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A neural crest and stem cell technology, applied in the field of stem cell acquisition, can solve problems affecting experiments and treatments, and achieve fast and efficient acquisition, strong stem cell characteristics, and simple operation
Inactive Publication Date: 2019-03-26
FEED VETERINARY MEDICINE RES INST SHANXI ACADEMY OF AGRI SCI
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Most of the neural stem cell lines currently established are derived from mice, and there are obvious species differences between mice and humans. For disease models, pigs are closer to humans than mice. Pig neural crest stem cell models are used for The effect of drug screening, human disease research, and stem cell therapy is better, but the separation technology and method of pig neural crest stem cells have not yet appeared in the prior art, which affects subsequent experiments and treatment research
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Embodiment 1
[0049] 1. Isolation of porcine neural crest stem cells:
[0050] Take 10 μL of type Ι collagen in a 35 mm petri dish, use a disposable scraper to spread the sample and hang it evenly, and place it in a 37°C, 5% carbon dioxide incubator for 1 hour before use; use a small skin puncher to drill the neck of the pig Adipose tissue, rinsed with 75% ethanol, take the tissue that has not been soaked in alcohol in the middle, wash with phosphate buffer saline for 3 times, and mince in DMEM culture solution with 10% fetal bovine serum; take 35mm petri dish and add 2 drops DMEM culture medium with 10% fetal bovine serum; put the minced porcine adipose tissue in the culture medium, cover with a sterilized cover glass, press lightly with the reverse side of tweezers to make it contact with the bottom, and observe under the microscope Whether there is connective tissue. After static cultivation in a 37°C, 5% carbon dioxide incubator for 1 hour, add 1ml of culture medium, and the tip of the...
Embodiment 2
[0057] 1. Isolation of porcine neural crest stem cells:
[0058] Take 20 μL of type Ι collagen in a 35 mm petri dish, use a disposable scraper to spread the sample and hang it evenly, and place it in a 37.5°C, 5% carbon dioxide incubator for 1 hour before use; use a small skin puncher to drill the neck of the pig Adipose tissue, rinsed with 75% ethanol, take the tissue that has not been soaked in alcohol in the middle, wash with phosphate buffer saline for 3 times, and mince in DMEM culture solution with 10% fetal bovine serum; take 35mm petri dish and add 2 drops DMEM culture medium with 10% fetal bovine serum; put the minced porcine adipose tissue in the culture medium, cover with a sterilized cover glass, press lightly with the reverse side of tweezers to make it contact with the bottom, and observe under the microscope Whether there is connective tissue. After static culture in a 37.5°C, 5% carbon dioxide incubator for 1 hour, add 1ml of culture solution, and the tip of t...
Embodiment 3
[0065] The difference from Reference Document 1 is that in Step 1, Step 2, and Step 3, a mixture containing final concentrations of 1mmol / L sodium pyruvate, 0.05mg / ml uridine, 50U / ml penicillin, 50U / ml streptomycin and 5% fetal DMEM medium with bovine serum.
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Abstract
The invention relates to a method for obtaining pig neural crest stem cells. The method comprises the following steps: taking pig neck adipose tissues and chopping in a culture solution; standing andculturing; separating to obtain primary pig neural crest stem cells; carrying out steps including primary culture, amplification culture of the pig neural crest stem cells and the like to obtain the pig neural crest stem cells. A lot of tests and optimization are carried out to establish a set of effective pig neural crest stem cell separation and purification method; the method provided by the invention has the advantages of simple stem cell separation operation, good repeatability and high survival rate; after the cells are subjected to in-vitro amplification culture for 10 times, the cellsstill have relatively strong stem cell properties, can meet the requirements of experiment researches and are easy to culture; the neural crest stem cells can be rapidly and efficiently obtained, andthe obtained stem cells have a relatively rapid proliferation growth speed and a relatively strong differentiation potential. The speed and efficiency of developing nerves and repairing damaged nervous tissues are improved when the stem cells are used for treating. The method is successfully implemented so that a firm foundation is laid for clinical popularization and application of the neural crest stem cells and the neural crest stem cells can be possibly used as a neural crest stem cell medicine for development and research.
Description
technical field [0001] The invention relates to a method for obtaining stem cells, in particular to a method for extracting and culturing pig neural crest stem cells. Background technique [0002] The neural crest is two longitudinal bands of cells that migrate from the dorsolateral neural tube in early vertebrate embryos and lie between the neural tube and the ectoderm. During biological development, the nerves from which neural crest cells migrate give rise to a variety of mammalian cell types such as neurons, glial cells or melanocytes. In the head region of the neural crest cells of vertebrates differentiate into ectoderm and mesoderm cells. The neuroectodermal cells between the ectoderm and the edge of the neural sulcus form two ribbon-shaped cell cords parallel to the neural tube on the dorsolateral side of the neural tube, extending from the midbrain plane to the caudal. The resulting emigration of neural crest stem cells is widespread. From a stem cell biology per...
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