Irrigating solution, enzymatic hydrolysate and method for isolating placenta hematopoietic stem cells

A technology of hematopoietic stem cells and enzymatic hydrolysis solution, applied in the field of stem cells, can solve the problems of low activity rate of hematopoietic stem cells, low content of hematopoietic stem cells, and large volume of digested tissues, so as to improve cell activity rate, simple components, increase quantity and The effect of viability

Active Publication Date: 2016-03-02
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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  • Irrigating solution, enzymatic hydrolysate and method for isolating placenta hematopoietic stem cells
  • Irrigating solution, enzymatic hydrolysate and method for isolating placenta hematopoietic stem cells

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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 containing 1×penicillin-streptomycin double antibody, 25U / mL heparin sodium, 1m / 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 two pla...

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 containing 2×double antibody, 25U / mL heparin sodium, 1m / 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 place...

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 containing 3× double antibody, 25 U / mL heparin sodium, 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 pla...

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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 I, collagenase II, heparin sodium, phentolamine, vitamin C and Z-VAD-FMK. The method comprises the following steps: cleaning blood inside a placenta with the irrigating solution; irrigating the enzymatic hydrolysate into the placenta and enzymatically hydrolyzing at room temperature; collecting the enzymatic hydrolysate, centrifuging and reserving cell sediments; re-suspending the cell sediments by virtue of the PBS, adding a hydroxyethyl starch solution, standing by, reserving and centrifuging upper liquid and discarding supernatant; re-suspending the cell sediments by virtue of a red blood cell lysis solution, promoting lysis to residual red cells, centrifuging and discarding supernatant; and re-suspending the cell sediments by virtue of a culture medium, centrifuging and discarding supernatant, so that the placenta hematopoietic stem cells are obtained. 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] Hematopoietic stem cells (Hemopoietic Stemcell, HSC) 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 f...

Claims

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Application Information

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Patent Type & Authority Applications(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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