Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, 132001, India.
Sci Rep. 2017 Apr 26;7(1):1203. doi: 10.1038/s41598-017-01355-6.
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.
家畜与人类有密切的相似之处，因此可能是比啮齿动物更好的原代肝细胞来源。在此，我们成功开发了用于原代绵羊和水牛肝细胞的三维（3D）培养系统。绵羊肝细胞的 3D 结构在第 5 天形成，并在涂有 polyHEMA 的平板和含有 William's E 培养基（HDW）的悬滴中维持到第 10 天。在培养细胞和新鲜细胞之间，我们观察到 GAPDH、HNF4α、ALB、CYP1A1、CK8 和 CK18 的表达相似。有趣的是，TAT、CPS、AFP、AAT、GSP 和 PCNA 的表达显著增加。在水牛肝细胞培养中，第 3 天在 polyHEMA 和 HDW 上形成 3D 样结构，并维持到第 6 天。HNF4α、GSP、CPS、AFP、AAT、PCNA 和 CK18 的表达在培养细胞和新鲜细胞之间相似。此外，TAT 和 CK8 的表达显著增加，而 GAPDH、CYP1A1 和 ALB 的表达则降低。在培养系统中，HDW 或多或少地维持了绵羊和水牛新鲜肝细胞的肝脏转录标志物，分别维持了 10 天和 6 天。综上所述，悬滴是一种有效的绵羊和水牛原代肝细胞 3D 培养方法。
