Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver

Authors


Abstract

We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1-10 × 106 cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 ± 1.0 vs. 3.1 ± 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 ± 3.3 vs. 4.6 ± 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 ± 63 vs. 92 ± 13 μmol/L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale-up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure. (Liver Transpl 2005;11:901–910.)

Hepatic failure is a serious problem that costs the lives of tens of thousands of Americans each year.1 While liver transplantation can provide a permanent solution to hepatic failure, it is limited by a shortage of donor organs that is unable to supply the demand for liver transplantation. Liver transplantation also imposes the long-term side effects of immunosuppression on those who might require only short-term hepatic support during recovery from acute hepatic injury. Thus, there has been much interest in developing an extracorporeal liver support device to replace the function of the failing liver, much as hemodialysis can replace the function of the failing kidney. A most promising source of auxiliary liver function is the bioartificial liver (BAL) composed of biologically active hepatocytes within an extracorporeal framework.2 A few BAL devices have reached clinical testing in the settings of acute (fulminant) and acute-on-chronic hepatic failure.3–6 However, none of these first-generation devices have gained U.S. Food and Drug Administration approval.

Limitations of first-generation BAL systems have included factors such as excess device complexity,7 insufficient number of hepatocytes at inoculation,8 premature death of hepatocytes,8, 9 and absence10 or loss of their differentiated function during treatment.11 The possibilities of tumor spread when using a transformed hepatocyte line10 and xeno-zoonosis when using porcine hepatocytes12 have been suggested. But neither of these latter two possibilities have been realized in clinical testing of first-generation BAL systems.3, 13–16

In consideration of all of these concerns, we have developed a novel hepatocyte bioreactor based on spheroid technology (Fig. 1). Spheroids of porcine hepatocytes are formed in this bioreactor by a novel oscillation-based technique. Prior studies using transmission electron microscopy have revealed that spheroids are composed predominately of differentiated hepatocytes with well-developed cytoplasmic and surface features.17 The maintenance of normal three-dimensional shape of hepatocytes within spheroids likely contributes to their enhanced functional activity in vitro.18, 19 Along with increasing the rate of spheroid formation, oscillation provides excellent mixing of oxygen and other nutrients within the bioreactor reservoir. By incorporating a reservoir within the hepatocyte compartment, our novel BAL system can support a significantly greater numbers of hepatocytes than using a hollow fiber cartridge alone, as in most first-generation systems.20–22 The purpose of this study was to characterize the influence of oscillation frequency, cell density, and storage condition (fresh, cryopreserved) of porcine hepatocytes on kinetics of spheroid formation. In addition, we hypothesized that culture of hepatocytes in the spheroid reservoir configuration would be associated with increased functional activity compared to standard monolayer culture conditions.

Figure 1.

Schematic representation of the spheroid reservoir BAL (A) under in vitro condition and (B) during extracorporeal application. These figures demonstrate the practical aspects of oscillatory agitation that leads to hepatocyte aggregation into spheroids. The design allows oxygenation of culture medium by both head-space exposure and through an oxygen-permeable membrane at the floor of the reservoir. Two sources of oxygen, along with vigorous oscillation at 15 Hz, avoids hypoxic conditions at high cell density. An access port is submerged within the media to allow intermittent sampling of both media and cells during continuous culture. Under extracorporeal conditions, a settling column is utilized to prevent flow of porcine hepatocyte spheroids out of the reservoir and into the hollow fiber cartridge. The hollow fiber cartridge serves as a barrier to the recipient patient's immune system. An exit pump and return pump in the reservoir circuit are used to control the rate of ultrafiltration across the hollow fiber membrane.

Abbreviations

BAL, bioartificial liver; LDH, lactate dehydrogenase; mRNA, messenger RNA.

Methods

All animal procedures were performed under the guidelines set forth by the Mayo Foundation Institutional Animal Care and Use Committee and are in accordance with those set forth by the National Institutes of Health.

Porcine Hepatocyte Isolation

Hepatocytes were isolated from 15-kg pigs by a 2-step collagenase perfusion technique as previously described.23 The viability of freshly isolated pig hepatocytes exceeded 93% after all isolations as determined by trypan blue exclusion.

Hepatocyte Cryopreservation

Freshly isolated porcine hepatocytes were suspended at 1.0 x 107 cells/mL in cryopreservation medium (William's E medium supplemented with 10% fetal calf serum and 10% dimethylsulfoxide) and transferred to a controlled rate freezer (Programmable Freezing Controller, Custom Biogenic Systems, Shelby Township, MI). After freezing to −90°C, bags of frozen hepatocytes were stored in liquid nitrogen. Hepatocytes were thawed rapidly, resuspended in an equal volume of William's E medium at 37°C, and then washed before final suspension in culture medium. The viability of washed cryopreserved pig hepatocytes exceeded 80% by trypan blue exclusion.

Spheroid and Monolayer Culture

Hepatocytes (freshly isolated or thawed after cryopreservation) were seeded in T-75 flasks (Becton Dickinson and Co., Franklin Lakes, NJ) at cell densities ranging from 1.0-5.0 or 10.0 × 106 cells/mL. Flasks were seeded with fresh or cryopreserved cells in 20 mL of culture medium (William's E medium, 10% fetal bovine serum, 10 U/mL penicillin G, 100 μg/mL streptomycin, 10 mU/mL insulin, 5 μg/mL transferrin, 6.25 ng/mL selenium) supplemented with 17.5 μmol/L diazepam. To determine rates of ammonia conversion to urea, 50 mL of deuterium-enriched (heavy) ammonia gas (Cambridge Isotope Laboratories, Inc, Andover, MA) was equilibrated in a closed container of 1 liter of culture medium overnight at atmospheric pressure and 37°C. Flasks in the monolayer group were placed on a stable rack, while flasks in the spheroid group were placed on a rocker platform (Bellco Technology, Vineland, NJ) oscillating at 0.125 Hz or 0.25 Hz (Fig. 1A). All flasks were kept in an incubator at 37°C and 5% CO2.

Sampling of Cells and Medium

Hepatocytes were cultured for up to 5 days with medium changes on day 1 and day 3. Samples of medium were collected after 1, 3, and 5 days of culture. Cells were collected for visual and biochemical examination after 4 hours, 1 day, 3 days, and 5 days of culture. Spheroids were obtained by centrifugation of medium from spheroid-containing flasks, while monolayer hepatocytes were obtained by scraping them off of the flask surface.

Viability of Porcine Hepatocytes

Live and dead hepatocytes were identified using a fluorescence viability stain (Fluoroquench, One Lambda, Canoga Park, CA) and an epifluorescence video microscopy system (Axiovert; Carl Zeiss, Inc., Thornwood, NY) configured for fluorescein isothiocyanate (450-490-nm excitation filter, 510-nm emission filter). The extent of cell death was also estimated by release of lactate dehydrogenase (LDH) into the culture medium.24 The concentration of LDH was determined by a standardized assay (Takara Mirus BioCorp, Madison, WI).

Measurement of Spheroid Diameter

Spheroid formation was observed directly by phase microscopy and by standard light microscopy after hematoxylin & eosin staining of formalin-fixed sections. Spheroid counts and spheroid diameters were measured in samples of spheroid medium using an automated counter (Coulter-Z2, Coulter Corp. Miami, FL). Samples diluted 100-fold in, and samples from each flask were repeated in triplicate to establish reproducibility.

Measurement of Porcine Albumin

Concentrations of porcine albumin were determined by using a goat anti-pig albumin antibody (Cappel/Oragano Teknika, Durham, NC) as part of a previously reported immunoassay.25

Measurement of Total RNA by Agilent Chip Analysis

Total RNA was first extracted from cell fractions of porcine hepatocytes by standardized kit (RNeasy Mini Kit; Qiagen, Waltham, MA) and then stored at −80°. Total RNA was measured in the Genomics Core Facility (Mayo Clinic, Rochester, MN) using a standardized reagent kit (RNA 6000 Nano LabChip Kit; Caliper Technologies Corporation, Mountain View, CA) and automated apparatus (Agilent 2100 Bioanalyzer, Caliper Technologies Corporation).

