Factors affecting in vitro growth of harvested enterocytes

Selective enterocyte transplantation may be an alternative to whole organ transplantation for increasing absorptive capacity. Our aim was to determine the effect of initial cell number and viability, proportion of intact crypts, and basement membrane components (BMC) on the in vitro growth of rabbit enterocytes. Enterocytes were harvested using warm trypsinization from ileal segments in 40 rabbits. Initial cell viability was 92 ± 4% (mean ± SD), cell yield was 7.7 ± 3.6 × 10⁶ cells/cm, and there were 0.51 ± 0.33 crypts/100 cells. Initial cell viability correlated with cell yield (r = -0.508, p < 0.001) and % crypts (r = 0.313, p < 0.05). Cell yield also correlated with % crypts (r = -0.645, p < 0.001). Enterocytes (5 × 10⁶) were incubated in growth media in plain or BMC coated growth culture vessels for 14 days. There was a correlation between both number of cells seeded (r = 0.824, p < 0.001) and cell viability (r = -0.696, p < 0.01) and % growth colonies containing epithelial cells at 14 days. Both total growth colonies (r = -0.565, p < 0.05) and colonies with epithelial cells (r = -0.589, p < 0.05) had a negative correlation with % crypts. Incubating cells in BMC coated vessels (n = 6) resulted in significantly more dispase liberated cells after 14 days than in plain vessels (n = 6) (6.6 ± 1.1 vs. 3.8 ± 1.0 × 10⁶, p < 0.05) but viability was similar (97 ± 2% vs. 96 ± 2%). Diamine oxidase (DAO) activity was significantly increased in the growth media of cells cultured on BMC compared to controls (0.76 ± 0.24 vs. 0.43 ± 0.28 units, p < 0.05) although DAO act/cell was similar. Conclusions: 1) cell yield using warm trypsinization is increased at the expense of viability and intact crypts; 2) in vitro growth is increased by increasing the number of cells but not intact crypts seeded; 3) BMC increase the yield of cells with viability and maturity similar to those cultured in plain vessels.