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Method for separating sub-totipotent stem cells from human placenta decidua

A technology for subpluripotent stem cells and human placenta, which is applied in the field of isolating subpluripotent stem cells from the decidua of human placenta, can solve the problems of affecting the enzymolysis effect, subpluripotent stem cells, miscellaneous cells, susceptible to infection of miscellaneous bacteria, etc., so as to improve the separation quality and avoid the Miscellaneous bacteria infection, the effect of ensuring the production efficiency

Pending Publication Date: 2020-08-07
辽宁和泽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The prior art has the following deficiencies: in the existing method for separating placental subtotipotent stem cells, the number of times of disinfection and washing is only one during the decidua peeling process, which is easy to infect miscellaneous bacteria and affects the effect of enzymatic hydrolysis. The human placental subtotipotent stem cells finally obtained There are many miscellaneous cells in the medium, resulting in the production of sub-totipotent stem cells with miscellaneous cells, which affects the production quality of sub-totipotent stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A method for isolating subpotent stem cells from human placental decidua, the specific operation method is as follows:

[0022] Step 1: Tissue extraction: select fresh placental tissue, disinfect and wash, cut the decidua to a size of 3 cm and put it into a centrifuge tube;

[0023] Step 2: Pre-digestion treatment: Add a trypsin solution with a mass volume ratio of 0.1% g / ml to the centrifuge tube in Step 1, let it stand for 20 minutes for pre-digestion, then take out the decidua, and perform disinfection and washing again;

[0024] Step 3: Digestion of decidua: Cut the pre-digested decidua in step 2 into 2mm size and put them in a centrifuge tube, add trypsin solution with a mass volume ratio of 0.1%g / ml and 0.2%g / ml to the centrifuge tube ml of collagenase solution, let stand for 30min to digest, and obtain the decidua digestive juice;

[0025] Step 4: Centrifugal purification: put the centrifuge tube loaded with the decidual digestive fluid in step 3 into a centrifu...

Embodiment 2

[0034] A method for isolating subpotent stem cells from human placental decidua, the specific operation method is as follows:

[0035] Step 1: Tissue extraction: select fresh placental tissue, disinfect and wash, cut the decidua to a size of 3 cm and put it into a centrifuge tube;

[0036] Step 2: Pre-digestion treatment: Add a trypsin solution with a mass volume ratio of 0.3% g / ml to the centrifuge tube in Step 1, let it stand for 20 minutes for pre-digestion, then take out the decidua, and perform disinfection and washing again;

[0037] Step 3: Digestion of decidua: Cut the pre-digested decidua in step 2 into pieces of 2mm size and put them in a centrifuge tube, add trypsin solution with a mass volume ratio of 0.3%g / ml and 0.3%g / ml to the centrifuge tube ml of collagenase solution, let stand for 30min to digest, and obtain the decidua digestive juice;

[0038] Step 4: Centrifugal purification: put the centrifuge tube loaded with the decidual digestive fluid in step 3 into ...

Embodiment 3

[0047] A method for isolating subpotent stem cells from human placental decidua, the specific operation method is as follows:

[0048] Step 1: Tissue extraction: select fresh placental tissue, disinfect and wash, cut the decidua to a size of 3 cm and put it into a centrifuge tube;

[0049] Step 2: Pre-digestion treatment: Add a trypsin solution with a mass volume ratio of 0.2% g / ml to the centrifuge tube in Step 1, let it stand for 15 minutes for pre-digestion, then take out the decidua, and perform disinfection and washing again;

[0050] Step 3: Digestion of decidua: Cut the pre-digested decidua in step 2 into pieces of 2mm size and put them in a centrifuge tube, add trypsin solution with a mass volume ratio of 0.2% g / ml and 0.25% g / ml to the centrifuge tube ml of collagenase solution, let stand for 25min to digest, and obtain the decidua digestive juice;

[0051] Step 4: Centrifugal purification: put the centrifuge tube loaded with the decidual digestive fluid in step 3 in...

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Abstract

The invention discloses a method for separating sub-totipotent stem cells from human placenta decidua. The invention particularly relates to the technical field of biomedicine. The method comprises the following steps: tissue extraction, pre-digestion treatment, decidua digestion, centrifugal purification, dilution and filtration, sub-totipotent stem cell separation liquid preparation and sub-totipotent stem cell culture preparation. The method utilizes two enzymolysis processes. Enzymolysis is carried out for the first time to remove miscellaneous cells on the amnion, enzymolysis is carried out on the placenta cells for the second time, a disinfecting and washing procedure is carried out once before each enzymolysis operation so that infectious microbe infections are avoided, human sub-totipotent stem cells with high purity are obtained, when the cell suspension is prepared, one-time centrifugal operation is firstly carried out in the last-time enzymolysis operation, and compared withthe traditional two-step centrifugal operation, the third operation is used, so that the sub-totipotent stem cell separation quality is improved, and the preparation efficiency of the human placentasub-totipotent stem cell is ensured.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for isolating subtotipotent stem cells from human placental decidua. Background technique [0002] Stem cells can be roughly classified into totipotent stem cells, pluripotent stem cells and tissue stem cells according to their differentiation potential. Whether adult tissues persist during human development and post-embryonic near-totipotent stem cell subsets. Professor Zhao Chunhua, a "Changjiang Scholar" of the Chinese Academy of Medical Sciences, first proposed the concept of "human subtotipotent stem cell theory", and obtained primitive stem cells from human fetal tissues such as bone marrow, pancreas, lung, skin, skeletal muscle, and fat before international counterparts. Stem cell population, the placenta is a temporary organ, which is the direct connection hub between the mother and the fetus. It is very important to the development and growth of the fetus t...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0607C12N2509/00
Inventor 肖强郭龙浩孙涛祁航张栋郭佳伟王晓磊宁志英高娜娜马宏梅徐玮琦
Owner 辽宁和泽生物科技有限公司
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