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Center for Engineering in Medicine/Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, Massachusetts, USA
1Correspondence: Center for Engineering in Medicine/Department of Surgery, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston, MA 02114, USA. E-mail: ireis{at}sbi.org
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.
3. Cellular viability in oxygen carrier-collagen cultures
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. 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.
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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.
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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). 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 µl 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.
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7. Gene expression during long-term culture
To detect whether the increase observed in hepatic function was due to altered gene expression, we analyzed 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,
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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:
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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 TGFßbeta;1 or TNF
, might have stimulated the apoptotic pathway in cells already challenged by oxidative stress.
An alternative approach is to increase the oxygen-carrying 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 µmol/ml. The addition of oxygen carrier-collagen increases available oxygen to 1.3 µmol/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 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:
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-fetoprotein, shown to inhibit proliferation of HepG2 cells in culture. Alternatively, serum can induce hepatic stress by exposing cells to proapoptotic cytokines TGFßbeta;1 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.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6192fje
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