The FASEB Journal • FJ Express Full-Length Article
A novel formulation of oxygen-carrying matrix enhances liver-specific function of cultured hepatocytes Yaakov Nahmias, Yiannos Kramvis, Laurent Barbe, Monica Casali, Francois Berthiaume, and Martin L. Yarmush1 Center for Engineering in Medicine/Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, Massachusetts, USA Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygencarrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function.—Nahmias, Y., Kramvis, Y., Barbe, L., Casali, M., Berthiaume, F., Yarmush, M. L. A novel formulation of oxygen-carrying matrix enhances liverspecific function of cultured hepatocytes. FASEB J. 20, E1828 –E1836 (2006)
ABSTRACT
Key Words: cytochrome P450 䡠 fluorocarbon Oxygen is an important component of the cellular microenvironment, regulating cell survival, differentiation, and function (1, 2). Adequate oxygen supply is critical for the function of highly metabolic tissues such as liver or muscle (3, 4). Oxygen is supplied to hepatocytes in vivo by a mixture of arterial and venous blood (4) at an estimated rate of ⬃1.2 nmol/s/106cells. Conversely, hepatocyte oxygen consumption has been measured in vitro to be 0.9 nmol/s/106cells during the first 48 h after seeding and 0.4 nmol/s/106cells during stable long-term cultures (5, 6). In both cases the in vivo E1828
oxygen supply is more than adequate to supply the measured cellular demand in vitro. Oxygen supply in standard tissue culture applications is significantly different. Typically hepatocytes are cultured below 1–1.5 mm of a stagnant medium layer under ambient oxygen partial pressure. Under these conditions, oxygen partial pressure on the cell surface is expected to reach 12 mmHg (7), resulting in an oxygen uptake/supply rate of 0.35 nmol/s/106cells in culture, ⬃ 3-fold less than the oxygen demand of freshly seeded hepatocytes (8). Although raising oxygen tension to 500 mmHg or above would meet the oxygen demand of the cells (Fig. 1), several studies have shown that supraphysiological oxygen levels can cause free radical formation that compromises cellular viability, especially during long-term culture (9, 10). One way to increase oxygen supply without raising oxygen tension is to enhance the solubility of oxygen in the culture medium by adding an oxygen carrier (Fig. 1) (11, 12). Hemoglobin (Hb) -based oxygen carriers can potentially reproduce the oxygen-carrying capacity of blood (12–15). However, outside the red blood cell Hb is unstable, degrading in hours (15, 16). Crosslinked or synthetic hemoglobins have significantly longer half-lives, making such materials potentially useful in clinical settings as a blood substitute (12, 14). Yet even stabilized Hb can auto-oxidize in hours to its toxic forms (Fe⫹3, Fe⫹4), which may induce cellular damage during long-term tissue culture applications (12, 16). Fluorocarbons, on the other hand, are highly stable molecules that can carry ⬎ 20-fold more oxygen than water (Fig. 1) (15, 17, 18). Fluorocarbons are not subject to oxidation or free radical reactions and cannot be metabolized; therefore, they are ideally suited for long-term tissue culture uses (11, 12, 15, 17). Although fluorocarbons are not miscible with water, they can be emulsified by a number of surfactants. 1 Correspondence: Center for Engineering in Medicine/ Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, MA 02114, USA. E-mail:
[email protected] doi: 10.1096/fj.06-6192fje
0892-6638/06/0020-1828 © FASEB
Life Technologies (Carlsbad, CA, USA). Fibronectin was obtained from BD Biosciences (San Jose, CA, USA). Glucagon was purchased from Eli Lilly (Indianapolis, IN, USA). Fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor (HGF) were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Egg yolk phospholipids, formulated as Lipoid E80s, were a generous gift from Lipoid LLC (Newark, NJ, USA). Perfluorooctyl bromide (perflubron) was purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Unless otherwise noted, all other chemicals, growth factors, and solutions were purchased from Sigma-Aldrich Chemicals. Formulation of oxygen carrier
Figure 1. Oxygen concentration as a function of oxygen partial pressure. Adapted from Lowe et al. (18). Normal oxygen supply in vivo (top left) between 70 to 25 mmHg is ⬃2000 nmol/ml. Plasma-like culture medium can only hold up to 1000 nmol/ml of oxygen at 760 mmHg. The addition of 20% fluorocarbon-based oxygen carrier to culture medium allows it to hold an additional 2000 nmol/ml of oxygen at partial pressures of 400 mmHg.
Emulsified fluorocarbons have been shown to increase bacterial proliferation and survival, mitotic revision, and perspiration of plant cells in suspension cultures (17, 18). However, the high density (1.98 g/ml) of fluorocarbons has limited their use in mammalian cell cultures. One solution has been to culture cells at the interface between fluorocarbon oil and culture medium on a thin film of denatured proteins (17, 19 –22) or collagen monomers (22). A similar two-layered system has been used extensively for organ preservation (23). Although these experiments showed increased cellular proliferation and function, the technique is difficult to use and cultures are subject to mechanical disturbances. Alternatively, fluorocarbon gels can be formed by the addition of a gelling agent such as perfluoroalkylated amine oxide (24, 25). Fluorocarbon gels can carry large amounts of oxygen, but differ widely from the native extracellular matrix (ECM) that cells can attach to and remodel. Here we describe a novel use for fluorocarbon-based oxygen carriers in mammalian cell culture consisting of embedding an oxygenated fluorocarbon emulsion in an ECM gel, thus preserving cell-matrix interactions (26). This technique increases oxygen delivery to freshly seeded cells undergoing attachment and spreading. Using this technique, we found that hepatocytes show significantly increased viability and cytochrome P450 detoxification activity, as well as significantly higher albumin and urea secretion.
