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Limbal stem cell (LSC) culture medium and culture method

A medium and stem cell technology, applied in the field of stem cell culture, can solve the problems of spontaneous differentiation of LSCs, exhaustion of LSCs expansion ability, etc.

Inactive Publication Date: 2019-04-26
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing in vitro culture methods of LSCs will face the situation of exhaustion of LSCs expansion ability and spontaneous differentiation of LSCs without exception.

Method used

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  • Limbal stem cell (LSC) culture medium and culture method
  • Limbal stem cell (LSC) culture medium and culture method
  • Limbal stem cell (LSC) culture medium and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Experimental materials:

[0034] DMEM / F12 medium, 0.25% trypsin, and fetal bovine serum were purchased from Invitrogen. Hydrocortisone, insulin-transferrin-selenium-ethanolamine (ITSE), and mitomycin C were purchased from Solarbio. Dispase II was purchased from Roche Company. Human SPARC and human epidermal growth factor were purchased from Peprotech. EdU cell proliferation detection kit was purchased from Ribobio Company. p63α, Bmi-1 and ABCG2 antibodies were purchased from Abcam. CK3 antibody was purchased from Santa Cruz Company.

[0035] 2. Experimental steps

[0036] 1) Preparation of 3T3 mouse fibroblast feeder layer: 3T3 cells were cultured and expanded in DMEM medium supplemented with 10% fetal bovine serum (fetal Bovine Serum, FBS). The 3T3 cells were pretreated with mitomycin at a final concentration of 10 μg / ml for 2 hours and then treated according to 2.4×10 4 cells / cm 2 The density was inoculated on the upper layer of the transwell chamber to pre...

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PUM

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Abstract

The invention belongs to the technical field of culture of stem cells, and specifically relates to a limbal stem cell (LSC) culture medium and a culture method. In the prior art, LSCs in-vitro culturemethods encounter with situations that LSCc amplification capacity is exhausted and LSCs are spontaneously differentiated without exception, so that LSCs cells meeting transplanting requirements cannot be acquired. In allusion to a technical problem, the invention provides a culture method capable of meeting autotransplantation cell number requirements. It is indicated, by research, that throughadding SPARC (secreted protein acidic and rich in cysteine) with certain concentration into the LSCs cell culture medium, amplification capacity exhaust in an in-vitro passage process can be effectively delayed; colony forming efficiency is increased; proliferative activity is increased; a stem cell number meeting the transplanting requirements is easily acquired; and a transplanting success rateis improved.

Description

technical field [0001] The disclosure belongs to the technical field of stem cell culture, and in particular relates to a culture medium and a culture method for expanding limbal stem cells (LSCs). Background technique [0002] The information disclosed in this Background section is only intended to increase the understanding of the general background of the disclosure, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes prior art that is already known to those skilled in the art. [0003] Limbal stem cells (LSCs) located at the junction of the cornea, conjunctiva and sclera are the basis for the self-renewal and repair of the corneal epithelium and the key to maintaining the normal physiological function of the cornea. Severe ocular surface injury such as corneal thermal and chemical injury, inflammation, immune disease, and contact lens-related ocular surface disease can lead to LSCs decompensation (limbal stem...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N5/0797
CPCC12N5/0621C12N5/0623C12N2500/25C12N2500/46C12N2501/11C12N2501/39C12N2501/405
Inventor 朱婧
Owner SHANDONG UNIV QILU HOSPITAL
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