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Method for isolating mesenchymal stem cells from placental vessel and used digestive enzyme composition

A technology of mesenchymal stem cells and digestive enzymes, applied in the field of isolating stem cells, can solve problems such as isolating mesenchymal stem cells

Active Publication Date: 2019-01-18
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] It is expected to have a new source of human mesenchymal stem cells, such as isolating mesenchymal stem cells from placental blood vessels, but there is no method for isolating mesenchymal stem cells from placental blood vessels in the prior art

Method used

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  • Method for isolating mesenchymal stem cells from placental vessel and used digestive enzyme composition
  • Method for isolating mesenchymal stem cells from placental vessel and used digestive enzyme composition
  • Method for isolating mesenchymal stem cells from placental vessel and used digestive enzyme composition

Examples

Experimental program
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Effect test

Embodiment 1

[0086] Embodiment 1. Isolation, subculture, and cryopreservation of placental MSCs

[0087] (1) Within four hours after delivery, soak the placenta in 75% alcohol for 30 seconds under aseptic conditions, and then wash it twice with PBS;

[0088] (2) Strip the placental blood vessels from the umbilical cord root of the placenta, and squeeze the blood vessels with surgical forceps to remove blood stains;

[0089] (3) Cut the blood vessels into pieces of about 1.5 mm^3, wash them with PBS, and then filter through a 300-mesh filter to remove residual blood stains to obtain placental vascular tissue (5.8 g of placental vascular tissue was obtained from one placenta in this embodiment);

[0090] (4) Add mixed enzyme solution (including: 0.2% collagenase II, 0.15% collagenase IV, 0.1% deoxyribonuclease I in PBS buffer) to digest for 1 hour;

[0091] (5) Add serum to stop the digestion, then filter through a 300-mesh filter, collect the tissue fluid, wash with PBS, and combine the ...

Embodiment 2

[0096] Embodiment 2, the isolation of placental MSC, subculture, cryopreservation

[0097] (1) Within four hours after delivery, soak the placenta in 75% alcohol for 30 seconds under aseptic conditions, and then wash it twice with PBS;

[0098] (2) Strip the placental blood vessels from the umbilical cord root of the placenta, and squeeze the blood vessels with surgical forceps to remove blood stains;

[0099] (3) Cut the blood vessels into 1mm^3 fragments, wash them with PBS, and then filter through a 300-mesh filter to remove residual blood stains to obtain placental vascular tissue (5.5 g of placental vascular tissue was obtained from one placenta in this embodiment);

[0100] (4) Add mixed enzyme solution (including: 0.3% collagenase II, 0.1% collagenase IV, 0.15% deoxyribonuclease I in PBS buffer) to digest for 0.5h;

[0101] (5) Add serum to stop the digestion, then filter through a 300-mesh filter, collect the tissue fluid, wash with PBS, and combine the cleaning sol...

Embodiment 3

[0106] Embodiment 3, the isolation of placental MSC, subculture, cryopreservation

[0107] (1) Within four hours after delivery, soak the placenta in 75% alcohol for 30 seconds under aseptic conditions, and then wash it twice with PBS;

[0108] (2) Strip the placental blood vessels from the umbilical cord root of the placenta, and squeeze the blood vessels with surgical forceps to remove blood stains;

[0109] (3) Cut the blood vessels into 2 mm^3 pieces, wash them with PBS, and then filter through a 300-mesh filter to remove residual blood stains to obtain placental vascular tissue (4.7 g of placental vascular tissue was obtained from one placenta in this embodiment);

[0110] (4) Add mixed enzyme solution (including: 0.1% collagenase II, 0.2% collagenase IV, 0.05% deoxyribonuclease I in PBS buffer) for digestion for 2 hours;

[0111] (5) Add serum to stop the digestion, then filter through a 300-mesh filter, collect the tissue fluid, wash with PBS, and combine the cleanin...

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Abstract

The present invention relates to a method for isolating mesenchymal stem cells from placental vessels and used digestive enzyme compositions. The method comprises the following steps: the placenta issterilized with alcohol; Dissecting placental blood vessels from the placenta; Cut into pieces, wash with PBS, filter the residual blood stains to obtain placental vascular tissue; Adding mixed enzymesolution to digest; End digestion, filter and collect tissue fluid; The placental mesenchymal stem cells (P0) were obtained by centrifugation and cultured with DMEM-F12 basal medium was resuspended and the number and viability of nucleated cells were counted. The obtained cells are cryopreserved with cryopreservation protection solution so as to be re-cultured before use, or the passages and / or the mesenchymal stem cells are continued to be subjected to cell identification and / or detection, cryopreservation, library establishment and the like. The method of the invention can effectively improve the efficiency of isolating mesenchymal stem cells from the blood vessels of the placenta.

Description

technical field [0001] The present invention relates to a method for isolating stem cells from the blood vessels of the placenta, in particular to a method for isolating mesenchymal stem cells from the blood vessels of the placenta, and more particularly relates to a method of using the digestive enzyme composition of the unique formula of the present invention to extract the mesenchymal stem cells from the placenta A method for isolating mesenchymal stem cells from blood vessels. Using the method of the present invention can effectively improve the efficiency of isolating mesenchymal stem cells from placental blood vessels. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) such as human mesenchymal stem cells were first isolated from bone marrow, a type of tissue stem cells derived from mesoderm with multi-lineage differentiation potential and self-renewal ability, in vivo and Under specific conditions in vitro, it has the ability to diffe...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N9/26C12N9/64C12N9/94
CPCC12N5/0665C12N9/2474C12N9/6491C12N9/94C12N2509/00C12Y301/11001C12Y302/01035
Inventor 王正肖海蓉
Owner BOYALIFE
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