Progressive maturation resistance to microcystin-LR cytotoxicity in two different hepatospheroidal models

To develop three-dimensional (3D) cytotoxicity models further, microcystin-induced cytoskeletal disruption was tested in two different models of multicellular hepatocyte aggregate formation (hepatospheroids). Rat hepatocyte suspensions were seeded either onto poly(2-hydroxyethylmethacrylate)-treated culture wells (poly-HEMA) or in a rotating wall vessel (RWV) device which provides minimal shear forces and enhances differentiated 3D growth. Ninety percent of spheroids forming on poly-HEMA tended to fuse and form nonhomogeneous multilobular structures by day 4 of incubation. In contrast, spheroids cultured in the low-shear environment formed homogeneous aggregates that averaged 126 + 10 microm diameter in size at day 7. Microcystin-LR (10(-6) mol/L) was put into contact (90 min in serum-free medium) with hepatocyte suspensions and spheroids formed in both systems for 1, 4 or 7 days. As already described, microcystin-LR (after 90 min), induced cytoskeletal disruptions (blebs) in 98% of the isolated primary hepatocytes maintained in suspension. In 3D cultures, blebs were detected only on poly-HEMA nonhomogeneous early prespheroids. All other mature spheroids (poly-HEMA or RWV) exposed to the toxin did not exhibit obvious morphological signs of toxicity. Moreover, microcystin-LR pre-incubation with hepatocyte suspension prevented the formation of conventional spheroids. In conclusion, the low-shear, simulated-microgravity environment generated high yields of regularly engineered spheroids. In both models, progressive resistance of mature spheroids to microcystin-LR-induced cell deformation developed with time in culture. Microcystin-LR inhibition of the formation of rat hepatospheroids in isolated hepatocyte suspension could be used as a complementary biological assay for detection of the presence of biologically active microcystin-LR in water samples.