Basement membrane components enhance isolated enterocyte growth

Isolated enterocyte transplantation may have a potential role in increasing intestinal surface area. However, enterocytes are notoriously difficult to grow. Basement membrane components (BMC) promote adherence, migration, and differentiation of enterocytes. Our aim was to determine if BMC enhance enterocyte growth. Twenty-nine rabbits had 5-cm ileal segments resected and serosal pouches (n =.22) created from the serosal surface of the colon. Harvesting of enterocytes by warm trypsinization resulted in 92 ± 6% cell viability and yielded 5.0 ± 2.4 10⁶ enterocytes/cm intestine. Enterocytes (10⁵) were cultured in vitro (n = 7) in 10 ml growth media in plain flasks and flasks coated with laminin alone or Matrigel (an extract of mouse basement membrane containing laminin plus Type IV collagen and heparan sulfate). The cells were subcultured at 2 weeks and examined after Geimsa staining at 4 weeks. Epithelial growth was confirmed by light microscopy and staining for cytokeratin and quantitated by image analysis (JAVA). Epithelial coverage of the flasks was greater with Matrigel (80 ± 15%) than laminin (66 ± 15%) which was greater than control (44 ± 20%) (P < 0.01). For in vivo studies 10⁵ harvested cells were infused into the serosal pouches either in growth media (n = 9) or media + Matrigel (n = 13). Epithelial growth in the pouch was evaluated by qualitative scoring of cytokeratin staining. Cytokeratin staining was similar on control colon serosa (n = 5) and serosa after infusion of cells in media alone. However, Matrigel significantly enhanced the area, depth, and intensity of staining. These effects were apparent at 1 and 2 weeks, but diminished at 4 weeks. BMC enhance enterocyte growth in vitro. A combination of components had a greater effect than laminin alone. BMC increased cytokeratin staining in serosal pouches suggesting that they also enhance enterocyte growth in vivo.