MATERIALS AND METHODS Reagents and antibodies FBS, PBS, Dulbecco’s modified Eagle medium (DMEM), penicillin, and streptomycin were obtained from Invitrogen OXYGEN-CARRYING MATRIX ENHANCES LIVER FUNCTION
Oxygen carrier formulation was similar to the description of Oxygent (11, 27, 28) with several important differences. The formulation is presented in Table 1. The oxygen carrier emulsion was prepared by dissolving all components except perflubron in a sterile environment, followed by 5 min of vortexing until a consistent gray-white emulsion formed. Perflubron was slowly added to the emulsion while agitating. Finally, the mixture was transferred to a 50 ml conical tube and homogenized for 2 min using a Branson Ultrasonic Sonifier 450 (Danbury, CT, USA). The oxygen carrier emulsion was stored at 4°C and used within 1–2 wk. The emulsion’s particle size distribution was analyzed by diluting it in PBS (1:500) and measuring its light scatter characteristics with a ZetaPlus Potential Analyzer (Brookhaven Instrument, Holtsville, NY, USA). Preparation of oxygen carrier-collagen Type I collagen stock solution was prepared from rat tail tendon as described by Dunn et al. (29). Briefly, isolated rat tail tendons were dissolved in 3% acetic acid overnight at 4°C. The solution was filtered, concentrated, and precipitated by 30% NaCl. The resulting pellet was rinsed and dialyzed against 1 mM HCl. Collagen gelling solution was prepared by mixing nine parts of collagen stock (1.25 mg/ml) with one part of 10 ⫻ DMEM on ice. Oxygen carrier-collagen was prepared by mixing two parts collagen gelling solution with one part oxygen carrier emulsion on ice. The solution was then bubbled with 100% oxygen for 10 min. Finally, 600 l of oxygen carrier-collagen was spread in each well of a 6-well plate and incubated at 37°C for 30 min. Oxygen partial pressure following incubation was measured as 400 mmHg (data not shown). To control for the presence of the emulsifying agents in the collagen gel, an identical procedure was carried out using the egg yolk dispersion without perflubron. Hepatocyte isolation and culture Rat hepatocytes were harvested from adult female Lewis rats purchased from Charles River Laboratories (Boston, MA, TABLE 1.
Oxygen carrier formulation
Component
Weight %
Purpose
60 3.6 34.7 1 0.5 0.15 0.05
Oxygen carrier Surfactant Continuous phase Antioxidant Osmolarity Buffer Chelating cations
Perflubron Egg yolk phospholipids Water RPMI vitamin solution NaCl NaHCO3 EDTA
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USA) weighing 150 –200 g by a two-step in situ collagenase perfusion technique, modified by Dunn et al. (30). Hepatocyte viability after the harvest was ⬎90% based on Trypan blue exclusion, and hepatocyte purity was ⬎95%. All animals were treated in accordance with National Research Council guidelines and approved by the Subcommittee on Research Animal Care at the Massachusetts General Hospital. Hepatocytes were seeded directly on collagen gel-coated 6-well plates at a density of 100,000 cells/cm2. Culture medium consisted of phenol red-free Williams E supplemented with 200 U/ml penicillin/streptomycin, 5 ng/ml epidermal growth factor (EGF), 0.8 ng/ml HGF, 5 pg/ml FGF-2, 0.1 M dexamethasone, 0.286 ng/ml glucagon, 0.5 g/ml insulintransferrin-selenium (ITS), 5 g/ml fibronectin, 1 U/ml heparin, 1 g/liter BSA, and 1 M ethanolamine. For serumcontaining media experiments, for the first 24 h after seeding, medium was supplemented with 5% serum to enhance cell attachment and 2% serum throughout the rest of the experiment. For serum-free experiments, medium was supplemented with 45 g/ml fibronectin for the first 24 h to promote cell attachment but otherwise remained unchanged. Hepatocyte cultures were maintained at 37°C, 10% CO2 humidified incubator. Oxygen uptake profile of spreading hepatocytes Oxygen uptake profile was measured as described previously (5, 6). Briefly, 1 ml of either oxygen carrier-collagen or collagen dispersion control was prepared as described above and spread on a 60 mm tissue culture dish. Freshly isolated rat hepatocytes were seeded at a density of 100,000 cells/cm2 and allowed to adhere for 45 min in a 10% CO2 humidified incubator at 37°C. Once cells had adhered, the dish was filled to a final volume of 11.5 ml of serum-free medium and hermetically sealed. A magnetic stir bar maintained a uniform oxygen pressure throughout the chamber monitored by an Ocean Optics (Dunedin, FL, USA), FOXY Fiber Optic Oxygen Sensor immersed in the chamber. Measurements were carried out at 37°C. Aspartate aminotransferase (AST) release
