation is maintained, and the amplified cells can be induced in vitro to form cartilage cells, osteoblasts and adipocytes. The culture medium and the culture method have the characteristics of no other animal sources, wide source and no limitation of ethics.
Description
technical field [0001] The invention belongs to a mesenchymal stem cell culture medium and a culture method thereof in the field of biotechnology, and specifically relates to a human amniotic mesenchymal stem cell serum-free culture medium and a culture method thereof. Background technique [0002] In recent years, regenerative medicine has developed vigorously, and stem cell research has made major breakthroughs. It has gradually become a reality to use stem cells to generate body tissues and organs to replace the organs and tissues that have lost function in the human body to achieve therapeutic purposes. Stem cells are likely to lead to revolutionary progress in the medical field, so they have inestimable medical value and have attracted widespread attention and research all over the world. It is the most promising field for development and application in the 21st century. Regenerative medicine needs to meet three necessary conditions: (1) stem cells, that is, cells with...
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