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Stem cell regenerating surface cornea and its application in corneal transplantation

A stem cell and superficial technology, applied in the field of stem cell regeneration superficial cornea and its application in corneal transplantation, can solve the problems of limited number of stem cells, few treatment opportunities, lack of stem cells, etc., to ensure long-term effectiveness, rich sources, and immune rejection light effect

Inactive Publication Date: 2003-02-26
北京科宇联合干细胞生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. Traditional corneal transplantation is mainly used to treat central corneal opacity caused by various reasons. Its advantage is that the corneal optical zone is transparent after surgery. The disadvantage is that only the central φ6-7mm optical zone is transplanted. It is ineffective for those who lack stem cells, and the cornea quickly regenerates Cloudy, additionally requiring long-term immunosuppressive therapy
[0005] 2. The advantage of autologous edge transplantation is that it does not require immunosuppressive treatment, but there is a risk of stem cell loss in healthy eyes, and it cannot be transplanted in a 360° circle, so the effect is limited to a certain extent
[0006] 3. Allogeneic edge transplantation is currently a relatively mature surgical method for the treatment of stem cell deficiency, but the donor materials are extremely limited, and treatment opportunities are very few; there is immune rejection, and the postoperative medication is at least one year, and the cost is very high
The advantage of this method is autologous cell culture, no rejection, and no trophoblast 3T3, but it is not suitable for binocular patients and the culture method of primary culture makes stem cells and differentiated cells mixed, and the number of stem cells is limited, so it may not be possible. Guaranteed long-term effectiveness

Method used

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  • Stem cell regenerating surface cornea and its application in corneal transplantation
  • Stem cell regenerating surface cornea and its application in corneal transplantation
  • Stem cell regenerating surface cornea and its application in corneal transplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Take the whole corneal epithelium from the eyes of aborted fetuses at 13 weeks of embryonic age, cut it into pieces, digest with 3ML 0.1% trypsin + 0.02% EDTA for 15 minutes, stop the digestion with the same amount of DMEM + 10% fetal bovine serum, and centrifuge at 1500RPM After 10 minutes, add culture medium to prepare single cell suspension, put 1.5ML single cell suspension in 35MM plastic Petri dish, add culture medium in 5% CO 2 , Culture at 37°C.

[0027] Medium composition: DMEM:F12=1:1, 10% fetal bovine serum, epidermal growth factor EGF (10ng / ml), basic fibroblast growth factor bFGF (20ng / ml), insulin (5ug / ml), transfection Ferritin (5ug / ml), triiodothyronine (2nM), glutamine 4mM, sodium selenate 5ug / ml.

[0028] Culture process: the culture medium is changed once every 2 days, and the cells of the 20th day are passaged when the cell confluence reaches 80-90%. On the treated amniotic membrane, after 14 days of culture under the same culture conditions, stem c...

Embodiment 2

[0031] The limbal epithelium of a 65-year-old male patient who underwent enucleation of the left eye due to left eye absolute glaucoma was immediately cut up and digested with 0.1% trypsin + 0.02% EDTA for 40 minutes. % fetal calf serum to stop the digestion, centrifuge at 1200RPM for 10 minutes, add the following medium to make a single cell suspension, transfer 1.5ML single cell suspension into a 35MM plastic culture dish and add the medium for culture. The culture condition is 5% CO 2 , 37°C.

[0032]Medium composition: DMEM:F12=3:1, 10% fetal calf serum, epidermal growth factor EGF (20ng / ml), basic fibroblast growth factor bFGF (10ng / ml), insulin (10ug / ml), transfection Ferritin (10ug / ml), triiodothyronine (1nM), glutamine 8mM.

[0033] Culture process: the cultured cells were changed every 3 days, passaged when the cells reached 80-90% on the 22nd day, the second generation was passed on the next 9 days, the third generation was passed on the next 8 days, and the third ...

Embodiment 3

[0037] Immediately after enucleation of the left eye of a 10-year-old male patient due to trauma, the limbal epithelium was harvested. The epithelium was cut into pieces and digested with 0.1% trypsin for 70 minutes. The digestion was terminated with an equivalent amount of 10% fetal bovine serum, and centrifuged at 1200RPM for 10 minutes. Minutes, add the following medium to make a single-cell suspension, and transfer 1.5ml of the single-cell suspension into a 35MM plastic culture dish for culture. The culture condition is 5% CO 2 , 37°C.

[0038] Culture medium composition: the culture medium includes: DMEM:Ham's F12 ratio 2:1, 10% fetal bovine serum, epidermal growth factor EGF15ng / ml.

[0039] Culture process: the medium of the cultured cells is changed every 1 day, and the cells of the 25th day are passaged when the confluence reaches 80-90%, the second generation is passed on the next 8 days, the third generation is passed on the next 7 days, and the third generation ce...

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Abstract

In the present invention an embryo or adult cornea epithelium is cut, digested with digesting liquid and centrifugated to prepare single cell suspension, and the cell suspension is cultured in culture dish or bottle with proper amount of culture medium and CO2 in 5% at 37 deg.c. The cultured cell is passed after cell converges to 80-90% and the second and sixth generation of cell is passed directly to amnion for culture for another 8-20 days to obtain the stem cell regenerating surface cornea of the present invention. The stem cell regenerating surface cornea may be used as material for treating corneal disease. The present invention provides a new material for treating corneal disease with rich material source, no danger of mouse-originated pollution, no or slight immunological rejection.

Description

field of invention [0001] The present invention relates to a stem cell regenerating superficial cornea and its application in corneal transplantation; in particular, it relates to stem cell regenerating superficial cornea and its application in corneal transplantation, which is made by using embryo or allogeneic cornea as raw material and non-differentiated culture on amniotic membrane after passage. on the application. Background technique [0002] The cornea is the outermost part of the eyeball, an avascular transparent structure located at the very front of the eyeball (the so-called black eyeball). It is an important refractive medium like a camera lens, and external light must first pass through the cornea to enter the eye. If the cornea becomes cloudy due to chemical burns, heat burns, infection, or other diseases, and the surface of the cornea invades blood vessels, it will seriously affect vision and even blindness. Severe corneal diseases require surgery to remove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61F2/14A61L27/22C12N5/071
Inventor 杜仪琴李凌松
Owner 北京科宇联合干细胞生物技术有限公司
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