Growth of a human colonic adenocarcinoma cell line (HT 29) on microcarrier beads: Metabolic studies by 31phosphorus nuclear magnetic resonance spectroscopy

Abstract

A method allowing the growth of a human colon adenocarcinoma cell line (HT 29) on beaded polystyrene microcarriers has been developed by modifying the culture conditions used in monolayer cultures. Under optimized conditions, the cells became confluent 7 days after seeding and reached a density of 2.8 x 10⁵ cells/cm of microcarrier (65% of the available area occupied). ³¹P NMR spectra were typically recorded on 300 x 10⁶ cells continuously perfused at a flow rate of 15 ml/min in a specially designed NMR chamber in which the micro-carrier beads were sequestered within the receiver coil volume. The in vivo spectrum displays a series of resonances assigned to nucleoside triphosphates (ATP and GTP), inorganic phosphate and various phosphomonoesters (mainly glucose-6-P and phosphorylcholine). Diphosphodiester resonances (DPDE, mainly UDP-N-acetyl-glucosamine and UDP-N-acetylgalactosamine) were not detected in the in vivo spectrum and were only apparent in the spectrum of the perchloric acid extract of the cells, indicating that these compounds have a restricted mobility in the intracellular compartment. The intracellular pH of HT 29 cells was 7.2 during the perfusion with a medium buffered at pH 7.3. The internal pH decreased slowly (2 x 10^-3 pH unit/min) during anoxic perfusion, but severe intracellular acidosis occurred after 40 min of ischemia (2.7 x 10^-1 pH unit/min). Sequential recording of ³¹P NMR spectra has shown that HT 29 cells are able to maintain their high energy phosphorylated compound levels (ATP) when subjected to 100 min of anoxia and 40 min of total ischemia.