Measurement of Pig Albumin messenger RNA (mRNA) by Quantitative-Competitive Reverse Transcription-Polymerase Chain Reaction

Transcription (i.e., copy number of mRNA) of the pig albumin gene was measured in cell fractions by a standardized quantitative-competitive reverse transcription-polymerase chain reaction with capillary electrophoresis detection.26, 27 Quantitative-competitive reverse transcription-polymerase chain reaction analysis was performed on frozen samples of extracted total mRNA (outlined in previous section) by the Immune Monitoring Lab Core Services (Mayo Clinic, Rochester, MN).

Quantitation of Ammonia and Isotopes of Urea

Ammonia was quantified using an automated analyzer (Vitros 250; Ortho-Clinical Diagnostics, Rochester, NY) in the clinical lab (Mayo Clinic, Rochester, MN). This assay utilizes a sensitive ammonia detector, bromphenol blue dye, with reflection density measured at 600 nm. The concentration of natural and deuterium-enriched (heavy) urea were quantified using an automated analyzer (Hitachi 912 Chemistry Analyzer; Hitachi, Life Sciences, San Jose, CA). The relative distribution of enriched species was determined by capillary gas chromatography/mass spectrometry.28

Diazepam Metabolism

Concentrations of diazepam and its 3 major metabolites (temazepam, nordiazepam, and oxazepam) were measured by high-performance liquid chromatography with UV detection by modification of a previously developed procedure.29 Conjugated forms of the 3 metabolites were measured by comparing the difference in their concentration with and without glucuronide hydrolysis. Standard curves for diazepam and metabolites (temazepam, oxazepam and des-methyl diazepam) showed an R2 ≥ 0.9988. The coefficient of variation was <10% at all concentrations of diazepam or the metabolites.

Statistical Testing

Unpaired Student t test was used to determine statistical significance between control and study groups. Significance was defined as P value <0.05 between groups. Variables for comparison include LDH release, albumin production, diazepam metabolism, ammonia detoxification, and urea formation. Average values (±standard deviation) are reported.

Results

General Description

Our set of experiments consisted of testing spheroid formation, viability, metabolic performance of fresh and cryopreserved porcine hepatocytes under oscillation (spheroid-forming) conditions compared to monolayer (control) conditions. Culture conditions were standardized as follows: T75 flasks containing 20 mL of media incubated in 5% CO2 environment at 37°C. An oscillating rocker plate was utilized for spheroid formation while monolayer cultures were placed on a stable rack within the incubator. All cultures were maintained for 5 days. Five T75 flask cultures were included in each group. All cells within an experiment were obtained from the same harvest. Harvests were repeated in triplicate to confirm reproducibility of the data. Representative data from 1 experiment is presented unless otherwise indicated.

Kinetics of Spheroid Formation

We were first interested in the influence of cell density and oscillation frequency on spheroid formation kinetics. To assess the effect of cell density on spheroid formation, fresh porcine hepatocytes were inoculated into T75 flasks at concentrations of 1.0, 5.0, and 10 × 106 cells per mL. Flasks were oscillated at a frequency of 0.125 or 0.25 Hz for 24 hours. As illustrated in Figure 2, we observed spontaneous aggregation of porcine hepatocytes within 4 hours of placing them in oscillation culture (Fig. 2A and 2B). Tight round spheroids were observed after 24 hours of culture as illustrated in Figures 2C and 2D. We observed that spheroid size after 24 hours was influenced by concentration of porcine hepatocytes at inoculation. At low density 1 × 106 cells per mL and 0.125 Hz, 99% of spheroids were less than 400 microns in diameter compared to 94% at medium density (5 × 106 cells per mL). This value decreased to 90% when cells were inoculated at 10 × 106 cells per mL. When we increased the oscillation frequency of the high concentration group (10 × 106 cells per mL) to 0.25 Hz, we observed that spheroid diameter was reduced such that 99% of spheroids were less than 400 microns in diameter 24 hours after inoculation. Spheroid diameter was not influenced by further increases in the rate of oscillation.

Figure 2.

