Co-regulation of primary mouse hepatocyte viability and function by oxygen and matrix

Abstract

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound.

Keywords: albumin secretion; hepatocyte; mouse; oxygen consumption; static culture.

Figure 1

Evaluation of PreSens OxoDish + SensorDish Reader. A. Real-time oxygen traces, recorded every 3 minutes over the first 48 h of culture for primary mouse hepatocytes cultured first for 5 h at 1.3mm medium depth, then subsequently either 1.3 mm or 2.6 mm medium depth. B. Representative phase contrast image of hepatocytes cultured adjacent to the edge of the sensor spot, after 48 h of culture. C. Representative fluorescent image of sensor surface on OxoDish culture plate.

Figure 2

Precise placement of oxygen sensors allow for real-time measurement of oxygen concentration near the cell surface. A. Schematic diagram showing a cross-section of the sensor lid with integrated sensors in a 12-well culture plate. Once the probes are secured flush with the bottom of the plate, 0.5 mm spacers are added to the legs to raise the sensors 0.5 mm above the well bottom. B. Diagram showing the cross-section of a single culture well with a 0.5 mm diameter probe situated in the center of the well, 0.5 mm above the well bottom, with 1.3 mm medium depth. C. A discrete diffusion-reaction model showing oxygen concentration in a radial cross-section of a culture well with the oxygen probe placed in the center, assuming steady-state oxygen consumption along the bottom of the well. D. Real-time oxygen traces, recorded every 3 minutes over the first 48 h of culture for primary mouse hepatocytes cultured first for 3 h at 1.3mm medium depth, then subsequently either 1.3 mm or 2.6 mm medium depth.

Figure 3

Pericellular oxygen concentration is a function of medium depth and gas phase oxygen saturation. A. Oxygen concentration at the cell surface, calculated from averaging probe measurements for Days 1 and 4. Data * indicates statistically significant difference from 20 % / 1.3 mm condition (p <0.001, n=2 ). B. Representative images of hoescht (blue) and hypoxyprobe (green) staining 24 h after initial seeding. All images have common exposure intensities and thresholding. Non-specific staining of dead cells is observed in all conditions.

Figure 4

Results of quantitative imaging following Hoechst/EtBr staining on Days 1 and 4. A. Dot plot showing density of viable cells calculated for each biological replicate, as a function of gas-phase oxygen saturation and medium depths. B. Percentage of live cells, defined as the fraction of non-EtBr stained nuclei. * indicates statistically significant difference from 20 % / 1.3 mm condition (p <0.05).

Figure 5

Modeling and predicting steady-state oxygen consumption based on experimental data. A. Day 1 oxygen consumption rates, calculated from cell-surface oxygen measurements and gas-phase oxygen saturation. B. 0^th order oxygen diffusion model fit to the Day 1 experimental averages of oxygen concentration at the cell surface.

Figure 6

Albumin secretion is influenced at early timepoints by oxygen and at late timepoints by Matrigel and oxygen. Conditioned media from hepatocyte cultures was collected/replaced with fresh medium ever 24 hours and albumin was quantified via ELISA. Standards and samples were tested in triplicate. All conditions shows statistically significant difference from 20% O₂/1.3mm conditions except * (p<0.05, n>2).