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The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes
Biochimica et Biophysica Acta 1526 (2001) 119^130
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The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes Konstantinos J. Dabos a; *, Leonard J. Nelson a , Timothy J. Bradnock a , John A. Parkinson b , Ian H. Sadler b , Peter C. Hayes a , John N. Plevris a a
Liver Cell Biology Laboratory, Department of Internal Medicine, University of Edinburgh, Edinburgh, UK b Department of Chemistry, University of Edinburgh, Edinburgh, UK Received 25 July 2000; received in revised form 18 January 2001; accepted 26 January 2001
Abstract The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 108 porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1 H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P 6 0.01), and significantly smaller pyruvate (day 1 vs day 14 P 6 0.03) and threonine consumption (day 1 vs day 10 P 6 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P 6 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P 6 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : High aspect ratio vessel; Bioreactor; Glycolysis ; Ketogenesis; Glutamine synthesis ; Succinate precursor synthesis
1. Introduction During the last decades our ability to culture isolated cells in vitro has been the basis for fundamental advances in cell biology. In spite of the amount of information gained in the traditional cell culture settings, it is believed that conventional tissue culture in two dimensions may be inadequate to model the complex cellular interactions that promote tissue-speci¢c di¡erentiation. Cells maintained as homotypic populations in 2D cultures quickly lose their di¡erentiated functions [18]. These functions can be main-
* Corresponding author. Koukouridi 1, Parodos Kalvokoresi, Evaggelistria, 821-00 Chios, Greece. Fax: +44-131-2292948; E-mail : [email protected]
tained by growing cells under the necessary environmental conditions for organ-like assembly, such as cell^cell interactions in three dimensions. Although 3D cell aggregates can be obtained in suspension cultures the high levels of £uid shear stress encountered in conventional stirred fermentors limit the level of tissue-speci¢c di¡erentiation of the aggregated cells [21]. Experiments in space £ight-induced microgravity can overcome the high shear forces. Indeed, cells cultured in suspension under those conditions have shown signi¢cant changes in cell functions. These include increased growth rate and antibiotic resistance in bacteria, increased substrate attachment in human kidney cells and increased hormone secretion by cultured lymphocytes and macrophages and many others. Even in the absence of any direct evidence for any speci¢c mechanisms, it has become increasingly clear that space £ight-induced microgravity has profound e¡ects on cells in vitro [25].
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 0 9 7 - 6
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Recently the in vitro generation of simulated microgravity has become possible with the high aspect ratio vessel (HARV). HARVs o¡er a revolutionary approach to study cell functions. HARVs were developed at the National Aeronautics and Space Administration (NASA)^Johnson Space Center. HARVs allow the cultivation of cells in an environment of low shear stress, low turbulence and no gas^£uid interface [1,2,24,25]. The principle of rotating the culture vessel allows cells to be maintained in perpetual suspension with the net gravity vector approaching zero all the time. The key to the utility of the HARV system is that the culture vessel and the £uid contained therein approximate a solid body during rotation [11]. When cells are added to the HARV they sink to the bottom of the vessel at a constant rate related to the gravitational ¢eld, the di¡erence in density between the particles and the medium, the size of the particles and other factors. Rotation of the HARV results in a path for the cells determined by the combination of sedimentation and movement along with the medium. By increasing the rotation speed we can prevent the cells from reaching the bottom of the vessel. The cells appear to describe an elliptical path in the medium relative to the observer. Further increases in the rotation rate result in the diminution of the elliptical path until the cells become essentially motionless relative to the medium. At this point the vessel, contents and medium approximate a solid body. The cells maintain 3D orientations relative to each other and the surrounding medium in an apparently low shear environment. These conditions are similar to those expected to prevail in true microgravity [10]. Primary porcine hepatocytes (PPHs) are currently considered the cells of choice in bioarti¢cial liver support systems (BALSS). PPHs are metabolically active with a biochemical pro¢le similar to that of human hepatocytes and can be obtained in the large numbers required for bioarti¢cial liver treatments. PPHs are currently being used in experimental settings and phase I clinical trials as biosubstrate in BALSS [7,32,33]. Unfortunately, so far we have been unable to provide on demand hepatocytes that exhibit the same metabolic functions as freshly isolated hepatocytes. Frozen and revived hepatocytes have been used but they clearly have an unacceptably low viability and some of their key metabolic functions are impaired [3], although they seem to retain the ability to metabolise drugs through their P450 system [26]. The microgravity environment presents an exciting new modality for continuous cultures of hepatocytes in suspension. They can be easily transferred out of the rotating vessels and loaded onto the bioreactors for BALSS treatments on demand. The aim of this study was to characterise key metabolic functions of PPHs cultured in a HARV and to assess cell aggregation and maintenance of proliferation of hepatocytes on 3D structures for a predetermined time period of 21 days.
2. Materials and methods 2.1. Chemicals Type IV collagen (from Clostridium histolyticum), Long epidermal growth factor (L-EGF), Williams E medium and Leibovitz L-15 medium were from Sigma. Hanks' balanced salt solution (HBSS) bu¡er, L-glutamine, penicillin^ streptomycin and amphotericin B were from ICN. All reagents were of cell culture grade. Other reagents were : gentamicin (non-proprietary), porcine insulin (Pork Velosulin, Novo Nordisk, Denmark), dexamethasone (David Bull Labs, UK) and aprotinin (Trasylol, Bayer). All chemicals were of Analar grade. 2.2. Hepatocyte isolation Hepatocytes were isolated from weanling piglets ( 6 15 kg) using our previously described ex vivo collagenase perfusion method [27]. Brie£y, piglets were killed with i.v. pentobarbital sodium, the infrahepatic inferior vena cava (IVC) was clamped and the suprahepatic IVC cannulated. The whole liver was retrogradely perfused in situ with ice-cold phosphate-bu¡ered saline, excised, then exsanguinated. This was followed by an ex vivo open-loop and recirculating collagenase perfusion in ¢ve steps. The liver was disrupted, sequentially ¢ltered in washing bu¡ers, puri¢ed by centrifugation and resuspended in E Williams medium. Initial cell viability was assessed by trypan blue exclusion and yield determined using a Neubauer haemocytometer (BDH, Merck). 2.3. Hepatocyte culture in simulated microgravity: RCCS^HARV Cells were seeded in a 55 ml capacity HARV (CellonSarl, Luxembourg) at a density of 2U106 viable cells/ml Williams E medium at 37³C. The medium consisted of serum-free, chemically de¢ned medium supplemented with 50 ng/ml L-EGF, 10 Wg/ml porcine insulin, 1 Wmol/l dexamethasone, 2 mmol/l L-glutamine, 50 mg/ml gentamicin and 50 mg/ml penicillin^streptomycin. The HARV was connected to the horizontally rotating axis of the RCCS (Rotary Cell Culture System) which pumped incubator gas via a central aperture, across a semi-permeable gas exchange membrane within the HARV. The RCCS^HARV assembly was placed in a humidi¢ed incubator under a 95% air:5% CO2 atmosphere. Next day (day 1), the HARV was aspirated and fresh medium added, which was subsequently changed every 3 days. The optimal rotational speed of the HARV was determined by adjusting the RCCS rotational speed so that the aggregate(s) maintained a freefall, quasi-stationary orbit within the culture vessel. The rotational speed varied according to both aggregate size and the corresponding sedimentation coe¤-
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Fig. 1. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 0^2.2 ppm.
cient ; however, by day 6 of culture, rotational speed was set at 10^15 rpm. For changes of supplemented Williams E medium a 0.5 cm ¢ll port was used using a suction aspirator. When changing the medium, the cells were covered by a small amount of medium to minimise cell damage. Medium samples were taken daily via a syringe attached to one of the luer-lock syringe ports and stored at 370³C until nuclear magnetic resonance (NMR) analysis.
scope (Zeiss, Germany) under phase contrast on both day 0 and day 21 of culture. Photomicrographs recorded hepatocytes in suspension and RCCS culture. Cell purity was determined by cytospin (200Ug, 5 min) of 5U105 freshly isolated cells and staining with haematoxylin+eosin (HpE)/periodic acid Schi¡ (PAS), followed by counting cells in three separate ¢elds for three separate isolations.
2.4. Urea synthesis rate
We used 1 H NMR spectroscopy in this study to try and elucidate patterns of consumption and production of individual amino acids by the cultured hepatocytes in the HARV. This technique has been developed in the last few years and it has been used to measure substances present in biological £uids [8,22,28]. 1 H NMR spectroscopy was used as it was possible to obtain spectra from the medium tested which exhibited no overlapping peaks and a very low signal to noise ratio. The absence of macromolecules in signi¢cant quantities in the medium made it easier to identify peaks and assign them to individual amino acids. Further advantages of NMR spectroscopy are the non-destructive nature of the technique for the samples tested, the possibility of acquiring the spectra of an array of substances at one experiment, the possibility of storing and reusing both the samples and the acquired spectra and the fact that if facilities are available the sample processing and testing is relatively cheap.
