Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver

Background. This study was designed to determine if an anti-necrotic compou nd glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viabilit y and function of hepatocytes in a bioartificial liver. Methods. Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:medi a) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk . The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat h epatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragme ntation studies) were determined for each condition. Results The viability of GERH declined gradually and then stabilized 12 hou rs after hepatocyte isolation. The rate of urea Production by GERH was dire ctly proportional to the number of viable hepatocytes. Apoptotic death pred ominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after expos ure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concent ration of 60 mu mol/L. After anoxic exposure or under high cell density cul ture, glycine demonstrated a maximum benefit of inhibiting necrosis at a co ncentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk wer e additive. Conclusions. The metabolic activity of a hepatocyte bioartificial fiver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine .