Bovine corneal endothelial cells

OTEIER EPITEIEUA Electrogenic and electroneutral ion transporters and their regulation in tracheal epithelium, 192, 549 transformation of airway epithelial cells with persistence of cystic fibrosis or normal ion transport phenotypes, 192, 565 cell culture of bovine corneal endothelial cells and its application to transport studies, 192, 571 methods for studying eccrine sweet gland function in vivo and in vitro, 192, 583. 

The growth rate of mammalian cells in the presence of Cell-Tak adhesive was assayed to evaluate any potential adverse effects caused by this protein. Bovine corneal endothelial cell (BCE) stocks were grown to confluency in MEM plus 15% calf serum, trypsinized, and washed several times by centrifugation on MEM. Suspensions (5 x 104 cells/mL) were seeded onto untreated 35-mm dishes (control) and dishes with Cell-Tak protein (5 fig/cm2) in MEM with 15% calf serum. At various time points during the incubation at 37 °C with 5% CO2, triplicate plates were removed. The attached cells were then trypsinized from the surface, washed, and counted in a hemacytometer. 

The mechanism by which Cell-Tak protein accomplishes this efficiency at the molecular level is addressed with the observation that different cell types attach with the same kinetics (Figure 2A). The lymphoma line (U-937), BHK cells, and bovine corneal endothelial cells attach with varying efficiencies to plastic (Figure 2B) but exhibit the same rate and overall efficiency on Cell-Tak protein. The attachment lacks the specificity that would indicate receptor-mediated attachment and supports the roll of nonspecific interactions. Moreover, other data (not shown) suggest that the attachment of cells is due to Cell-Tak and not to any component such as fibronectin that is found in serum. This is seen in experiments involving the preincubation of Cell-Tak-coated dishes with either fibronectin or serum. Cell-Tak-coated plates were preincubated with cell culture medium containing 20% calf serum for 30 min, and fibronectin was dried onto other plates coated with Cell-Tak before U-937 cells were plated. Cell attachment on fibronectin-coated Cell-Tak plates was identical to attachment to Cell-Tak alone, and attachment efficiency was inhibited by 40% on plates preincubated with serum. 

Fig. 4. Fibrin zymography for detection of PA induction in cell culture media. Samples 1-7 were from conditioned media of bovine corneal endothelial cells at different growth stages and in the presence of various PA inducers (R4).

Fig. 5. Reverse fibrin zymography for detection of PAIs. The fibrin overlay zymogram was supplemented with uPA (see Section 3.6). Samples 1-5 were from conditioned media of bovine corneal endothelial cells at different growth stages and in the presence of various PA inducers (X2). Increased amido black staining indicating position of PAI-1 (55-kDa) is shown (lanes 1 and 2). A small amount of uPA (45-kDa) and tPA (70-kDa) in lanes 3-5 is also noted.

Nguyen LK, Yee RW, Sigler SC, Ye H-S. Use of in vitro models of bovine corneal endothelial cells to determine the relative toxicity of viscoelastic agents. J Cataract Refract Surg 1992 18 7-13 Nimrod A, Ezra E, Ezov N, Nachum G, Parisada B. Absorption, distribution, metabolism, and excretion of bacteria-derived hyaluronic acid in rats and rabbits. J Ocular Pharmacol 1992 8 161-172 Norn MS. Peroperative protection of cornea and conjunctiva. Acta Ophthalmol (Copenh) 1981 59 587-594... 

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. 

These invasion steps are most easily studied on amnion complete with epithelium, or in bioengineered cell monolayers. These can be easily produced by seeding vascular endothelial cells (EC) onto reconstituted extracellular matrix (Matrigel ), or corneal EC onto bovine lens capsule mounted in a combi-ring dish. 

Ravalico G, Tognetto, Baccara F, Lovisato A. Corneal endothelial protection by different viscoelastics during phacoemulsification. J Cataract Refract Surg 1997 23 433-439 Raymond L, Jacobson B. Isolation and inhibitory cell growth factors in bovine vitrous. Exp Eye Res 1982 34 267-286... 

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