Microscopy of aggregation of porcine hepatocytes into spheroids. (A) At 4 hours after inoculation, porcine hepatocytes have aggregated into precursors of spheroids. (B) At 4 hours after inoculation, precursors of spheroids are formed by viable porcine hepatocytes (stain green), while rare dead cells (orange nuclei) remain in isolated suspension. (C) At 24 hours after inoculation, a cross-section of an intact spheroid is demonstrated after fixation and staining with hematoxylin & eosin. Rare pyknotic nuclei representing apoptotic hepatocytes primarily within the deep aspects of the spheroid. Cord-like structures consistent with normal hepatocyte architecture are identified within the spheroids. (D) Gross demonstration of a representative spheroid 24 hours after inoculation. The vast majority of hepatocytes observed in this representative spheroid are viable and stained green. Only rare dead hepatocytes with orange nuclei are identified. (E) Three days after inoculation, spheroids began clumping into larger tissue-like structures under the conditions utilized this experimentation. Very few dead hepatocytes were observed in these larger aggregates of spheroids. (F) At 5 days of culture, spheroids aggregated into large, well-formed tissue-like structures still maintaining high cell viability based on viability staining and low LDH release into the media.

Viability of Porcine Hepatocyte Spheroids

As illustrated in Figure 2, viability of fresh porcine hepatocytes within spheroids remained high throughout 5 days of culture. Very few dead cells (orange nuclei) were observed in these aggregates. Over time, spheroids gradually aggregated into larger spheroids and eventually clumped into tissue-like structures by 5 days in culture (Fig. 2E and 2F). Viability remained high in tissue-like structures based on fluorescence vital staining.

Influence of cell density on cell death was addressed using LDH release into the culture media. We observed a direct correlation between number of fresh porcine hepatocytes inoculated and LDH release into the media of both spheroid and monolayer cultures (Table 1). At low cell density (1 × 106 cells per mL), LDH release was similar in spheroid and monolayer cultures. However, at higher cell density (5 and 10 × 106 cells per mL), LDH levels were significantly higher after 24 hours of monolayer conditions suggesting an advantage of oscillation culture for maintaining cell viability. On day 3 and day 5, media levels of LDH were similar under spheroid and monolayer conditions suggesting that the majority of cell death occurred during the first 24 hours of culture.

Table 1. LDH Release as a Measure of Cell Death
Day of CultureCell Density (× 106 cells/mL)LDH Release (U/mL)
SpheroidMonolayer
  • *

    P < 0.05 vs. day 1, 5.0 × 106 cells/mL, same culture condition.

  • P < 0.05 vs. spheroid group, same day and cell density.

11.00.6 ± 0.20.9 ± 0.6*
 5.00.9 ± 0.52.2 ± 0.6
 10.01.3 ± 1.03.1 ± 0.7*
35.00.7 ± 0.40.8 ± 0.7
55.00.6 ± 0.20.7 ± 0.4

Gene Expression and Hepatocellular Function Improved by Spheroidal Growth

Albumin Production: Influence of Cryopreservation

We were next interested in the influence of cryopreservation and spheroid culture on the gene expression of porcine hepatocytes. The albumin gene was selected for these studies, which compared 5 × 106 cells per mL of cryopreserved under monolayer and spheroid conditions (oscillation at 0.125 Hz).

Cryopreservation for 7 days at −80° was associated with a significant decline in viability of porcine hepatocytes (96.8 ± 0.4% vs. 82.4 ± 4.1%; P < 0.001) and reduced levels of albumin mRNA compared to freshly isolated porcine hepatocytes (Table 2). Levels of albumin mRNA were undetectable in both monolayer and spheroid samples after 24 hours of culture. Low levels of albumin production were detected by a sensitive immunoassay specific for porcine albumin in cultures of cryopreserved porcine hepatocytes. Albumin production by spheroids of cryopreserved porcine hepatocytes was higher than monolayer controls (0.42 ± 0.11 vs. 0.30 ± 0.04 fg/cell/h; P < 0.001) on day 1 of culture. Albumin production by cryopreserved porcine hepatocytes declined over the subsequent 5 days of culture. Due to the low rates of albumin production observed in cultures of cryopreserved porcine hepatocytes, only cultures of fresh porcine hepatocytes were utilized in the diazepam and ammonia/urea metabolism studies.

