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

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.

Figure 1

Primary sheep hepatocytes cultured in hanging drops at 200x magnification. Primary sheep hepatocytes formed spheroids in hanging drops with Hepatozyme-SFM (HDH) and William’s E media (HDW) on the fifth day and those spheroids were maintained until the tenth day.

Figure 2

Primary buffalo hepatocytes cultured in hanging drops at 200x magnification. Primary buffalo hepatocytes formed spheroids in hanging drops with Hepatozyme-SFM (HDH) and William’s E media (HDW) on the third day and those were maintained until the sixth day.

Figure 3

Fluorescent imaging of the primary sheep hepatocytes cultured on collagen-coated and polyHEMA-coated plates on the twelfth day at 200x magnification. The nucleus of the primary sheep hepatocytes were stained with DAPI and the cytoskeletal protein F-actin was stained with Phalloidin-TRITC stain. Hepatocytes formed patches on collagen-coated plates (CH) on the twelfth day of the culture. However, different layers of cells could be observed in the intact spheroids formed on the polyHEMA coated dishes on the twelfth day (PH).

Figure 4

Fluorescent imaging of the primary buffalo hepatocytes cultured on collagen-coated and polyHEMA-coated plates on the sixth day at 200x magnification. The nucleus of the primary buffalo hepatocytes were stained with DAPI and the cytoskeletal protein F-actin was stained with Phalloidin-TRITC stain. Hepatocytes formed patches on collagen-coated plates (CH) on the sixth day of the culture. However, different layers of cells could be observed in the intact spheroids formed on the polyHEMA coated dishes on the sixth day (PH).

Figure 5

Heat map for the fold change of selected gene markers in different culture systems for primary sheep hepatocyte 3D culture. The expression of the markers, CYP1A1, GAPDH, ALB, HNF4α, and CK18, was not significantly different between fresh and cultured cells. However, the expression of the markers, AFP, GSP, CPS, PCNA, AAT, TAT, and CK8, was significantly (P < 0.05, indicated as ★) different between fresh and cultured cells. The green and red colours indicate higher and lower fold change in the gene expression, respectively, than that of fresh cells. The circled culture systems are considered as best than other culture methods on the fifth, tenth and twelfth days of culture.

Figure 6

Heat map for the fold change of selected gene markers in different culture systems for primary buffalo hepatocyte 3D culture. The expression of the markers, HNF4α, AAT, PCNA, AFP, GSP, CK18, and CPS, was not significant between fresh and cultured cells. However, the expression of the markers, GAPDH, ALB, CK8, CYP1A1, and TAT, was significantly (P < 0.05, indicated as ★) different between fresh and cultured cells. The green and red colours indicate higher and lower fold change in the gene expression, respectively, than that of fresh cells. The circled culture systems are considered as best than other culture methods for culturing buffalo hepatocytes for 6 days in a 3D manner.