Effect of Thermal stress on Functional Properties of Caprine Hepatocytes Culture in Vitro

Abstract

Liver is involved in several vital functions such as synthesis, secretion, storage, metabolism of plasma proteins, detoxification of ammonia to urea. Digestion of liver tissue with collagenase to separate hepatocytes was done by non perfusion technique. The activity of enzyme Lactate Dehydrogenase (LDH) as a marker for assessing the integrity of cell membrane, concentration of albumin, urea and transforming growth factor beta (TGF-β) in the supernatant of hepatocyte culture were used as markers of functionality of hepatocytes. The present study was conducted to evaluate the effect of thermal stress on caprine hepatocytes culture on all the above mentioned parameters at different time intervals. At first, caprine hepatocyte culture was optimized at 37°C. After optimization, the hepatocytes culture was subjected to three different temperatures i.e. 37°C, 40°C and 42°C in different CO2 incubators under controlled humidified conditions. The culture was performed in 6 well plastic culture dishes. Supernatant was harvested at 24, 48 and 72 hours respectively from the hepatocyte cultured plates for estimation of the different mentioned parameters. It was observed that LDH activity at 37°C was greater at 48 hours and decreased by 72 hours of culture, when hepatocytes culture incubated at 40 and 42°C, activity of LDH temporally increased significantly (P<0.01) by 48 hours and was maintained till 72 hours, there was a significant increase in the concentration of albumin (P<0.01) and urea (P<0.05) at 72 hours of incubation at 37°C, whereas at 40°C and 42°C decrease in the secretion of urea and albumin was observed. Least Square Mean concentration of hepatocytes TGF-β was significantly greater (P<0.01) at 42°C when compared with LSM value at 37°C.The net effect observed was that when caprine hepatocytes were subjected to hyperthermic conditions, the function of hepatocytes decreased and TGF β secretion was significantly greater at 40°C and 42°C, indicating that caprine hepatocytes suffered from thermal stress over a period of 48-72 hours of incubation at temperatures higher than 37oC. It was also reflected by the significant decrease in the viability the cells at 42°C post 48 hours of incubation.