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Human corneal endothelial cell culture solution as well as preparation method and application thereof

A technology of endothelial cells and corneal endothelium, which is applied in the field of human corneal endothelial cell culture medium and its preparation, can solve problems such as no obvious results, and achieve the effects of maintaining shape, promoting proliferation, and improving quantity and quality

Active Publication Date: 2009-12-09
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have confirmed that the conditioned medium of human corneal stromal cells contains basic fibroblast growth factor (bFGF) secreted by stromal cells, which can promote the proliferation of corneal endothelial cells. Cell growth factor (bFGF), epidermal growth factor (EGF) or nerve growth factor (NGF), etc. to promote the proliferation of corneal endothelial cells, but the long-term culture of corneal endothelial cells in the culture medium added with the above-mentioned long factors can maintain the shape and inhibit aging. No obvious effect on apoptosis

Method used

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preparation example Construction

[0010] The preparation method or steps of the present invention are as follows:

[0011] Step 1, first culture bovine corneal endothelial cells in vitro; step 2, prepare bovine corneal endothelial cell lysates, and finally use bovine corneal endothelial cell lysates with a protein content of 10-200 μg / ml as the main active ingredient, and fetuses with a content of 10% Bovine serum, penicillin, and streptomycin were each 100 U / ml, and the rest were supplemented with D / F12 basal culture medium to make it specially used for culturing human corneal endothelial cells in vitro.

[0012] One of the steps:

[0013] (1) Obtain fresh bull's eyeballs from the slaughterhouse, after removing the attached tissues outside the eyeballs, rinse the bull's eyeballs with PBS to remove the residual tissue on the bull's eyeballs;

[0014] (2) Move the bull's eyeball into the ultra-clean workbench, rinse it again with PBS, and soak it in water containing 1000U / ml tobramycin for 10-15 minutes, then ...

Embodiment 1

[0026] Analysis of the proliferation of human corneal endothelial cells by different concentrations of bovine corneal endothelial cell lysate by MTT method

[0027] (1) Human fetal corneal endothelial cells were seeded on a 96-well plate with the same number of starting cells (cell seeding density was 4-5×10 4 / ml), placed at 37°C, 5% CO 2 cultivated under conditions;

[0028] (2) After the cells adhere to the wall the next day, remove the original solution and replace it with D / F12 culture solution containing 0, 10, 20, 50, 100, 200 μg / ml protein bovine corneal endothelial cell lysate (10% fetal bovine serum), and each group had 6 replicate wells;

[0029] (3) After continuing to culture for 4-5 days, the proliferation of human embryonic corneal endothelial cells in each group was measured by MTT method.

[0030] The results showed that the bovine corneal endothelial cell lysate group with a protein content of 10-200 μg / ml was better than the group containing only fetal bo...

Embodiment 2

[0032] MTT assay to analyze the effect of different culture medium on promoting the proliferation of human corneal endothelial cells

[0033] (1) Human fetal corneal endothelial cells were seeded on a 96-well plate with the same number of starting cells (cell seeding density was 4-5×104 / ml), placed at 37°C, 5% CO 2 cultivated under conditions;

[0034] (2) After the cells adhere to the wall the next day, remove the original solution and replace with D / F12 culture medium, bovine corneal endothelial cell lysate containing 50 μg / ml protein concentration, and bovine corneal epithelial cell lysate with 50 μg / ml protein concentration respectively , 50 μg / ml protein concentration of bovine corneal stromal cell lysate, human fetal corneal stromal cell conditioned culture medium and D / F12 culture medium of bovine corneal endothelial cell conditioned medium (the content of 10% fetal bovine Serum) 5 replicate wells were set up for each group;

[0035] (3) After continuing to culture f...

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Abstract

The invention relates to a human corneal endothelial cell culture solution as well as a preparation method and an application thereof. The solution comprises bovine corneal endothelial cell lysis solution with 10-200 mug / ml of protein as the main active component, fetal calf serum with the content being 10%, 100U / ml of penicillin, 100U / ml of streptomycin and the remainder being supplemented by D / F12 basal culture solution. The method comprises of: firstly culturing bovine corneal endothelial cell in vitro; preparing bovine corneal endothelial cell lysis solution and formulating in a disinfection chamber to obtain the product. The invention, for the first time, prepares bovine corneal endothelial cell lysis solution into cell culture solution and applies to in vitro culture of human corneal endothelial cells, can promote great augmentation of human corneal endothelial cells cultured in vitro, maintain form feature of corneal endothelial cells, inhibit cells from ageing and apoptosis, and facilitate the subculture of endothelial cells; wherein, the raw material bovine corneal endothelial cell has abundant source and is easy for in vitro culture, the preparation method of the culture solution is simple, easy to implement, conducive for industrialization, and has wide application and development prospect.

Description

technical field [0001] The invention relates to a culture medium for culturing and expanding human corneal endothelial cells in vitro, in particular to a human corneal endothelial cell culture medium formulated with bovine corneal endothelial cell lysate as the main active ingredient and its preparation method and application. Background technique [0002] Corneal endothelial cell (CEC) is a single layer of cells located in the inner surface of the cornea. After its external attachment, the aqueous humor in the elastic membrane plays an important role in maintaining corneal transparency and visual function. When CEC dysfunction occurs (such as bullous keratopathy, Fuch corneal endothelial dystrophy, endothelial decompensation after intraocular surgery, etc.), CEC cannot regenerate and only rely on the expansion of surrounding normal endothelial cells to fill the lost cells and play a role in metabolism. When it exceeds its compensatory capacity, it will seriously affect norm...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 史伟云高彦周庆军杨玲玲
Owner SHANDONG EYE INST
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