Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro.

In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma C(max) levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4α (HNF-4α)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ~4-fold and urea ~5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 ± 10.3; CYP1A2: 64.0 ± 15.1; CYP2B1: 15.2 ± 2.9; CYP2B2: 2.7 ± 0.8; CYP3A2: 4.0 ± 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 ± 2.41 vs. 0.42 ± 0.015; CYP1B: 3.47 ± 1.66 vs. 0.4 ± 0.09; CYP3A: 11.65 ± 4.70 vs. 2.43 ± 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A = 27.33 and CYP3A = 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.

Bone marrow transplantation in canine mucopolysaccharidosis I. Effects within the central nervous system.

Five dogs with mucopolysaccharidosis I, a model of human Hurler/Scheie syndrome, were transplanted with marrow from phenotypically normal littermates at 5 mo of age. At 3 and 9 mo posttransplantation, biopsies of cerebral cortex, liver, and cerebrospinal fluid were obtained. The alpha-L-iduronidase levels in these tissues were 0.8-7.4, 26-45, and 6.3-14.9% of the paired donor tissues, respectively. Although iduronidase was present in relatively low levels in the recipients' brains and cerebrospinal fluid at both biopsy times, reduction in brain glycosaminoglycan (GAG) was comparable to that observed in liver. Ultrastructural studies of cells within the transplanted dogs' brains showed less lysosomal distension and storage product than in affected, nontransplanted, littermate controls. The most marked clearing of stored GAG was in cells surrounding blood vessels, but decreased lysosomal storage in neurons and glial cells was also observed. Urinary GAG excretion also decreased to near normal levels by 5 mo posttransplantation.

Tritiated thymidine incorporation into canine cyclic hematopoietic bone marrow in vitro.

To investigate changes in the proliferative activity of bone marrow cells in canine cyclic hematopoiesis, nonadherent cells were incubated for 1 h with tritiated thymidine either immediately after the cultures were established or following an 18-h preincubation period. The data suggest that changes in thymidine incorporation show a 12- to 14-day cycle that consists of two distinct phases. During the first six days of the cycle (from peripheral neutropenia to relative neutrophilia), two peaks of incorporation were observed. During the second phase (corresponding to the neutrophilia and oncoming neutropenia), thymidine incorporation was uniformly lower than control values. The change from an apparently cyclical process to a low stable value occurred after the wave of marrow myelopoiesis and close to a time point (days 8-10 of the cycle) at which we have recently suggested significant changes in cell release and/or proliferation take place. The data can be interpreted in the context of a periphery-to-stem-cell feedback loop through an intermediate cell population, probably of myeloid precursors.

Fluorometric assay of alpha-L-iduronidase in serum for detection of affected and carrier animals in a canine model of mucopolysaccharidosis I.

We investigated measuring serum alpha-L-iduronidase (EC 3.2.1.76) by a sensitive fluorometric assay in 28 members of a canine family with mucopolysaccharidosis I. If assayed on the day of collection, serum was an acceptable specimen for identifying affected, carrier, and normal dogs. The overall correlation (r) between iduronidase activity in serum and mononuclear leukocytes was 0.966. However, iduronidase was extremely labile in serum refrigerated or frozen for 48 h.