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

Scott Nyberg, Joseph A. Hardin, Lisa E. Matos, Douglas J. Rivera, Sri P. Misra, Gregory James Gores

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Background. This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. Methods. Isolated rat hepatocytes were entrapped in collagen gel (1.0 - 10.0 x 106 cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (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 fragmentation studies) were determined for each condition. Results: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure 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 concentration of 60 μmol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. Conclusions. The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

Original languageEnglish (US)
Pages (from-to)447-455
Number of pages9
JournalSurgery
Volume127
Issue number4
StatePublished - 2000

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Artificial Liver
Glycine
Hepatocytes
Gels
Cell Count
Urea
Cell Death
Necrosis
Cytoprotection
Ethidium
Serum-Free Culture Media
DNA Fragmentation
Cultured Cells
Cell Survival
Electron Microscopy
Collagen
Cell Culture Techniques

ASJC Scopus subject areas

  • Surgery

Cite this

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver. / Nyberg, Scott; Hardin, Joseph A.; Matos, Lisa E.; Rivera, Douglas J.; Misra, Sri P.; Gores, Gregory James.

In: Surgery, Vol. 127, No. 4, 2000, p. 447-455.

Research output: Contribution to journalArticle

Nyberg, Scott ; Hardin, Joseph A. ; Matos, Lisa E. ; Rivera, Douglas J. ; Misra, Sri P. ; Gores, Gregory James. / Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver. In: Surgery. 2000 ; Vol. 127, No. 4. pp. 447-455.
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abstract = "Background. This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. Methods. Isolated rat hepatocytes were entrapped in collagen gel (1.0 - 10.0 x 106 cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (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 fragmentation studies) were determined for each condition. Results: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure 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 concentration of 60 μmol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. Conclusions. The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.",
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T1 - Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver

AU - Nyberg, Scott

AU - Hardin, Joseph A.

AU - Matos, Lisa E.

AU - Rivera, Douglas J.

AU - Misra, Sri P.

AU - Gores, Gregory James

PY - 2000

Y1 - 2000

N2 - Background. This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. Methods. Isolated rat hepatocytes were entrapped in collagen gel (1.0 - 10.0 x 106 cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (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 fragmentation studies) were determined for each condition. Results: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure 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 concentration of 60 μmol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. Conclusions. The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

AB - Background. This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. Methods. Isolated rat hepatocytes were entrapped in collagen gel (1.0 - 10.0 x 106 cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (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 fragmentation studies) were determined for each condition. Results: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure 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 concentration of 60 μmol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. Conclusions. The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

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