In vitro culture method of limbal stem cell stability

A corneal limbal stem cell and in vitro culture technology, which is applied in the field of stem cell culture, can solve the problems of easy differentiation of cells, poor ability of limbal stem cells to maintain stemness, and inability to stably culture limbal stem cells. Clinical needs, the effect of maintaining stability

Inactive Publication Date: 2019-02-12
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More importantly, the limbal stem cells obtained by existing methods have poor ability to maintain stemness, and the cells are prone to differentiation, and most of them can only be used for one-time transplantation, and cannot continue to stably culture limbal stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0029] 2. Preparation of limbal tissue cell suspension: put the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into about 0.5-1 mm in size with ophthalmic scissors 3 Then add 4.5 ml of tissue digestion solution, place it in a constant temperature shaker at 37°C, digest at 60 rpm for 2-4 hours, and obtain a corneal limbal tissue cell suspension;

[0030] 3. Pretreatment of the cell culture plate: Aspirate the limbal stem cell coating solution and add it to the bottom of the cell culture plate. After the bottom of the plate is completely wet, aspirate the excess limbal stem cell coating solution, and then place the cell culture plate in the ultra Air-dry on a clean bench for 1 h;

[0031] 4. Primary culture of limbal stem cells: filter the digested limbal tissue cell suspension with a 200-mesh cell screen, then centrifuge the filtrate at 500 rpm for 10 min, remove the supernatant, and use limbal stem cells for primary culture The tissue cell pellet was re...

Embodiment 1

[0034] First, the limbal tissue was washed with sterile 0.9% (w / v) normal saline until there were no impurities, then 10 ml of DMEM / F12 medium was taken, and 10 mg of gentamicin (titer 1000 U / mg) was added to dissolve it completely. Filter sterilize with a 0.22 μm syringe filter, and then make up to 100 ml with DMEM / F12 medium to prepare a gentamicin solution. Then, the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, and then rinsed 3 times with sterile PBS balanced salt solution and 3 times with DMEM / F12 medium;

[0035] Then take 10 ml DMEM / F12 medium, add 24 mg Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve and filter sterilization with a 0.22 μm syringe filter, then add 5 ml fetal bovine serum, and finally use DMEM / F12 medium was adjusted to 100 ml, and prepared into tissue digestion solution. The limbal tissue was placed in 500 μl of tissue digestion solution, and the tissue was cut into approximately 1 mm in size...

Embodiment 2

[0040] First, the limbal tissue was washed with sterile 0.9% (w / v) normal saline until there were no impurities, then 10 ml of DMEM / F12 medium was taken, and 10 mg of gentamicin (titer 1000 U / mg) was added to dissolve it completely. Filter sterilize with a 0.22 μm syringe filter, and then make up to 100 ml with DMEM / F12 medium to prepare a gentamicin solution. Then, the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, and then rinsed 3 times with sterile PBS balanced salt solution and 3 times with DMEM / F12 medium;

[0041] Then take 10 ml of DMEM / F12 medium, add 48 mg of Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve and sterilize with a 0.22 μm syringe filter, then add 5 ml of fetal bovine serum, and finally use DMEM / F12 medium was adjusted to 100 ml, and prepared into tissue digestion solution. The limbal tissue was placed in 500 μl of tissue digestion solution, and the tissue was cut into a size of about 0.5 mm with ...

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Abstract

The invention relates to an in vitro culture method of limbal stem cell stability. The method is characterized in that after treated by a gentamicin solution, the limbal tissue is cut into small blocks, and then placed in a tissue digestion solution for digestion and filtration, and tissue cell precipitate is collected, and then the precipitate is resuspended by a primary culture solution of limbal stem cells and is inoculated to a cell culture plate pretreated with a limbal stem cell coating solution for primary culture, after a cell cloned sphere appears, the cell cloned sphere is digested with the tissue digestion solution, then centrifugation is performed, the cell precipitate is collected, and the cells are resuspended by a subculture solution of the limbal stem cells, then are inoculated to the pretreated cell culture plate for subculture to obtain stable limbal stem cells. In order to obtain more stable limbal stem cells, multi-generation expansion culture can be carried out after the subculture, and a large number of stem cells with good stability and high safety can be obtained so as to meet the requirement of clinical application.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to an in vitro culture method for corneal limbal stem cell stability (that is, the corneal limbal stem cell does not differentiate during the proliferation process and can maintain its stem cell properties). Background technique [0002] Limbal stem cells are located in the unique wavy structure "Vogt fence" of the limbal basal layer and are the source of corneal epithelial cell renewal. The corneal limbal stem cell renewal can maintain the continuous horizontal centripetal and vertical upward movement of the corneal limbal epithelial cells, thereby ensuring the structural integrity and normal function of the corneal epithelial layer. In this process, on the one hand, limbal stem cells undergo self-renewal through symmetrical division, that is, the way the cells proliferate and maintain their stem cell properties ensure that the number of stem cells in the stem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/079
CPCC12N5/0621C12N5/0623C12N2500/25C12N2501/11C12N2501/115C12N2501/2306C12N2501/235C12N2501/39C12N2533/52C12N2533/54C12N2533/70C12N2533/80
Inventor 徐彬樊廷俊郑明月田成磊
Owner OCEAN UNIV OF CHINA
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