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

Abstract

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.

Keywords: hemodynamics; hepatocyte; metabolism; organotype; phenotype.

Fig. 1.

A: microenvironmental factors contributing to hepatocyte phenotype in vivo. Polarized morphology with resultant 3-dimensional cell-cell interactions and biliary canalicular formation, extracellular matrix-mediated signaling, oxygen, and nutrient transport as well as physiological concentration profiles mediated by sinusoidal and interstitial flow, is known to impact hepatocyte phenotype. B: controlled hemodynamic hepatocyte monoculture system. Cone and plate technology was previously described by Hastings et al. (20) using primary human smooth muscle and endothelial cocultures plated in a Transwell configuration and physiological hemodynamics to restore vascular phenotype. We adapted the technology by plating hepatocytes in a collagen gel configuration within the device across a semipermeable membrane and operationally regulating physiological attributes of the environment such as hemodynamics, shear, and transport of solutes (including oxygen).

Fig. 2.

Cultures under controlled hemodynamics maintain in vivo like morphology relative to nonflow cultures over 2 wk. A: primary rat hepatocytes were plated in nonflow collagen gel configurations and in the devices under controlled hemodynamics (0.6 dyn/cm² shear across the membrane) and followed up over 2 wk. By day 7, hepatocytes in nonflow cultures displayed higher levels of cytoplasmic E-cadherin (green) confirmed and quantified by morphometric analysis (adjacent graphs) and irregular peripheral membrane distribution. B: under controlled hemodynamics, hepatocytes exhibited a more differentiated morphology characterized by distinct peripheral membrane localization and lower cytoplasmic levels of E-cadherin. C: transmission electron microscopy images of day 7 cultures under controlled hemodynamics demonstrate the presence of bile canaliculi (white arrow) and confirm the presence of tight junctions (black arrows) as well as retention of abundant mitochondria (D). Polarized morphology and canalicular localization of the transporter multidrug-resistant protein-2 (Mrp-2, green) that appears after 5–7 days of culture is lost in nonflow cultures by day 14 (E), but the networks are stable and extensive under controlled hemodynamics (F). Day 14 cultures from devices costained for Mrp-2 and hepatocyte nuclear factor-4α (HNF-4α; H) along side sections from rat in vivo liver (G) show very similar staining patterns of canalicular localization of Mrp-2 and nuclear localization of HNF-4α demonstrating the stable retention of in vivo like polarized morphology.

Fig. 3.

Controlled hemodynamics results in retention of hepatocyte-specific function in rat hepatocytes relative to nonflow cultures over 2 wk. Hepatocytes were plated in nonflow collagen gel configurations and in the devices under 0.6 dyn/cm² shear. Medium was changed and collected over 2 wk, and measured albumin and urea levels were normalized to μg/10⁶ cells/day. Albumin levels (A) in the medium show a decline over time in nonflow cultures but are retained at 3- to 4-fold higher levels (*P < 0.01) under controlled hemodynamics in the device over the course of 14 days. A similar trend is seen with urea (B) as well.

Fig. 4.

Controlled hemodynamics upregulates the expression of phase I and phase II metabolic genes and proteins. Hepatocytes were plated in nonflow collagen gel configurations and in the devices under 0.6 dyn/cm² shear. Quantitative RT-PCR was performed for select metabolic genes on RNA samples at day 7 and normalized corresponding nonflow cultures. A: hepatocytes cultured under controlled hemodynamics resulted in expression levels of cytochrome P450 (CYP)1A1, 1A2, and 2B1 that are 10- to 60-fold higher than nonflow cultures; *P < 0.05. B: CYP2B2 and CYP3A2 were also consistently expressed at higher levels than nonflow cultures at 3- to 4-fold higher levels. C: expression levels of glutathione S-transferase (GST) pi were lower in hepatocytes cultured under controlled hemodynamics (−2.3-fold; P = 0.025) compared with nonflow cultures. Transwells were taken down at days 4, 7, 11, and 14, lysed for proteins and separated on SDS page gels before probing with antibodies for UDP glucuronosyltransferase-1 (UGT1) and β-actin. C: Western blots show that controlled hemodynamics upregulate the expression of UGT-1 at all time points. A black line demarcates noncontiguous gel lanes, and no information contained therein has been altered.

Fig. 5.

Controlled hemodynamics results in higher level of CYP activity compared with nonflow cultures while exhibiting extensive concomitant biliary efflux activity. Hepatocytes were cultured in nonflow collagen gel configurations and in the devices under 0.6 dyn/cm² shear. On day 7, the devices were taken down and filter segments excised to perform cytochrome activity assays using commercial kits as per protocols. A: basal activity level of the CYP enzymes in untreated cultures is seen to be upregulated by controlled hemodynamics compared with nonflow controls (CYP1A1: ∼15-fold; CYP1B1: ∼9-fold; and CYP3A1/2: ∼5-fold; *P < 0.05). B: Transwell filter segments from the devices incubated with the substrate carboxy-2,7-dichlorofluorescein diacetate (CDFDA), which gets broken down to carboxy-2,7-dichlorofluorescein (CDF) and actively secreted out into the canalicular spaces, lighting up the networks, demonstrating active canalicular transport and biliary efflux activity.

Fig. 6.

Hepatocytes maintained under controlled hemodynamics display toxic and enzyme induction responses at closer to in vivo levels compared with nonflow cultures. Primary rat hepatocytes were cultured in nonflow collagen gel sandwiches in the devices under controlled hemodynamics for 5 days before being treated with different concentrations of dexamethasone or vehicle control for 48 h and harvested at day 7 for thiazolyl blue tetrazolium bromide (MTT) assay. A: dose-related increase in toxicity under controlled hemodynamics was noted with over 75% decrease in mitochondrial activity at 50 μM while none of the concentrations tested displayed marked toxicity in nonflow cultures. B: to test induction, hepatocytes were cultured in nonflow cultures or controlled hemodynamics for 5 days before being treated with 0.1% DMSO, 3-methyl cholanthrene (1 μM in nonflow cultures and 0.1 μM under controlled hemodynamics) for CYP1A induction, or dexamethasone (50 μM in nonflow and 2.5 μM under controlled hemodynamics) for CYP3A induction. On day 7, the devices were taken down and filter segments were excised to perform cytochrome activity assays using commercial kits as per protocols. B: under controlled hemodynamics, responses to classical inducers were well maintained (P < 0.05) in spite of high basal levels and were seen at concentrations much closer to those seen in vivo.

Fig. 7.

Controlled hemodynamics and transport restore and maintain integrated metabolic and transporter activity, concurrently, and may restore in vitro drug responses to in vivo drug concentrations for efficacy and toxicity. A: primary hepatocytes require 3–7 days to regain the canalicular transporter expression (red solid line) and activity in polarized cultures, during which period there is a decline in phase I and phase II enzymatic expression and activity (blue solid line). The system developed herein results in integrated stability of these parameters over time (blue dashed line). B: such a system may have the potential to restore in vitro hepatocyte responsiveness to drug concentrations observed in vivo, thereby decreasing the gap that exists between preclinical or clinical in vivo responsiveness and in vitro measurements.