The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes.
The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 10(8) porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P < 0.01), and significantly smaller pyruvate (day 1 vs day 14 P < 0.03) and threonine consumption (day 1 vs day 10 P < 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P < 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P < 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.
Dabos
K J
KJ
Liver Cell Biology Laboratory, Department of Internal Medicine, University of Edinburgh, Edinburgh, UK. kostasophia@yahoo.com
Nelson
L J
LJ
Bradnock
T J
TJ
Parkinson
J A
JA
Sadler
I H
IH
Hayes
P C
PC
Plevris
J N
JN
D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002449:Cell Aggregation; D002455:Cell Division; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D005947:Glucose / Q000032:analysis / Q000378:metabolism; D019344:Lactic Acid / Q000032:analysis / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D019289:Pyruvic Acid / Q000032:analysis / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014893:Weightlessness
D000596:Amino Acids; D003470:Culture Media; D019344:Lactic Acid; D019289:Pyruvic Acid; D005947:Glucose