Liver defatting: an alternative approach to enable steatotic liver transplantation.

Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.

Hepatocyte viability and adenosine triphosphate content decrease linearly over time during conventional cold storage of rat liver grafts.

INTRODUCTION: The gold standard in organ preservation is static cold storage (SCS) using University of Wisconsin solution (UW). Although it is well-known that there is a finite limit to SCS preservation, and that there is a correlation between the adenosine triphosphate (ATP) levels and organ function post-preservation, a quantitative relationship has not been established, which is important in understanding the fundamental limitations to preservation, minimizing cold ischemic injury, and hence maximizing use of the donor organ pool. AIM: This study determines the time limits of cellular viability and metabolic function during SCS, and characterizes the relationship between cellular viability and energetic state using clinically relevant techniques in organ preservation. METHODS: Rat livers were procured and stored using conventional storage in UW solution at 4 °C. Viability was assessed by determining the amount of viable hepatocytes and intracellular ATP content after 0, 24, 48, 72, and 120 hours of storage. RESULTS: Numbers of viable hepatocytes that were isolated from these livers decreased steadily during SCS. After 5 days, viable hepatocytes decreased from 25.95 × 10(6) to 0.87 × 10(6) cells/gram tissue. Intracellular ATP content decreased from 9.63 to 0.93 moles/g tissue. Statistical analysis of variance established a linear relation for both parameters as a function of time (P < .05). CONCLUSION: The linear correlation between hepatocyte viability, ATP content, and storage time suggests a shared physiological foundation. These findings confirm ATP as direct predictor for organ quality in the context of liver preservation, which will aid quantitative assessment of donor organs for various applications.

Diluted blood reperfusion as a model for transplantation of ischemic rat livers: alanine aminotransferase is a direct indicator of viability.

Donors after cardiac death present a significant pool of untapped organs for transplantation, and use of machine perfusion strategies has been an active focus area in experimental transplantation. However, despite 2 decades of research, a gold standard has yet to emerge for machine perfusion systems and protocols. Whole blood reperfusion has been used as a surrogate for organ transplantation, especially as a model for the short-term response posttransplantation, and for optimization of perfusion systems. Although it is known that there is a strong correlation between liver function in whole-blood reperfusion and survival, the exact nature of these correlations, and to what extent they can be considered as an indicator of viability for transplantation/recipient survival, remain unclear. In this work, we demonstrate that diluted whole-blood reperfusion can be used as a direct model for transplantation of ischemic rat liver grafts. Specifically, we show that recipient survival can be predicted based simply on the value of alanine aminotransferase during perfusion, providing quantitative criteria of viability for use in this animal model. These results indicate that in the rat model graft survival is highly correlated with hepatocellular damage.

Selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation: an in vitro study.

This work describes the selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation. We observed: (1) single pulsed laser irradiation caused cellular damages on pigmented, and not on non-pigmented RPE cells at laser radiant exposure up to 2550 mJ/cm(2); (2) in the mixture of pigmented and non-pigmented RPE cells, single pulsed laser-induced damage was confined to pigmented RPE cells. This study demonstrates that the pigmented RPE cells can be selectively damaged, using a single pulsed laser irradiation, without thermal coagulation to adjacent non-pigmented RPE cells.

Sequential cold storage and normothermic perfusion of the ischemic rat liver.

Extending transplant criteria to include livers obtained from donors after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and, therefore, is perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N = 10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N = 6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.

Isolation rearing impairs wound healing and is associated with increased locomotion and decreased immediate early gene expression in the medial prefrontal cortex of juvenile rats.

In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test [Levine JB, Youngs RM, et al. (2007) Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience 145(1):42-55]. In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After 4 weeks of either isolation or group rearing (a period that encompasses post-wearing and early adolescence), rats were killed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post-burn injury. We found that: 1) gross wound healing was significantly impaired in isolation-reared rats compared with group-reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation-reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group-reared rats than for uninjured group-reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation-reared rats during burn injury compared with uninjured isolation-reared rats. Thus, in the mPFC, behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.

Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex.