Cytochrome P450 activity To probe for cytochrome P450 IA1 (CYPIA1) activity in situ, we measured ethoxyresorufin-O-deethylase (EROD) activity as described by Behnia et al. (31). Briefly, 5 M of nonfluorescent ethoxyresorufin, a specific substrate for rat CYPIA1, was added to the culture medium. The rate of appearance of the fluorescent resorufin product secreted into the surrounding medium, which reflects the activity of CYPIA1 in the culture, was measured using an Fmax fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 530 nm excitation and 590 nm emission wavelengths. Albumin and urea analysis Media samples were collected daily and stored frozen at –20°C for subsequent analysis of albumin and urea content. Albumin concentration was measured by ELISA, as reported by Dunn et al. (30). Purified rat albumin and anti-rat albumin antibody (Ab) were purchased from Cappel Laboratories (Aurora, OH, USA). Urea synthesis was analyzed via its specific reaction with diacetyl monoxime by a commercially available Blood Urea Nitrogen Assay Kit (Stanbio Laboratory, Boerne, TX, USA). Standard curves were generated using purified rat albumin (Cappel Laboratories) or urea dissolved in culture medium. Reverse transcription polymerase chain reaction (PCR) RNA template was acquired from crude lysates by boiling samples at 95°C for 20 min. Reverse transcription and amplification were performed using a one-step PCR kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s directions. The reaction was carried on an Eppendorf MasterCycler (Hamburg, Germany) using the following primers: rat albumin forward 5⬘-GAGAAGGTCACCAAGTGCTGTAGT-3⬘; reverse 5⬘-CTGGGAGTGTGTGCAGATATCAGAGT-3⬘; rat Cytochrome P450 IA1 forward 5⬘- GATCATGCCTTCTGTGTATGGATTCC-3⬘; reverse 5⬘TGGAGAAACTCTTCAGCGCATTCT-3⬘. -Actin was used for load control. Separation was carried out on a 2% agarose gel and images were captured using the MultiAnalyst® software (Bio-Rad Laboratories, Hercules, CA, USA).
Collected media were analyzed for levels of aspartate aminotransferase (AST) using Thermo Electron Infinity™ AST Reagent (Louisville, CO, USA). Medium samples (15 l/well) were loaded onto a 96-well plate in duplicate, followed by 150 l/well of the AST liquid reagent. Absorbance decay was measured at 340 nm wavelength in 15 s intervals in a Bio-Rad (Hercules, CA, USA) Benchmark Plus microplate spectrophotometer. Values were normalized to the total amount of AST available per culture, which was determined by running the assay after total cell lysis induced by 1% Triton X-100 for 20 min at room temperature.
RESULTS
Fluorescence live-dead staining
Oxygen carrier stability
After the first day of culture, cells were stained using a Live/Dead Viability assay (Molecular Probes, Eugene, OR, USA) according to manufacturer’s directions. In this assay, the cytoplasm of live cells accumulates green fluorescent calcein due to esterase activity, while the nucleus of dead cells is labeled red by ethidium homodimer due to loss of membrane integrity. To quantify cellular viability, random images were taken under identical camera gain and exposure settings. More than 100 cells were counted in every field. The number of live cells was divided by the number of live plus dead cells to give percent viability 24 h postseeding.
Long-term tissue culture applications require stable materials that will not degrade over time. To characterize the oxygen carrier emulsion and its stability, we measured the emulsion particle size distribution over time using forward light scattering. Figure 2A shows the oxygen carrier particle diameter distribution after 3, 18, and 75 days of storage at room temperature. Average particle size was measured to be 300 ⫾ 34 nm at day 3, 320 ⫾ 34 nm at day 18, and 370 ⫾ 90 nm at day 75. Little change was observed, suggesting that the
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Statistical analysis Results are expressed as mean ⫾ sd. N states the number of cell culture wells. Statistical analysis was performed using the Student’s t test, with a P value of ⬍0.05 considered significant.
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out oxygen carrier, the oxygen concentration dropped below the published value of Km (2.6 mmHg) (6) within 27 min. The average oxygen uptake rate was 0.52 nmol/s/106cells, which agrees with published results (5, 6). Hepatocytes seeded on oxygen carrier-collagen substrates reached the oxygen-limiting region of Km after 38 min under the same conditions. By comparing the area under the curve, we calculate an 87% increase in available oxygen, giving a final concentration of 2.2 mol of oxygen. Theoretically, the source of the oxygen increase is 0.2 ml of fluorocarbon embedded in the gel, storing 11 mol/ml of oxygen at 400 mmHg. Therefore, the experimental result agrees with the theoretical increase of 2.2 mol of oxygen. Cellular viability in oxygen carrier-collagen cultures
Figure 2. Oxygen carrier and system characterization. A) Particle size distribution in oxygen carrier emulsion after 3, 18, and 75 days of storage at room temperature. Oxygen carrier emulsion was diluted in PBS prior to analysis. B) Oxygen uptake profile of hepatocytes 45 min after seeding on collagen mixed with oxygen carrier emulsion (oxygen carrier) or collagen mixed with egg yolk dispersion with no oxygen carrier (control). Cells cultured on oxygen carriercollagen have 87% more oxygen than those cultured on emulsion collagen control.
oxygen carrier emulsion is stable. The slight drift in particle size may be due to Oswald ripening (32) but was not found to be significant over the course of our studies (P⫽0.094, n⫽4). Oxygen uptake profile in oxygen carrier-collagen cultures Oxygen supply in tissue culture cannot meet the elevated demand of spreading hepatocytes during the first 24 h of culture (33). To increase available oxygen in culture, the oxygen carrier emulsion was mixed with the collagen substrate and equilibrated with 100% oxygen before coating the culture surfaces. Freshly isolated hepatocytes were seeded onto the oxygen carrier-collagen at a density of 105 cells/cm2 and allowed to adhere for 45 min, after which the cultures were transferred to a closed, well-mixed chamber where oxygen concentration was monitored. Figure 2B shows the oxygen uptake profile of spreading hepatocytes seeded on oxygen carrier-collagen compared with those cultured on dispersion-collagen controls. WithOXYGEN-CARRYING MATRIX ENHANCES LIVER FUNCTION
To assess the viability of hepatocytes cultured on oxygen carrier-collagen., release of the intracellular enzyme aspartate aminotransferase (AST) was measured in culture medium 24 h postseeding. Figure 3A shows the fraction of dead cells based on AST release in both serum-supplemented and serum-free cultures seeded on oxygen carrier-collagen or dispersion-collagen controls. The data show that cultures seeded on oxygen carrier-collagen experienced significantly less cell death (P⫽0.030, n⫽9) in both serum-free and (P⫽0.033, n⫽9) and serum-containing media. To further confirm the benefit of the oxygen carrier on cell viability, cultures were double-labeled using calcein (live stain) and ethidium homodimer (dead stain) 24 h postseeding. Consistent with AST release data, the fraction of live cells was higher in hepatocyte cultures seeded on oxygen carrier-collagen both in serum-free and serum-containing media (Fig. 3B). The morphology of hepatocytes 24 h after seeding on the collagen gels is shown in Fig. 3C. Viable hepatocytes appear to be well spread, creating typical plate-like structures that fluoresce green due to esterase activity. Cytochrome P450 (CytP450) activity in oxygen carrier-collagen cultures Cytochrome P450s are an important family of hepatic enzymes mediating xenobiotic metabolism in hepatocytes (31, 34, 35). Maintaining the activity of cytochrome P450 enzymes is especially important for drug toxicity studies. Cytochrome P450IA1 activity was measured by the EROD assay. Figure 4A shows that hepatocytes cultured on oxygen carrier-collagen had a significantly higher CytP450 activity than hepatocytes cultured on dispersion-collagen controls in both serumcontaining and serum-free culture media. Oxygenated hepatocytes in serum-free culture medium had a 140 ⫾ 10% (P⫽0.016, n⫽9) higher CytP450 function, and hepatocytes in serum-containing medium showed an 820 ⫾ 20% (P⫽0.011, n⫽9) higher CytP450 function. Similar enhancement of cytochrome P450 enzymes in the presence of oxygen was found using the penE1831
Figure 3. Hepatocyte morphology and viability 24 h after seeding on oxygen carrier-collagen. Cells are seeded at 105 cells/cm2 either on oxygen carrier-collagen or collagen control gels. A) AST release by hepatocytes. Values are presented as percent of total AST in the cells. B) Number of adhering calcein-stained live cells, data normalized to the total number of adherent cells. C) Live-dead staining of hepatocytes in serum-free medium cultured on collagen gel control. D) Live-dead staining of hepatocytes in serum-free medium cultured on oxygen carrier-collagen. E) Phase contrast image of the same field of view as panel D. Bar ⫽ 50 m.
toxyresorufin O-deethylase and benzyloxyresorufin O-deethylase assays as well (data not shown). Short-term albumin and urea secretion in oxygen carrier-collagen cultures Albumin and urea secretion are liver-specific functions used to evaluate hepatocyte differentiation (29). Secretion rates of albumin and urea measured 24 h postseeding are shown in Fig. 4B and Fig. 4C, respectively. The data show that hepatocytes cultured on oxygen carrier-collagen secrete significantly more albumin than the dispersion-collagen controls (P⫽0.004, n⫽12). Albumin secretion was 121 ⫾ 7% and 93 ⫾ 8% higher in serum-free and serum-containing medium, respectively. Hepatic urea secretion on oxygen carriercollagen was also significantly higher than the dispersion-collagen controls (P⫽0.033, n⫽12). Urea secretion was 20 ⫾ 5% and 38 ⫾ 6% higher in serum-free and serum-containing medium, respectively. There was no statistically significant difference in albumin or urea secretion between serum-free and serum-containing cultures. E1832
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Long-term albumin and urea secretion in oxygen carrier-collagen cultures To assess long-term hepatocyte survival and function in our system, cells were seeded on a layer of oxygen carrier-collagen, and 1 day later overlaid with a second layer of oxygen carrier-collagen. To further increase the oxygen-carrying capacity of the medium, 50 l of oxygen carrier was added to the culture medium daily after that. Long-term function of hepatocytes cultured in a “sandwich” of oxygen carrier-collagen layers is shown in Fig. 5. Long-term albumin secretion was increased by 350 ⫾ 54% in serum-free cultures and by 166 ⫾ 33% in serum-containing culture (Fig. 5A). Long-term urea secretion was increased by 79 ⫾ 25% in serum-free cultures and by 76 ⫾ 10% in serum-containing cultures (Fig. 5B). Gene expression during long-term culture To detect whether the increase observed in hepatic function was due to an altered gene expression, we analyzed day 11 cultures for albumin and CytP450 IA1 mRNA levels. We observed no difference in the expres-
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Figure 4. Short-term function of hepatocytes 24 h after seeding on oxygen carrier-collagen compared with collagen gel controls. A) Cytochrome P450 activity measured by the EROD assay. B) Albumin secreted during the first 24 h of culture as measured by ELISA. C) Urea produced during the first 24 h of culture. D) Schematic of the oxygen carrier-collagen tissue culture configuration.
sion of albumin or CytP450 in hepatocytes cultured in a sandwich of oxygen carrier-collagen compared with dispersion-collagen controls (Fig. 6).
DISCUSSION An extensive body of literature has been devoted to the study of ECM, culture medium, and cell-cell interactions. However, relatively little attention has been paid to the role of oxygen in tissue culture despite its known effects on cellular differentiation and function. There is a striking difference between the oxygen supply in the liver and the in vitro oxygen supply in tissue culture. The liver is supplied by both arterial and venous blood, which release ⬃ 2000 nmol/ml of oxygen by passing through the sinusoid. Thus, the in vivo oxygen supply can be estimated by dividing the oxygen release in the hepatic sinusoid by the number of hepatocytes per sinusoid length, OXYGEN-CARRYING MATRIX ENHANCES LIVER FUNCTION
OxSupply ⫽
DS 䡠 VS 䡠 CO2 䡠 AH 4 䡠 LS
where DS is the sinusoid diameter (11.4 m) (36, 37), LS is sinusoid length (275 m) (36, 37), VS is the average flow velocity (144 m/s) (38), AH is the average hepatocyte area (400 m2) (39), and CO2 is 2000 nmol/ml. Therefore, we calculate the in vivo oxygen supply to be 1.2 nmol/s/106cells. Fittingly, hepatocyte oxygen consumption has been measured in vitro to be 0.9 nmol/s/106cells during the first 48 h after seeding and 0.4 nmol/s/106cells during stable long-term cultures (5, 6). In both cases the in vivo oxygen supply is more than adequate to supply the measured cellular demand. Conversely, in standard culture hepatocytes are limited by diffusion of atmospheric oxygen. Under these conditions oxygen supply can be calculated from the oxygen partial pressure on the cell surface given by (7): E1833
surface of the cells, D is the diffusivity of oxygen in medium (2⫻10⫺5 cm2/s), ⌬x is the thickness of the air-liquid interface (0.1 cm), Vm and Km are the Michaelis-Menten constants (0.4 nmol/s/106cells and 2.6 mmHg, respectively), Nc is the cell seeding density (105cells/cm2), and k is the oxygen solubility in medium (1.19 nmol/ml/mmHg). Under these conditions oxygen partial pressure on the cell surface is expected to reach 12 mmHg, resulting in an oxygen uptake/ supply rate of 0.35 nmol/s/106cells, well below the initial oxygen demand of hepatocytes (8). Attempts to increase the ambient oxygen partial pressure met with mixed results. Since oxygen transport rates needed to be increased by 3-fold, partial oxygen pressures of ⬎500 mmHg were required. At these supraphysiological oxygen concentrations, cellular viability was reduced, likely as a result of the generation of intermediate oxygen species and free radicals (9, 10). Another possibility is that cytokines found in serum, such as TGF1 or TNF␣, might have stimulated the apoptotic pathway in cells challenged by the oxidative stress (40 – 42). An alternative approach is the addition of an oxygen carrier that will increase the oxygen-carrying capacity of the medium. Previous work demonstrated that fluorocarbon emulsions can be excellent oxygen carriers for organ perfusion and cell culture (11, 17). However, technical problems, namely lack of solubility in aqueous solutions and high density, made it difficult to use fluorocarbons as oxygen carriers for these applications (17). Here we present a simple formulation of an oxygen carrier emulsion that is stable for several weeks at room temperature. The slow increase in average particle size suggests that the oxygen carrier emulsion can be stabilized further, perhaps with the addition of perfluorodecyl bromide, a component of Oxygent that was omitted from our formulation (15, 32). Embedding the oxygen carrier in collagen gels provides a means to increase oxygen supply to cells in culture. Under standard conditions, oxygen concentration in tissue culture is 0.22 mol/ml. The addition of oxygen carriercollagen increases available oxygen to 1.3 mol/ml.
Figure 5. Long-term function of hepatocytes seeded in oxygen carrier-collagen matrices compared with collagen gel controls. A) Albumin secretion measured daily. B) Urea production measured daily. C) Schematic of oxygen release in oxygen carrier-collagen sandwich tissue culture configuration.
D
Pg ⫺ Pc VmPc Nc 䡠 ⫽ ⌬x Km ⫹ Pc k
where Pg is the ambient oxygen partial pressure (mmHg), Pc is the oxygen partial pressure on the top E1834
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Figure 6. Albumin and Cyt P450 IA1 mRNA levels in long-term hepatocyte cultures (day 11). Cells were cultured in a collagen sandwich containing either the oxygen carrier or the egg yolk dispersion. Freshly isolated hepatocytes were used as control.
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Hence, culture of cells on oxygen carrier-collagen matrices increases available oxygen by 6-fold during the critical stages of cell attachment and spreading. The use of oxygen carriers embedded in collagen substrate significantly increased hepatocyte viability, cytochrome P450 activity, albumin secretion, and urea production. However, cytochrome P450 and albumin gene expression remained unchanged through day 11, suggesting that the increase in hepatic function was not due to an altered phenotype. Taken together, these results further demonstrate that oxygen is a limiting factor for hepatocytes cultured under standard conditions (7). The notable high increase in cytochrome P450 activity, 140 to 820%, could be due to the participation of molecular oxygen in the enzymatic reaction: R-H ⫹ O2 ⫹ NADPH ⫹ H⫹ 3
ing cardiomyocytes have a high oxygen demand that normally is not met by tissue culture techniques (45). In addition, pancreatic islets increase their oxygen demand when they are stimulated to secrete insulin (46), thus the oxygen carrier-collagen could be used to “buffer” oxygen fluctuations in these cultures.
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R-OH ⫹ NADP⫹ ⫹ H2O
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Serum can also be shown to play a role in the cellular reaction to oxygen. Hepatocytes cultured in serumcontaining media show a much lower cytochrome P450 activity than those cultured in serum-free conditions. Serum also had a significant effect on the long-term secretion of albumin in our cultures. Cells cultured in oxygen carrier-collagen sandwich secreted 87% more albumin in serum-free conditions than in serum-containing media. One reason for the poor performance of serum-containing media could be the presence of fetal signals in the serum such as alpha-fetoprotein, which has been shown to inhibit the proliferation of HepG2 cells in culture (43). Alternatively, serum can induce hepatic stress by exposing cells to proapoptotic cytokines TGF1 or TNF␣ (40, 41). There are two distinct phases of oxygen delivery mediated by the oxygen carrier. The first is the release of a bolus of oxygen, stored in the ECM, which occurs during the critical stage of cell spreading when oxygen demand is maximal. The high level of liver-specific function on the first day of culture suggests that this bolus of oxygen may be especially useful for short-term applications such as drug toxicity screening. Freshly isolated hepatocytes seeded on oxygen carrier-collagen could be studied the very next day, eliminating the lengthy wait typically required for hepatic functional recovery (29). The second phase of oxygen delivery is a steady-state phase where oxygen diffusion is enhanced by the presence of oxygen carrier in the overlaying ECM and the culture medium (Fig. 5C). The high level of albumin and urea secretion during long-term culture (⬎day 3) suggests that the oxygen carrier-collagen provides a long-term increase in oxygen supply. This could be useful in bioartificial liver devices, where hepatocytes are normally embedded in collagen through which oxygen must diffuse to reach the cells (44). Embedding the hepatocytes in oxygenated collagen would significantly increase oxygen delivery in such a device, improving survival and function. Other cell types might also benefit from the use of the oxygen carrier-collagen matrix. For example, beat-
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The FASEB Journal
Received for publication March 29, 2006. Accepted for publication July 6, 2006.
NAHMIAS ET AL.
The FASEB Journal • FJ Express Summary
A novel formulation of oxygen-carrying matrix enhances liver specific function of cultured hepatocytes Yaakov Nahmias, Yiannos Kramvis, Laurent Barbe, Monica Casali, Francois Berthiaume, and Martin L. Yarmush1 Center for Engineering in Medicine/Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, Massachusetts, USA To read the full text of this article, go to http://www.fasebj.org/cgi/doi/ 10.1096/fj.06-6192fje
SPECIFIC AIMS Oxygen is an important component of the cellular microenvironment mediating cell survival, differentiation, and function. Oxygen supply is severely limited during culture of highly metabolic cells such as hepatocytes. Our aim was to enhance hepatic survival and function by increasing oxygen concentration in culture using a fluorocarbon-based oxygen carrier embedded in the extracellular matrix (ECM). PRINCIPAL FINDINGS 1. Oxygen carrier stability Long-term tissue culture applications require stable materials that will not degrade over time. Fluorocarbon-based oxygen carrier was characterized using forward light scatter. Oxygen carrier particle diameter remained relatively unchanged after 75 days of storage at room temperature, suggesting the emulsion is stable. 2. Oxygen uptake profile in oxygen carrier-collagen cultures Oxygen supply in tissue culture cannot meet the demand of spreading hepatocytes during the first 24 h of culture. To increase available oxygen in culture, the oxygen carrier emulsion was mixed with collagen and equilibrated with 100% oxygen prior to coating the culture surfaces. Freshly isolated hepatocytes were seeded onto the oxygen carrier-collagen at a density of 105 cells/cm2 and allowed to adhere for 45 min, after which cultures were transferred to a closed, well-mixed chamber where oxygen concentration was monitored as a function of time. Without oxygen carrier, the oxygen concentration dropped below the published value of Km (2.6 mmHg) within 27 min. The average oxygen uptake rate was 0.52 nmol/s/106cells, in agreement with published results. Hepatocytes seeded on oxygen carrier-collagen substrates reached the oxygen limiting region of Km after 38 min under the same conditions. By comparing the area under the curve, we calculate an 87% increase in available oxygen.
aspartate aminotransferase (AST) was measured in culture medium 24 h postseeding. The data show that cultures seeded on oxygen carrier-collagen experienced significantly less cell death (P⫽0.030, n⫽9) in serum-free and (P⫽0.033, n⫽9) and serum-containing media, based on AST release. To further confirm the benefit of the oxygen carrier on cell viability, cultures were double-labeled using live-dead staining 24 h postseeding. Consistent with AST release data, the fraction of live cells was higher in hepatocyte cultures seeded on oxygen carrier-collagen in serum-free and serum-containing media (Fig. 1). Viable hepatocytes appear to be well spread, creating typical plate-like structures that fluoresce green due to esterase activity. 4. Cytochrome P450 (CytP450) activity in oxygen carrier-collagen cultures Cytochrome P450s are an important family of hepatic enzymes mediating xenobiotic metabolism in hepatocytes. Cytochrome P450IA1 activity was measured by the EROD assay. Figure 2 shows that hepatocytes cultured on oxygen carrier-collagen had a significantly higher CytP450 activity than hepatocytes cultured on dispersion-collagen controls in serum-containing and serum-free culture media. Oxygenated hepatocytes in serum-free culture medium had a 140 ⫾ 10% (P⫽0.016, n⫽9) higher CytP450 function whereas hepatocytes in serum-containing medium showed an 820 ⫾ 20% (P⫽0.011, n⫽9) higher CytP450 function. Similar enhancement of cytochrome P450 enzymes in the presence of oxygen were found using PROD and BROD assays. 5. Short-term albumin and urea secretion in oxygen carrier-collagen cultures Albumin and urea secretion are liver-specific functions used to evaluate hepatocyte differentiation. Secretion rates of albumin and urea were measured 24 h postseeding. The data show that hepatocytes cultured on oxygen carrier-collagen secrete significantly more albumin than dispersion-collagen controls (P⫽0.004, n⫽12). 1
3. Cellular viability in oxygen carrier-collagen cultures To assess the viability of hepatocytes cultured on oxygen carrier-collagen, release of the intracellular enzyme 0892-6638/06/0020-2531 © FASEB
Correspondence: Center for Engineering in Medicine/ Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, MA 02114, USA. E-mail:
[email protected] doi: 10.1096/fj.06-6192fje 2531
Figure 1. Hepatocyte morphology and viability 24 h after seeding on oxygen carrier-collagen. Cells are seeded at 105 cells/cm2 either on oxygen carrier-collagen or collagen control gels. The number of adhering calcein-stained live cells is shown. Data are normalized to the total number of adherent cells.
Albumin secretion was 121 ⫾ 7% and 93 ⫾ 8% higher in serum-free and serum-containing medium, respectively. Hepatic urea secretion on oxygen carrier-collagen was also significantly higher than the dispersion-collagen controls (P⫽0.033, n⫽12). Urea secretion was 20 ⫾ 5% and 38 ⫾ 6% higher in serum-free and serum-containing medium, respectively. There was no statistically significant difference in albumin or urea secretion values between serum-free and serum-containing cultures. 6. Long-term albumin and urea secretion in oxygen carrier-collagen cultures To assess long-term hepatocyte survival and function in our system, cells were seeded on a layer of oxygen carrier-collagen and 1 day later overlaid with a second layer of oxygen carrier-collagen; 50 l of oxygen carrier was added to the culture medium daily after that. Long-term function of hepatocytes cultured in a “sandwich” of oxygen carrier-collagen layers is shown in Fig. 3. Long-term albumin secretion was increased by 350 ⫾ 54% in serum-free cultures and by 166 ⫾ 33% in serum-containing culture. Long-term urea secretion was increased by 79 ⫾ 25% in serum-free cultures and by 76 ⫾ 10% in serum-containing cultures. 7. Gene expression during long-term culture To detect whether the increase observed in hepatic function was due to altered gene expression, we ana-
Figure 3. Long-term function of hepatocytes seeded in oxygen carrier-collagen matrices compared with collagen gel controls. A) Albumin secretion measured daily. B) Schematic of oxygen release in an oxygen carrier-collagen sandwich tissue culture configuration.
lyzed day 11 cultures for albumin and CytP450 IA1 mRNA levels. We noted no difference in the expression of albumin or CytP450 in hepatocytes cultured in a sandwich of oxygen carrier-collagen than with dispersion-collagen controls. CONCLUSIONS AND SIGNIFICANCE An extensive body of literature has been devoted to the study of ECM, culture medium, and cell-cell interactions, but little attention has been paid to the role of oxygen in tissue culture despite its known effects on cellular differentiation and function. There is a striking difference between the oxygen supply in the liver and the in vitro oxygen supply in tissue culture. The liver is supplied by arterial and venous blood, which release ⬃ 2000 nmol/ml of oxygen by passing through the sinusoid. Thus, the in vivo oxygen supply can be estimated by dividing the oxygen release in the hepatic sinusoid by the number of hepatocytes per sinusoid length,
Figure 2. Short-term function of hepatocytes 24 h after seeding on oxygen carrier-collagen compared with collagen gel controls: cytochrome P450 activity measured by the EROD assay. 2532
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OxSupply ⫽
DS 䡠 VS 䡠 CO2 䡠 AH 4 䡠 LS NAHMIAS ET Al.
where DS is the sinusoid diameter (11.4 m), AH is the average hepatocyte area (400 m2), and CO2 is 2000 nmol/ml. Therefore, we calculate the in vivo oxygen supply to be 1.2 nmol/s/106cells. Fittingly, hepatocyte oxygen consumption has been measured in vitro to be 0.9 nmol/s/106cells during the first 48 h after seeding and 0.4 nmol/s/106cells during stable long-term cultures. In both cases the in vivo oxygen supply is more than adequate to supply the measured cellular demand. Conversely, standard culture hepatocytes are limited by diffusion of atmospheric oxygen. Under these conditions oxygen supply can be calculated from the oxygen partial pressure on the cell surface given by: Pg ⫺ Pc VmPc Nc 䡠 D ⫽ ⌬x Km ⫹ Pc k where Pg is the ambient oxygen partial pressure (mmHg), Pc is the oxygen partial pressure on the top surface of the cells, D is the diffusivity of oxygen in medium (2⫻10⫺5 cm2/s), ⌬x is the thickness of the air-liquid interface (0.1 cm), Vm and Km are the Michaelis-Menten constants (0.4 nmol/s/106cells and 2.6 mmHg, respectively), Nc is the cell seeding density (105cells/cm2), and k is the oxygen solubility in medium (1.19 nmol/ml/mmHg). Under these conditions, oxygen partial pressure on the cell surface is expected to reach 12 mmHg, resulting in an oxygen uptake/supply rate of 0.35 nmol/s/106cells, well below the initial oxygen demand of hepatocytes. Attempts to increase the ambient oxygen partial pressure met with mixed results. Since oxygen transport rates needed to be increased by 3-fold, partial oxygen pressures of ⬎500 mmHg were required. At these supraphysiological oxygen concentrations, cellular viability was reduced likely as a result of the generation of intermediate oxygen species and free radicals. Another possibility is that cytokines found in serum, such as TGF1 or TNF␣, might have stimulated the apoptotic pathway in cells already challenged by oxidative stress. An alternative approach is to increase the oxygencarrying capacity of the medium by adding an oxygen carrier. Earlier work demonstrated that fluorocarbon emulsions can be excellent oxygen carriers for organ perfusion and cell culture, but technical problems made it difficult to use fluorocarbons as oxygen carriers for these applications. Here we present a simple formulation of an oxygen carrier emulsion that is stable for several weeks at room temperature. Embedding the oxygen carrier in collagen gels provides a means to increase oxygen supply to cells in culture. Under standard conditions, oxygen concentration in tissue culture is 0.22 mol/ml. The addition of oxygen carriercollagen increases available oxygen to 1.3 mol/ml. Hence, culture of cells on oxygen carrier-collagen matrices increases available oxygen by 6-fold during the critical stages of cell attachment and spreading. The use of oxygen carriers embedded in collagen
OXYGEN-CARRYING MATRIX ENHANCES LIVER FUNCTION
substrate significantly increased hepatocyte viability, CytP450 activity, albumin secretion, and urea production. However, cytochrome P450 and albumin gene expression remained unchanged through day 11, suggesting the increase in hepatic function was not due to an altered phenotype. Taken together, these results further demonstrate that oxygen is a limiting factor for hepatocytes cultured under standard conditions. The notable high increase in CytP450 activity could be due to the participation of molecular oxygen in the enzymatic reaction: R-H ⫹ O2 ⫹ NADPH ⫹ H⫹ 3 R-OH ⫹ NADP⫹ ⫹ H2O Serum can also be shown to play a role in the cellular reaction to oxygen. Hepatocytes cultured in serumcontaining media show a much lower CytP450 activity than those cultured under serum-free conditions. Serum also had a significant effect on long-term secretion of albumin in our cultures. Cells cultured in an oxygen carrier-collagen sandwich secreted 87% more albumin in serum-free conditions than in serum-containing media. One reason for the poor performance of serumcontaining media could be the presence of fetal signals in the serum such as ␣-fetoprotein, shown to inhibit proliferation of HepG2 cells in culture. Alternatively, serum can induce hepatic stress by exposing cells to proapoptotic cytokines TGF1 or TNF␣. There are two distinct phases of oxygen delivery mediated by the oxygen carrier. The first is the release of a bolus of oxygen, stored in the ECM, which occurs during the critical stage of cell spreading when oxygen demand is maximal. The high level of liver-specific function on the first day of culture suggests that this bolus of oxygen may be especially useful for short-term applications such as drug toxicity screening. Freshly isolated hepatocytes seeded on oxygen carrier-collagen could be studied the very next day, eliminating the lengthy wait typically required for hepatic functional recovery. The second phase is a steady-state phase where oxygen diffusion is enhanced by the presence of oxygen carrier in the overlaying ECM and the culture medium (Fig. 3C). The high level of albumin and urea secretion during long-term culture (⬎day 3) suggests that the oxygen carrier collagen provides a long-term increase in oxygen supply. This could be useful in bioartificial liver devices. Embedding the hepatocytes in oxygenated collagen would significantly increase oxygen delivery, improving survival and function. Other cell types might also benefit from use of the oxygen carrier-collagen matrix. For example, beating cardiomyocytes have a high oxygen demand that normally is not met by tissue culture techniques. In addition, pancreatic islets increase their oxygen demand when they are stimulated to secrete insulin; thus, the oxygen carrier-collagen could be used to “buffer” oxygen fluctuations in these cultures.
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