The urea synthesis rate (USR) was determined in 0.5 ml samples taken at t = 0 and t = 2 h using a modi¢ed colorimetric urea nitrogen kit (Sigma kit 535-B). The di¡erences in absorbance for the chromogenic reaction were measured at each time point at V525 in a Pye Unicam spectrophotometer and USR calculated. The coe¤cient of variation for this assay was less than 5%. 2.5. Ethanol synthesis To con¢rm the presence of ethanol in our culture media we also measured ethanol levels by a conventional biochemical assay. That con¢rmed the presence of ethanol in all our samples and the interassay variability was less than 6%. Morphology was assessed using a Zeiss inverted micro-
2.6. Proton NMR spectroscopy
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Fig. 2. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 2.2^4.2 ppm.
1 H NMR spectra were measured on control Williams E medium, and supernatants of culture medium from the HARV on days 1, 2, 4, 7, 10, 14 and 21. Three parts of a typical 1 H NMR spectrum obtained in our experiments are shown in Figs. 1^3. Data were acquired on a Varian ANOVA 600 NMR spectrometer operating at 600 MHz for protons. All spectra were acquired at ambient probe temperature (298 þ 0.2 K). For each sample 128 transients were acquired into 32 000 complex data points over a spectral width of 6 kHz. 30³ pulses were applied with an acquisition time of 4.0 s to achieve better resolution followed by a recovery delay of 12 s to allow for complete relaxation and recovery of the magnetisation. Water signal suppression was achieved by applying a gated secondary irradiation ¢eld at the water resonance frequency. Spectral assignments were made by reference to literature values of chemical shifts in various media and biological £uids [8,28] and coupling constants. Quantitation of compounds present in the culture supernatants was achieved by two alternative means. (a) Integrals were measured relative to that of a known quantity of sodium 3-[trimethylsilyl-2,2,3,3-2 H4 ]1-propionate (TSP, 10 mg/ml) present as an internal standard to the solution. (b) When signals partially overlapped, peak height measurements were used, taking into account the appropriate coupling pattern of intensities of the non-overlapped lines.
2.7. NMR spectroscopy monitoring We opted to measure the following substances from the culture medium : glucose, lactate, pyruvate, alanine, threonine, histidine, arginine, glutamate, glutamine, leucine, isoleucine, phenylalanine, valine, methionine, acetate and ethanol. Glucose, alanine and threonine concentrations were measured as a measure of gluconeogenesis and to monitor the cells' energy requirements [5,30]. Lactate and pyruvate concentrations were measured as a measure of active aerobic and anaerobic glycolysis [5,30]. Concentrations of glutamate, glutamine, histidine and arginine were measured as indices of active transamination and urea synthesis [20,29]. Leucine, isoleucine, phenylalanine and tyrosine concentrations were measured as the key ketogenic amino acids used by hepatocytes [5,8] and valine, isoleucine and methionine concentrations were measured as they are succinate precursors and possible substrates for the Krebs cycle [4,16]. Acetate concentration was measured as this is a ketone body mainly exported by liver cells. Ethanol found within supernatants of all samples was also measured. 2.8. Sample preparation for NMR spectroscopy Samples were prepared by adding a D2 O solution (150 Wl) to culture supernatants (500 Wl) providing an internal
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Fig. 3. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 4.2^8 ppm.
¢eld frequency lock for the spectrometer. Chemical shifts were referenced internally to the singlet methyl resonance of TSP at 0 ppm. 2.9. Statistical analysis At di¡erent time points of the experiments, comparisons between cells maintained in the HARV environment were made. Six consecutive cultures were used as the basis of our experiments. The concentration results obtained represent the mean of those six experiments. Results are expressed as mean þ S.E.M. 3. Results Table 1 shows the concentrations of the di¡erent amino acids present in Williams E medium measured by reverse phase HPLC and as determined by our experimental 1 H NMR methods. The interassay variability was less than 10% at all experiments. The intraassay variability for the NMR measurements was less than 3%. Cell viability, determined by trypan blue exclusion, following isolation (day 0) was 85 þ 6%. The cell suspension was composed of single cells and small aggregates of 5^20
cells showing bright cytoplasm, sharply de¢ned borders, commonly with a round or polygonal shape and little physical damage (blebbing) under phase contrast microscopy. Hepatocyte purity for fresh cells was 95 þ 3% (n = 3). HpE staining showed gross morphology of cell aggregates on days 13 and 21; PAS staining revealed hepatocyte glycogen content at each time point. Cell aggregates stained with the £uorescent dye acridine orange on day 21 showed viable cell aggregates. Table 1 Composition of the Williams E medium used as culture medium for our experiments Leucine Isoleucine Valine Threonine Alanine Glutamine Glutamate Methionine Tyrosine Histidine Phenylalanine
HPLC
NMR
0.42 þ 0.02 0.25 þ 0.03 0.32 þ 0.03 0.22 þ 0.02 0.40 þ 0.03 1.42 þ 0.02 0.21 þ 0.02 0.06 þ 0.01 0.17 þ 0.03 0.07 þ 0.01 0.12 þ 0.02
0.44 þ 0.04 0.28 þ 0.01 0.31 þ 0.03 0.21 þ 0.01 0.43 þ 0.03 1.38 þ 0.04 0.21 þ 0.02 0.07 þ 0.01 0.16 þ 0.01 0.06 þ 0.01 0.13 þ 0.01
Comparison of concentrations of the medium substances measured using NMR spectroscopy to those stated by the manufacturers.
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Fig. 4. Comparison between concentrations of glucose and lactate in the culture medium from day 1 to day 21 during our culture experiments.
3.1. Glucose metabolism Concentrations of glucose, lactate, pyruvate, alanine and threonine were measured. Figs. 4 and 5 show the results of di¡erent measurements at the time points sampled. 3.1.1. Glucose (Fig. 4) Cells in the HARV consumed glucose during the experiments up to day 21 when experiments were terminated. The concentration of glucose in every sample tested was signi¢cantly reduced when compared to the concentration of the medium (P 6 0.01 for all measurements). Using onefactor independent-measures ANOVA we did not ¢nd any signi¢cant di¡erences between concentrations of glucose on di¡erent days. 3.1.2. Lactate (Fig. 4) Cultured hepatocytes in the HARV produced lactate during the experiments up to day 21. The production increased from day 1 to day 4 when it reached a peak and then decreased smoothly to day 21 (see Fig. 4). Lactate concentrations on day 21 were similar to lactate concen-
trations on day 1. There were statistically signi¢cant di¡erences in lactate concentrations between day 4 and day 1 (P 6 0.0029), day 4 and day 21 (P 6 0.017) and day 7 and day 1 (P 6 0.033). 3.1.3. Pyruvate (Fig. 5) Pyruvate is present in our culture medium at a concentration of 0.2 mmol/l. There was net consumption of pyruvate from cultured hepatocytes on all days except day 14 when a net production of pyruvate was observed. The lowest concentration was measured on day 1 of the experiments. There was a statistically signi¢cant di¡erence between the baseline concentration of pyruvate and the concentration on day 1 which was signi¢cantly lower (P 6 0.018). The ANOVA test showed statistical signi¢cance between concentrations on di¡erent days (F 6 0.03). The Tukey test con¢rmed that there were statistically signi¢cant di¡erences between day 1 and day 14 (P 6 0.03) and day 10 and day 14 (P 6 0.02). 3.1.4. Alanine (Fig. 5) Cells in the HARV consumed alanine during the experiments up to day 21. The measured concentration of ala-
Fig. 5. Comparison between concentrations of pyruvate, alanine and threonine in the culture medium from day 1 to day 21 during our culture experiments.
