In vitro screening and amplification technology of mouse pancreatic stem/progenitor cells
A technology of progenitor cells and stem cells, applied in the field of expanding adult mouse pancreatic stem/progenitor cells with strong proliferation and differentiation capabilities, can solve the problems of lack of marker molecules, mixed cells, difficult screening and enrichment, etc.
Inactive Publication Date: 2012-06-27
NORTHEAST FORESTRY UNIVERSITY +2
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Problems solved by technology
However, adult pancreatic stem / progenitor cells as the source of transplantation donor cells currently have the following obstacles: (1) The ratio of stem / progenitor cells in adult pancreatic tissue is extremely low, lacks recognized marker molecules, and is difficult to screen by flow cytometry and enrichment; (2) In the process of screening pancreatic stem / progenitor cells using in vitro culture methods, various cells are mixed, especially fibroblasts grow vigorously, which makes it difficult for pancreatic stem / progenitor cells derived from epithelium to be purified and expanded in large quantities. increase
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[0026] 1. Ultra-clean bench and sterile room are sterilized by ultraviolet radiation for 40 minutes, then turn off the ultraviolet equipment, open the exhaust system, and enter the sterile room after 5 minutes.
[0027] 2. Take 3 petri dishes with a diameter of 3.5cm and place them on a clean bench, add 2mL cold PBS (4°C) to two of them, put them on ice packs, and set them aside.
[0028] 3. Prepare two sets of sterile surgical instruments, put them in the ultra-clean bench, and set them aside.
[0029] 4. One mouse was killed by cervical dislocation, soaked in 75% ethanol for 3 minutes, and brought into the sterile room after disinfection.
[0030] 5. Place the left abdomen of the mouse upwards and place it flat in a clean 10cm glass dish. Open the abdominal cavity of the mouse on the ultra-clean table to expose the internal organs. Carefully remove the pancreas and place it in a 3.5cm petri dish filled with cold PBS. middle.
[0031] 6. Move the small dish containing pancr...
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The invention discloses a novel and high-efficient in vitro screening and amplification technology of mouse pancreatic stem / progenitor cells, which comprises the steps of taking mouse pancreatic tissues, separating pancreatic single cells, enriching and purifying the pancreatic stem / progenitor cells, greatly amplifying the pancreatic stem / progenitor cells and the like. The key of the technology is to partially digest the enriched pancreatic stem / progenitor cells through collagenase, effectively screen fibroblasts through the two-transit method, finally purify the pancreatic stem / progenitor cells through the method of picking cell colonies by trypsin spotted digestion, greatly amplify and finally screen, thereby obtaining a group of homogenous pancreatic epithelial stem / progenitor cells with strong proliferation capacity and multi-lineage differentiation potential.
Description
technical field [0001] The present invention relates to a new type of high-efficiency in vitro screening and expansion of mouse pancreatic stem / progenitor cells. Specifically, it involves the use of collagenase to partially digest and transfer twice to enrich pancreatic stem / progenitor cells, and single-cell colonies are picked and purified. And a method for further expanding adult mouse pancreatic stem / progenitor cells with strong proliferation and differentiation capabilities. Background technique [0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by lack or dysfunction of pancreatic β cells. In recent years, with the development of social economy and the improvement of residents' living standards in various countries in the world, the incidence and prevalence of diabetes have increased year by year. Diabetes has become one of the three major medical problems today. Diabetes not only affects the quality of life of patients, but also br...
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IPC IPC(8): C12N5/071A61K35/39A61P3/10
Inventor 滕春波王法张丽新
Owner NORTHEAST FORESTRY UNIVERSITY