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Separation and culture method of bovine umbilical cord mesenchymal stem cells

A culture method and stem cell technology, applied in the field of life sciences, can solve the problems of increasing the cost of the separation process, cumbersome enzyme digestion process, prolonging the separation time, etc., and achieve the effects of low cost, promotion of in vitro expansion, and simple operation

Active Publication Date: 2015-05-20
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic digestion method for isolating umbilical cord mesenchymal stem cells has some disadvantages. The enzymatic digestion process takes a certain amount of time, which prolongs the separation time; and the use of trypsin or collagenase to digest the umbilical cord also greatly increases the cost of the separation process; Thirdly, the process of enzymatic digestion method is more cumbersome, and the separation effect is less stable

Method used

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  • Separation and culture method of bovine umbilical cord mesenchymal stem cells
  • Separation and culture method of bovine umbilical cord mesenchymal stem cells
  • Separation and culture method of bovine umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Isolation and cultivation of bovine umbilical cord MSC of embodiment 1

[0041] 1. Collect fetal bovine umbilical cord

[0042] (1) Take a 4-month-old fetal cow with a uterus, store it in a low-temperature transport box, and send it to the laboratory within 3 hours.

[0043] (2) Take the fetal calf out of the uterus in an ultra-clean bench, cut the umbilical cord with scissors, and put it into a glass plate filled with pre-cooled 75% ethanol for 10 seconds.

[0044] (3) Transfer the umbilical cord to another glass plate containing pre-cooled PBS solution, clamp one end of the umbilical cord with a hemostat, squeeze out the blood in the blood vessel with flat forceps, and clean the blood stains on the surface of the umbilical cord. Repeat washing once.

[0045] 2. Separation of bovine umbilical cord

[0046] (1) Cut the umbilical cord into small pieces of 1 cm, peel off the outer membrane of the umbilical cord with tissue forceps, remove the arteries and veins, and te...

Embodiment 2

[0057] Example 2 Bovine umbilical cord MSC cell identification

[0058] 1. Morphological observation

[0059] Regularly observe the growth and morphological changes of the primary cells and each generation of cells after passage under an inverted microscope, and initially determine the type and purity of the cells. Cell morphology see figure 2 and image 3 ; figure 2 The primary cells crawled out after the tissue block adhered to the wall, and grew in colonies near the tissue block. The cell morphology was spindle-shaped and fibrous, which conformed to the morphological characteristics of mesenchymal stem cells.

[0060] image 3 It is bovine umbilical cord MSC P3 generation cells. It can be seen from the figure that the cells are in good condition, fusiform and fibroblast, and the uniform shape can preliminarily determine that the cells have high purity.

[0061] 2. Flow detection of surface markers

[0062] The bovine umbilical cord MSC cells of the P3 generation wer...

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Abstract

The invention discloses a separation and culture method of bovine umbilical cord mesenchymal stem cells. The method comprises the following steps: taking fetal bovine umbilical cord and separating cells by a tissue block adherence method; after the cells are separated, cultivating the cells in a complete culture medium; when cell fusion degree reaches 80%-90%, digesting for 1-3 minutes, terminating the digestion and then blowing and beating to obtain the bovine umbilical cord mesenchymal stem cells, wherein the complete culture medium is a serum-free culture medium added with EGF and PDGF, concentration of the EGF is 5-10ng / mL and concentration of the PDGF is 5-10ng / mL. According to the invention, the tissue block adherence method is utilized to perform a primary separation to the bovine umbilical cord MSC and EGF and PDGF are added into the serum-free culture medium to perform culture amplification, so as to successfully obtain bovine umbilical cord MSC with self renewal and differentiation potentials. Addition of EGF and PDGF in the culture system not only promotes in-vitro amplifications of the cells, but also inhibits differentiation of the cells in in-vitro cultivation.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a method for separating and culturing bovine umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. MSCs have the common characteristics of stem cells, that is, the ability of self-renewal, multilineage differentiation and homing. In a specific in vitro differentiation environment, it can be induced to differentiate into various tissue cells such as nerve, heart, liver, bone, cartilage, tendon, fat, and epithelium. [0003] Due to the potential of self-renewal and multi-directional differentiation, MSC has become a research hotspot in recent years. A large number of studies have confirmed that MSCs can be isolated from bone marrow, but they also exist in different tissues and organs, such as muscle, adipose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 葛啸虎陈海佳王一飞陈洁鸿罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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