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Culture and cryopreservation method of amniotic mesenchymal stem cells

A technology of amniotic mesenchymal stem cells and culture methods, applied in the field of culture and cryopreservation of amniotic mesenchymal stem cells, can solve the problems of long operation time, low yield, complicated process, etc., achieve good repeatability and increase yield , the effect of effective separation

Active Publication Date: 2020-05-12
赛瑞诺(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing method for extracting amniotic mesenchymal stem cells is to separate the amniotic membrane from the chorion, digest hAEC from the basement membrane with different concentrations of trypsin, neutral protease or other digestive enzymes for different times, and then use collagenase The process of digesting and separating mostly hAMSC is complicated, the operation time is long, and the yield is not high

Method used

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  • Culture and cryopreservation method of amniotic mesenchymal stem cells
  • Culture and cryopreservation method of amniotic mesenchymal stem cells
  • Culture and cryopreservation method of amniotic mesenchymal stem cells

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Embodiment 1

[0035] This embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

[0036](1) Separation of amniotic membrane tissue: Soak the taken placental tissue in PBS buffer solution, sample blood water as the sample source microbial detection sample, then soak the placental tissue for 2 minutes, and then soak the placental tissue in 75% alcohol for about 1 minute, and use After washing the placental tissue repeatedly with normal saline, the amniotic tissue was gently torn off with tissue forceps, soaked in PBS, and used for the separation of amniotic mesenchymal stem cells, and the normal saline that washed the placenta was finally sampled to detect microorganisms;

[0037] (2) Isolation of amniotic membrane mesenchymal stem cells: cut the amniotic membrane tissue into 1-2mm 2 Add small pieces into a 50mL sterile centrifuge tube, and add an equal volume of mixed enzyme digestion solution (dissolve neutral protease, deoxyribonuclease...

Embodiment 2

[0045] This embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

[0046] (1) Separation of amniotic membrane tissue: Soak the taken placental tissue in PBS buffer, sample blood water as the sample source microbial detection sample, then soak the placental tissue for 3 minutes, then soak the placental tissue with 75% alcohol for about 2 minutes, and use After washing the placental tissue repeatedly with normal saline, the amniotic tissue was gently torn off with tissue forceps, soaked in PBS, and used for the separation of amniotic mesenchymal stem cells, and the normal saline that washed the placenta was finally sampled to detect microorganisms;

[0047] (2) Isolation of amniotic membrane mesenchymal stem cells: cut the amniotic membrane tissue into 1-2mm 2 Add small pieces into a 50mL sterile centrifuge tube, and add 0.8 times the volume of amniotic membrane tissue mixed enzyme digestion solution (dissolve neutral protea...

Embodiment 3

[0055] This embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

[0056] (1) Separation of amniotic membrane tissue: Soak the taken placental tissue in PBS buffer solution, take a sample of blood and water as the sample source microbial detection sample, then soak the placental tissue for 3 minutes, and then soak the placental tissue with 75% alcohol for about 2 minutes, and use After repeatedly washing the placental tissue with normal saline, gently tear off the amniotic membrane tissue with tissue forceps, soak it in PBS, and use it for the separation of amniotic mesenchymal stem cells, and take samples of the normal saline that rinses the placenta at the end to detect microorganisms;

[0057] (2) Isolation of amniotic membrane mesenchymal stem cells: cut the amniotic membrane tissue into 1-2mm 2 Add small pieces into a 50mL sterile centrifuge tube, and add 1.2 times the volume of amniotic membrane tissue mixed enzyme d...

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Abstract

The invention relates to a culture and cryopreservation method of amniotic mesenchymal stem cells. The culture method comprises amniotic tissue separation, amniotic mesenchymal stem cell separation, P0 generation amniotic mesenchymal stem cell culture and amplification culture. According to the culture method of the amniotic mesenchymal stem cells, at a separating stage of the amniotic mesenchymalstem cells, a special mixed enzyme digestive juice system (the final concentrations of components in the digestive juice are as follows: 1.5-2U / mL of neutral protease, 0.5mg / mL of deoxyribonuclease Iand 1mg / mL of collagenase IV) is adopted for digestion, so that the amniotic mesenchymal stem cells can be more effectively separated from amniotic tissues, and then the yield of P0 generation cellsis obviously improved. Compared with conventional two-step digestion, the culture method provided by the invention has the advantages that a one-step digestion method is adopted, operation is more convenient, and the cultured amniotic mesenchymal stem cells have good activity, high yield, high purity and good repeatability.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a method for culturing and freezing amniotic mesenchymal stem cells. Background technique [0002] In recent years, with the in-depth study of the functional characteristics of mesenchymal stem cells, mesenchymal stem cells have become a research hotspot in cell replacement therapy. Human amniotic mesenchymal stem cells have the same characteristics as the widely used bone marrow mesenchymal stem cells, such as multi-directional differentiation potential, immune regulation, and hematopoietic support. Advantage. It has a high survival rate and a long survival time in various tissues and organs. Amniotic mesenchymal stem cells derived from placental amniotic tissue are adult stem cells with obvious plasticity and multi-lineage differentiation potential. Under the regulation of different growth factors, they can differentiate into different tissue cell types ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A01N1/02
CPCC12N5/0668A01N1/0284A01N1/021C12N2509/00C12N2500/90C12N2500/84C12N2501/11C12N2501/115C12N2500/32
Inventor 李哲浩
Owner 赛瑞诺(北京)生物科技有限公司
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