Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell

A technology of corneal limbal stem cells and corneal epithelium, applied in biochemical equipment and methods, tissue culture, microorganisms, etc., can solve problems such as restrictions on widespread use, spread of xenogeneic animal diseases, and great concerns of patients, and achieve the effect of wide application potential

Inactive Publication Date: 2005-02-16
西北农业科技大学
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Problems solved by technology

However, when using 3T3 cells as feeder layer culture and constructing corneal limbal epithelial grafts, since 3T3 cells are fibroblasts from fetal mice, there is a risk of spreading xenogeneic animal diseases to humans in clinical application. Due to the concerns of patients large enough to limit its widespread use

Method used

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  • Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell
  • Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell
  • Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell

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Embodiment 1

[0021] Preservation of limbal stem cells and construction of corneal epithelium without feeder layer (taking Guanzhong dairy goat as an example)

[0022] Guanzhong dairy goats were anesthetized with 2.4ml mixture of 846 mixture (Veterinary Medicine Factory of PLA Agriculture and Animal Husbandry University), rinsed the conjunctival sac and corneal surface with double-antibiotic saline (self-made), 5ml lidocaine hydrochloride (Shanghai Hefeng Pharmaceutical Co., Ltd.) 5ml, Bubi Caine (Shanghai Hefeng Pharmaceutical Co., Ltd.) 5ml retrobulbar anesthesia. Lay a sterile drape, open the eyelids with an eyelid speculum (Jiangsu Liuliu Vision Co., Ltd.), and under an ophthalmic operating microscope (Jiangsu Liuliu Vision Co., Ltd.), aseptically cut the upper limbal epithelium (with a small amount of limbal stroma), Place in 1.2IU dispase (Gibco Lot No.1126857) for cold digestion for 14-16h, transfer to DMEM / F12 (Gibco Lot No.1107011) + 10% NBS (product of Beijing Yuanheng Shengma Bio...

Embodiment 2

[0028] Preservation of human limbal stem cells and feeder-free construction of corneal epithelium

[0029] Take 2-4mm from the ipsilateral or contralateral side of patients with limbal defects 2 The size of the limbal epithelium, at 200 x 10 -3 Soak in U / L penicillin and 200mg / L streptomycin saline for 15-20min, and bring it into the sterile room. Rinse several times with PBS(-). Under a stereomicroscope (Leica Company), after further lamellar separation of the corneal epithelium, place in 1.2IU dispase for cold digestion for 12-14h, put in PBS(-) to wash once, transfer to DMEM / F12+10%NBS culture medium to terminate digestion Gently scrape off the digested epithelial layer with a cell scraper (Nunc.Inc.USA), and blow the discrete cells repeatedly with a pipette. Centrifuge twice at 1000r / min for 5min each time to collect the cells.

[0030] Subsequent implementation methods are the same as implementation 1. image 3 Human corneal limbal stem cells after cryopreservation.

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Abstract

The invention publishes a kind of method to stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer. stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer. It can stores self stem cell of corneal limbus for long time in order to satisfy the request of transporting, storing and proliferating stem cell of corneal limbus under different experimental conditions. The invention stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer, to make that corneal epithelium rebuild self when stem cell of corneal limbus colobomas come true in clinic. The invention can successfully form planting piece of artificial corneal epithelium which can autoplastic graft to misshapen surface of cornea of corneal limbus stem cell and the effect to get eyesight back is obvious. So it has a wide application potency in clinic.

Description

technical field [0001] The invention belongs to the technical field of stem cells and tissue engineering, and specifically relates to a method for isolating and cloning limbal stem cells in vitro, storing them under different conditions in vitro, and using them for feeder-free construction of corneal epithelium and artificial cornea after thawing. Background technique [0002] The surface of the normal eyeball is covered with three types of epithelium: cornea, limbus, and conjunctiva, which are completely different in cell phenotype, and form a complete ocular surface with the tear film on the front surface. Among them, the limbal stem cells in the basal layer of the limbal epithelium not only play an important role in maintaining the integrity of the corneal epithelium, but also have the following characteristics: (1) proliferative; (2) self-renewal ability; (3) can produce a large number of Progeny cells in terminal undifferentiated state and function; (4) have the ability...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 屈雷王馨窦忠英
Owner 西北农业科技大学
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