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Perfusate, enzymatic solution and method for isolating placental hematopoietic stem cells

A technology of hematopoietic stem cells and enzymatic solution, applied in the field of stem cells, can solve the problems of low content of hematopoietic stem cells, low activity rate of hematopoietic stem cells, and large volume of digested tissues, so as to inhibit the initiation of apoptosis of cells, increase the activity rate of cells, The effect of increasing the number and survival rate

Active Publication Date: 2019-09-24
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although mechanical enzymatic hydrolysis can obtain a sufficient number of cells, the content of miscellaneous cells is high, the content of hematopoietic stem cells is low, and the viability of hematopoietic stem cells is low
In addition, in the prior art, the placental chorion is mainly digested by enzymolysis, which requires a large amount of enzymatic solution and high production cost; the whole extraction process is complicated and time-consuming, and the number of extracted cells fluctuates Larger, the probability of pollution is greatly increased, which is not conducive to industrialization

Method used

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  • Perfusate, enzymatic solution and method for isolating placental hematopoietic stem cells

Examples

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Effect test

Embodiment 1

[0036] The placenta obtained after childbirth was soaked in placenta preservation solution, preserved and transported at 4°C. The formula of the placenta preservation solution is: PBS contains 1× penicillin-streptomycin double antibody, 25 U / mL heparin sodium, and 1 m / v% low molecular weight dextran-40. Placental hematopoietic stem cells were isolated within 6 hours after delivery as follows.

[0037] S1. The amnion is peeled off, and the placenta is preliminarily washed with perfusate at 0-4°C. The specific method of cleaning the placenta is as follows: use a disposable syringe to absorb perfusate from the two placental arteries and perfuse it into the placenta until the blood in the placenta is cleaned and the transparent perfusate flows out.

[0038] The formula of the lavage solution is: PBS containing 1×penicillin-streptomycin double antibody, 0.05mg / mL phentolamine, and 3mg / mL vitamin C.

[0039] S2. Use a disposable syringe to absorb the enzymatic solution from the tw...

Embodiment 2

[0045] The placenta obtained after childbirth was soaked in placenta preservation solution, preserved and transported at 4°C. The formula of the placenta preservation solution is: PBS contains 2× double antibody, 25 U / mL heparin sodium, and 1 m / v% low molecular weight dextran-40. Placental hematopoietic stem cells were isolated within 12 hours after delivery as follows.

[0046] S1. The amnion is peeled off, and the placenta is preliminarily washed with perfusate at 0-4°C. The specific method of cleaning the placenta is as follows: use a disposable syringe to absorb perfusate from the two placental arteries and perfuse it into the placenta until the blood in the placenta is cleaned and the transparent perfusate flows out.

[0047] The formula of the lavage solution is: PBS containing 2× double antibody, 0.05 mg / mL phentolamine, and 3 mg / mL vitamin C.

[0048] S2. Use a disposable syringe to absorb the enzymatic solution from the two placental arteries and infuse it into the ...

Embodiment 3

[0054] The placenta obtained after childbirth was soaked in placenta preservation solution, preserved and transported at 4°C. The formula of the placenta preservation solution is: PBS contains 3× double antibody, 25 U / mL heparin sodium, and 1 m / v% low molecular weight dextran-40. Placental hematopoietic stem cells were isolated within 6 hours after delivery as follows.

[0055] S1. The amnion is peeled off, and the placenta is preliminarily washed with perfusate at 0-4°C. The specific method of cleaning the placenta is as follows: use a disposable syringe to absorb perfusate from the two placental arteries and perfuse it into the placenta until the blood in the placenta is cleaned and the transparent perfusate flows out.

[0056] The formula of the lavage solution is: PBS containing 3× double antibody, 0.05 mg / mL phentolamine, and 3 mg / mL vitamin C.

[0057] S2. Use a disposable syringe to absorb the enzymatic solution from the two placental arteries and infuse it into the p...

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Abstract

The invention discloses an irrigating solution, an enzymatic hydrolysate and a method for isolating placenta hematopoietic stem cells. The irrigating solution consists of a penicillin-streptomycin double-antibody, phentolamine, vitamin C and PBS. The enzymatic hydrolysate consists of a basal culture medium, collagenase VIII, pancreatin, 1*heparin sodium, phentolamine, vitamin C and Z-VAD-FMK. The method comprises the following steps: irrigating and cleaning a preprocessed placenta with the irrigating solution; irrigating the enzymatic hydrolysate from placental artery until the enzymatic hydrolysate flows out of placental vein, ligating placental artery and vein and standing by; loosening the placental artery and vein and irrigating the irrigating solution from the artery, collecting all liquid from the vein, centrifuging and reserving cell sediments, and recovering and purifying mononuclear cells containing the hematopoietic stem cells sequentially by virtue of a hydroxyethyl starch method and a red blood cell lysis method; re-suspending and obtaining the placenta hematopoietic stem cells. The irrigating solution, the enzymatic hydrolysate and the method disclosed by the invention can significantly improve separation and extraction efficiencies, and a great amount of cells are obtained; the hematopoietic stem cells are high in content, high in survival rate and quite low in cell pollution rate.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a perfusate, an enzymolysis solution and a method for isolating placental hematopoietic stem cells. Background technique [0002] Hemopoietic Stem cells (HSCs) are a group of primitive hematopoietic cells that exist in hematopoietic tissues. They are not tissue-fixed cells and can exist in hematopoietic tissues and blood. Hematopoietic stem cells are the origin of all hematopoietic and immune cells. [0003] With the deepening of research, HSC transplantation has become an effective means of treating many malignant hematological diseases and other tumors. At present, the main source of HSC is bone marrow, peripheral blood and cord blood. The proliferation and differentiation ability of bone marrow and peripheral blood HSC is reduced, and it is easily affected by the health status of the donor. However, the number of cord blood HSC is limited. Increasing demand for clinical application...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
Inventor 陈海佳王一飞葛啸虎卢瑞珊王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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