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Fig. 6. Comparison between concentrations of histidine, arginine, glutamate and glutamine in the culture medium from day 1 to day 21 during our culture experiments.
nine on days 1, 2, 4 and 10 was signi¢cantly reduced when compared to the concentration in the medium (P 6 0.03 for all measurements). There were statistically signi¢cant di¡erences in alanine concentration between day 2 and day 14 (P 6 0.009) and day 2 and day 21 (P 6 0.029) (see Fig. 5). 3.1.5. Threonine (Fig. 5) Cultured hepatocytes consumed threonine from day 1 up to day 21. The threonine concentration decreased between day 1 and day 4 and was lowest on day 4. Thereafter it started steadily increasing again up to day 21 (see Fig. 5). If we look at the one-way ANOVA test between days there were highly signi¢cant di¡erences in the concentration of threonine (F 6 0.00003). Table 2 shows the statistically signi¢cant di¡erences between concentrations on di¡erent days using the Tukey test. 3.2. Glutamine synthesis Fig. 6 shows the overall results for histidine, which is a glutamate precursor in the cell, arginine, an amino acid Table 2 Shown are statistically signi¢cant di¡erences between concentrations of threonine on di¡erent days of the experiments using the Tukey test ANOVA
Signi¢cance 0.000023
Day Day Day Day Day Day Day Day Day Day Day Day
P 6 0.022 P 6 0.00053 P 6 0.002 P 6 0.022 P 6 0.00000012 P 6 0.011 P 6 0.0037 P 6 0.0015 P 6 0.0000028 P 6 0.000039 P 6 0.0033 P 6 0.002
1 vs day 2 1 vs day 7 1 vs day 10 2 vs day 14 4 vs day 1 4 vs day 2 4 vs day 7 4 vs day 10 4 vs day 14 4 vs day 21 7 vs day 14 10 vs day 14
involved in urea synthesis, glutamate and glutamine, which can be produced from glutamate and could be utilised by the cells in DNA synthesis. 3.2.1. Histidine Using the one-way ANOVA test we could not ¢nd any statistically signi¢cant di¡erences between the concentrations of histidine in the samples measured. Overall there was a trend for consumption of histidine by the cultured cells. On days 7, 10 and 14 there was total consumption of histidine present in the medium. 3.2.2. Arginine There was net production of arginine by hepatocytes cultured in the HARV from day 1 up to and including day 21. We found two peaks of arginine concentration, on day 1 and on day 14. Between those days the concentrations of arginine measured were marginally higher than the concentrations in the cultured medium (see Fig. 6). One-way ANOVA con¢rmed that there were statistically signi¢cant di¡erences between days (F 6 0.043). Using the Tukey test we found statistically signi¢cant di¡erences in arginine concentrations between day 14 and day 4 (P 6 0.002), day 14 and day 7 (P 6 0.000016), day 14 and day 10 (P 6 0.0007), day 21 and day 7 (P 6 0.02) and day 1 and day 7 (P 6 0.037). 3.2.3. Glutamate The pattern of glutamate concentrations in the culture media was similar to the pattern of arginine concentrations. There was net production of glutamate by hepatocytes cultured in the HARV from day 1 up to and including day 21. There was one statistically signi¢cant di¡erence in glutamate concentrations, between day 1 and day 10 (P 6 0.031). The day 1 concentration was the highest and the day 10 concentration was the lowest, with high concentrations of glutamate shown in samples from days 7, 14 and 21.
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Fig. 7. Comparison between concentrations of leucine, isoleucine, phenylalanine and tyrosine in the culture medium from day 1 to day 21 during our culture experiments.
3.2.4. Glutamine Cells in the HARV consumed glutamine during the experiments up to day 21. The concentration of glutamine in every sample tested was signi¢cantly reduced when compared to the concentration of the culture medium (P 6 0.01 for all measurements). The pattern of glutamine concentrations we identi¢ed was of almost identical glutamine consumption in all samples measured. Using onefactor independent-measures ANOVA we con¢rmed that there was no signi¢cant di¡erence between concentrations of glutamine on di¡erent days. 3.3. Urea production Using a conventional biochemical assay we estimated the urea production rate after an ammonium chloride challenge and also the levels of urea in the HARV throughout our experiments. There was signi¢cant urea production during the experiments at all time points sampled. Urea synthesis rate was 196 þ 36 nmol/h/mg protein on day 0 (n = 5) and increased signi¢cantly to 220 þ 10 nmol/h/mg protein (n = 5) on day 21 (P 6 0.01, Table 3A). The rate of urea production after the ammonium challenge showed a statistically signi¢cant increase after day 10 which was sustained until day 21 (P 6 0.03 at all time points) (Table 3A). Measurement of urea levels in the HARV showed that there was urea production by the cultured cells, which diminished towards the end of the Table 3A Urea production rate during a challenge with ammonium chloride at a concentration of 10 mmol/l
experiments ^ but the di¡erences were not statistically signi¢cant (Table 3B). 3.4. Ketogenesis Fig. 7 shows the overall results for phenylalanine, tyrosine, leucine and isoleucine which together with tryptophan and lysine are the acetyl-CoA precursors. 3.4.1. Phenylalanine There was production of phenylalanine by the cultured hepatocytes on all days except day 1 of the experiments when there was a small consumption of phenylalanine. One-way ANOVA showed statistically signi¢cant di¡erences between days (F 6 0.02). The Tukey test con¢rmed statistically signi¢cant di¡erences in phenylalanine concentrations between day 1 and day 7 (P 6 0.028), day 1 and day 10 (P 6 0.0012) and day 1 and day 14 (P 6 0.0008). 3.4.2. Tyrosine The tyrosine concentration showed an inverse pattern to the concentration of phenylalanine. There was consumption of tyrosine by the cultured hepatocytes on all days except day 1 when there was a small production. On day 21 the consumption of tyrosine was minimal (see Fig. 7). One-way ANOVA con¢rmed statistically signi¢cant di¡erences between days (F 6 0.038). There were statistically signi¢cant di¡erences in concentrations of tyrosine between day 10 and day 1 (P 6 0.0001), day 10 and day 21 Table 3B Urea levels in the HARV from day 1 to day 21 during our culture experiments
Time point
Urea production (nmol/h/106 cells)
Time point
Urea level (mmol/l)
Day Day Day Day Day Day Day
196 þ 36 160 þ 12 165 þ 31 130 þ 15 210 þ 21 216 þ 30 220 þ 10
Day Day Day Day Day Day Day
3.652 þ 0.21 3.162 þ 0.26 3.048 þ 0.15 2.484 þ 0.14 2.318 þ 0.19 2.549 þ 0.09 2.326 þ 0.13
1 2 4 7 10 14 21
1 2 4 7 10 14 21
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Fig. 8. Comparison between concentrations of acetate in the culture medium from day 1 to day 21 during our culture experiments.
(P 6 0.021), day 14 and day 1 (P 6 0.0029) and day 14 and day 21 (P 6 0.0051). 3.4.3. Leucine Cells in the HARV consumed leucine during the experiments up to day 21 when experiments were terminated. The concentration of leucine at every sample tested was signi¢cantly reduced when compared to the concentration in the medium (P 6 0.01 for all measurements). Using onefactor independent-measures ANOVA we did not ¢nd any signi¢cant di¡erences between concentrations of leucine on di¡erent days. 3.4.4. Isoleucine Cells in the HARV did not use isoleucine for their needs. Overall there were no signi¢cant changes from the concentration in the medium in our samples throughout the 21 days of the experiments. Isoleucine concentrations remained relatively stable with small amounts of isoleucine consumed or released on di¡erent days (see Fig. 7). No statistically signi¢cant di¡erences were found between days.
3.5. Acetate Fig. 8 shows the overall production of acetate by the cultured cells during the experiments. Hepatocytes in the HARV produced acetate on all days until day 21. Acetate peaked on day 2 and then decreased on day 4 to reach a minimum by day 14 (see Fig. 8). One-way ANOVA con¢rmed statistically signi¢cant di¡erences between days. Using the Tukey test we found statistically signi¢cant di¡erences in acetate concentrations between day 14 and day 1 (P 6 0.013), day 14 and day 2 (P 6 0.00014), day 14 and day 7 (P 6 0.009) and day 21 and day 2 (P 6 0.0130). 3.6. Succinate precursors Succinate is an essential intermediate acid of the Krebs cycle. Valine, isoleucine and methionine are some of the amino acids that are transformed to succinate by the hepatocytes to feed the Krebs cycle. Fig. 9 shows the concentrations of valine and methionine during our experiments.
Fig. 9. Comparison between concentrations of valine and methionine in the culture medium from day 1 to day 21 during our culture experiments.
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Fig. 10. Comparison between concentrations of ethanol in the culture medium from day 1 to day 21 during our culture experiments.
