2.2.1. Rat hepatocyte isolation and 2D cultures maintenance
Hepatocytes were isolated from rats using the two-step collagenase perfusion–based method described by Seglen (Seglen 1976) with slight modifications. Briefly, the rats were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg body weight) and xylazine (10 mg/kg body weight) solutions. The liver was perfused via the vena portae for 10 min with perfusion buffer I (0.14 M sodium chloride, 6.7 mM potassium chloride and 10 mM HEPES), adjusted to pH 7.5 with 2.4 mM EGTA at 37ºC. Subsequently, perfusion was continued with a collagenase buffer, consisting of 200 mg/L collagenase P (Roche applied Science), 67 mM sodium chloride, 6.7 mM potassium chloride, 100 mM HEPES, Bovine Serum Albumin (BSA, 200 g/L) adjusted to pH 7.6, and 4.8 mM calcium chloride dihydrated, at 37ºC for 7 min. The flow rate for the perfusion buffers was 10 mL/min. After perfusion the liver was removed from the animal and dissociated in cold perfusion buffer I with 10 g/L of albumin. The resultant cell suspension was filtered through gauze, centrifuged for 10 min at 50 g, washed once with medium, centrifuged again,
and resuspended in medium in a final concentration of 3.5x106 cells/mL. An
additional Percoll-step was included for hepatocytes purification thereby eliminating both non-viable and non-parenchymal cells, by layering 5 mL of cell suspension over a 25% Percoll solution. After centrifuging at 1300 g at 4ºC for 20 min, the pellet of hepatocytes was diluted in PBS, centrifuged for 10 min at 50 g, and washed twice with PBS for removing the Percoll solution. Finally, the cells were resuspended in complete Williams’ E medium (described below). The viability of the freshly isolated hepatocytes was assessed by counting the cells in a haemaocytometer using the trypan blue exclusion method; values within an 85–95% range were routinely obtained.
For 2D cultures, freshly harvested hepatocytes were seeded onto matrigel®
pre-coated culture plates at a density of 5x104 cells/cm2 total cells. Hepatocytes were
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hydrocortisone, 0.032 U/mL insulin, 15mM HEPES, 1mM sodium pyruvate, 1mM non-essential aminoacids, and antibiotics - 100 U/mL penicillin, 100 mg/mL streptomycin and 40 ug/mL gentamicin (WE complete medium). In 2D cultures, hepatocytes expressed liver-specific functions during approx. 7 days.
2.2.2. Fibroblasts cultures
Three types of fibroblasts were tested: mouse embryonic fibroblasts (mef), human foreskin fibroblasts (hff) and NIH 3T3 fibroblasts. Mouse embryonic fibroblasts were isolated after fibrous tissue trypsin digestion of fetuses with 12 to 14 gestation days using the methods described by Robertson (Robertson 1987). Both hff and NIH 3T3 fibroblasts were purchased from ATCC.
Fibroblasts were routinely cultured in tissue culture flasks and maintained in medium MEM supplemented with 10% (v/v) FBS and 100 U/ml (Penicillin/Streptomycin). The culture medium was exchanged every 3 days and fibroblasts were diluted 1:2 (first passage) and 1:3 upon (first passage) confluence. For co-cultures, confluent fibroblasts were growth arrested by adding 100 µM of Mytomicin for 3 hours into the culture medium. Afterwards, cells were rinsed three times in PBS, trypsinized, gently mixed with the hepatocytes suspension and inoculated in spinner vessels or bioreactors as described below.
2.2.3. 2D Co-cultures of Hepatocytes and Fibroblast
Freshly harvested hepatocytes and fibroblasts were seeded simultaneously
onto matrigel® pre-coated culture plates at a density of 1.25x105 cells/cm2, according
to Bhatia (Bhatia et al. 1998). After seeding, cultures were kept untouched for at least
12h at 37°C in a humidified atmosphere with 95% air and 5% CO2, to allow cell
attachment. Unattached cells were then removed by medium exchange. The culture medium was renewed every 24h and cells were routinely examined under phase contrast microscopy before every culture medium renewal. Culture supernatant and cells were collected according to the protocol at the same time points as 3D cultures
and stored at -20°C for further assay. In 2D co-cultures, hepatocytes expressed liver- specific functions during approx. 10 days.
2.2.4. 3D Co-cultures of Hepatocytes and Fibroblast
3D co-cultures of hepatocytes/fibroblasts were performed in bioreactor and spinner vessels. Spinner cultures were maintained inside an incubator at 37ºC with a
humidified atmosphere of 5% CO2 in air. The inoculation of both spinner and
bioreactor vessels was performed as follows: single-cell suspensions of hepatocytes and fibroblasts were mixed together at an agitation rate of 60 rpm in 70/200 ml (spinner/bioreactor) of Williams E complete medium supplemented with 15% (v/v) FBS to promote cell aggregation. Three hours post inoculation, the agitation rate was increased to 80 rpm to avoid sedimentation of the cell spheroids and after 24 h, half of the culture medium was replaced by fresh medium (supplemented with FBS, to obtain a final concentration of 10% (v/v)) resulting a final culture volume of 125 ml in the spinners and of 300 ml in the bioreactors. Three inoculums were used in the
spinner vessel cultures, namely 1.2x105, 2.4x105 or 3.6x105 cells/mL.
