Purpose:
To enable direct observation of human corneal fibroblast (HCF) integration with loaded or unloaded scaffolds continuously, at high-magnification and over long periods of time (weeks).
Methods:
A low-volume mechanical bioreactor was designed and used to continuously observe HCF behavior on collagen-based substrates (liquid crystalline and disorganized - PureCol© Advanced Biomaterials, San Diego, CA). The device comprises a low working volume (1.8mL) environmentally-controlled transparent chamber housing stainless steel tissue grips driven by a precision actuator and coupled to a sensitive load cell. FBS supplemented cell culture media flowed through the chamber at a rate of 4µL per minute. HCF/matrix interaction was observed with Differential Interference Contrast microscopy.
Results:
Liquid Crystal. HCFs attached readily to the collagen substrate, elongated and aligned in generally the same direction (Fig. A). Most cells were aligned perpendicular to applied load. However, post-hoc SEM indicated that the HCFs were generally aligned with the collagen substrate as well. Disorganized Collagen. Cells colonized the disorganized collagen at low density and exhibited no consistent alignment (Fig. B). A subset of morphologically distinct, rounded cells appeared to be degrading the collagenous substrate. These cells were interspersed with more elongated spindle-shaped fibroblasts. In addition, fibroblasts were continuously attempting to "connect" with one another via filopodia which could extend over hundreds of microns.
Conclusions:
The rich set of data provided by this unique bioreactor included measurements of cell motility, morphology and alignment all as a function of time and load. The low-volume design permits live cell staining which we have demonstrated as well. The device is likely to provide an unprecedented view of in vitro matrix remodeling.
Keywords: cornea: stroma and keratocytes • cornea: basic science • extracellular matrix