In Situ Collagen Gelation: A New Method for Constructing Large Tissue in Rotary Culture Vessels

Abstract

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.

Transplantation of engineered bone tissue using a rotary three-dimensional culture system

“…Osteoblast-like calvarial cells were obtained from the calvaria of 20 Wistar newborn rats via sequential digestion with collagenase (Wako, Osaka, Japan) as described previously (Su et al 2003). The culture medium contained an alpha modification of minimum essential medium (already containing 50 μg/ ml of L-ascorbic acid; GIBCO, Grand Island, NY), 15% fetal bovine serum (Cansera, Canada), 50 μg/ml penicillin, 50 μg/ml streptomycin, and 100 μg/ml neomycin (GIBCO).…”

“…CG-RC system. We had previously created the CG-RC system by combining the collagen solution embedding method with the HARV bioreactor (Su et al 2003). The HARV bioreactor was purchased from Synthecon, Inc. (TX, USA).…”

“…This promotes cell growth in the interior of the scaffold. As a result, living cells are distributed uniformly in the engineered 3-D tissue (Su et al 2003;Yu et al 2004). The dynamic culture conditions were also shown to be useful for growing tissues and organs, such as cartilage, (Baker and Goodwin 1997), skin (Doolin et al 1999), and bone (Qiu et al 2001;Yu et al 2004).…”

“…However, it is difficult for the rotating bioreactor to produce large tissues because microbeads (Qiu et al 2001) and small synthetic constructs (Freed et al 1998;Pei et al 2002) are the common scaffolds that are used. We previously tried to produce large 3-D tissue in vitro, and we created the 3-D culture system (CG-RC: in situ collagen gelation with rotary 3-D culture system) by combining the bovine type I collagen (Col-I) solution with the HARV bioreactor; this challenges the limitation of scaffold size and minimizes the disrupting effect of increased tissue density (Su et al 2003). Using this in vitro culture system, a 3-D tissue, measuring about 28.0 mm in diameter and 2.5 mm in thickness, can be engineered by the end of 3 wk (Su et al 2003).…”

“…We previously tried to produce large 3-D tissue in vitro, and we created the 3-D culture system (CG-RC: in situ collagen gelation with rotary 3-D culture system) by combining the bovine type I collagen (Col-I) solution with the HARV bioreactor; this challenges the limitation of scaffold size and minimizes the disrupting effect of increased tissue density (Su et al 2003). Using this in vitro culture system, a 3-D tissue, measuring about 28.0 mm in diameter and 2.5 mm in thickness, can be engineered by the end of 3 wk (Su et al 2003). However, in our previous study, we did not fully investigate osteogenesis.…”