Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells

Abstract

The invention relates to a method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells after reviving the umbilical cord, and the step comprises the following steps of preparing umbilical cord tissue freezing liquid; sterilizing and washing the umbilical cord tissues; cutting the tissues into blocks; placing the tissue blocks and the freezing liquid into afreezing tube, cold storing the tissue blocks for 0.5 hour under the temperature condition of 4 DEG C and then storing the tissue blocks for one day under the temperature condition of minus 80 DEG C,and then freezing the tissue blocks in liquefied nitrogen; and taking the umbilical cord tissues out of the liquefied nitrogen to use, thawing the umbilical cord tissues in constant-temperature waterbath, utilizing mesenchymal stem cell culture base to washing the umbilical cord tissues through a drop method, and separating and increasing the mesenchymal stem cells of the revived umbilical cord tissues through a tissue attachment method. The method can effectively protect the frozen umbilical cord tissue, so that the umbilical cord tissues can be revived to use, and the method is particularly suitable for separating and increasing the mesenchymal stem cells after the umbilical cord tissue is revived.

Description

technical field

[0001] The invention relates to a method for processing umbilical cord tissue, in particular to a method for freezing fresh tissue of umbilical cord, recovering and separating and expanding stem cells after recovery, in particular to a method for isolating and expanding mesenchymal stem cells from fresh tissue of umbilical cord. 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 potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused b...

Examples

Embodiment 1

[0099] Example 1. Umbilical Cord Tissue Cryopreservation, Resuscitation and Method for Isolation and Expansion of Stem Cells after Resuscitation

[0100] The method of cryopreservation of umbilical cord tissue includes the following steps:

[0101] (1) Prepare umbilical cord tissue cryopreservation solution: the umbilical cord tissue cryopreservation solution contains 80 parts by weight of human serum albumin and 10 parts by weight of DMSO (dimethyl sulfoxide, dimethyl sulfoxide), prepared cryopreservation solution Store in a 4°C refrigerator until use;

[0102] (2) Disinfection and cleaning: In a biological safety cabinet, disinfect the surface of the umbilical cord tissue with alcohol, cut the umbilical cord from the middle, spread it on a sterile 10cm cell culture plate, and clean the tissue with PBS to reduce the size of the tissue surface. red blood cells;

[0103] (3) Umbilical cord tissue processing: transfer the umbilical cord tissue obtained in step (2) to another ...

Embodiment 2

[0116] Example 2, Umbilical Cord Tissue Cryopreservation, Resuscitation, and Method for Separation and Expansion of Stem Cells after Resuscitation

[0117] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the revived umbilical cord tissue on the 7th day of culture, and the cell fusion rate reached 60% by the 13th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 19. After 3 passages, the cell purity was greater than 90%.

Embodiment 3

[0118] Example 3, Umbilical Cord Tissue Cryopreservation, Resuscitation, and Method for Isolation and Expansion of Stem Cells after Resuscitation

[0119] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the revived umbilical cord tissue on the 8th day of culture, and the cell fusion rate reached 60% by the 15th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 18. After 3 passages, the cell purity was greater than 90%.

technical field

[0001] The invention relates to a method for processing umbilical cord tissue, in particular to a method for freezing fresh tissue of umbilical cord, recovering and separating and expanding stem cells after recovery, in particular to a method for isolating and expanding mesenchymal stem cells from fresh tissue of umbilical cord. 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 potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused b...

Examples

Embodiment 1

[0099] Example 1. Umbilical Cord Tissue Cryopreservation, Resuscitation and Method for Isolation and Expansion of Stem Cells after Resuscitation

[0100] The method of cryopreservation of umbilical cord tissue includes the following steps:

[0101] (1) Prepare umbilical cord tissue cryopreservation solution: the umbilical cord tissue cryopreservation solution contains 80 parts by weight of human serum albumin and 10 parts by weight of DMSO (dimethyl sulfoxide, dimethyl sulfoxide), prepared cryopreservation solution Store in a 4°C refrigerator until use;

[0102] (2) Disinfection and cleaning: In a biological safety cabinet, disinfect the surface of the umbilical cord tissue with alcohol, cut the umbilical cord from the middle, spread it on a sterile 10cm cell culture plate, and clean the tissue with PBS to reduce the size of the tissue surface. red blood cells;

[0103] (3) Umbilical cord tissue processing: transfer the umbilical cord tissue obtained in step (2) to another ...

Embodiment 2

[0116] Example 2, Umbilical Cord Tissue Cryopreservation, Resuscitation, and Method for Separation and Expansion of Stem Cells after Resuscitation

[0117] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the revived umbilical cord tissue on the 7th day of culture, and the cell fusion rate reached 60% by the 13th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 19. After 3 passages, the cell purity was greater than 90%.

Embodiment 3

[0118] Example 3, Umbilical Cord Tissue Cryopreservation, Resuscitation, and Method for Isolation and Expansion of Stem Cells after Resuscitation

[0119] Carried out with reference to the method of Example 1. Adherent cells began to crawl out of the revived umbilical cord tissue on the 8th day of culture, and the cell fusion rate reached 60% by the 15th day of culture. After passage to T25 culture flasks, the fusion rate reached 90% on day 18. After 3 passages, the cell purity was greater than 90%.