3.6.1. Valine Hepatocytes consumed small amounts of valine from day 1 up to day 21 when the experiments were terminated. There were no statistically signi¢cant di¡erences either between concentrations of valine on di¡erent days or between concentrations of valine sampled and the baseline concentration of valine in Williams E medium. 3.6.2. Methionine There was net production of methionine from the cultured hepatocytes on days 1, 2 and 21. On the remaining days there was net consumption of methionine. The consumption peaked on day 14 when all available methionine was consumed by the hepatocytes. One-way ANOVA did not reveal statistically signi¢cant di¡erences between concentrations on di¡erent days. 3.7. Ethanol There was signi¢cant ethanol production by the hepatocytes on all days of the experiments (see Fig. 10). On day 1 and day 21 the highest concentrations of ethanol were measured. There was a statistically signi¢cant di¡erence in ethanol concentrations between day 1 and day 4 (P 6 0.02). 4. Discussion During the last decade with the advent of the HARV system many di¡erent types of cells and cell lines have been successfully cultured in simulated microgravity conditions [13,15,23]. Our paper examined the maintenance of di¡erentiated metabolic functions of PPHs in a simulated microgravity environment. We were able to show that cells in HARVs not only survive for long periods of time but are also able to maintain key metabolic functions. Successful long-term cultures of hepatocytes from di¡erent species have been established in 3D constructs in nor-
mal gravity using either microbeads as anchoring surfaces for the hepatocytes or di¡erent foamy materials to entrap the hepatocytes in [6,14,19,31,34]. These successful studies have demonstrated urea synthesis, ammonia removal, albumin production, secretion of other hepatocyte-speci¢c proteins or capability to detoxify substances noxious for the hepatocyte. In our study we tried to dissect metabolic pathways that would lead to the formation of end products like proteins and urea from the hepatocytes. Our ¢ndings were entirely consistent with those from a published paper [17], where the authors showed that human liver cells cultured in simulated microgravity were viable for 60 days and aggregated to tissue-like structures. They also showed that albumin and urea were produced and glucose was consumed. Another study of interest [6] assessed amino acid metabolism on hepatocytes incubated in a bioreactor and entrapped in a porous material. Hepatocytes were incubated for 14 h/day in supplemented Williams E medium for 3 consecutive days. Compared to control bioreactors which were incubated without any cells there was a signi¢cant increase in glutamate concentration and a signi¢cant decrease in glutamine, arginine, alanine, methionine, lysine, asparagine, glycine, aromatic amino acids and branched-chain amino acids. There were also lower concentrations of lactate and pyruvate observed in the loaded bioreactors. Our ¢ndings are essentially compatible with this study as we have observed a reduction in the concentrations of most amino acids studied with the exception of glutamate, phenylalanine and arginine. Our study showed that hepatocytes not only consume glucose but there is active urea synthesis and Krebs cycle for aerobic glycolysis. Looking at glucose metabolism in particular, a pattern emerged showing that during the ¢rst 7^10 days there was lactate production, pyruvate consumption, alanine consumption and threonine consumption. This is a pattern observed in cells breaking down glucose anaerobically for their energy requirements and showing active gluconeogenesis. From day 10 onwards another pattern emerged. There was signi¢cantly less lac-
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tate production and signi¢cantly less pyruvate, alanine and threonine consumption. This is a pattern observed in cells breaking down their glucose aerobically for their energy requirements and not very actively using glucogenic amino acids for gluconeogenesis. This observation and similar others on key metabolic pathways detailed below have enabled us to put forward the hypothesis that cells grow quite happily in the RCCS. After an initial period of acclimatisation, at the time point when static cell cultures drift towards cell death, cell aggregates formed in the RCCS switch from the anaerobic pattern of metabolism to the more e¤cient aerobic pattern. They exhibited the same pattern of metabolism until the experiments were terminated. Looking at glutamine synthesis it is well established that glutamate can be produced either from the urea cycle through arginine breakdown, or directly from histidine or through transamination from K-ketoglutarate, which produces the bulk of intracellular glutamate. Our results showed that glutamate production was evident. There was histidine consumption, which could partially account for glutamate production. This only produces a small proportion of the overall glutamate produced by the cells. We suggest that transamination activity, and production of glutamate overall, was proportional to glutamate production from histidine. The hypothesis of cells acclimatising in the microgravity environment for the ¢rst 10 days and then continuing their life cycles in a friendly environment is suggested by those results too. The glutamine consumption pattern being inverse to the glutamate production pattern suggested that after day 7 cells were consuming glutamine for their DNA synthesis. They were as such dividing until day 21 when our experiments were terminated. Looking at ketogenesis we observed acetate production by the hepatocytes which diminished after day 10 of the experiments. We also observed that, contrary to what has been reported on static cultures and in bioreactor cultures [6], there was phenylalanine production from protein breakdown in our experiments. It is well established that hepatocytes tend to convert phenylalanine to tyrosine which is further converted to acetyl-CoA and fumarate. The increased concentration of phenylalanine in the medium and in the cells accelerates the turnover of tyrosine and its conversion to metabolites more useful to the cell. The results are entirely in keeping with this hypothesis. Overall the pattern of ketogenesis proves again the theory of cells acclimatising in the RCCS environment. Ketone bodies are the export products of liver cells to other organs that need ketone bodies to survive. The microgravity cultures exhibited high acetate production at the beginning. As only hepatocyte aggregates existed in the system there was no target organ to export ketone bodies. We believe this is the reason why acetate production diminished after day 10, reached its minimum on day 14 and remained low until the end of the experiments.
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There is no evidence of big succinate production as all precursors of succinate seemed not to be taken up in large quantities. Overall it does not seem that the Krebs cycle was fed from succinate production under our experimental conditions. Looking at the branched-chain amino acids as an entity it becomes clear that cells preferentially use leucine for their requirements. The most plausible explanation is that as all three branched-chain amino acids use the same transporter to enter the hepatocyte, leucine is preferentially taken up because its concentration in the extracellular milieu is the highest. Until the three branchedchain amino acids are in concentration equilibrium in the medium leucine is almost exclusively taken up and as a net result the concentrations of the three branched-chain amino acids in the extracellular supernatant are almost identical. High levels of ethanol were observed in all culture supernatants. Their presence is enigmatic. The obvious theory of bacterial and fungal contamination of the culture medium is not supported by the sterility of the medium on conventional testing. We introduced an ethanolfree environment after the ¢rst culture experiments and it was clear that there was evidence of ethanol in the culture medium. These observations have been previously reported in biological £uids of animals [8,12]. One plausible explanation is that as threonine can be used as a substrate for alcohol synthesis porcine hepatocytes have the ability to produce ethanol under conditions of alcohol dehydrogenase inhibition. Overall our results show that PPHs can be happily grown in microgravity conditions for up to 3 weeks. This may have implications for BALSS as they can provide an active biosubstrate for the systems. We have shown that this culture modality overcomes the initial disorder in cellular amino acid metabolism which is critical in long-term cultures of porcine hepatocytes [9]. Further studies are urgently required to scale up this model for growth of substantial quantities of hepatocytes and studies without time limits need to be performed to ascertain the feasibility of long-term culture and maintenance of these hepatocytes in culture.
References [1] R.E. Akins, N.A. Schroedl, S.R. Gonda, C.H. Hartzell, Neonatal rat heart cells cultured in simulated microgravity, In Vitro Cell. Dev. Biol. Animal 33 (1997) 337^343. [2] T.L. Baker, T.J. Goodwin, Three-dimensinal culture of bovine chondrocytes in rotating wall vessels, In Vitro Cell. Dev. Biol. Animal 33 (1997) 358^365. [3] T.B. Darr, A. Hubel, Freezing characteristics of isolated pig and human hepatocytes, Cell Transpl. 6 (1997) 173^183. [4] I. De Blaaw, N.E.P. Deutz, W. Boers, M.F. von Meyenfeldt, Hepatic amino acid and protein metabolism in non anorectic, moderately cachectic tumour bearing rats, J. Hepatol. 26 (1997) 396^408.