To perform cultures under environment-controlled conditions, commercially available bioreactors (Sartorius-Stedim Biostat Q-Plus system) with volume capacities between 200 and 500 ml were used. The agitation in the bioreactors was provided by marine impellers (in contrast to the paddle impellers in the spinners). The top of the bioreactors has multiple ports for different applications, such as sampling, addition or removal of medium or other supplements or solutions and
placement of the temperature, pH and pO2 sensors. Both pH and oxygen were
controlled through the addition of gases via surface aeration. The pH of the culture
was controlled at 7.4 with CO2 and the pO2 at 30% (of air saturation) with air and N2.
The temperature was kept at 37ºC by water recirculation in the vessel jacket controlled by a thermocirculator bath. Data acquisition and process control were performed using MFCS/Win Supervisory Control and Data Acquisition (SCADA)
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software (B-Braun Biotech International GmbH, Melsungen, Germany). The inoculum
used in the bioreactor cultures was 1.2x105 cells/mL.
To avoid nutrient limitations and the accumulation of products of cellular metabolism that can be toxic to the cells, 50% of the culture medium was replaced every 4 days (in both spinner vessel and bioreactor cultures). To evaluate the effect of oxygen concentration in hepatocyte functionality and inducibility, three bioreactors were run in parallel using different oxygen levels (5%, 30% and 70 % of air saturation).
2.2.5. Sampling
Samples of cell spheroids and culture medium were collected at several time points to evaluate cell morphology and the synthesis/secretion and biotransformation capacities of hepatocytes. The samples were centrifuged at 50 g, and the supernatants were stored at -20ºC for albumin and urea quantification and at 4ºC for Lactate dehydrogenase (LDH) analysis as described by Racher (Racher 1998). Cell spheroid pellets were used for incubation with the proper substrates of ECOD and UGT to assess biotransformation capacities (described below in detail). Although some cellular death occurs mainly during the first days of culture (probed by LDH analysis), due to difficulties in disaggregating cell spheroids to assess the exact cell number, the ECOD and UGT activities are expressed by seeded hepatocytes (this is also a more accurate comparison between mono- and co-cultures since feeder cells have no hepatocyte specific functional capacities).
2.2.6. Fluorescent cell staining
To evaluate co-aggregation between hepatocytes and fibroblasts, both cells were stained separately (hepatocytes in green and fibroblasts in red), using a kit for general cell membrane labelling (PKH67 Fluorescent Cell Linker Mini Kit) following the instructions of the manufacturer. Briefly, cells were washed and then incubated
for 15 min in a dye at a concentration of 2.5x105 cell/µl. Afterwards, cells were
washed with culture medium and put together in culture.
2.2.7. Determination of albumin secretion and urea synthesis
Albumin secretion was measured by an enzyme-linked immunosorbent assay (NEPHRAT, Exocell, Philadelphia, USA). The urea synthesis rate was determined using a quantitative colorimetric kit (QuantiChromTM Urea Assay Kit, DIUR-500, ref DIUR-500; BioAssay Systems), according to the manufacturer’s instructions.
2.2.8. 7-ethoxycoumarin-O-deethylase (ECOD) activity
7-ethoxycoumarin-O-deethylase (ECOD) activity was measured according to Gomez-Lechon et al. (1997) with slight modifications. Salicylamide (1.5 mM) was added to the medium to prevent conjugation of 7-hydroxy metabolites of 7-
ethoxycoumarin. Therefore, hydrolyzation treatment with β-
glucuronidase/arylsulfatase was avoided. The activity is expressed as µmol of 7-
hydroxycoumarin formed per hour and per 106 hepatocytes inoculated. Day zero
assay corresponds to the enzymatic activity of hepatocytes immediately after isolation. The activity refers to 7-hydroxycoumarin formed and is expressed as
µmol/hour/106 hepatocytes.
2.2.9. Uridine diphosphate glucuronoltransferase (UGT) activity
Uridine diphosphate glucuronoltransferase (UGT) activity was determined by quantification of the substrate, 4-methylumbelliferone (4-MU), before and after cell incubation with the substrate. The procedure was performed according to Gomez- Lechon et al. (Gomez-Lechon et al. 1997) with slight modifications. Briefly, 100 µM solution of 4-MU in 0.01M PBS was incubated with cells for an hour at 37ºC. Samples fluorescence was analyzed at an excitation wavelength of 320 nm and emission of 450 nm. The 4-MU remaining concentration was determined based on a
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standard curve generated in PBS spiked with 0, 1.5, 3, 6, 12.5, 25, 50 and 100 µM.
The activity is expressed as µmol of 4-MU metabolized per hour and per 106
hepatocytes inoculated. Day zero assay corresponds to the enzymatic activity of hepatocytes immediately after isolation. The activity refers to 4-MU metabolized and
is expressed as µmol/hour/106 hepatocytes.
2.2.10. Induction Assay
For the induction assay, the compound beta-Naphthoflavone (BNF), a CYP 1A2 inducer, was added to the bioreactor co-cultures at day 3, to a final concentration of 25 µM. Cultures were exposed to the inducer for a 72 h period. Samples were collected daily and enzymatic activity of ECOD was then measured as described above.
2.2.11. Statistics
Error bars correspond to the standard deviation of the average values. Data comparison between systems was performed using a two-way ANOVA test. Level of confidence was set at 0.05 for all tests, with p-values lower than 0.05 considered statistically significant. The albumin, urea, ECOD and UGT quantifications were performed in triplicate. The complete experiment (1 co-culture bioreactor, 1 co- culture spinner vessel, 1 mono-culture bioreactor, 1 monoculture spinner vessel, 1 2D co-culture and 1 2D mono-culture) was run twice (n=2).
3. RESULTS