Table 2. Influence of Cryopreservation and Spheroid Culture on Pig Albumin RNA and Protein Production at Intermediate Cell Density (5 × 106 cells/mL)
CryopreservedTime PointViabilityAlbumin RNA (copies/μg RNA)Albumin Production (fg/cell/h)
MonolayerSpheroidMonolayerSpheroid
  • Abbreviations: t, time; ND, non detected.

  • *

    P < 0.0001 spheroid vs. monolayer.

  • P < 0.001 spheroid vs. monolayer.

No (fresh)t = 096.8 ± 0.4%644 (100%)  
 Day 1 561 (87%)346 (53%)4.6 ± 1.414.7 ± 3.3*
 Day 3 195 (30%)239 (37%)7.7 ± 2.023.6 ± 3.8*
 Day 5 93 (14%)315 (49%)10.0 ± 1.724.7 ± 5.9*
Yes (−80° × 7 days)t = 082.4 ± 4.1%136 (21%)  
 Day 1 ND (<5%)ND (<5%)0.30 ± 0.040.42 ± 0.10
 Day 3 ND (<5%)ND (<5%)0.26 ± 0.020.27 ± 0.02
 Day 5 ND (<5%)ND (<5%)0.05 ± 0.020.06 ± 0.02

Albumin Production: Fresh Porcine Hepatocytes

In contrast to cultures of cryopreserved cells, significant levels of both albumin mRNA and albumin production were detected in comparable cultures of fresh porcine hepatocytes on day 1, day 3, day 5 (Table 2). Levels of albumin mRNA in spheroid culture remained stable (346 vs. 315 copies/μg RNA) over the 5-day culture period, while levels of albumin mRNA in monolayer culture declined significantly (561 vs. 93 copies/μg RNA) over the same time course. Higher levels of albumin mRNA in spheroid cultures vs. monolayer cultures were associated with higher levels of albumin production. Albumin production increased from day 1 to day 5 under both conditions, though the extent of increase was higher on day 5 of spheroid conditions compared to monolayer conditions (24.7 ± 5.9 vs. 10.0 ± 1.7 fg/cell/h; P < 0.001). Levels of total RNA remained relatively stable (day 1: 73 ng/mL; day 5: 60 ng/mL) in spheroid cultures of fresh porcine hepatocytes. The ratio of 28S vs. 18S ribosomal RNA increased slightly from 1.90 to 2.59 between day 1 and day 5 suggesting a relative effect of spheroid culture on ribosomal gene expression (or RNA stability) over this time frame.

Albumin Production: Influence of Cell Density

Albumin production by cultures of fresh porcine hepatocytes was also measured under low, medium, and high cell density conditions over a 5-day culture interval. The cellular rate of albumin production remained relatively stable over 5 days of culture at high cell density. At low cell density (1 × 106 cells/mL), the cellular rate of albumin production started low and increased over 5 days of culture. The highest cellular rate of albumin production was achieved by cultures of fresh porcine hepatocytes on day 5 of culture at 1 × 106 cells/mL (36.2 ± 5.1 fg/cell/h). Total albumin production (product of cell density × cellular rate of albumin production) was greatest in high cell density spheroid culture on both day 1 (0.20 μg/h) and day 5 (0.19 μg/h).

Diazepam Metabolism: Influence of Cell Density in Spheroid Culture

The rate of diazepam metabolism to its conjugated and nonconjugated metabolites was studied using cultures of fresh porcine hepatocytes. As illustrated in Figure 3, diazepam elimination from culture medium was rapid when spheroids of fresh hepatocytes were utilized in the system. No diazepam was detected after 7 hours of culture under medium or high cell density conditions. Using data from low cell density cultures (1 × 106 cells/ mL), we estimated a diazepam elimination rate of 1.4 μmol/L/h/106 cells. Two conjugated metabolites of diazepam (conjugated nordiazepam, conjugated temazepam) were detected with essentially no conjugated oxazepam or unconjugated metabolites detected. Percent conversion of diazepam to its conjugated metabolites ranged from 83% at low cell density to 90% at high cell density, reflecting a gradual increase in the production of conjugated temazepam from low to high cell density. Interestingly, although diazepam was rapidly eliminated from the culture media by spheroids of fresh porcine hepatocytes, the appearance of conjugated metabolites lagged considerably. As illustrated in Figure 3, levels of conjugated temazepam and conjugated nordiazepam, to a lesser extent, were detected in the culture media at 8 hours. However, by 24 hours of culture, levels of these conjugated metabolites were significant and in combination approached those of the initial concentration of diazepam.