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[5] R.M. Denton, A.P. Halestrap, Regulation of pyruvate metabolism in mammalian tissues, Essays Biochem. 15 (1978) 37^44. [6] L.M. Flendrig, J.W. LaSoe, J.J.A. Jorning, A. Steenbeek, O.T. Karlsen, W.M.M.J. Bovee, N.C.J.J. Ladiges, A.A. TeVelde, R.A.F.M. Chamuleau, In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates, J. Hepatol. 26 (1997) 1379^1392. [7] L.M. Flendrig, R.A.F.M. Chamuleau, M.A.W. Maas, J. Daalhuisen, B. Hasset, C.G. Kilty, S. Doyle, N.C.J.J. Ladiges, J.J.A. Jorning, J.W. LaSoe, D. Sommeijer, A.A. TeVelde, Evaluation of a novel bioarti¢cial liver in rats with complete liver ischaemia: treatment e¤cacy and species-speci¢c alpha-GST detection to monitor hepatocyte viability, J. Hepatol. 30 (1999) 311^320. [8] P.J.D. Foxall, R.G. Price, J.K. Jones, G.H. Neild, F.D. Thompson, J.K. Nicholson, High resolution proton magnetic resonance spectroscopy of cyst £uids from patients with polycystic kidney disease, Biochim. Biophys. Acta 1138 (1992) 305^331. [9] J.C. Gerlach, M. Fuchs, M.D. Smith, R. Bornemann, J. Encke, P. Neuhaus, E. Riedel, Is a clinical application of hybrid liver support systems limited by an initial disorder in cellular amino acid and Kketo acid metabolism rather than by later gradual loss of primary hepatocyte function?, Transplantation 62 (1996) 224^228. [10] T.J. Goodwin, T.L. Prewett, D.A. Wolf, G.F. Spaulding, Reduced shear stress: a major component in the ability of the mammalian tissue to form three-dimensional assemblies in simulated microgravity, J. Cell. Biochem. 51 (1993) 310^311. [11] T.J. Goodwin, T.L. Prewett, G.F. Spaulding, J.L. Becker, Three dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics, In Vitro Cell. Dev. Biol. Animal 33 (1997) 366^374. [12] R.G. Gosden, I.H. Sadler, D. Reed, R.H.F. Hunter, Characterisation of ovarian follicular £uids of sheep, pigs and cows by using proton nuclear magnetic resonance spectroscopy, Experientia 46 (1990) 1012^1015. [13] M. Ingram, G.B. Techy, R. Saroufeem, O. Yazan, K.S. Narayan, T.J. Goodwin, G.F. Spalding, Three dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor, In Vitro Cell. Dev. Biol. Animal 33 (1997) 459^466. [14] H.O. Jauregui, S. Naik, H. Santangini, J. Pan, D. Trenkler, C. Mullon, Primary cultures of rat hepatocytes in hollow ¢bre chambers, In Vitro Cell. Dev. Biol. Animal 30A (1994) 23^29. [15] J.M. Jessup, D. Brown, W. Fitzgerald, R.D. Ford, A. Nachman, T.J. Goodwin, G. Spaulding, Induction of carcinoembryogenic antigen expression in a three dimensional culture system, In Vitro Cell. Dev. Biol. Animal 33 (1997) 352^357. [16] L.W. Kinsell, H.A. Harper, H.C. Barton, M.E. Hutchin, J.R. Hess, Studies in methionine and sulfur metabolism. The fate of i.v. administered methionine in normal individuals and patients with liver damage, J. Clin. Invest. 27 (1948) 677^688. [17] V.L. Khaoustov, G.J. Darlington, H.E. Soriano, B. Krishnan, D. Risin, N.R. Pellis, B. Yo¡e, Induction of three dimensional assembly of human liver cells by simulated microgravity, In Vitro Cell. Dev. Biol. Animal 35 (1999) 501^509. [18] S. Kloth, C. Edenbeck, M. Kubitza et al., Stimulation of renal orga-
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The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes Konstantinos J. Dabos a; *, Leonard J. Nelson a , Timothy J. Bradnock a , John A. Parkinson b , Ian H. Sadler b , Peter C. Hayes a , John N. Plevris a a
Liver Cell Biology Laboratory, Department of Internal Medicine, University of Edinburgh, Edinburgh, UK b Department of Chemistry, University of Edinburgh, Edinburgh, UK Received 25 July 2000; received in revised form 18 January 2001; accepted 26 January 2001
Abstract The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 108 porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1 H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P 6 0.01), and significantly smaller pyruvate (day 1 vs day 14 P 6 0.03) and threonine consumption (day 1 vs day 10 P 6 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P 6 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P 6 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : High aspect ratio vessel; Bioreactor; Glycolysis ; Ketogenesis; Glutamine synthesis ; Succinate precursor synthesis
1. Introduction During the last decades our ability to culture isolated cells in vitro has been the basis for fundamental advances in cell biology. In spite of the amount of information gained in the traditional cell culture settings, it is believed that conventional tissue culture in two dimensions may be inadequate to model the complex cellular interactions that promote tissue-speci¢c di¡erentiation. Cells maintained as homotypic populations in 2D cultures quickly lose their di¡erentiated functions [18]. These functions can be main-
* Corresponding author. Koukouridi 1, Parodos Kalvokoresi, Evaggelistria, 821-00 Chios, Greece. Fax: +44-131-2292948; E-mail : [email protected]
tained by growing cells under the necessary environmental conditions for organ-like assembly, such as cell^cell interactions in three dimensions. Although 3D cell aggregates can be obtained in suspension cultures the high levels of £uid shear stress encountered in conventional stirred fermentors limit the level of tissue-speci¢c di¡erentiation of the aggregated cells [21]. Experiments in space £ight-induced microgravity can overcome the high shear forces. Indeed, cells cultured in suspension under those conditions have shown signi¢cant changes in cell functions. These include increased growth rate and antibiotic resistance in bacteria, increased substrate attachment in human kidney cells and increased hormone secretion by cultured lymphocytes and macrophages and many others. Even in the absence of any direct evidence for any speci¢c mechanisms, it has become increasingly clear that space £ight-induced microgravity has profound e¡ects on cells in vitro [25].
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 0 9 7 - 6
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Recently the in vitro generation of simulated microgravity has become possible with the high aspect ratio vessel (HARV). HARVs o¡er a revolutionary approach to study cell functions. HARVs were developed at the National Aeronautics and Space Administration (NASA)^Johnson Space Center. HARVs allow the cultivation of cells in an environment of low shear stress, low turbulence and no gas^£uid interface [1,2,24,25]. The principle of rotating the culture vessel allows cells to be maintained in perpetual suspension with the net gravity vector approaching zero all the time. The key to the utility of the HARV system is that the culture vessel and the £uid contained therein approximate a solid body during rotation [11]. When cells are added to the HARV they sink to the bottom of the vessel at a constant rate related to the gravitational ¢eld, the di¡erence in density between the particles and the medium, the size of the particles and other factors. Rotation of the HARV results in a path for the cells determined by the combination of sedimentation and movement along with the medium. By increasing the rotation speed we can prevent the cells from reaching the bottom of the vessel. The cells appear to describe an elliptical path in the medium relative to the observer. Further increases in the rotation rate result in the diminution of the elliptical path until the cells become essentially motionless relative to the medium. At this point the vessel, contents and medium approximate a solid body. The cells maintain 3D orientations relative to each other and the surrounding medium in an apparently low shear environment. These conditions are similar to those expected to prevail in true microgravity [10]. Primary porcine hepatocytes (PPHs) are currently considered the cells of choice in bioarti¢cial liver support systems (BALSS). PPHs are metabolically active with a biochemical pro¢le similar to that of human hepatocytes and can be obtained in the large numbers required for bioarti¢cial liver treatments. PPHs are currently being used in experimental settings and phase I clinical trials as biosubstrate in BALSS [7,32,33]. Unfortunately, so far we have been unable to provide on demand hepatocytes that exhibit the same metabolic functions as freshly isolated hepatocytes. Frozen and revived hepatocytes have been used but they clearly have an unacceptably low viability and some of their key metabolic functions are impaired [3], although they seem to retain the ability to metabolise drugs through their P450 system [26]. The microgravity environment presents an exciting new modality for continuous cultures of hepatocytes in suspension. They can be easily transferred out of the rotating vessels and loaded onto the bioreactors for BALSS treatments on demand. The aim of this study was to characterise key metabolic functions of PPHs cultured in a HARV and to assess cell aggregation and maintenance of proliferation of hepatocytes on 3D structures for a predetermined time period of 21 days.
2. Materials and methods 2.1. Chemicals Type IV collagen (from Clostridium histolyticum), Long epidermal growth factor (L-EGF), Williams E medium and Leibovitz L-15 medium were from Sigma. Hanks' balanced salt solution (HBSS) bu¡er, L-glutamine, penicillin^ streptomycin and amphotericin B were from ICN. All reagents were of cell culture grade. Other reagents were : gentamicin (non-proprietary), porcine insulin (Pork Velosulin, Novo Nordisk, Denmark), dexamethasone (David Bull Labs, UK) and aprotinin (Trasylol, Bayer). All chemicals were of Analar grade. 2.2. Hepatocyte isolation Hepatocytes were isolated from weanling piglets ( 6 15 kg) using our previously described ex vivo collagenase perfusion method [27]. Brie£y, piglets were killed with i.v. pentobarbital sodium, the infrahepatic inferior vena cava (IVC) was clamped and the suprahepatic IVC cannulated. The whole liver was retrogradely perfused in situ with ice-cold phosphate-bu¡ered saline, excised, then exsanguinated. This was followed by an ex vivo open-loop and recirculating collagenase perfusion in ¢ve steps. The liver was disrupted, sequentially ¢ltered in washing bu¡ers, puri¢ed by centrifugation and resuspended in E Williams medium. Initial cell viability was assessed by trypan blue exclusion and yield determined using a Neubauer haemocytometer (BDH, Merck). 2.3. Hepatocyte culture in simulated microgravity: RCCS^HARV Cells were seeded in a 55 ml capacity HARV (CellonSarl, Luxembourg) at a density of 2U106 viable cells/ml Williams E medium at 37³C. The medium consisted of serum-free, chemically de¢ned medium supplemented with 50 ng/ml L-EGF, 10 Wg/ml porcine insulin, 1 Wmol/l dexamethasone, 2 mmol/l L-glutamine, 50 mg/ml gentamicin and 50 mg/ml penicillin^streptomycin. The HARV was connected to the horizontally rotating axis of the RCCS (Rotary Cell Culture System) which pumped incubator gas via a central aperture, across a semi-permeable gas exchange membrane within the HARV. The RCCS^HARV assembly was placed in a humidi¢ed incubator under a 95% air:5% CO2 atmosphere. Next day (day 1), the HARV was aspirated and fresh medium added, which was subsequently changed every 3 days. The optimal rotational speed of the HARV was determined by adjusting the RCCS rotational speed so that the aggregate(s) maintained a freefall, quasi-stationary orbit within the culture vessel. The rotational speed varied according to both aggregate size and the corresponding sedimentation coe¤-
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Fig. 1. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 0^2.2 ppm.
cient ; however, by day 6 of culture, rotational speed was set at 10^15 rpm. For changes of supplemented Williams E medium a 0.5 cm ¢ll port was used using a suction aspirator. When changing the medium, the cells were covered by a small amount of medium to minimise cell damage. Medium samples were taken daily via a syringe attached to one of the luer-lock syringe ports and stored at 370³C until nuclear magnetic resonance (NMR) analysis.
scope (Zeiss, Germany) under phase contrast on both day 0 and day 21 of culture. Photomicrographs recorded hepatocytes in suspension and RCCS culture. Cell purity was determined by cytospin (200Ug, 5 min) of 5U105 freshly isolated cells and staining with haematoxylin+eosin (HpE)/periodic acid Schi¡ (PAS), followed by counting cells in three separate ¢elds for three separate isolations.
2.4. Urea synthesis rate
We used 1 H NMR spectroscopy in this study to try and elucidate patterns of consumption and production of individual amino acids by the cultured hepatocytes in the HARV. This technique has been developed in the last few years and it has been used to measure substances present in biological £uids [8,22,28]. 1 H NMR spectroscopy was used as it was possible to obtain spectra from the medium tested which exhibited no overlapping peaks and a very low signal to noise ratio. The absence of macromolecules in signi¢cant quantities in the medium made it easier to identify peaks and assign them to individual amino acids. Further advantages of NMR spectroscopy are the non-destructive nature of the technique for the samples tested, the possibility of acquiring the spectra of an array of substances at one experiment, the possibility of storing and reusing both the samples and the acquired spectra and the fact that if facilities are available the sample processing and testing is relatively cheap.
The urea synthesis rate (USR) was determined in 0.5 ml samples taken at t = 0 and t = 2 h using a modi¢ed colorimetric urea nitrogen kit (Sigma kit 535-B). The di¡erences in absorbance for the chromogenic reaction were measured at each time point at V525 in a Pye Unicam spectrophotometer and USR calculated. The coe¤cient of variation for this assay was less than 5%. 2.5. Ethanol synthesis To con¢rm the presence of ethanol in our culture media we also measured ethanol levels by a conventional biochemical assay. That con¢rmed the presence of ethanol in all our samples and the interassay variability was less than 6%. Morphology was assessed using a Zeiss inverted micro-
2.6. Proton NMR spectroscopy
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Fig. 2. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 2.2^4.2 ppm.
1 H NMR spectra were measured on control Williams E medium, and supernatants of culture medium from the HARV on days 1, 2, 4, 7, 10, 14 and 21. Three parts of a typical 1 H NMR spectrum obtained in our experiments are shown in Figs. 1^3. Data were acquired on a Varian ANOVA 600 NMR spectrometer operating at 600 MHz for protons. All spectra were acquired at ambient probe temperature (298 þ 0.2 K). For each sample 128 transients were acquired into 32 000 complex data points over a spectral width of 6 kHz. 30³ pulses were applied with an acquisition time of 4.0 s to achieve better resolution followed by a recovery delay of 12 s to allow for complete relaxation and recovery of the magnetisation. Water signal suppression was achieved by applying a gated secondary irradiation ¢eld at the water resonance frequency. Spectral assignments were made by reference to literature values of chemical shifts in various media and biological £uids [8,28] and coupling constants. Quantitation of compounds present in the culture supernatants was achieved by two alternative means. (a) Integrals were measured relative to that of a known quantity of sodium 3-[trimethylsilyl-2,2,3,3-2 H4 ]1-propionate (TSP, 10 mg/ml) present as an internal standard to the solution. (b) When signals partially overlapped, peak height measurements were used, taking into account the appropriate coupling pattern of intensities of the non-overlapped lines.
2.7. NMR spectroscopy monitoring We opted to measure the following substances from the culture medium : glucose, lactate, pyruvate, alanine, threonine, histidine, arginine, glutamate, glutamine, leucine, isoleucine, phenylalanine, valine, methionine, acetate and ethanol. Glucose, alanine and threonine concentrations were measured as a measure of gluconeogenesis and to monitor the cells' energy requirements [5,30]. Lactate and pyruvate concentrations were measured as a measure of active aerobic and anaerobic glycolysis [5,30]. Concentrations of glutamate, glutamine, histidine and arginine were measured as indices of active transamination and urea synthesis [20,29]. Leucine, isoleucine, phenylalanine and tyrosine concentrations were measured as the key ketogenic amino acids used by hepatocytes [5,8] and valine, isoleucine and methionine concentrations were measured as they are succinate precursors and possible substrates for the Krebs cycle [4,16]. Acetate concentration was measured as this is a ketone body mainly exported by liver cells. Ethanol found within supernatants of all samples was also measured. 2.8. Sample preparation for NMR spectroscopy Samples were prepared by adding a D2 O solution (150 Wl) to culture supernatants (500 Wl) providing an internal
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Fig. 3. Representative 1 H NMR spectrum from a HARV sample on day 10. Region shown: 4.2^8 ppm.
¢eld frequency lock for the spectrometer. Chemical shifts were referenced internally to the singlet methyl resonance of TSP at 0 ppm. 2.9. Statistical analysis At di¡erent time points of the experiments, comparisons between cells maintained in the HARV environment were made. Six consecutive cultures were used as the basis of our experiments. The concentration results obtained represent the mean of those six experiments. Results are expressed as mean þ S.E.M. 3. Results Table 1 shows the concentrations of the di¡erent amino acids present in Williams E medium measured by reverse phase HPLC and as determined by our experimental 1 H NMR methods. The interassay variability was less than 10% at all experiments. The intraassay variability for the NMR measurements was less than 3%. Cell viability, determined by trypan blue exclusion, following isolation (day 0) was 85 þ 6%. The cell suspension was composed of single cells and small aggregates of 5^20
cells showing bright cytoplasm, sharply de¢ned borders, commonly with a round or polygonal shape and little physical damage (blebbing) under phase contrast microscopy. Hepatocyte purity for fresh cells was 95 þ 3% (n = 3). HpE staining showed gross morphology of cell aggregates on days 13 and 21; PAS staining revealed hepatocyte glycogen content at each time point. Cell aggregates stained with the £uorescent dye acridine orange on day 21 showed viable cell aggregates. Table 1 Composition of the Williams E medium used as culture medium for our experiments Leucine Isoleucine Valine Threonine Alanine Glutamine Glutamate Methionine Tyrosine Histidine Phenylalanine
HPLC
NMR
0.42 þ 0.02 0.25 þ 0.03 0.32 þ 0.03 0.22 þ 0.02 0.40 þ 0.03 1.42 þ 0.02 0.21 þ 0.02 0.06 þ 0.01 0.17 þ 0.03 0.07 þ 0.01 0.12 þ 0.02
0.44 þ 0.04 0.28 þ 0.01 0.31 þ 0.03 0.21 þ 0.01 0.43 þ 0.03 1.38 þ 0.04 0.21 þ 0.02 0.07 þ 0.01 0.16 þ 0.01 0.06 þ 0.01 0.13 þ 0.01
Comparison of concentrations of the medium substances measured using NMR spectroscopy to those stated by the manufacturers.
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Fig. 4. Comparison between concentrations of glucose and lactate in the culture medium from day 1 to day 21 during our culture experiments.
