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Abstract
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.
Highlights
3D cultures provide advantages such as 1) it mimics the in vivo cellular microenvironment; 2) the 3D culture can act as a model to study different pathophysiological states; and 3) it is more realistic to grow the cells in 3D to study the effect of drug dosages as the layers of cells and the tight junctions between the cells in 3D cell culture instead of the monolayer culture act as natural barriers, which affect the diffusion of drugs[13]
Our data suggest that hanging drops with William’s E media (HDW) is a best method for 3D culturing of primary sheep hepatocytes for ten days
3D culturing of primary sheep and buffalo hepatocytes was performed in collagen-coated plates, collagen sandwich, polyHEMA-coated plates and hanging drop method with an objective to establish a 3D culture system for primary hepatocytes of livestock which are considered as better alternative animal models than rodents
Summary
3D cultures provide advantages such as 1) it mimics the in vivo cellular microenvironment; 2) the 3D culture can act as a model to study different pathophysiological states; and 3) it is more realistic to grow the cells in 3D to study the effect of drug dosages as the layers of cells and the tight junctions between the cells in 3D cell culture instead of the monolayer culture act as natural barriers, which affect the diffusion of drugs[13]. Most spheroid culture studies have employed HepG2 cell-line and not primary human hepatocytes. Development of 3D cell culture for ruminant hepatocytes is useful as in vitro models for toxicological studies, and to understand the physiological processes of hepatocytes in farm animal biology. We chose sheep and buffalo as model ruminants in this study, as they are common livestock animals with an easy availability of the tissue samples from slaughterhouses in developing countries, like India. These are the closest species to cows, whose genome is similar to humans. This study describes the characterization of the 3D cell culture systems using hepatocyte-specific RNA markers for sheep and buffalo hepatocytes
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