Culture solution for retinal pigment epitheliums and preparation method and application thereof

A technique for retinal pigment and epithelial cells, which is applied to the culture medium for retinal pigment epithelial cells and the fields of preparation and application thereof, can solve the problems of undisclosed chemical composition, hidden safety hazards of hESC-RPE, etc., and achieves clear components and low cost. , the effect of promoting cell proliferation

Active Publication Date: 2016-08-10
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, KOSR is still derived from animals, and its chemical composition has not been disclosed so far, which brings safety risks to the clinical application of hESC-RPE

Method used

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  • Culture solution for retinal pigment epitheliums and preparation method and application thereof
  • Culture solution for retinal pigment epitheliums and preparation method and application thereof
  • Culture solution for retinal pigment epitheliums and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Culture medium preparation

[0058] 1) According to 2mg / L L-carnitine, 1mg / L ethanolamine, 16mg / L putrescine, 0.016mg / L sodium selenite, 1mg / L linoleic acid, 1mg / L linolenic acid , 5 mg / L of transferrin was dissolved in sterilized deionized water to prepare a 50× stock solution (Table 1), filtered and aliquoted using a 0.22 μm filter, and stored at -20°C.

[0059] 2) Calculate the final medium preparation volume, add 1% non-essential amino acid, 1% L-glutamic acid, 0.5%-2% N2 additive according to the volume, and add KODMEM to the required preparation total volume;

[0060] 3) Add the corresponding amount of the 50× stock solution described in 1) according to the required preparation volume;

[0061] 4) According to the concentrations of vitamin C acid of 50-200mg / L or vitamin C acid / magnesium salt of vitamin C acid phosphate of 50-200mg / L, reduced glutathione of 1mg / L, and insulin concentrations of 4ng / mL, 1000 × stock solution (table 2);

[0062] 5) Ad...

Embodiment 2

[0069] Example 2 Cultivating retinal pigment epithelial cells derived from human embryonic stem cells using the medium of the present invention

[0070] Specific implementation steps:

[0071] 1) Obtain hESC-RPE cells using the traditional self-differentiation method, and use Tryple TM Enzyme digestion for about 10 minutes, the cells are detached or about to detach from the bottom of the dish, the enzyme solution is collected, washed once with phosphate buffered saline (PBS), centrifuged at 1200 rpm for 3 minutes. Discard the supernatant, resuspend with the medium of the present invention, and count the number of cells. Take 0.5-1×10 5 cells / cm 2 density inoculation. Taking a 12-well plate as an example, the amount of conventional medium added to one well of a 12-well plate is 1 mL, which means that one well of a 12-well plate can be inoculated with 0.5-1×10 5 cells / cm 2 hESC-RPE cells.

[0072] 2) The cells inoculated in step 1) are cultured with the medium of the p...

Embodiment 3

[0073] Example 3 Use culture medium of the present invention to cultivate ARPE-19 cells

[0074] Specific implementation steps:

[0075] 1) Use the culture medium of the present invention to cultivate APRE-19 cells, and after about 15 days, the cells cover the culture dish, and use Tryple TM Enzyme digestion for about 5 minutes, the cells are detached or about to detach from the bottom of the dish, the enzyme solution is collected, washed once with PBS, centrifuged at 1200rpm for 3 minutes. Discard the supernatant, resuspend with the medium of the present invention, and count the number of cells. Taking a 6-well plate as an example, the amount of conventional medium added to one well of a 6-well plate is 2 mL.

[0076] 2) The cells inoculated in step 1) are cultured with the medium of the present invention, the medium is replaced every other day, and the cells are passaged when the cells are full.

[0077] figure 1 The clinical-grade human embryonic stem cell line (Q-CT...

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Abstract

The invention provides a culture solution for retinal pigment epitheliums (hESC-RPE). The culture solution is characterized in that the culture solution contains, 96-97.5 volume fractions of a basic culture medium, 1 volume fraction of non-essential amino acids (NEAA), 1 volume fraction of L-glutamine, 0.5-21 volume fractions of an N2 additive, a component a and a component b. The invention further provides a preparation method of the culture solution and a method for culturing the retinal pigment epitheliums (hESC-RPE) by using the culture solution. Compared with existing culture media, the culture medium of the culture solution is simple composition, the costs are lower, no ethical problem is related, and cell proliferation can be promoted.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture medium for culturing retinal pigment epithelial cells (RPE), the retinal pigment epithelial cells include ARPE-19 cell lines, primary isolated retinal pigment epithelial cells, and human embryonic stem cell sources Human induced pluripotent stem cell-derived retinal pigment epithelial cells (hiPSC-PRE) and pluripotent stem cell-derived RPE. Specifically, the present invention relates to the use of an animal-free medium with fully defined chemical components in maintaining the morphology and function of RPE cells, promoting cell proliferation, and performing large-scale expansion and any form of clinical use. Background technique [0002] Retina-related diseases such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and congenital macular degeneration (SMD) seriously affect the quality of life of human beings, but there is still a lack of effective treatmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/02
CPCC12N5/0621C12N2500/05C12N2500/24C12N2500/30C12N2500/32C12N2500/36C12N2500/38C12N2500/46C12N2501/11C12N2501/33C12N2501/392
Inventor 周琪祝贺谭元卿郝捷王磊王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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