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Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William’s E media (HDW). Between the cultured and fresh cells, we observed a similar expression of  GAPDH, HNF4α, ALB, CYP1A1, CK8  and  CK18 . Interestingly, a statistically significant increase was noted in the  TAT, CPS, AFP, AAT, GSP  and  PCNA  expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of  HNF4α, GSP, CPS, AFP, AAT, PCNA  and  CK18  was similar between cultured and fresh cells. Further, a statistically significant increase in the  TAT  and  CK8  expression, and a decrease in the  GAPDH, CYP1A1  and  ALB  expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes