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Pancreatic stem cell preparation method and special culture medium composition therefor

A medium and stem cell technology, applied in non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc., to achieve high efficiency and easy operation.

Active Publication Date: 2014-04-09
微能生命科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of individual development, there are various forms of stem cells, such as embryonic stem cells, pluripotent stem cells, and committed stem cells in various tissues, such as hematopoietic stem cells, neural stem cells, etc., but they face ethical challenges or limitations in practical application. limit

Method used

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  • Pancreatic stem cell preparation method and special culture medium composition therefor
  • Pancreatic stem cell preparation method and special culture medium composition therefor
  • Pancreatic stem cell preparation method and special culture medium composition therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 Experimental material

[0056] Penicillin, streptomycin and trypsin-EDTA were purchased from GIBCO. Trizol was purchased from Invitrogen Company of the United States, and Oligo dT, M-MLV reverse transcriptase, Taq DNA polymerase, dNTP and RNase inhibitor were purchased from Takara Company of Japan.

[0057] Mouse anti-human Foxa2 monoclonal antibody, rabbit anti-human Insulin monoclonal antibody, rabbit anti-human C-peptide polyclonal antibody, and rabbit anti-human AMY monoclonal antibody were purchased from Abcam, USA. Goat anti-human Sox17 monoclonal antibody and goat anti-human PDX1 monoclonal antibody were purchased from American R&D Company. Isothiocyanate-labeled chicken anti-mouse IgG, isothiocyanate-labeled chicken anti-rabbit IgG, rhodamine-labeled donkey anti-goat IgG were purchased from Santa Cruz Company, rhodamine-labeled goat anti-mouse IgG, rhodamine-labeled Goat anti-rabbit IgG and isothiocyanate-labeled mouse anti-goat IgG were purchased...

Embodiment 2

[0058] Embodiment 2, the preparation of culture medium

[0059] DMEM / F12 medium (also known as DF12 medium) and high-glucose DMEM medium were purchased from GIBCO. Fetal calf serum (FCS) was purchased from GIBCO. Nicotinamide was purchased from Sigma. Glutamine was purchased from GIBCO Company, the product catalog number is 25030-081. Retinoic acid was purchased from Sigma Company, the product catalog number is R2625.

[0060] Human Activin A (ActivinA) was purchased from Pepro Tech, UK, with a catalog number of 120-14.

[0061] EGF (epidermal growth factor, epidermal growth factor) was purchased from Pepro Tech Company, the product catalog number is 100-15.

[0062] bFGF (basic fibroblast growth factor, basic fibroblast growth factor) was purchased from Sigma Company, the product catalog number is F0291.

[0063] exendin-4 was purchased from Sigma, the product catalog number is E7144.

[0064] FGF7 was purchased from Pepro Tech Company, the product catalog number is 100...

Embodiment 3

[0075] Example 3, Sequential induction and culture of human insulin-secreting cells

[0076] Step 1. Preparation of third generation mesenchymal stem cells

[0077] 1. Wash the adipose tissue collected by liposuction with D-Hanks’ (PBS can also be used) to remove blood cells and anesthetics, digest with 0.2% type II collagenase at 37°C for 30 minutes, filter through a 100-mesh sieve and collect the filtrate.

[0078] 2. Resuspend the cells obtained in step 1 with D-Hank's solution and wash twice to remove collagenase, centrifuge at 1200rpm at room temperature for 10min, and collect the cells.

[0079] 3. Divide the cells from step 2 in a 2×10 6 The density of individual / ml is inoculated in the expansion culture medium (one of the examples of the formula is containing 58% DF12, 40% MCDB, 2% fetal bovine serum, 1 × 10 -9 MITS, 1×10 -9 M dexamethasone, 1×10 -4 M2-ascorbic acid phosphate, 20ng / mL interleukin-6, 10ng / mL EGF, 10ng / mL PDGF-BB, 100U / mL penicillin and 100μg / mL stre...

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Abstract

The invention relates to a pancreatic stem cell preparation method and a special culture medium composition therefor, and concretely discloses a method for obtaining pancreatic stem cells by inducing human mesenchymal stem cells, and a matched culture medium composition thereof. The method is characterized in that a two-step induction process is used to obtain definitive endoderm cells and pancreatic stem cells in vitro once, cells in all stages can express corresponding specific genes, and the obtained pancreatic stem cells differentiate to become functional pancreatic endocrine cells and pancreatic exocrine cells. The obtained pancreatic stem cells can be used for treating the pancreatic injury caused by diabetes and the like, provides experiment bases and clinic research evidences for the cell treatment of the pancreatic injury diseases, and provides a new way for the cell transplant treatment.

Description

technical field [0001] The invention relates to a method for preparing pancreatic stem cells and a special medium combination thereof. More specifically, the present invention relates to a method for inducing mesenchymal stem cells into pancreatic stem cells by using a special medium combination and the special medium combination. Background technique [0002] Diabetes has become a frequent and refractory metabolic disease in the world. It is a disease in which glucose in the blood tends to accumulate too much. The alias given to it abroad is "Silent Killer" (Silent Killer). Especially "adult type diabetes", the infection rate of middle-aged people over forty years old is particularly high. In Japan, 10% of the population over the age of 40 has the disease, that is, there is one "diabetic" patient among ten people. Once suffering from "diabetes", life expectancy will be reduced by as much as ten years, and possible complications can spread throughout the body. [0003] O...

Claims

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Application Information

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IPC IPC(8): C12N5/074
Inventor 赵春华
Owner 微能生命科技集团有限公司
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