The effect of hypothermia on primary porcine hepatocyte metabolism monitored by (1H) nuclear magnetic resonance spectroscopy

Objective
The aim of our study was to use (1H) nuclear magnetic resonance (NMR) spectroscopy as a tool to assess metabolic functions of hepatocytes and to monitor major metabolic pathways of these cells during culture following hypothermic preservation.

Methods
After isolation, 2 × 107 primary porcine hepatocytes were preserved at 4°C in supplemented Leibovitz L-15 medium for 48 h. Viability was assessed at isolation, 24 and 48 h. At isolation and at 48 h cells were plated and cultured with serum free supplemented Williams E medium. 1H NMR spectroscopy was used to assess indices of glucose metabolism, ammonia clearance indices and ketone bodies precursors at 48 h post-plating. Peak integration was applied with sodium 3-(trimethylsilyl-2,2,3,3-2H4)-1-propionate as an internal standard to obtain quantitative results.

Results
Results were obtained from six isolations. Viability was 78.1 ± 1.2% at isolation, 69 ± 3.4% at 24 h and 58.9 ± 3.8% at 48 h of hypothermia. Plating efficiency was 87 ± 4% for freshly isolated cells and 33.6 ± 7.6% for hypothermically preserved cells. Glucose consumption was comparable in both groups. Hypothermically preserved cells consumed more threonine, produced more pyruvate and alanine but less lactate. They also produced less acetate and consumed less tyrosine. Glutamate and glutamine concentrations were similar under both conditions.

Conclusion
1H NMR spectroscopy is a valid method for assessing metabolic pathways of cultured primary porcine hepatocytes. Although hypothermically preserved cells had a reduced plating efficiency, they were still metabolically active. Thus, hypothermia can be used as a temporary preservation technique for primary porcine hepatocytes.