Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

Abstract

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDPglucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP-U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.

Culture of Primary Bovine Chondrocytes on a Continuously Expanding Surface Inhibits Dedifferentiation

“…11,12 Analyses of cell morphology, cartilagespecific gene expression, fibrotic gene expression, and cell numbers strongly indicated that CE culture preserves the chondrocyte phenotype during population expansion. SD culture chondrocytes exhibited a spindle-like morphology that was characteristic of a more fibroblast-like phenotype [27][28][29] FIG. 7.…”

Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes

“…The protocols for the conventional collagen-coated single gel layer model and the organotypical double gel layer are described in our previous reports [20][21][22], based on the original methods [23,24].…”