Method for freezing and thawing placental whole cells and separating and expanding stem cells

A whole-cell, qualitative stem cell technology, applied in the field of placental whole cell cryopreservation, resuscitation, isolation and expansion of stem cells, can solve the problem that the method and technology are not fully mature

A whole-cell, qualitative stem cell technology, applied in the field of placental whole cell cryopreservation, resuscitation, isolation and expansion of stem cells, can solve the problem that the method and technology are not fully mature

CN102807966AActive Publication Date: 2012-12-05BOYALIFE
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1, placental whole cell cryopreservation, resuscitation and the method of stem cell separation and expansion after resuscitation Law

[0114] The placental tissue cryopreservation method includes the following steps:

[0115](1) Placental tissue cleaning: The placental tissue is processed in a biological safety cabinet. According to the size of the placenta, an appropriate amount of PBS buffer is used to wash the placental tissue 2-3 times, so that the residual blood on the surface of the placental tissue is washed clean, and there is no blood clot on the surface of the placenta. piece;

[0116] (2) Digestion of placenta tissue: use surgical scissors to cut placental leaflets from the placental tissue obtained in step (1), transfer the leaflets to a petri dish, add 25ml PBS buffer solution and cut the placental leaflets as much as possible, add 25ml 0.25 % trypsin (Gibco) (the volume ratio of trypsin to PBS buffer is 1:1) and mix the tissue, put the cultu...

Embodiment 2

[0133] Embodiment 2, placental whole cell cryopreservation, resuscitation and the method of stem cell separation and expansion after resuscitation Law

[0134] Carried out with reference to the method of Example 1. The recovered placental whole cells began to have adherent cells on the 7th day of culture, and the cell fusion rate reached 80% by the 19th day of culture. After 3 passages, the cell purity was greater than 90%.

Embodiment 3

[0135] Embodiment 3, placental whole cell cryopreservation, resuscitation and the method of stem cell separation and expansion after resuscitation Law

[0136] Carried out with reference to the method of Example 1. The recovered placental whole cells began to have adherent cells on the 8th day of culture, and the cell fusion rate reached 80% by the 21st day of culture. After 3 passages, the cell purity was greater than 85%.

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Abstract

The invention relates to a method for freezing and thawing placental whole cells and separating and expanding stem cells. The method comprises the steps as follows: disinfecting and washing a placenta tissue; cutting off placental lobules from the tissue, and carrying out digestive treatment for 20 min; preparing a placenta tissue freezing solution for standby application; adding whole cells obtained by digestive treatment and the freezing solution into a freezing tube, refrigerating for 0.5 h at the temperature of 4 DEG C, freezing for 1 day at the temperature of subzero 80 DEG C, and freezing in liquid nitrogen for standby application; taking out the placental whole cells from the liquid nitrogen when needed, thawing in a thermostatic water bath, carrying out drop-method washing by a culture medium for mesenchymal stem cells, removing red cells by a red cell lysis solution, and expanding the mesenchymal stem cells by the thawed placental whole cells through cell culture and cell passage. According to the method, the frozen placenta tissue can be effectively protected and is convenient to thaw and use; and the method is in particular suitable for separating and expanding the mesenchymal stem cells after thawing the frozen placental tissue.

Description

technical field [0001] The invention relates to a method for processing placental whole cells, in particular to a method for freezing and resuscitating placental whole cells, and a method for isolating and amplifying stem cells from the resuscitated placental whole cells, in particular to freezing, storing, and resuscitating placental whole cells. A method of resuscitating, then isolating and expanding mesenchymal stem cells therefrom. Background technique [0002] Mesenchymal stem cells (MSCs) originate from mesoderm and ectoderm in the early stages of development, and have the characteristics of multilineage differentiation potential, immune regulation and self-replication, and have attracted increasing attention. Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. It has multi-directional differentiation pote...

Claims

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Application Information

Patent Timeline
05 Dec 2012
Publication
CN102807966A
IPC
C12N5/073; C12N5/0735; C12N5/0775; A01N1/02; G06F17/30
Inventors
林卓衡; 朱业峰