Preparation method of placenta hematopoietic stem cells

Abstract

The invention relates to the technical field of biology, and discloses a preparation method of placenta hematopoietic stem cells. The method comprises the following steps: cleaning a pretreated placenta by injection with an irrigating solution; injecting an enzymolysis solution from the placenta artery to the placenta vein until the enzymolysis solution flows out, tying the placenta artery and vein, and standing; loosening the placenta artery and vein, injecting the irrigating solution from the artery, collecting all the liquid at the vein, centrifugating, taking the cell precipitate, recovering and purifying single karyocytes comprising hematopoietic stem cells sequentially by a hydroxyethyl starch process and an erythrocyte cracking process, and resuspending to obtain the placenta hematopoietic stem cells. The method improves the enzymolysis injection technique, selects multiple mutually-suitable components to constitute the novel enzymolysis solution which is used in the placenta hematopoietic stem cell preparation process, can mobilize the hematopoietic stem cells inside the placenta to quickly enter the blood circulation system within a short time, and enhances the single karyocyte quantity comprising hematopoietic stem cells, the living cell quantity and the CD34 positive cell quantity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing 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, with self-renewal and multilineage differentiation capabilities. Its basic feature is self-renewal ability, that is, after a cell cycle activity, it can produce two hematopoietic stem cells with the same properties as before division, and it also has multi-directional differentiation ability, that is, under certain environmental conditions, hematopoietic stem cells have The ability to differentiate into various blood cell lines. [0003] With the in-depth research on hematopoietic stem cells (HSC), HSC transplantation has beco...

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Problems solved by technology

[0004] At present, the separation and extraction methods of placental HSC mainly adopt mechanical method and mechanical enzymatic hydrolysis method, etc., but the extraction process of these two methods is complicated, time-consuming and costly, and is not suitable for large-scale production and preparation.

[0005] Chinese patent CN103789262A discloses a preparation method of clinically applied placental hematopoietic stem cells, which uses enzymatic hydrolysis perfusion to prepare hematopoietic stem cells, and the number of CD34 positive cells finally obtained is 1.2-1.6×10 9 , however, under the premise that the enzymatic hydrolysis time is extremely short, the effect data is basically impossible to reach the billion level, and after the test and verification of the patented method, the total number of mononuclear cells is up to 10 8 order of magnitude, the viability of mononuclear cells is low at 60-65%, and CD34 positive cells are only in 10 5 order of magnitude, obviously inconsistent with the recorded test results

[0007] Although the above-mentioned existing patent uses the enzymatic hydrolysis perfusion method to prepare placental hematopoietic stem cells, although it is more convenient, time-consuming and low-cost than the mechanical method and mechanical enzymatic hydrolysis method, the number and vitality of the cells finally obtained are not ideal. make further improvements

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