Figure 3.

Diazepam metabolism in spheroid culture— influence of cell density. Diazepam was rapidly eliminated (by 8 hours) from cultures of porcine hepatocyte spheroids at a cell density of 5 × 106 cells/ mL. Metabolism by phase 1 and phase 2 pathways accounted for 83-90% of diazepam biotransformation. Low total levels of diazepam plus metabolite at 8 hours suggest a significant difference in the rate of diazepam uptake into the cell and the combined rate of metabolism and release of diazepam from the cell. Open bars, diazepam; light grey bars, conjugated temazepam; dark grey bars, conjugated nordiazepam.

Ammonia Detoxification and Ureagenesis Increased in Spheroid Culture

Since ammonia and its detoxification to urea is of relevance to the treatment of clinical liver failure and the prevention of hepatic encephalopathy, we were interested in the influence of spheroid formation on these metabolic activities by cultured porcine hepatocytes. We utilized culture medium supplemented with heavy ammonia to measure the true rate of ureagenesis. As illustrated in Figure 4, detoxification of ammonia was greatest under spheroid conditions compared to monolayer conditions on both day 1 and day 5. Monolayer cultures of porcine hepatocytes eliminated 80% of ammonia on day 1. However, this percentage declined to 64% on day 5. In contrast, porcine hepatocytes in spheroid culture eliminated 95% of ammonia on day 1 and maintained a high level of 88% ammonia elimination on day 5. Both of these differences were highly significant (P < 0.001).

Figure 4.

Ammonia detoxification was significantly greater by porcine hepatocytes in spheroid culture compared to monolayer culture on both day 1 and day 5 of continuous culture. Cell density of 5 / 106 cells/mL was used for both conditions. Ammonia concentration, grey bars.

The elimination of heavy ammonia from the medium was associated with production of heavy urea, as illustrated in Figure 5. Greater amounts of both natural and heavy urea were produced by porcine hepatocytes in spheroid culture compared to monolayer culture on both day 1 and day 5 (P < 0.001). The percentage of urea formed by conversion of heavy ammonia to heavy urea was similar in monolayer and spheroid cultures of porcine hepatocytes on day 1 (40.8% vs. 40.1%). On day 5, the percentage of heavy urea produced by porcine hepatocytes in spheroid culture was higher (44.9%) compared to monolayer culture (41.5%) suggesting that porcine hepatocytes exhibited greater function and more intact ureagenesis than monolayer cultures later in culture.

Figure 5.

Urea formation was significantly greater in spheroid culture compared to monolayer culture. Cell density of 5 × 106 cells/mL was used for both conditions. Urea formation from ammonia was confirmed by a radiolabeled technique, which accounted for approximately 40% of total urea formation. Heavy urea, light shaded bars; natural urea, dark shaded bars.

Discussion

We have developed a novel bioreactor composed of porcine hepatocyte spheroids in batch reservoir culture for use as a BAL: the spheroid reservoir BAL. This new technology was based on the serendipitous observation that isolated porcine hepatocytes spontaneously aggregate into spheroids under conditions of gentle oscillation. The reservoir design of our novel spheroid bioreactor is simple, as illustrated in Figure 1, yet capable of supporting 250-500 grams of nontransformed hepatocytes (2- to 5-fold over most first-generation devices). Spheroid formation has previously been recognized to provide a favorable microenvironment associated with high viability and stable function of primary hepatocytes in static culture,17, 30 rotational culture,18, 31, 32 and during extracorporeal treatment.11 The spheroid reservoir bioreactor, composed of an oscillating suspension of porcine hepatocytes, is unique from rotational-style spheroid bioreactors in that it provides greater mixing of cells and nutrients such as oxygen. In our bench studies, which utilized T75 flasks loaded with hepatocytes at a cell density of 1-10 × 106 cells/mL, gassing the headspace was sufficient to prevent hypoxia. The scaled-up spheroid reservoir BAL also includes a gas permeable membrane at the bottom of the reservoir (Fig. 1). In unpublished studies using the scaled-up device loaded with fresh porcine hepatocytes at 20 × 106 cells/mL, 2 sources of oxygen along with the mixing of continuous oscillation prevented hypoxia and supported a stable rate of oxygen consumption (0.88-0.94 mmol/h) for 24 hours of continuous operation. Oscillation mixing also increased hepatocyte-hepatocyte contact that was felt to contribute to rapid spheroid formation.

We also observed that oscillation frequency was inversely associated with spheroid diameter, while cell density was directly associated spheroid diameter on day 1 after inoculation of cells into the reservoir. Since cell number in the spheroid reservoir BAL is the product of cell density and reservoir volume, the option of low cell density is unacceptable, since it would require an excessive reservoir volume to provide a sufficient number of hepatocytes for clinical application. Our current data indicate that an oscillation frequency of 0.25 Hz was associated with stable cell viability and therefore should be acceptable for future clinical application of the scaled-up spheroid reservoir BAL.

Smaller spheroids are preferred in the reservoir culture to allow diffusion of nutrients throughout the spheroid and maintain metabolic activity without hypoxia. Other investigators have noted that spheroid formation is influenced by species of hepatocyte (pig > dog),33 extracellular matrix components (spheroid formation inhibited by dermatan sulfate, heparin, fibronectin, and laminin),30, 34 and inhibitory molecules (cytochalasin D was shown to block actin filaments in spheroid formation).35 These agents and techniques may be useful in the regulation of spheroid aggregation into tissue-like structures that were observed in the spheroid reservoir from day 3 to day 5 of culture (Fig. 2). The ability of porcine hepatocytes to aggregate into tissue-like structures is fascinating and worthy of further investigation. However, spheroids of greater than 200 microns in diameter are undesirable to a BAL, since a large diameter may impair nutrient delivery within the spheroid and overall metabolic performance during clinical application of the spheroid reservoir BAL.

Another benefit of smaller spheroids is that they may tolerate cryopreservation better than larger aggregates of hepatocytes or isolated hepatocytes. Our current data suggests that cryopreservation of isolated porcine hepatocytes by freezing leads to significant injury and loss of metabolic activity (i.e., albumin gene expression and albumin production) compared to freshly isolated cells. Data from porcine hepatocytes are consistent with preliminary data from murine hepatocytes showing an adverse impact of freezing on gene expression.36 Using high-density microarrays (MG-U74A gene chip; Affymetrics, Santa Clara, CA), we observed that freezing of isolated murine hepatocytes was associated with a significant reduction in expression of numerous genes of detoxification (phase 1, 8 genes; phase 2, 5 genes; urea cycle, 4 genes) and serum protein synthesis (5 genes including albumin). Of note, hepatocyte viability following cryopreservation exceeded 80% in both sets of experiments. These studies suggest a poor correlation between postthaw viability of isolated hepatocytes and their metabolic activity.

Despite the drawbacks of frozen cryopreservation, our studies indicated a significant benefit of spheroid formation when fresh porcine hepatocytes were utilized. Our studies are consistent with the results of improved differential gene expression and hepatocyte function reported by others.18, 19, 31 Diazepam was utilized as a substrate for measuring metabolic activity, since endogenous benzodiazepine receptors are associated with development of hepatic encephalopathy,37 and diazepam metabolism requires intact (phase 1 oxidation and phase 2 conjugation) pathways of metabolism.38, 39 Our data suggest that diazepam is taken up rapidly into the cell, but that intracellular metabolism followed by release of newly formed conjugates into the media was a somewhat slower process. Specifically, 3-hydroxylation of diazepam to temazepam requires intact CYP3A activity while N-demethylation to nordiazepam requires a CYP2C activity.40 Conjugation of these metabolites is catalyzed by a UDP-glucuronosyltransferase.41 Therefore, a demonstration of rapid conversion of diazepam to its primary conjugated metabolites confirms the presence of both phase 1 and phase 2 metabolism by freshly isolated porcine hepatocytes under spheroid culture conditions.

