In situ collagen gelation: a new method for constructing large tissue in rotary culture vessels.

In situ collagen gelation is a method that combines a static three-dimensional culture technique with rotating bioreactors. This method was designed for large dense tissue engineering ex vivo. To challenge the current limitations on size, we combined the static collagen gel embedding method with high-aspect ratio rotating bioreactors. Rat calvarial cells in gelated collagens were cultured in rotating vessels with 5 mM beta-glycerophosphate-containing medium for 1, 2, or 3 wk and then analyzed for cell morphology, cell distribution, and viability, as well as for contraction of the collagen gel. The size of collagen gels with rat calvarial cells averaged 2.8 cm in diameter x 0.25 cm in thickness at the end of 3 wk. Scanning electron microscopy and laser scanning confocal microscopy of collagen gels revealed a homogeneous distribution of living cells. Despite the barrier effects from induced calcification, in collagen gels, cell metabolic activity (alkaline phosphatase assay and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay) increased over the 3 wk, and cell viability (trypan blue exclusion and flow cytometry analysis) remained at about 90% at the end of 3 wk. Based on our results, we determined that in situ collagen gelation provides a feasible method for engineering large dense tissue ex vivo.