Method for separation and purification of human embryonic trophoblast cells and placental mesenchymal stem cells

A technology for nourishing cells and stem cells, applied in the field of biomedicine, can solve the problem of high cell success rate, achieve high cell survival rate, reduce cell damage, and improve cell activity.

Active Publication Date: 2019-07-26
广州杜德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The thermosensitive chitosan copolymer film has good temperature-sensitive characteristics, so that it can achieve the purpose of artificially controlled cell adsorption and desorption only by changing the temperature without the need for enzymatic hydrolysis. Compared with immunomagnetic The bead method is gentler, less damage to the cells, and the success rate of the cells is high, and it is easier to obtain a large number of primary cells at one time. There are few reports on the separation and purification of cells by combining them with specific antibody proteins.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A method for separating and purifying human embryonic trophoblast cells and placental mesenchymal stem cells, comprising the following steps:

[0036] (1) Placenta treatment: After the placenta is cleaned and sterilized, the placenta tissue is obtained, and then the placenta tissue is digested with digestive juice to obtain a cell suspension;

[0037](2) Cell suspension pre-separation: Take a certain amount of the primary cell suspension and transfer it to a centrifuge tube, and slowly add the cell separation liquid into the centrifuge tube. The volume ratio of the cell separation liquid to the primary cell suspension is 1:9 , at 37°C, 1300g centrifugal force, horizontal rotor centrifugation for 30min, take the cell layer, and resuspend the cells with PBS to obtain a secondary cell suspension; use the primary pre-separation to extract the mixed cells of human embryonic trophoblast cells and placental mesenchymal stem cells layer, to exclude interfering substances; the c...

Embodiment 2

[0046] This embodiment is basically the same as Example 1, except that the specific temperature-sensitive chitosan copolymer film is filled in the glass tube after rolling, and the rolling is placed compared to just laying on the inner wall of the glass tube. Greatly increased the surface contact rate of placental mesenchymal stem cells with specific temperature-sensitive chitosan copolymer membranes. It is proved by experiments that under the same other conditions, the adsorption rate of the coiled specific temperature-sensitive chitosan copolymer film is 18% higher than that of the adherent placental mesenchymal stem cells.

Embodiment 3

[0048] This example is basically the same as Example 2, except that the antibody protein is CD44.

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PUM

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Abstract

The invention discloses a method for separating and purifying human embryonic trophoblast cells and placental mesenchymal stem cells. The method comprises the following steps: (1) placenta treatment;(2) pre-separation of a cell suspension; (3) flow adsorption separation; (4) desorption and separation of the placental mesenchymal stem cells; and (5) purification of the human embryonic trophoblastcells. In the invention, an antibody protein is planted on the outer surface of a temperature-sensitive chitosan copolymer membrane, the surface molecules of the placental mesenchymal stem cells are recognized by the antibody to achieve the purpose of specific binding, and a decreasing flow rate circulating manner is adopted to reserve the placental mesenchymal stem cells on the specific temperature-sensitive chitosan copolymer membrane. The desorption and separation of the placental mesenchymal stem cells and a protein antibody carried out by cooling and eluent addition. The method has the advantages of mild separation way, high cell survival rate and high purity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for separating and purifying human embryo trophoblast cells and placental mesenchymal stem cells. Background technique [0002] It is difficult to culture and passage trophoblasts in vitro, and many researchers adopt the scheme of direct treatment or intervention after separating cells, so obtaining a large number of trophoblasts at one time is very beneficial to the experiment. The abundance of placental mesenchymal stem cells in placental tissue is low, and a lot of placental tissue is needed to obtain a certain number of primary cells. [0003] The conventional methods currently used for trophoblast cells and placental mesenchymal stem cells include the following four: [0004] (1) Adhesive separation method. This method mainly utilizes the characteristics of stem cells to adhere to the wall in plastic culture flasks to separate stem cells from other...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073C12N5/0735
CPCC12N5/0603C12N5/0606C12N2509/00
Inventor 夏凡张洁
Owner 广州杜德生物科技有限公司
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