One of the most important observations of this study was that ammonia detoxification to urea was present in cultures of porcine hepatocytes and that detoxification activity was increased by spheroid formation. To assess the true rate of ureagenesis (i.e., detoxification of ammonia to urea by the urea cycle), we utilized a heavy isotope gas of ammonia solubilized in culture media. Our data conclusively demonstrate that over 40% of urea production by porcine hepatocytes occurred via urea cycle activity. Our data also suggest that alternative mechanisms for urea production exist in cultures of porcine hepatocytes, such as the single-step conversion of arginine to ornithine and urea, a reaction catalyzed by arginase.42 The distinction between these pathways of urea production is important, since only true ureagenesis is associated with direct detoxification of ammonia.

To predict whether a BAL can provide sufficient metabolic activity to a patient during extracorporeal activity, an engineering mass transfer analysis was performed. Such an analysis was useful to determine the rate-limiting step of the process—namely, whether metabolic performance of the spheroid BAL will be limited by transport of molecules across the membrane or limited by metabolic activity of the hepatocytes. First-generation BAL devices have been limited by the latter (i.e., metabolic activity), which likely contributed to an inability to demonstrate efficacy in clinical trials. We chose to examine the detoxification of ammonia into urea for our engineering analysis, since ammonia detoxification is strongly associated with hepatic encephalopathy,43 formation of cerebral edema,44 and herniation under clinical conditions.45 Assuming that 2 molecules of heavy ammonia were consumed to form 1 molecule of heavy urea, we estimated an ammonia detoxification rate of 5.4 mmol/h for a T-flask containing 1 × 108 cells. If the T-flask system were scaled up such that 500 grams of porcine hepatocytes (5 × 1010 cells) were inoculated into a 2-liter reservoir at 2.5 × 107 cells/mL, an ammonia detoxification rate of 2.1 mmol/h would be possible. These conditions are realistic and comparable to clinical application of our novel spheroid reservoir BAL. In unpublished bench studies from our laboratory using a polysulfone hollow fiber membrane with 100 kD nominal molecular weight cutoff and surface area of 0.8 m2, a transmembrane ammonia flux rate of 2.0 mmol/h was observed. Experimentally derived values of ammonia flux and ammonia detoxification are of similar magnitude and are large enough to be of clinical significance. Together these estimates suggest that the spheroid reservoir BAL should not be limited by detoxification activity or transmembrane flux under clinical conditions.

In summary, first-generation BAL devices have failed due to a number of factors that have included premature death and loss of differentiated function by hepatocytes within the device. In consideration of these limitations, we have developed a novel bioreactor configuration based on spheroid technology and the observation that rapid spheroid formation is possible through oscillation-based cell culture. We observed that formation of spheroids composed of viable, metabolically active porcine hepatocytes was possible and optimized at high cell density if oscillation frequency was accelerated at 0.25 Hz. Our ammonia detoxification studies were noteworthy since they confirm intact urea cycle activity by high-density cultures of porcine hepatocyte spheroids. More importantly, our results suggest that a scaled-up version of the spheroid reservoir BAL is feasible and without mass transport or biochemical limitations (i.e., capable of the detoxification needs of the clinical state). Therefore, further investigation of this device in a preclinical model of liver failure is warranted. 3

Table 3. Influence of Cell Density on Cellular Rate of Albumin Production by Spheroids of Fresh Porcine Hepatocytes
Cell Density (× 106 cells/mL)Albumin Production (fg/cell/h)
Day 1Day 5
  • *

    P < 0.0001 vs. Day 1.

  • P < 0.05 vs. 1 × 106.

  • P < 0.05 vs. Day 1.

  • §

    P < 0.0001 vs. 1 × 106.

1.010.7 ± 4.336.2 ± 5.1*
5.014.7 ± 4.324.7 ± 4.6,§
10.020.0 ± 4.5§18.5 ± 2.8§

Ancillary