Mesenchymal stem cell separated from placenta blood vessel with 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: 2019-04-16
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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  • Mesenchymal stem cell separated from placenta blood vessel with digestive enzyme composition
  • Mesenchymal stem cell separated from placenta blood vessel with digestive enzyme composition
  • Mesenchymal stem cell separated from placenta blood vessel with digestive enzyme composition

Examples

Experimental program
Comparison scheme
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 invention relates to a mesenchymal stem cell separated from the placenta blood vessel with a digestive enzyme composition, in particular to the digestive enzyme composite used for a method for separating the placenta mesenchymal stem cell from the placenta blood vessel. A buffering solution containing tissue digestive enzymes is provided with added digestive enzymes which include pancreatic enzymes, deoxyribonuclease I, collagenase II, collagenase IV and hyaluronidase. In addition, the invention further relates to the method for separating the mesenchymal stem cell from the placenta bloodvessel, and the method includes the steps that the placenta is sterilized; the placenta vessel is separated from the placenta; cutting, cleaning and bloodiness filtering removal are conducted, so thatplacenta blood vessel tissue is obtained; mixed enzyme liquor is added for digestion; digestion is stopped, and interstitial fluid is filtered and collected; cell sediments obtained through centrifuging are original placenta mesenchymal stem cells, re-suspending is conducted, sampling is conducted, and the number nucleated cells and the survival rate of the nucleated cells are calculated; the obtained cells are subjected to refrigeration preservation or continuous passage and / or identified, detected and subjected to refrigeration preservation and database creation. Through the method, the efficiency of separating the mesenchymal stem cell from the placenta blood vessel can be improved effectively.

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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 王正肖海蓉
Owner BOYALIFE
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