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Method for improving culture of human skin pluripotent stem cells (SKPs)

A technology of pluripotent stem cells and culture methods, which is applied in the field of improvement of human skin pluripotent stem cell culture, can solve the problems of limited research and application, long culture period, and small quantity, and achieve broad application prospects, reduce costs, and shorten culture cycle effect

Pending Publication Date: 2017-07-28
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the current methods for isolating and culturing skin pluripotent stem cells need to be improved due to their small number, long culture period, high cost, and limited research and application.

Method used

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  • Method for improving culture of human skin pluripotent stem cells (SKPs)
  • Method for improving culture of human skin pluripotent stem cells (SKPs)
  • Method for improving culture of human skin pluripotent stem cells (SKPs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Method for culturing skin pluripotent stem cells of the present invention

[0042] 1 Skin and dermal adherent cell culture and expansion, that is, the culture and expansion of FBs

[0043] 1.1 Take a fresh human foreskin specimen, sterilize it with 75% alcohol, rinse with PBS for 3 times, remove the subcutaneous fat layer, and retain the dermis and epidermis.

[0044] 1.2 Cut the skin into 5×5mm 2 / Tablet, use 0.25% pancreatin in a refrigerator at 4°C overnight.

[0045] 1.3 Stop the digestion with an equal volume of DMEM medium containing 10% fetal bovine serum (FBS), and rinse twice with PBS. The epidermis is detached and washed away during the washing process, and the unseparated epidermis is manually separated and removed with tweezers.

[0046] 1.4 Cut the dermal tissue into 2-3mm 2 / Tablet, add 1mg / mL type I collagenase and place it in a 37°C incubator for 2-3 hours, causing the tissue to be almost completely digested. Add 5 times the volume of DMEM high glucose ...

Embodiment 2

[0054] Example 2 Long-term storage of the present invention

[0055] The fibroblast FBs obtained in step 1 of Example 1 of the present invention can be frozen, thawed when actually needed, and then transformed into skin pluripotent stem cells (SKPs cells). The freezing and thawing methods are as follows:

[0056] 1 After centrifuging the expanded skin adherent cells, collect the cell pellet, add it to the cryopreservation solution (10% DMSO + 20% FBS + 70% DMEM), mix well, and then freeze.

[0057] 2 Take out the freezing tube and immediately put it in a 37°C water tank to quickly thaw it, gently shake the freezing tube to make it all melt within 1 minute, wipe the outside of the preservation tube with 70% alcohol, and move it into the aseptic operating table.

[0058] 3 Take out the thawed cell suspension, slowly add it to a culture dish with adherent medium (dilution ratio is 1:9), mix well, and put in CO 2 Culture in an incubator and change the medium every 3 days until the cells ar...

experiment example 1

[0062] Experimental example 1 Comparison of the method of the present invention and the existing method

[0063] 1. Experimental method

[0064] (1) The method of the present invention: the cultured pluripotent stem cells obtained according to the method of Example 1 of the present invention, the culture time is 1 week.

[0065] (2) Existing methods: According to the method disclosed by Jeffrey A Biernaskie1, etc., Isolation of skin-derivedprecursors (SKPs) and differentiation and enrichment of their Schwanncell progeny, NATURE PROTOCOLS VOL.1, NO. 6, 2006, 2803, pluripotent stem cells are cultivated (The primary cells isolated from the dermis are directly cultured in the suspension medium), the culture time is 2-4 weeks.

[0066] 2. Detection method

[0067] The cells cultured by the two methods were used for cell observation and the results of adipogenic bone formation were tested.

[0068] The skin pluripotent stem cells cultured by the method of the present invention are compared an...

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Abstract

The invention discloses a method for improving culture of human skin pluripotent stem cells (SKPs) and application of the method. The method comprises the following steps: (1) cutting corium layer tissue of skin, digesting collagenase, performing dissociation, and centrifuging, removing supernate so as to obtain cells; (2) performing adherent culture on the cells obtained in the step (1) by using a adherent culture medium so as to obtain fibroblast; (3) performing resuspension on the fibroblast obtained in the step (2) by using a suspension culture medium, inoculating into a culture bottle coated by Poly-HEMA, and culturing, thereby obtaining the human skin pluripotent stem cells. By adopting the method disclosed by the invention, the human skin pluripotent stem cells can be effectively cultured, a great number of cells can be cultured, the culture cycle is short, the cost of the method is low when being compared with that of a conventional method, and good application prospects are achieved.

Description

Technical field [0001] The invention belongs to the field of cell culture, and specifically is an improved method for culturing human skin pluripotent stem cells (SKPs). Background technique [0002] Skin-derived precursors (SKPs), which have characteristics similar to embryonic neural stem cells, can differentiate into various neural and mesodermal cell phenotypes, including peripheral neurons, glial cells, smooth muscle cells, bone, and cartilage And lipid cells. [0003] SKPs still have high proliferation activity and multi-directional differentiation potential after continuous and long-term cell culture, suggesting that their performance is stable, and they are expected to become ideal seed cells for cell replacement therapy. In addition, the skin is the largest organ of the human body, and it is also one of the tissues with the fastest self-renewal and regeneration and repair in the body. It is simple and convenient to obtain materials, has sufficient sources, and has no obvi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0656
Inventor 华薇戴茹李利李祎铭熊丽丹谢恒陈伟
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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