Rotating-Wall Vessel Coculture of Small Intestine as a Prelude to Tissue Modeling: Aspects of Simulated Microgravity

A new low shear stress, low turbulence microcarrier culture system has been developed at NASA's Johnson Space Center that permits large-scale three-dimensional tissue culture. Tissue culture bioreactors called rotating-wall vessels were used in conjunction with multicellular cocultivation to develop a unique in vitro tissue-modeling system. Normal small intestine epithelium and mesenchymal cells were cocultivated on Cytodex-3 microcarriers and were initiated in two phases. Normal small intestine mesenchymal cells were inoculated into the rotating-wall vessel at 2 × 10⁵ cells/ml and allowed to attach and proliferate for 2 to 3 days. Normal small intestine epithelium was then added at an innoculum of 2 × 10⁵ cells/ml and cultivation continued for 30 to 40 days. These cocultures attained cell numbers of 4-6 × 10⁶ cells/ml and differentiated to form tissue-like masses of 0.4-0.5 cm with minimal necrosis. The masses displayed apical brush borders, differentiated epithelial cells, cellular polarity, extracellular matrix, and basal lamina. Verification of mesenchymal and epithelial cell expression was determined by immunocytochemistry and scanning electron microscopy. These data suggest that the rotating-wall vessel affords a new tissue culture model for investigation of growth, regulatory, and differentiation processes within normal tissues.