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The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells