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Artificial CeUs. Blood Substitutes, and Biotechnolosy, 33: 297 306, 2005 Copyrighl Taylor & Francis, Inc.

. ISSN: 1073-1199 print/lS32-ll84 online DOI: 10.108I/BIO-200066626

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Impact of Increased Oxygen Delivery Via Bovine Red Blood Cell Supplementation of Culturing Media on Select Metabolic and Synthetic Functions of C3A Hepatocytes Maintained within a Hollow Fiber Bioreactor
Jason Gordon and Andre F. Palmer Department of Chemical and Blomolecular Engineering, University of Notre Dame, Notre Dame. IN. USA

Abstract: Hepatocytes are highly dependent upon appropriate oxygen provision for activity and viability. However, oxygen delivery to hepatocytes cultured within a hollow fiber bioreactor is believed to be problematic because of large diffusion distances, a high hcpatocyte oxygen consumption rate and low aqueous media oxygen solubility. Supplementation of bioreactor media with bovine red blood cells (bRBCs) is one means of improving oxygen delivery to hepatocytes as hemoglobin contained within bRBCs binds oxygen. The impact of supplementing hepatocyte culturing media with bRBCs (-5 x lO^bRBCs/ml) on hepatocyte activity (albumin and lactate production and glucose consumption) was studied. Decreased hepatocyte lactate production to glucose consumption ratios were found for the case when bRBCs were added to circulating culturing media, which indicated the presence of a more aerobic environment in comparison to the control (no bRBC supplementation). Additionally, albumin synthesis was found to be improved when the circulating media was supplemented with bRBCs. Our results thus support the use of bRBCs to improve oxygen delivery to hepatocytes maintained within a hollow fiber bioreactor.

INTRODUCTION Acute liver failure (ALF) remains a clinically frustrating medical condition that carries a high mortality rate [1,2]. Currently, the only effective
We would like to thank Jenny Craig and Matthew Dare from the University of Notre Dame for their assistance in performing much of the assay work. Address correspondence to Andre F. Palmer, 171 Fitzpatrick Engineering Hall, Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA. E-mail: apalmer@nd.edu 297

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treatment for ALF is orthotopic liver transplantation (OLF), which requires available organs, sufficient patient stability, and is relatively expensive. OLT is most effective when carried out during the early stages of ALF; however, the patient's liver retains some ability to self-regenerate after ALF in many cases [3,4]. Allowing a patient's liver to regenerate on its own alleviates concerns of organ rejection, and reduces the demand on an already limited supply of organs available for transplantation. Consequently, there is clearly a need for a liver assist device that can be utilized to sustain the patient while the native liver regenerates or until the point of OLT. Currently, bioartificial liver assist devices (BLADs) are receiving significant attention, as it is believed that these devices are most capable of providing a full range of required liver functionsdetoxification, metabolic, and synthetic functions. The BLAD, which typically employs a hollow fiber (HF) bioreactor, makes use of an artificial chamber housing hepatoeytes to provide global liver support. It is generally believed that such a device must support at minimum 20-40% of the in vivo hepatocyte cell mass, which translates to several hundred grams of cells [5,6], The two types of cells that could be most realistically utilized within a BLAD are primary cells and cell lines. In the study described here, the C3A cell line was chosen. It is our belief that the use of a cell line is advantageous due to its ability to proliferate, and lack of harvesting requirements. Additionally, the C3A cell line has been fairly well characterized, produces high levels of albumin, and displays a strong nitrogen metabolizing activity [14,15]. One significant problem still facing the development of the BLAD involves oxygen delivery to the cells [7-10]. Hepatoeyte activity {synthetic and metabolic) and viability is highly dependent on the provision of appropriate oxygen levels, and thus maintaining suitable oxygen deUvery to these cells within a bioreactor is of importance. Within the liver, hepatic sinusoids bring blood into close contact with hepatocytes, since generally no more than one to two hepatoeytes separate each sinusoid [11]. This minimizes the required oxygen diffusion distances for oxygen delivery to hepatocytes within the liver. However, the HF bioreactor is incapable of providing similar fiber surface areas for cell attachment, which results in increased oxygen diffusion distances. When run in vitro, this is problematic because oxygen is sparingly soluble in aqueous culturing media. This in combination with a packed hepatoeyte cell spaee that maintains a high oxygen consumption rate can result in the formation of hypoxic and anoxic regions within the cell space. One potential means of delivering inereased concentrations of oxygen to hepatocytes is by supplementation of the circulating aqueous culturing media with bovine red blood cells (bRBCs). Tetrameric hemoglobin (Hb) contained within bRBCs serves to cooperatively bind oxygen, which greatly increases the amount of oxygen that can be delivered to the hepatocytes via the media. In this study, bRBC

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supplementation of the circulating culturing media within a C3A hepatoeyte-con tain ing hollow fiber bioreactor is described, along with the consequences on select metabolic and synthetic functions of the hepatocytes. MATERIALS AND METHODS HF Bioreactor The HF bioreactor system employed in this study was the commercially available CellMax'-' system (Spectrum Labs, Rancho Dominguez, CA). Essentially, culturing medium was pumped from a reservoir into the HF cartridge via silicone tubing that permitted gas transfer between the incubator's ambient atmosphere and the circulating media. The media then passed through the HF cartridge fibers, delivering nutrients and oxygen and carrying away metabolic byproducts. A schematic of the HF system is displayed in Figure 1. The hollow fiber cartridge used in these studies was composed of a cellulosic membrane containing pores with a 95% 50 kDa molecular weight cutoff. The cartridge was maintained at 37''C and 5% CO2 within a Heraueus (Kendro Laboratory Products, Hanau, Germany) incubator throughout the duration of the study.
Cell Inoculation and Maintenance

C3A hepatocytes (ATCC, Manassas, VA, Cat. #CRL-10741) were thawed in a 37C water bath, counted using a hemaeytometer (--7.4x10'' eells), and stained via trypan blue for viability (-^93%
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viability). The cells were then inoculated into 2 separate HF cartridge systems via the extracapillary space (ECS) ports H 3 . 7 x 10^ cells per cartridge). Culturing media was circulated at '-33mL/min through --2 feet of silicone tubing; and was composed of 90% Eagle's Minimum Essential Medium (MEM) (ATCC, Cat. #30-2003), 10% fetal bovine serum (FBS) (ATCC, Cat. #30-2020), and 0.2% Normocin (an antibiotic/antimycotic agent) (Invivogen, San Diego, CA, Cat. #ant-nr-o) in both cases. In one of the two HF systems, the medium was supplemented with sterile, washed bRBCs (Quad Five, Ryegate, MT, Cat. #943) at 10% of the human in vivo concentration (--5 x 10^ cells per mL). The bRBC supplemented system is referred to as the experimental culture, while the other system is referred to as the control culture. A third system was employed to provide a gauge as to the extent of metabolic functions occurring within bRBCs. This system utilized the same medium formulation as the experimental culture (including bRBCs). but lacked a HF bioreactor containing hepatocytes. Therefore, it was simply medium circulating through silieone tubing. Throughout the study, the reservoir (125-250mL) was replaced with fresh medium every 24-48 hours (fresh bRBCs were replaced at the same time within the experimental culture), and at the same time an aliquot was taken from both the spent medium and the fresh medium being taken/placed into the reservoir. The ECS space was flushed with complete medium every 2-4 days (lacking bRBCs in both cases) to allow for determination of albumin production. Metabolic Assays All samples collected were stored at -20C until analyzed. Commercially available colorimetric kits were utilized to determine glucose (Diagnostic Chemicals Limited, Oxford, CT, Cat. #220-32) and lactate (Biomedical Research Service Center, SUNY at Buffalo, Buffalo, NY, Cat. #A-108) concentrations within each of the samples. Initial and fmal sample aliquots were each analyzed in triplicate for glucose concentration, and in duplicate for lactate concentration. Initial and fmal aliquots for determination of albumin synthesis were taken from the ECS space and each was analyzed in duplicate. Albumin concentration was determined using an enzyme linked immunosorbent sandwich assay kit (Bethyl Laboratories, Montgomery, TX, Cat. #E80-129). Finally, all sample analysis was conducted using a plate reader (Bio-tek, Winooski, VT). RESULTS AND DISCUSSIONS Metabolic Assays Glucose and lactic acid concentrations in the circulating media were monitored throughout the study (Figure 2). As can be seen, the bRBCs

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by themselves consumed little glucose, and produced little lactic aeid throughout the study. The average bRBC glucose consumption rate throughout the study was found to be 0.1 0.3 mg/hr, while the average lactie acid production rate was 0.2 0.1 mg/hr. In both cases, this value was subtracted from all of the experimental culture data to correct

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for approximate bRBC metabolic activity. After hepatocyte inoculation, relatively little glucose was consumed in either the control or experimental case until day 9 of the study, at which point the glucose consumption rate began to increase rapidly (to a maximum of '-^3 mg/hr by day 16). Throughout the study, no significant difference was detected in the glucose consumption rate between either culture. In a similar manner, little lactic acid was produced within either culture until approximately the 12th day of the study. However, it was consistently found from the 6th day on (with the exception of days 12-13) that the experimental culture produced significantly less lactic acid. Most likely, the lack of glucose consumption and lactic acid production at the beginning of the study reflects a lag (or adjustment) phase of the cultured hepatocytes as they became acclimated to their new environment. However, a 9-day lag phase seems like an abnormally lengthy lag period. This might be a result of the relatively small number of cells inoculated into the HF cartridge (seeding density of ^2.5 x 10^ cells/mL). It is conceivable that this low seeding density might have impaired cell-to-cell communication during the early stages of culture, and consequently slowed down proliferation and function during this period. The ratio of lactic acid production to glucose consumption (Figure 3) provides a gauge as to the oxygen level provided to the hepatocytes within the culture. This can be understood by considering the metabolic cycles available to hepatocytes. Glycolysis first results in the conversion of 1 mole of glucose into 2 moles of pyruvate. Under anaerobic

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conditions., this pyruvate will be converted into lactate, and thus the ratio of lactic acid produced to glucose consumed will be 2. Under aerobic conditions, pyruvate (from glycolysis) is used within the citric acid cycle and oxidative phosphorylation, and consequently the ratio of lactic acid produced to glucose consumed will be reduced. Aerobic metabolism is more efficient (38 moles of ATP produced per mole of glucose consumed as opposed to 2 moles produced under anaerobic metabolism) and thus desirable, but requires sufficiently aerobic conditions as 6 moles of oxygen are required to completely metabolize each mole of pyruvate formed in glycolysis. It is clear that this ratio is generally smaller for the experimental culture, thus supporting the conclusion that the experimental culture was exposed to improved oxygenation. A significant difference is seen for days 6-16, with the exception of days 12-13. The average ratio for the control data was found to be 1.4 0.2, while that for the experimental culture is 0.8 0.6. The variability seen in the experimental data is Hkely a reflection of the bRBCs settling out within the reservoir bottle with time. Attempts were made to prevent this by gentle swirling of the reservoir bottle during the morning and night, but bRBC settling remained a problem throughout the study. The increase in the lactic acid production to glucose ratio on days 12-13 is likely a reflection of this problem. The implementation of some sort of an in-line shaker should alleviate this problem, and it is expected that this will result in more consistent ratios for the experimental culture. The reduced ratios found for the experimental culture, however, supports the conclusion that this culture was subjected to a more aerobic environment than was the control culture.

Albumin Synthesis Conservation of albumin synthesis is considered to be important in designing a BLAD, as albumin, which is synthesized by hepatocytes, is important for delivering many toxins to hepatocytes and for regulating pH and osmotic pressure within the blood [12]. Additionally, in vitro, albumin synthesis can be utilized as an indicator of a culture's attainment of confiuency/maturation [13]. In vivo, fetal hepatocytes synthesize relatively low levels of albumin; however, upon reaching a mature state, albumin secretion increases dramatically [13]. Similarly, albumin synthesis by C3A hepatocytes significantly increases once the culture proliferates to the point of confiuency, and thus albumin synthesis is an indicator of culture maturation [13]. While this study was not maintained long enough for the cells to reach confiuency, hepatocyte albumin production during the study is still of importance and is displayed in Figure 4. Throughout the course of the study, more albumin was produced by the hepatocytes maintained within the experimental culture, and the difference between the two

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1-4 4-8 8-12 12-16 Measurement period during study (days) Figure 4. Albumin production over the course of the study. Values are an average of triplicate measurements from each sample, and standard deviations are indicated by error bars. A significant difference from the experimental culture is indicated by an asterisk over the respective time point {'P < 0.005).

cultures was found to be significant for days 8-16. Therefore, the results seem to indicate that improved oxygen delivery resulted in improved albumin production, which is in agreement with that previously reported within other studies [14]. This result would seem to make sense given the fact that albumin production is an energy dependent process. For instance, assuming that the energy for albumin production comes solely from complete glucose oxidation (requiring aerobic metabolism), then 75^moi of glucose and 450fimol of oxygen are required to synthesize 1 iimol of albumin in vivo [15]. It therefore seems reasonable that albumin production would be improved under increased oxygen delivery conditions.

CONCLUSIONS The results that we obtained within this study thus support the hypothesis that the addition of bRBCs to medium circulating within a hepatoctyecontaining HF bioreactor can increase the amount of oxygen delivered to the cells. This was indicated by the reduced lactate production to glucose consumption ratios (Figure 3) seen within the experimental culture. As discussed earlier, increased oxygen delivery allows for more efficient metabolism, which should consequently result in a better functioning BLAD as energy production is increased while specific substrate requirements is decreased. One indication of improved function witnessed within

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this study was the improvement in albumin synthesis by the experimental culture (Figure 4). The results of this study have encouraged us to design further experiments that examine the impact of oxygenation not only on hepatocyte metabolic and synthetic functions, but also on biotransformation and detoxification functions. Future studies will also utilize a shaking device to prevent bRBC settling within the media reservoir bottle. Additionally, we are currently working to implement inline dissolved oxygen probes within the HF system to allow for a measure of hepatocyte oxygen consumption, which could then be correlated with the amount of oxygen provided. It is our expectation that improved oxygen dehvery to hepatocytes will result in improved function within a HF bioreactor. REFERENCES 1. Shito. M., Titles, A.W., Tompkins, R.G., Yarmush, M.L., Toner, M. (2003). EfTicacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure. / Surg. Res. Ill: 53-62. 2. Riordan, S., Williams, R. (1997). Bioartificial liver support: Developments in hepatocyte culture and bioreactor design. Br. Med Bull. 53: 730-744. 3. Sussman, N.L., Gislason, G.T., Kelly, J.H. (1994). Extracorporeal liver support-Application to fulminant hepatic-failure. / Clin. Gastroenterol. 18: 320-324. 4. Tzanakakis, E.S., Hess, D.J., Sielaff, T.D.. Hu, W.S. (2000). Extracorporcal tissue engineered liver-assist devices. Annu. Rev. Biomed Eng. 02: 607 632. 5. Tsiaoussis, J., Newsomc, P.N., Nelson, L.J., Hayes, P.C, Plevds, J.N. (2001). Which Hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems. Liver TranspL 7: 2-iO. 6. Scotto, J., Opolon, P., Eteve, J. (1973). Liver Biopsy and prognosis in acute liver failure. Gui, 14: 927-933. 7. Hay, P.D., Veitch, A.R., Smith, M.D., Cousins, R.B., Gaylor, J.D.S. (2000). Oxygen transfer in a diffusion-limited hollow fiber bioartificial liver. Artif. Organs 24: 278-288. 8. Hay, P.D., Veitch, A.R., Gaylor, J.D.S. (2001). Oxygen transfer in a convection-enhanced hollow fiber bioartificial liver. Artif. Organs 25: 119-130. 9. Catapano, G. (1996). Mass transfer limitations to the performance of membrane bioartificial liver support devices. Ini. J. Artif. Organs 19: 18-35. 10. Catapano. G., DeBartolo, L., Lombardi, C.P., Drioli, E. (1996). The effect of oxygen transport resistances on the viability and functions of isolated rat hepatocytes. Int. J. Ariif Organs 19: 61-71. 11. Fausto, N., Campbell, J.S. (2003). The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech. Dev. 120: 117-130. 12. Fournier, R.L. (1999). Basic Transport Phenomena in Biomedical Engineering, Taylor & Francis, Philadelphia, p. 90. 13. Kelly, J.H. (1994). Permanent Human Hepatocyte Cell Line and its Use in a Liver Assist Device (LAD). 5,290,684, March I.

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14. McClelland, R.E., Coger, R.N. (2004). Effects of enhanced 0-2 transport on hepatocytes packed within a bioartificial liver device. Tissue Eng. 10: 253-266. 15. Seifter, S., Englard, S. (1994). Energy metabolism, in The Liver Biology and Pathobiology, 3rd Ed., l.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby, D. Schachter, D.A. Shafritz, Eds., Raven Press: New York, pp. 323-364.