SMALL-INTESTINAL SUBMUCOSA - A SUBSTRATE FOR IN-VITRO CELL-GROWTH

The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and th e external muscular layers. The remaining 80 mu m thick sheet was disi nfected and sterilized by methods which removed all cellular component s. The SIS-ECM, retaining its native 3-dimensional microarchitecture a nd composition, was evaluated for its ability to support in vitro cell growth. Six separate cell types were seeded either alone or in cocult ure with other cells upon this matrix, grown in selected media, and ex amined daily for time periods ranging from 48 h to 2 weeks. The six ce ll types tested were NIH Swiss mouse 3T3 fibroblasts, NIH 3T3/J2 fibro blasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteos arcoma (ROS) cell line. All cell types showed the ability to attach an d proliferate. All fibroblast cell lines and the keratinocytes; prolif erated and/or migrated into the 3-dimensional scaffold of the SIS matr ix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts an d primary human keratinocytes resulted in a distinctive spatial orient ation of the two cell types. The fibroblasts populated the mid-substan ce of the 3-dimensional matrix and the keratinocytes formed an epiderm al structure with rete ridge-like formation and stratification when th e composite was lifted to an air liquid interface in culture. In summa ry, SIS provides a substratum with a 3-dimensional scaffold that allow s for cell migration and spatial organization. This substratum is suit able for in vitro, studies of the interaction between epithelial or me senchymal cells and a naturally occurring extracellular matrix.