Environmental deprivation contributes in important ways to the development of a wide range of psychiatric disorders. Isolation rearing of rodents, a model for environmental deprivation in humans, consistently produces hyperlocomotion, which provides a measurable parameter to study the underlying mechanisms of early adverse psychosocial stressors. Male Sprague-Dawley rat pups were separated from dams at postnatal (PN) day 20 and reared either in groups of three or in isolation. On PN 38, locomotion was assessed in the open field. On PN 46, rats were killed and gene expression patterns examined in the medial prefrontal cortex (mPFC). Isolation-reared rats displayed increased locomotor activity and decreased resting time in the open field. Specific gene expression patterns in the mPFC were associated with both isolation rearing and hyperlocomotive behavior in the open field. Genes involved in these expression patterns included immediate early genes (IEGs) and genes that regulate cell differentiation and apoptosis. The study of these genes could provide important insights into how abnormal early psychosocial events affect brain function and behavior.

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Expression profiling analysis of the metabolic and inflammatory changes following burn injury in rats.

Burn injury initiates an inflammatory response as part of the healing process that is associated with extensive metabolic adjustments. While most studies have focused on understanding these changes from a biochemical perspective, not much work has been done to characterize these processes at the gene expression level. As a first step, we have comprehensively analyzed changes in gene expression in rat livers during the first 24 h after burn injury using Affymetrix GeneChips, which showed 339 genes to be differentially expressed at a statistical significance of P < 0.05 and changed at least twofold. Functional classification based on gene ontology terms indicated that two categories, metabolism (28%) and inflammation (14%), accounted for nearly 42%. Detailed analysis of the metabolism group of genes indicated that fatty acid (FA) and triglyceride (TG) biosynthesis in the liver were unchanged, whereas TG utilization, FA import, and beta-oxidation increased after burn injury. The increased FA pools after burn injury appear to serve as substrates for ATP production. Following burn injury, the cholesterol biosynthetic pathway was suppressed while cholesterol was increasingly imported and converted into bile acids. The inflammatory genes that were altered included several classic acute phase response markers, as well as genes involved in the complement, kinin, clotting, and fibrinolytic protein systems. These temporally coordinated changes in gene expression were also corroborated by biochemical measurements for FA, TG, cholesterol, and ATP. Together, these data indicate that FA are increasingly imported and oxidized in the liver to meet the enhanced energy demands arising from an inflammatory response during the first 24 h after burn injury.

Critical issues in bioartificial liver development.

End-stage liver disease accounts for over 30,000 deaths annually in the United States. Orthotopic liver transplantation is the only clinically proven treatment for patients with end-stage liver failure. A limitation of this therapy is a shortage of donor organs available. This donor organ shortage is exacerbated by the fact that the number of patients listed for transplantation has continued to increase. As a result, there has been a continuing increase in the number of patients who die waiting for a donor liver. Extracorporeal bioartificial liver devices consisting of viable hepatocytes have the potential to provide temporary support for patients with fulminant hepatic failure, thereby serving as a "bridge" to transplantation. In some patients, this temporary support would allow the native liver to regenerate function, eliminating the need for transplantation and the resulting life-long immunosuppressive therapy, all of which translates into a cost savings to the health care system. Although the bioartificial liver device is a promising technology for the treatment of liver failure, significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. An overview of the critical issues in the development of bioartificial liver devices is discussed.

Long-term maintenance of cytochrome P450 activities by rat hepatocyte/3T3 cell co-cultures in heparinized human plasma.

Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.

Poloxamer-188 improves capillary blood flow and tissue viability in a cutaneous burn wound.

After cutaneous burn injury, an area of tissue 1-2 mm thick surrounding the wound is the site of a pronounced inflammatory response where blood flow is reduced. This "zone of stasis" undergoes progressive necrosis within 24-48 h, resulting in an expansion of the burn wound. Poloxamer-188 (P-188) is a surfactant that has been shown to prevent cell death due to electrical injury in vivo and heat shock in vitro. In this study, we investigated the effect of P-188 on blood flow within and around a burn wound and on the expansion of the wound area within 24 h after administration of a full-thickness burn injury. Results show that immediately (0-2 h) after the burn, red blood cell speed decreased to zero in a zone extending up to 1 mm from the center of the burn in both P-188 (200 mg/kg)- and saline (0.9%)-treated animals. Between 1 and 3 mm from the center of the burn, red blood cell speed decreased to 50% of preburn levels in saline controls (n = 5), while no decrease occurred in P-188-treated animals (n = 5). Beyond 3 mm from the center of the burn, red blood speed was equal to the preburn levels in saline controls, while it increased by about 10% in P-188 animals. Twenty-four hours after administration of burn, the "zero red blood cell speed zone," termed as the zone of coagulation, became smaller in P-188-treated animals, with an area of 2.4 +/- 0.5 mm(2) (n = 5) compared to 3.5 +/- 0.5 mm(2) (n = 4) in saline controls (P < 0.01). These results suggest that P-188 prevented the formation of a zone of stasis within 2 h after the burn injury and reduced the area of coagulation observed 24 h after cutaneous burn injury.

Expression of liver-specific functions by rat hepatocytes seeded in treated poly(lactic-co-glycolic) acid biodegradable foams.

Techniques of liver replacement would benefit patients awaiting donor livers and may be a substitute for transplantation in patients whose livers can regenerate. Poly(lactic-co-glycolic acid) (PLGA) copolymers are biodegradable and have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, foam disks were prepared from PLGA (lactic-to-glycolic mole ratio of 85:15) by lyophilization of benzene (5% w/v) solutions. These disks were then used as scaffolds for rat hepatocyte culture. Foams were coated with either a type I collagen gel (0.1% w/v), coated with gelatin (5% w/v), or treated with oxygen plasma (25 W, 90 s) to modify their surface chemistry and wettability. The disks were then seeded with rat hepatocytes (10(6)/mL) and cultured for a period of 2 weeks. All surface treatments resulted in increased hydrophilicity, the greatest being obtained by collagen treatment (contact angle < 10 degrees ), and a minimal decrease in void fraction (5%). DNA content after a 2-week culture period increased proportionally with the wettability of the treated foam surface. Urea synthesis in untreated foams averaged 15.3 +/- 2.3 microg/h/microg DNA, which was significantly higher than that for controls, whereas gelatin and collagen treated foams exhibited urea synthetic rates below the control levels at all times. The DNA content decreased significantly by about 50% between days 1 and 12. PLGA foams, treated and untreated, represent a promising scaffold for scaling up hepatocyte cultures.

Intrahepatic amino acid and glucose metabolism in a D-galactosamine-induced rat liver failure model.

A better understanding of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and other nonsurgical medical therapies for FHF. We used an isolated perfused liver system in combination with a mass-balance model of hepatic intermediary metabolism to generate a comprehensive map of metabolic alterations in the liver in FHF. To induce FHF, rats were fasted for 36 hours, during which they received 2 D-galactosamine injections. The livers were then perfused for 60 minutes via the portal vein with amino acid-supplemented Eagle minimal essential medium containing 3% wt/vol bovine serum albumin and oxygenated with 95% O(2)/5% CO(2). Control rats were fasted for 36 hours with no other treatment before perfusion. FHF rat livers exhibited reduced amino acid uptake, a switch from gluconeogenesis to glycolysis, and a decrease in urea synthesis, but no change in ammonia consumption compared with normal fasted rat livers. Mass-balance analysis showed that hepatic glucose synthesis was inhibited as a result of a reduction in amino acid entry into the tricarboxylic acid cycle by anaplerosis. Furthermore, FHF inhibited intrahepatic aspartate synthesis, which resulted in a 50% reduction in urea cycle flux. Urea synthesis by conversion of exogenous arginine to ornithine was unchanged. Ammonia removal was quantitatively maintained by glutamine synthesis from glutamate and a decrease in the conversion of glutamate to alpha-ketoglutarate. Mass-balance analysis of hepatic metabolism will be useful in characterizing changes during FHF, and in elucidating the effects of nutritional supplements and other treatments on hepatic function.

Metabolic flux analysis of postburn hepatic hypermetabolism.

The hepatic response to severe injury is characterized by a marked upregulation of glucose, fatty acid, and amino acid turnover, which, if persistent, predisposes the patient to progressive organ dysfunction. To study the effect of injury on liver intermediary metabolism, metabolic flux analysis was applied to isolated perfused livers of burned and sham-burned rats. Intracellular fluxes were calculated using metabolite measurements and a stoichiometric balance model. Significant flux increases were found for multiple pathways, including mitochondrial electron transport, the TCA and urea cycles, gluconeogenesis, and pentose phosphate pathway (PPP). The burn-induced increase in gluconeogenesis did not significantly increase glucose output. Instead, glucose-6-phosphate was diverted into the PPP. These changes were paralleled by increases in glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities. Given that G6PDH and GR are the most significant NADPH producers and consumers in the liver, respectively, and that GR is responsible for recycling the free radical scavenger glutathione, these data are consistent with the notion that hepatic metabolic changes are in part due to the induction of liver antioxidant defenses.

Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma.

Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbecco's Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.

Optimization of rat hepatocyte culture in citrated human plasma.

BACKGROUND: Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma. MATERIALS AND METHODS: Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated. RESULTS: Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml). CONCLUSIONS: Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.

Dynamics of tissue neutrophil sequestration after cutaneous burns in rats.

BACKGROUND: Neutrophil recruitment in organs after burns may cause local vascular damage, which can be reduced by agents blocking neutrophil adhesion to the vascular wall. Because these agents may increase susceptibility to infection, it is important to characterize the dynamics of neutrophil sequestration in order to optimize an eventual anti-adhesion therapy. MATERIALS AND METHODS: Rats were scald burned over 20 or 40% of their total body surface area (TBSA) and saline resuscitated. Sham controls were used. Myeloperoxidase (MPO) activity was measured in lungs, liver, kidney, gut, and burned skin up to 1 week postburn. Extravascular accumulation of (125)I-labeled bovine serum albumin ((125)I-BSA) was measured at 12 h postburn. RESULTS: MPO activity in lungs, liver, and kidney was increased within 3 h postburn and returned to normal within 24-48 h. Peak MPO levels occurred at 6-12 h postburn and were similar for both burn sizes. No MPO increase was observed in gut. MPO levels in burned skin did not increase before 6 h, peaked at 24 h, decreased at 48 h, but remained elevated for up to 7 days. Neutrophil recruitment in lungs and liver was confirmed histochemically. No neutrophils were found in kidneys. Extravascular (125)I-BSA was increased in lungs, liver, kidneys, and gut, in the 40% TBSA group only. CONCLUSIONS: Neutrophil sequestration in remote organs is a transient phenomenon while neutrophil homing into the wound site is sustained. Neutrophil accumulation dynamics are independent of burn size, although a minimum size is required to trigger vascular damage. Temporary early anti-adhesion therapy to reduce lung and liver neutrophil sequestration with little impact on neutrophil homing into the burn wound may be possible.

Age- and disease-related decline in immune function: an opportunity for "thymus-boosting" therapies.

The thymus is the site of production of mature T lymphocytes and thus is indispensable for the development and maintenance of the T cell-mediated arm of the immune system. Thymic production of mature T cells is critically dependent on an influx of bone marrow-derived progenitor T cells that undergo replication and selection within the thymus. Thymus cellularity and thymic hormone secretion reach a peak during the first year of life and then decline gradually until the age of 50-60 years, a process known as "thymic involution." A rapid reduction of thymus cellularity occurs in young patients following injuries, chemotherapy, and other forms of stress. The mechanisms underlying the involution process appear to be dependent on factors intrinsic to the thymic tissue, such as the local production of cytokines and chemoattractants, promoting the recruitment, growth, and differentiation of bone marrow-derived T cell progenitors in the thymus, as well as extrinsic factors, such as systemic levels of endocrine hormones and mediators released by intrathymic nerves of the autonomic nervous system. Knowledge of these factors provides a rational basis for the development of an approach based on tissue engineering that could be used to provide either temporary or permanent reconstitution of thymic function.

Metabolic flux analysis: a powerful tool for monitoring tissue function.

In recent years, metabolic flux analysis has been widely used in bioprocess engineering to monitor cell viability and improve strain activity. Metabolic flux analysis refers to a methodology for investigating cellular metabolism whereby intracellular fluxes are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of reaction networks. By considering the stoichiometry of biochemical reactions, it is possible to estimate the degree of engagement of each pathway participating in overall cellular activity, and hence obtain a comprehensive view of a cell s metabolic state. Given the potential impact of cellular energy metabolism on the function of engineered tissues, such comprehensive analysis of metabolic activity can be an extremely useful tool for tissue engineers. Estimates of intracellular fluxes under various environmental conditions could be used to optimize function in vivo as well as culture conditions in vitro. In this review, we provide a brief theoretical background of metabolic flux analysis and summarize the most widely used experimental approaches to obtain flux data. This review is intended as an overview of the field and as a starting point for tissue engineers wishing to learn about and eventually employ this methodology.