Special culture medium and method for culturing porcine trophoderm stem cells

A technology for culturing trophoblast stem cells and stem cells, which is applied in the field of stem cell technology research, can solve the problems of low culture efficiency and failure to prove the characteristics of stem cells, and achieve the effects of low cell apoptosis rate, long-term stable stem cell characteristics, and strong proliferation and passaging ability

Active Publication Date: 2015-09-30
WENS FOOD GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the experiment, the culture efficiency was low, and about 30 blastocysts could obtain one cell clone group, which reflected the characteristics of trophoblast stem cells in morphology, but failed to prove its stem cell characteristics, that is to say, no real porcine trophoblast had been obtained. stem cell

Method used

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  • Special culture medium and method for culturing porcine trophoderm stem cells
  • Special culture medium and method for culturing porcine trophoderm stem cells
  • Special culture medium and method for culturing porcine trophoderm stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Isolation, culture and identification of porcine trophoblast stem cells from blastocysts derived from artificial insemination (AI)

[0048] 1. Establishment of the culture system of porcine trophoblast stem cells derived from AI-derived blastocysts

[0049] 1.1 Obtaining method of blastocyst:

[0050] The common estrous sows were bred by AI method, and the blastocysts were collected by the method of in vitro punching after the bridging. The specific operation is: select sows with high gestational age to be slaughtered on the sixth or seventh day after mating, take out the uterus, fix the front end of the uterine horn with a hemostatic forceps, inject egg flushing solution (DPBS+PVA) from the umbrella of the oviduct, and use The catheter is collected at the uterine horn, filtered and cleaned with the egg washing solution, and the blastocysts are collected under a stereo microscope, and transported to the laboratory at a constant temperature of 39°C (artif...

Embodiment 2

[0078] Example 2: Isolation, culture and identification of porcine trophoblast stem cells derived from somatic cell nuclear transfer (SCNT) blastocysts

[0079] 1. Establishment of culture system for porcine trophoblast stem cells isolated from SCNT blastocysts

[0080] 1.1 Acquisition of SCNT blastocysts: using a micromanipulator to perform nuclear transfer of porcine fibroblasts to obtain cloned embryos, acquisition and maturation of porcine oocytes, cultivation of donor fibroblasts, enucleation and injection of oocytes, The fusion and activation of oocytes and somatic cells were performed according to existing literature standards (Li et al., 2013, PMID: 24033142). The embryo culture medium PZM-3 was used to culture the reconstituted embryos until the sixth or seventh day, and the cysts were collected. Embryo. Compared with AI-derived blastocysts, its volume is smaller, the total number of cells in blastocysts is less, and the quality of embryos is poor (SCNT blastocysts a...

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Abstract

The invention discloses a special culture medium and method for culturing porcine trophoderm stem cells. The special culture medium is composed of bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol. The culture method comprises the following steps: previously coating a stem cell culture dish with an extracellular matrix; digesting porcine blastula with proteinase to remove the zona pellucida, transferring into the stem cell culture dish, and adding the special culture medium; and culturing until a trophoderm stem cell cloning group with the total cell count of 1000-2000 is formed, digesting into unicells, transferring into a new extracellular-matrix-coated culture dish, and carrying out cell subculture. The special culture medium has the advantages of definite components and high use safety, and avoids the pollution of heterogenous cells. The culture method is simple and efficient, and has the advantages of high cloning formation rate, low apoptosis rate, high proliferation and subculture capacity, high safety and high stability when being used for porcine trophoderm stem cell culture.

Description

technical field [0001] The invention belongs to the field of stem cell technology research, in particular to a special culture medium and method for cultivating porcine trophoblast stem cells. Background technique [0002] Stem cells are a kind of primitive undifferentiated cells with multi-directional differentiation potential and self-replication ability, and are the original cells that form various tissues and organs of mammals. The medical profession calls it "universal cell". [0003] The first cell differentiation occurs when the animal embryo develops to the blastocyst in the early stage, forming two kinds of inner cell mass and trophoblast with different characteristics and functions. Trophoblast stem cells are the progenitor cells of placental tissue cells, and gradually differentiate into trophoblast precursor cells, chorionic trophoblast, syncytiotrophoblast and multinucleated giant cells. During the apparent reprogramming process in early embryonic development,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 吴珍芳贺晓燕孟繁明李紫聪石俊松周荣张献伟
Owner WENS FOOD GRP CO LTD
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