Corneal cell culture models

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. 

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Cell Culture Models of the Corneal Epithelium and Reconstructed Cornea Equivalents for In Vitro Drug Absorption Studies... 

Table 12.1 Immortalized human corneal epithelial cell culture models.

Reconstituted HCE (order no. RHC/S/5) is available from Skinethic, Nice, France. It consists of transformed human corneal epithelial cells immortalized by Roger Beuerman from the Louisiana State University-Eye Center, New Orleans, USA. This reconstituted human corneal epithelium forms a multilayered cell culture model that does not exhibit tight junctions and is therefore unsuitable for in vitro drug transport studies. Main focus of the model is eye irritation and toxicity testing and it is commonly used in this area with good prediction qualities. Major advantage of this model is the high reproducibility and uniform appearance. The commercial availability provides a ready-to-use model that is easy to handle. 

Thus far, a wide array of useful cell culture models of the corneal epithelium has been established. Many of these cell culture models focus on toxicity testing and ocular irritation, but some cell layer models for drug permeation studies are also available. Indispensable for successful drug penetration testing is a cell layer that exhibits a tight epithelial barrier. This latter requirement of tight barrier properties disqualifies some of the models that were established as substitutes for the Draize test. At least two cell lines are available for pharmaceutical studies and some newer models may qualify as a useful tool, once they are characterized for their barrier properties. 

Reichl S, Bednarz J, Miiller-Goymann CC. Human corneal equivalent as cell culture model for in vitro drug permeation studies. Br J Ophthalmol 88 560-565 (2004). 

Ramesh GT, Manna SK, Aggarwal BB, Jadhav AL (1999) Lead activates nuclear transcription factor-kappaB, activator protein-1, and amino-terminal c-Jun kinase in pheochro-mocytoma cells. Toxicol Appl Pharmacol 155 280-286 Xu KP, Li XF, Yu FS (2000) Corneal organ culture model for assessing epithelial responses to surfactants. Toxicol Sci 58(2) 306-314... 

The corneal epithelial barrier and cell culture models... 

The Corneal Epithelial Barrier and Cell Culture Models... 

The Clonetics cell line from Cambrex Bio Science represents a cultured human corneal epithelial model (order no. CMS-2015 Cambrex Bio Science, Walkersville, MD). The culture model was generated by isolation of cells from normal human corneal tissues. The cells were then infected with an amphotropic recombinant retrovirus containing HPV-16 E6/E7 genes to extend the useful cell life span. The Clonetics cell model is a very recent entry into the immortalized corneal cell line field, but it has been proved to be useful for toxicity testing as well as in vitro drug permeation studies so far. Because of its very recent introduction, further examinations have to be undertaken to... 

Geerling, G. Daniels, J. X Dart, J. K. Cree, I. A. Khaw, P. X. Toxicity of natural tear substitutes in a fiiUy defined culture model of human corneal epithehal cells. Invest. Ophthalmol. Vis. Sci. 2001, 42, 948-956. 

The EpiOcular corneal model involves culturing normal, human-derived epidermal keratinocytes to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell-culture inserts using serum-free medium, differentiate to form a multi-layered structure that closely parallels the corneal epithelium. EpiOcular is mitotically and metabolically active and releases many of the pro-inflammatory agents (cytokines) known to be important in ocular irritation and... 

The HCE-T Tissue Construct (Gillette model) uses a transfected human corneal epithelial cell line (Kahn et al., 1993) cultured on collagen-membrane cell culture inserts, which, at the air-liquid interface, stratify to form a four- to six-layer epithelium, known as the HCE-T model. Transepithelial permeabiUty (TEP) to sodium fluorescein and transepitheUal electrical resistance (TER) have been identified as physiologically relevant parameters to evaluate the barrier function of the corneal epithelium. Cell viability can be determined by the MTT assay, and histomorphology can also be used as an endpoint (Kruszewski et al., 1997). 

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. 

The Cytosensor Microphysiometer (CM) test method is currently under discussion (draft test guideline on the Cytosensor Microphysiometer) at OECD level [34], It is a cytotoxicity and cell-function based in vitro assay that is performed on a sub-confluent monolayer of adherent mouse L929 fibroblasts cultured in a sensor chamber using a pH-meter to detect changes in acidity [35]. The CM test method serves as an in vitro model system for the cytotoxic action of a test chemical on the cell membranes of the corneal and conjunctival epithelium where the irritant chemical would be... 

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