A kind of construction method of human corneal stromal cell line

Abstract

The invention discloses a method for building human corneal stromal cell line and particularly relates to a method for preparing a human corneal stromal carrier bracket of tissue engineering by utilizing seawater fish collagen. The method comprises the steps of fully dissolving non-enzymatic hydrolysis codfish skin collagen lyophilized powder in hydrochloric acid, centrifuging to remove sediment, subpackaging supernatant into holes of a culture plate, freeze drying, adding 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide / N-hydroxysuccinimide cross-linking agent containing chondroitin sulfate to carry out crosslinking, stabilizing the solution by alkaline Na2HPO4 (disodium hydrogen phosphate) solution, cleaning by 2M of NaCl (sodium chloride), 1M of NaCl (sodium chloride) and distilled water, and freeze drying. The method disclosed by the invention has scientific and rational process; the prepared carrier bracket can be applied to volume production, meets heavy demand of large-scale reconstruction of artificial corneal stroma of tissue engineering on the carrier bracket and creates conditions for recovery sight of blind patients suffering from corneal stroma diseases through clinical corneal transplantation, and the carrier bracket preparation and application in in-vitro reconstruction and clinical treatment of the artificial corneal stroma of the tissue engineering are low in cost.

Description

technical field [0001] The invention belongs to the technical field of corneal cell culture, and in particular relates to a method for constructing a human corneal stromal cell line. Background technique [0002] The human corneal stroma (corneal stroma) is composed of human corneal stromal cells and collagen fibers, about 50-60 microns thick, accounting for about 90% of the thickness of the entire cornea. The collagen fibers form 200-250 lamellar layers, which are parallel to each other They overlap and have a certain radius of curvature, and are parallel to the surface of the cornea, ensuring the transparency of the cornea. Current studies have shown that many severe corneal diseases in clinical practice, such as deep thermal burns, mechanical or surgical trauma, chemical burns, and infection by various pathogenic microorganisms, can cause damage to human corneal stromal cells, usually leading to corneal edema and scar formation. Impaired vision, severe cases will lead to...

AI Technical Summary

Problems solved by technology

Subsequently, scholars successively studied the culture medium and in vitro culture conditions of human corneal stromal cells, and found that some growth factors (BorderieV et al., 1998), placental polydeoxynucleotides (MuratoreO et al., 2003), heparin (DenkPO and KnorrM, 1999 ) and so on have a certain mitogenic effect on corneal stromal cells, but it is still far from the establishment of cell lines

In the 1990s, many scholars have used human papillomavirus (PetersDM et al., 1996), telomerase (JesterJV et al., 2003), simian red herpes virus (ManzerAK et al., 2009), adenovirus (YoshikoK et al., 2010) ) conducted transfection research on human corneal stromal cells, obtained corneal stromal cells that can be subcultured, and even obtained several immortalized cell lines, but because the cells carry tumor virus oncogenes, they have potential tumorigenicity, It cannot be used for in vitro reconstruction of tissue engineered human corneal stroma and its clinical application

But so far, there is no research report on the successful construction of non-transfective, non-tumorigenic human corneal stromal cell lines using human corneal stromal tissue