A method for constructing tissue-engineered human corneal stroma in vitro

Abstract

The invention relates to an in vitro construction method of tissue engineered human corneal stroma. It is characterized in that the human corneal stromal cells are made into a cell suspension with special culture medium A for in vitro construction of tissue engineered human corneal stroma, and then the cell suspension is injected into the decellularized porcine corneal stroma carrier after rehydration with a special rehydration solution for carrier scaffolds Put the scaffold into a 24-well culture plate, add the special culture medium A for in vitro construction to culture in vitro, replace the special culture medium B for in vitro construction during the culture, and obtain the tissue-engineered human corneal stroma. The invention has low production cost and less time-consuming, and better maintains the integrity of the carrier bracket structure. The constructed tissue-engineered human corneal stroma is similar to the normal human corneal stroma in terms of morphology, structure and transparency, and has the characteristics of normal corneal stroma. Function, it can be used as a substitute for donated cornea for the repair of abnormal corneal stroma, so it can be used in mass production to meet the high-quality demand for tissue-engineered human corneal stroma.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering corneal stroma construction, in particular to an in vitro construction method of tissue engineering human corneal stroma. Background technique [0002] The human corneal stromal layer is composed of human corneal stromal cells and 200-250 collagen fiber lamina, which plays an irreplaceable role in maintaining corneal transparency and corneal thickness. Once the cornea is infected, deep thermal burns, deep chemical burns or surgical trauma, etc., it will cause different degrees of damage to the human corneal stromal cells, which will lead to corneal edema and scar formation, resulting in impaired vision, and severe cases will lead to corneal opacity, that is, Cause corneal blindness, common collagen plaques, opacity of subepithelial matrix and reticular corneal opacity. At present, human corneal stromal transplantation is the only way to cure corneal stromal blindness. There are nearly ...

AI Technical Summary

Problems solved by technology

Scholars at home and abroad have used immortalized human corneal stromal cells transfected with oncogenes (Peters DM et al., 1996; Doillon CJ et al., 2003; Reichl S et al., 2004; Manzer AK et al., 2009; Yoshiko K et al., 2010), original Subcultured human corneal stromal cells (Barbaro V et al., 2009; Proulx S et al., 2010) and subcultured 3-8 generations of human corneal stromal cells (Crabb RA et al., 2005; Builles N et al., 2007; Vrana E et al., 2008) as seed cells, using collagen-chondroitin sulfate gel (Griffith M et al., 1999), collagen-polyN-isopropylacrylamide blend polymer (Shimmura S et al., 2003), collagen- Glycosaminoglycan-chitosan foams (Builles N et al., 2007), polyglycolic acid undisassembled fibers (Liu and Cao, 2007), hydrogels (Mimura T et al., 2008), transparent human corneal sheets for transplantation ( Barbaro V et al., 2009), multilayer collagen fibers (Builles N et al., 2010), extracellular matrix secreted by human corneal stromal cells (Proulx S et al., 2010) and decellularized porcine corneal stroma (Yoeruek E et al., 2011) etc. as Carrier scaffolds can construct human corneal stroma tissue analogues in vitro whose shape and tissue structure are similar to normal corneal stroma, which opens up the way for the in vitro construction of tissue-engineered human corneal stroma. This leads to contact inhibition of cells due to excessive density, and the method takes a long time to withdraw from in vitro culture. Moreover, the seed cells used are either immortalized cells transfected with oncogenes that are potentially tumorigenic, or derived from The primary cultured cells of the eye bank cornea or the subcultured cells of only 3-8 passages, that is, the number of human corneal stromal cells obtained is very limited, which greatly limits the large-scale in vitro construction of tissue engineered human corneal stroma and its clinical application. application

The existing research reports on the in vitro construction of tissue-engineered human corneal stroma are still limited to experimental research and cannot meet the large number of blind clinical needs of many corneal stroma diseases.