3.1. Glucose metabolism Concentrations of glucose, lactate, pyruvate, alanine and threonine were measured. Figs. 4 and 5 show the results of di¡erent measurements at the time points sampled. 3.1.1. Glucose (Fig. 4) Cells in the HARV consumed glucose during the experiments up to day 21 when experiments were terminated. The concentration of glucose in every sample tested was signi¢cantly reduced when compared to the concentration of the medium (P 6 0.01 for all measurements). Using onefactor independent-measures ANOVA we did not ¢nd any signi¢cant di¡erences between concentrations of glucose on di¡erent days. 3.1.2. Lactate (Fig. 4) Cultured hepatocytes in the HARV produced lactate during the experiments up to day 21. The production increased from day 1 to day 4 when it reached a peak and then decreased smoothly to day 21 (see Fig. 4). Lactate concentrations on day 21 were similar to lactate concen-
trations on day 1. There were statistically signi¢cant di¡erences in lactate concentrations between day 4 and day 1 (P 6 0.0029), day 4 and day 21 (P 6 0.017) and day 7 and day 1 (P 6 0.033). 3.1.3. Pyruvate (Fig. 5) Pyruvate is present in our culture medium at a concentration of 0.2 mmol/l. There was net consumption of pyruvate from cultured hepatocytes on all days except day 14 when a net production of pyruvate was observed. The lowest concentration was measured on day 1 of the experiments. There was a statistically signi¢cant di¡erence between the baseline concentration of pyruvate and the concentration on day 1 which was signi¢cantly lower (P 6 0.018). The ANOVA test showed statistical signi¢cance between concentrations on di¡erent days (F 6 0.03). The Tukey test con¢rmed that there were statistically signi¢cant di¡erences between day 1 and day 14 (P 6 0.03) and day 10 and day 14 (P 6 0.02). 3.1.4. Alanine (Fig. 5) Cells in the HARV consumed alanine during the experiments up to day 21. The measured concentration of ala-
Fig. 5. Comparison between concentrations of pyruvate, alanine and threonine in the culture medium from day 1 to day 21 during our culture experiments.
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Fig. 6. Comparison between concentrations of histidine, arginine, glutamate and glutamine in the culture medium from day 1 to day 21 during our culture experiments.
nine on days 1, 2, 4 and 10 was signi¢cantly reduced when compared to the concentration in the medium (P 6 0.03 for all measurements). There were statistically signi¢cant di¡erences in alanine concentration between day 2 and day 14 (P 6 0.009) and day 2 and day 21 (P 6 0.029) (see Fig. 5). 3.1.5. Threonine (Fig. 5) Cultured hepatocytes consumed threonine from day 1 up to day 21. The threonine concentration decreased between day 1 and day 4 and was lowest on day 4. Thereafter it started steadily increasing again up to day 21 (see Fig. 5). If we look at the one-way ANOVA test between days there were highly signi¢cant di¡erences in the concentration of threonine (F 6 0.00003). Table 2 shows the statistically signi¢cant di¡erences between concentrations on di¡erent days using the Tukey test. 3.2. Glutamine synthesis Fig. 6 shows the overall results for histidine, which is a glutamate precursor in the cell, arginine, an amino acid Table 2 Shown are statistically signi¢cant di¡erences between concentrations of threonine on di¡erent days of the experiments using the Tukey test ANOVA
Signi¢cance 0.000023
Day Day Day Day Day Day Day Day Day Day Day Day
P 6 0.022 P 6 0.00053 P 6 0.002 P 6 0.022 P 6 0.00000012 P 6 0.011 P 6 0.0037 P 6 0.0015 P 6 0.0000028 P 6 0.000039 P 6 0.0033 P 6 0.002
1 vs day 2 1 vs day 7 1 vs day 10 2 vs day 14 4 vs day 1 4 vs day 2 4 vs day 7 4 vs day 10 4 vs day 14 4 vs day 21 7 vs day 14 10 vs day 14
involved in urea synthesis, glutamate and glutamine, which can be produced from glutamate and could be utilised by the cells in DNA synthesis. 3.2.1. Histidine Using the one-way ANOVA test we could not ¢nd any statistically signi¢cant di¡erences between the concentrations of histidine in the samples measured. Overall there was a trend for consumption of histidine by the cultured cells. On days 7, 10 and 14 there was total consumption of histidine present in the medium. 3.2.2. Arginine There was net production of arginine by hepatocytes cultured in the HARV from day 1 up to and including day 21. We found two peaks of arginine concentration, on day 1 and on day 14. Between those days the concentrations of arginine measured were marginally higher than the concentrations in the cultured medium (see Fig. 6). One-way ANOVA con¢rmed that there were statistically signi¢cant di¡erences between days (F 6 0.043). Using the Tukey test we found statistically signi¢cant di¡erences in arginine concentrations between day 14 and day 4 (P 6 0.002), day 14 and day 7 (P 6 0.000016), day 14 and day 10 (P 6 0.0007), day 21 and day 7 (P 6 0.02) and day 1 and day 7 (P 6 0.037). 3.2.3. Glutamate The pattern of glutamate concentrations in the culture media was similar to the pattern of arginine concentrations. There was net production of glutamate by hepatocytes cultured in the HARV from day 1 up to and including day 21. There was one statistically signi¢cant di¡erence in glutamate concentrations, between day 1 and day 10 (P 6 0.031). The day 1 concentration was the highest and the day 10 concentration was the lowest, with high concentrations of glutamate shown in samples from days 7, 14 and 21.
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Fig. 7. Comparison between concentrations of leucine, isoleucine, phenylalanine and tyrosine in the culture medium from day 1 to day 21 during our culture experiments.
3.2.4. Glutamine Cells in the HARV consumed glutamine during the experiments up to day 21. The concentration of glutamine in every sample tested was signi¢cantly reduced when compared to the concentration of the culture medium (P 6 0.01 for all measurements). The pattern of glutamine concentrations we identi¢ed was of almost identical glutamine consumption in all samples measured. Using onefactor independent-measures ANOVA we con¢rmed that there was no signi¢cant di¡erence between concentrations of glutamine on di¡erent days. 3.3. Urea production Using a conventional biochemical assay we estimated the urea production rate after an ammonium chloride challenge and also the levels of urea in the HARV throughout our experiments. There was signi¢cant urea production during the experiments at all time points sampled. Urea synthesis rate was 196 þ 36 nmol/h/mg protein on day 0 (n = 5) and increased signi¢cantly to 220 þ 10 nmol/h/mg protein (n = 5) on day 21 (P 6 0.01, Table 3A). The rate of urea production after the ammonium challenge showed a statistically signi¢cant increase after day 10 which was sustained until day 21 (P 6 0.03 at all time points) (Table 3A). Measurement of urea levels in the HARV showed that there was urea production by the cultured cells, which diminished towards the end of the Table 3A Urea production rate during a challenge with ammonium chloride at a concentration of 10 mmol/l
experiments ^ but the di¡erences were not statistically signi¢cant (Table 3B). 3.4. Ketogenesis Fig. 7 shows the overall results for phenylalanine, tyrosine, leucine and isoleucine which together with tryptophan and lysine are the acetyl-CoA precursors. 3.4.1. Phenylalanine There was production of phenylalanine by the cultured hepatocytes on all days except day 1 of the experiments when there was a small consumption of phenylalanine. One-way ANOVA showed statistically signi¢cant di¡erences between days (F 6 0.02). The Tukey test con¢rmed statistically signi¢cant di¡erences in phenylalanine concentrations between day 1 and day 7 (P 6 0.028), day 1 and day 10 (P 6 0.0012) and day 1 and day 14 (P 6 0.0008). 3.4.2. Tyrosine The tyrosine concentration showed an inverse pattern to the concentration of phenylalanine. There was consumption of tyrosine by the cultured hepatocytes on all days except day 1 when there was a small production. On day 21 the consumption of tyrosine was minimal (see Fig. 7). One-way ANOVA con¢rmed statistically signi¢cant di¡erences between days (F 6 0.038). There were statistically signi¢cant di¡erences in concentrations of tyrosine between day 10 and day 1 (P 6 0.0001), day 10 and day 21 Table 3B Urea levels in the HARV from day 1 to day 21 during our culture experiments
Time point
Urea production (nmol/h/106 cells)
Time point
Urea level (mmol/l)
Day Day Day Day Day Day Day
196 þ 36 160 þ 12 165 þ 31 130 þ 15 210 þ 21 216 þ 30 220 þ 10
Day Day Day Day Day Day Day
3.652 þ 0.21 3.162 þ 0.26 3.048 þ 0.15 2.484 þ 0.14 2.318 þ 0.19 2.549 þ 0.09 2.326 þ 0.13
1 2 4 7 10 14 21
1 2 4 7 10 14 21
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Fig. 8. Comparison between concentrations of acetate in the culture medium from day 1 to day 21 during our culture experiments.
