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Separation method for decidua basalis stem cells of placenta

A technology of placental decidua and separation method, which is applied in the field of regenerative medicine and biology, can solve the problems of large fluctuations in the number of extracted cells, large volume of digested tissue, unfavorable industrial production, etc., and achieve sufficient cell number and experimental operation The effect of short time and high activity rate

Active Publication Date: 2020-08-25
GUANGDONG CHINAHEALTH LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the enzymatic digestion and extraction of the decidua basalis of the placenta has a large volume and takes a long time, and the number of cells obtained is large, but the activity rate of stem cells is low; stem cells are extracted from the decidua basalis, and the obtained cells contain a large amount of impurities. Cells, causing trouble for subsequent purification; the entire decidua basal stem cell extraction process, the steps are cumbersome, the number of extracted cells fluctuates greatly, and the probability of contamination is greatly increased, which is not conducive to industrial production

Method used

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  • Separation method for decidua basalis stem cells of placenta
  • Separation method for decidua basalis stem cells of placenta
  • Separation method for decidua basalis stem cells of placenta

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Experimental program
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Embodiment 1

[0037] This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:

[0038] (1) Collect the placenta and soak it in the placenta preservation solution: soak the placenta obtained after delivery within 6 hours in PBS containing basal medium containing 1×-2× double antibody and volume concentration of 10% high-glucose DMEM solution, stored at 4°C for transportation;

[0039] (2) The placenta described in step (1) was peeled off to obtain the decidua basalis, and the placental decidua basalis was initially cleaned with 0°C PBS, and then the cleaning solution was sucked into the tissue with a 10mL disposable pipette until The decidua basalis blood is cleaned;

[0040] (3) Use scissors to cut up the decidua basalis treated in step (2), with an area of ​​3×3cm2 , the shredded decidua basalis was mixed with decidua basalis at 10: 1 volume ratio, 200R enzymatic hydrolysis for 0.5h, 1500rpm centrifugation for 5mi...

Embodiment 2

[0045] This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:

[0046] (1) Collect the placenta and soak it in the placenta preservation solution: Soak the placenta obtained after delivery in the PBS preservation solution containing the basal medium containing 1×-2× double antibody and 10% high-glucose DMEM within 6 hours , stored and transported at 4°C;

[0047] (2) The placenta described in step (1) was peeled off to obtain the decidua basalis, and the placental decidua basalis was initially cleaned with 4°C PBS, and then the cleaning solution was sucked into the tissue with a 10mL disposable pipette until The decidua basalis blood is cleaned;

[0048] (3) Use scissors to cut up the decidua basalis treated in step (2), with an area of ​​3×3cm 2 , the shredded decidua basalis was mixed with decidua basalis at 10: 1 volume ratio, 200R enzymatic hydrolysis for 0.5h, centrifugation at 1500rpm for 5mi...

Embodiment 3

[0053] This embodiment is a method for separating placental stem cells according to the present invention, comprising the following steps:

[0054] (1) Collect the placenta and soak it in the placenta preservation solution: soak the placenta obtained after delivery in PBS containing basal medium containing 1×-2× double antibody and 10% high-sugar DMEM within 6 hours. solution, stored at 4°C for transportation;

[0055] (2) The placenta described in step (1) was peeled off to obtain the decidua basalis, and the placental decidua basalis was initially cleaned with 2°C PBS, and then the cleaning solution was sucked into the tissue with a 10mL disposable pipette until The decidua basalis blood is cleaned;

[0056] (3) Use scissors to cut up the decidua basalis treated in step (2), with an area of ​​3×3cm 2 , the shredded decidua basalis was mixed with decidua basalis at 10: 1 volume ratio, 200R enzymatic hydrolysis for 0.5h, 1500rpm centrifugation for 5min; 15ng / ml survivin in...

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Abstract

The invention relates to a separation method for decidua basalis stem cells of a placenta, and belongs to the technical field of regenerative medicine and biology. The separation method for the decidua basalis stem cells of the placenta comprises the following steps of collecting the placenta, and soaking the placenta in a placenta preservation solution which is a basic culture medium PBS containing 1x-2xdouble antibodies and 10% high-sugar DMEM; stripping the decidua basalis and carrying out cleaning; cutting the decidua basalis into pieces, carrying out enzymolysis by using enzymatic hydrolysate a which is a DMEM high-sugar basal culture medium containing 0.25% of pancreatin and 0.02% of EDTA, carrying out centrifuging, and discarding supernate, carrying out enzymolysis by using enzymatic hydrolysate b which is a DMEM high-sugar basic culture medium containing 0.1 to 0.2 mg / mL of collagenase IV, 0.01 to 0.05 mg / mL of neutral enzyme, 5 to 10 ng / ml of a Fas ligand inhibitor and 5 to 10ng / ml of a survivin inhibitor; and carrying out subpackaging, counting and inoculating. According to the separation method for the decidua basalis stem cells of the placenta, the decidua basalis stemcells of the placenta are extracted by adopting a double-enzymolysis method, and the method is simple in operation step, high in cell acquisition rate, sufficient in extracted cell number and high incell viability. In addition, the invention aims to comprehensively extract the decidua basalis stem cells of the placenta.

Description

technical field [0001] The invention relates to a method for separating decidua stem cells at the base of the placenta, and belongs to the technical fields of regenerative medicine and biology. Background technique [0002] With the deepening research on mesenchymal stem cells (MSC), MSC transplantation has become an effective method for treating many systemic diseases. In recent years, the placenta has become an important source of MSCs that many scholars have paid attention to. Some scholars have successfully isolated MSCs with transplantation potential from the amnion and chorion on the fetal side of the placenta, but little research has been done on the decidua basalis on the maternal side. The decidual basal stem cells extracted from the placenta not only have the advantages of rich content, low rejection after transplantation, and no ethical barriers. Decidual basal mesenchymal stem cells have strong proliferative ability, and their doubling time is faster than that o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0605C12N2509/00
Inventor 李文东宋云庆黎波卢瑞珊
Owner GUANGDONG CHINAHEALTH LIFE SCI CO LTD
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