Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Cold storage of rat hepatocyte spheroids.

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 µM cyclosporin A (CsA); SFM + 1 mM Def + 1 µM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

Preliminary report of the Hepatic Encephalopathy Assessment Driving Simulator (HEADS) score.

INTRODUCTION: Audiovisual simulations of real-life driving (ie, driving simulators) have been used to assess neurologic dysfunction in a variety of medical applications. However, the use of simulated driving to assess neurologic impairment in the setting of liver disease (ie, hepatic encephalopathy) is limited. OBJECTIVES: The aim of this analysis was to develop a scoring system based on simulated driving performance to assess mild cognitive impairment in cirrhotic patients with hepatic encephalopathy. METHODS: This preliminary analysis was conducted as part of the Hepatic Encephalopathy Assessment Driving Simulator (HEADS) pilot study. Cirrhotic volunteers initially underwent a battery of neuropsychological tests to identify those cirrhotic patients with mild cognitive impairment. Performance during an audiovisually simulated course of on-road driving was then compared between mildly impaired cirrhotic patients and healthy volunteers. A scoring system was developed to quantify the likelihood of cognitive impairment on the basis of data from the simulated on-road driving. RESULTS: Mildly impaired cirrhotic patients performed below the level of healthy volunteers on the driving simulator. Univariate logistic regression and correlation models indicated that several driving simulator variables were significant predictors of cognitive impairment. Five variables (run time, total map performance, number of collisions, visual divided attention response, and average lane position) were incorporated into a quantitative model, the HEADS scoring system. The HEADS score (0-9 points) showed a strong correlation with cognitive impairment as measured by area under the receiver-operator curve (.89). CONCLUSION: The HEADS system appears to be a promising new tool for the assessment of mild hepatic encephalopathy.

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver.

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.