(P 6 0.021), day 14 and day 1 (P 6 0.0029) and day 14 and day 21 (P 6 0.0051). 3.4.3. Leucine Cells in the HARV consumed leucine during the experiments up to day 21 when experiments were terminated. The concentration of leucine at every sample tested was signi¢cantly reduced when compared to the concentration in the medium (P 6 0.01 for all measurements). Using onefactor independent-measures ANOVA we did not ¢nd any signi¢cant di¡erences between concentrations of leucine on di¡erent days. 3.4.4. Isoleucine Cells in the HARV did not use isoleucine for their needs. Overall there were no signi¢cant changes from the concentration in the medium in our samples throughout the 21 days of the experiments. Isoleucine concentrations remained relatively stable with small amounts of isoleucine consumed or released on di¡erent days (see Fig. 7). No statistically signi¢cant di¡erences were found between days.
3.5. Acetate Fig. 8 shows the overall production of acetate by the cultured cells during the experiments. Hepatocytes in the HARV produced acetate on all days until day 21. Acetate peaked on day 2 and then decreased on day 4 to reach a minimum by day 14 (see Fig. 8). One-way ANOVA con¢rmed statistically signi¢cant di¡erences between days. Using the Tukey test we found statistically signi¢cant di¡erences in acetate concentrations between day 14 and day 1 (P 6 0.013), day 14 and day 2 (P 6 0.00014), day 14 and day 7 (P 6 0.009) and day 21 and day 2 (P 6 0.0130). 3.6. Succinate precursors Succinate is an essential intermediate acid of the Krebs cycle. Valine, isoleucine and methionine are some of the amino acids that are transformed to succinate by the hepatocytes to feed the Krebs cycle. Fig. 9 shows the concentrations of valine and methionine during our experiments.
Fig. 9. Comparison between concentrations of valine and methionine in the culture medium from day 1 to day 21 during our culture experiments.
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Fig. 10. Comparison between concentrations of ethanol in the culture medium from day 1 to day 21 during our culture experiments.
3.6.1. Valine Hepatocytes consumed small amounts of valine from day 1 up to day 21 when the experiments were terminated. There were no statistically signi¢cant di¡erences either between concentrations of valine on di¡erent days or between concentrations of valine sampled and the baseline concentration of valine in Williams E medium. 3.6.2. Methionine There was net production of methionine from the cultured hepatocytes on days 1, 2 and 21. On the remaining days there was net consumption of methionine. The consumption peaked on day 14 when all available methionine was consumed by the hepatocytes. One-way ANOVA did not reveal statistically signi¢cant di¡erences between concentrations on di¡erent days. 3.7. Ethanol There was signi¢cant ethanol production by the hepatocytes on all days of the experiments (see Fig. 10). On day 1 and day 21 the highest concentrations of ethanol were measured. There was a statistically signi¢cant di¡erence in ethanol concentrations between day 1 and day 4 (P 6 0.02). 4. Discussion During the last decade with the advent of the HARV system many di¡erent types of cells and cell lines have been successfully cultured in simulated microgravity conditions [13,15,23]. Our paper examined the maintenance of di¡erentiated metabolic functions of PPHs in a simulated microgravity environment. We were able to show that cells in HARVs not only survive for long periods of time but are also able to maintain key metabolic functions. Successful long-term cultures of hepatocytes from di¡erent species have been established in 3D constructs in nor-
mal gravity using either microbeads as anchoring surfaces for the hepatocytes or di¡erent foamy materials to entrap the hepatocytes in [6,14,19,31,34]. These successful studies have demonstrated urea synthesis, ammonia removal, albumin production, secretion of other hepatocyte-speci¢c proteins or capability to detoxify substances noxious for the hepatocyte. In our study we tried to dissect metabolic pathways that would lead to the formation of end products like proteins and urea from the hepatocytes. Our ¢ndings were entirely consistent with those from a published paper [17], where the authors showed that human liver cells cultured in simulated microgravity were viable for 60 days and aggregated to tissue-like structures. They also showed that albumin and urea were produced and glucose was consumed. Another study of interest [6] assessed amino acid metabolism on hepatocytes incubated in a bioreactor and entrapped in a porous material. Hepatocytes were incubated for 14 h/day in supplemented Williams E medium for 3 consecutive days. Compared to control bioreactors which were incubated without any cells there was a signi¢cant increase in glutamate concentration and a signi¢cant decrease in glutamine, arginine, alanine, methionine, lysine, asparagine, glycine, aromatic amino acids and branched-chain amino acids. There were also lower concentrations of lactate and pyruvate observed in the loaded bioreactors. Our ¢ndings are essentially compatible with this study as we have observed a reduction in the concentrations of most amino acids studied with the exception of glutamate, phenylalanine and arginine. Our study showed that hepatocytes not only consume glucose but there is active urea synthesis and Krebs cycle for aerobic glycolysis. Looking at glucose metabolism in particular, a pattern emerged showing that during the ¢rst 7^10 days there was lactate production, pyruvate consumption, alanine consumption and threonine consumption. This is a pattern observed in cells breaking down glucose anaerobically for their energy requirements and showing active gluconeogenesis. From day 10 onwards another pattern emerged. There was signi¢cantly less lac-
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tate production and signi¢cantly less pyruvate, alanine and threonine consumption. This is a pattern observed in cells breaking down their glucose aerobically for their energy requirements and not very actively using glucogenic amino acids for gluconeogenesis. This observation and similar others on key metabolic pathways detailed below have enabled us to put forward the hypothesis that cells grow quite happily in the RCCS. After an initial period of acclimatisation, at the time point when static cell cultures drift towards cell death, cell aggregates formed in the RCCS switch from the anaerobic pattern of metabolism to the more e¤cient aerobic pattern. They exhibited the same pattern of metabolism until the experiments were terminated. Looking at glutamine synthesis it is well established that glutamate can be produced either from the urea cycle through arginine breakdown, or directly from histidine or through transamination from K-ketoglutarate, which produces the bulk of intracellular glutamate. Our results showed that glutamate production was evident. There was histidine consumption, which could partially account for glutamate production. This only produces a small proportion of the overall glutamate produced by the cells. We suggest that transamination activity, and production of glutamate overall, was proportional to glutamate production from histidine. The hypothesis of cells acclimatising in the microgravity environment for the ¢rst 10 days and then continuing their life cycles in a friendly environment is suggested by those results too. The glutamine consumption pattern being inverse to the glutamate production pattern suggested that after day 7 cells were consuming glutamine for their DNA synthesis. They were as such dividing until day 21 when our experiments were terminated. Looking at ketogenesis we observed acetate production by the hepatocytes which diminished after day 10 of the experiments. We also observed that, contrary to what has been reported on static cultures and in bioreactor cultures [6], there was phenylalanine production from protein breakdown in our experiments. It is well established that hepatocytes tend to convert phenylalanine to tyrosine which is further converted to acetyl-CoA and fumarate. The increased concentration of phenylalanine in the medium and in the cells accelerates the turnover of tyrosine and its conversion to metabolites more useful to the cell. The results are entirely in keeping with this hypothesis. Overall the pattern of ketogenesis proves again the theory of cells acclimatising in the RCCS environment. Ketone bodies are the export products of liver cells to other organs that need ketone bodies to survive. The microgravity cultures exhibited high acetate production at the beginning. As only hepatocyte aggregates existed in the system there was no target organ to export ketone bodies. We believe this is the reason why acetate production diminished after day 10, reached its minimum on day 14 and remained low until the end of the experiments.
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There is no evidence of big succinate production as all precursors of succinate seemed not to be taken up in large quantities. Overall it does not seem that the Krebs cycle was fed from succinate production under our experimental conditions. Looking at the branched-chain amino acids as an entity it becomes clear that cells preferentially use leucine for their requirements. The most plausible explanation is that as all three branched-chain amino acids use the same transporter to enter the hepatocyte, leucine is preferentially taken up because its concentration in the extracellular milieu is the highest. Until the three branchedchain amino acids are in concentration equilibrium in the medium leucine is almost exclusively taken up and as a net result the concentrations of the three branched-chain amino acids in the extracellular supernatant are almost identical. High levels of ethanol were observed in all culture supernatants. Their presence is enigmatic. The obvious theory of bacterial and fungal contamination of the culture medium is not supported by the sterility of the medium on conventional testing. We introduced an ethanolfree environment after the ¢rst culture experiments and it was clear that there was evidence of ethanol in the culture medium. These observations have been previously reported in biological £uids of animals [8,12]. One plausible explanation is that as threonine can be used as a substrate for alcohol synthesis porcine hepatocytes have the ability to produce ethanol under conditions of alcohol dehydrogenase inhibition. Overall our results show that PPHs can be happily grown in microgravity conditions for up to 3 weeks. This may have implications for BALSS as they can provide an active biosubstrate for the systems. We have shown that this culture modality overcomes the initial disorder in cellular amino acid metabolism which is critical in long-term cultures of porcine hepatocytes [9]. Further studies are urgently required to scale up this model for growth of substantial quantities of hepatocytes and studies without time limits need to be performed to ascertain the feasibility of long-term culture and maintenance of these hepatocytes in culture.
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