Primary Corneal Cell Cultures

Keywords Transcorneal drug absorption Corneal epithelium Corneal cell lines Primary culture of rabbit corneal epithelium Cornea equivalents Human cornea construct... 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

Cell culture is one of the primary methods being studied for animal replacement. Primary cultures of heart cells, liver, keratinocytes, corneal cells, and many other tissues are actively being studied [28], Much work has been done for decades in some cases to maintain the functions of the parent tissue close to those in vivo while the cells are in culture. Eventually most differentiated function of the cells is lost. The art is to maintain such function for as long as possible so that longer in vitro exposures can mimic in vivo dosing. 

Solubility/miscibility Soluble in water, ethanol, acetone, ether Biological considerations Oral LD50 (rats) = 17.9 ml kg. Repeated dermal exposure can defat skin. Repeated oral exposure can produce corneal opacities. Not cytotoxic to cells in primary culture. Intraperitoneal LD50 (mice) = 11.6 ml kg-1... 

Another 3-D cornea model, comprising rabbit primary cultures of epithelial and stromal cells as well as mouse immortalized endothelial cells, was described in 1994 by Zieske and coworkers [70], They showed the influence of endothelial cells on the formation of a tightly packed, multilayered epithelium as well as the expression of laminin, type VII collagen, a6 integrin, keratin K3, and a-enolase. Furthermore, their findings suggested that the formation of an in vivo-like epithelium requires the cultivation of the 3-D corneal construct under AIC conditions. By contrast, LCC methods of cultivating corneal equivalents in the absence of endothelial cells failed to promote the expression of differentiation markers and basement membrane components. 

In another approach, Parnigotto and coworkers reconstructed corneal structures in vitro by using corneal stroma containing keratocytes to which corneal epithelial cells from bovine primary cultures were overlaid [73], However, this particular corneal model did not contain an endothelial layer. This model was histochemically characterized and the toxicity of different surfactants was tested using MTT methods. This stroma-epithelium model has been reported to show a cornea-like morphology, where a multilayered epithelial barrier composed of basal cells (of a cuboidal shape) and superficial cells (of a flattened shape) is noted. Furthermore, the formation of a basement membrane equivalent and expression of the 64-kDa keratin were reported, indicating the presence of differentiated epithelial cells. The toxicity data for various surfactants obtained with this model correlate well with those seen by the Draize test [73], However, this corneal equivalent was not further validated or used as a model for permeation studies. 

In 1999, Germain and coworkers developed a corneal stroma-epithelium equivalent without endothelial cell coculture, using human primary cultures... 

Chang-Lin JE, Kim KJ, Lee VH. Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface. Exp Eye Res 80 827-836 (2005). 

DNA binding moiety has been synthesized and evaluated as a receptor-mediated gene delivery system. The system was able to transfect about 30% of corneal endothelial cells of rabbit, pig and human in the presence of chloroquine (Shewring et al., 1997). Associated with the fusogenic peptide of influenza, this peptide transfected 25-30% of primary cultures of vascular smooth muscle cells of man, rabbit and rat (Collins et al., 2000 Li et al., 2000). The molossin-based gene delivery system represents an interesting system in transplantation because the molossin peptide does not bind to vascular endothelium and pancreatic islets. 

Yang, W., and D. Acosta. 1994. Cytotoxicity potential of surfactant mixtures evaluated by primary cultures of rabbit corneal epithelial cells. Toxicol Lett 70 309. 

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