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Growth of a human colonic adenocarcinoma cell line (HT 29) on microcarrier beads: Metabolic studies by 31 phosphorus nuclear magnetic resonance spectroscopy
Author(s) -
Fantini Baetselier Jacques,
Marvaldi Jacques,
Galons JeanPhilippe,
Cozzone Patrick J.,
Canioni Paul
Publication year - 1987
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.2910390222
Subject(s) - phosphomonoesters , microcarrier , intracellular ph , tetramethylammonium , chemistry , cell culture , intracellular , phosphocreatine , nigericin , nuclear magnetic resonance spectroscopy , biophysics , nuclear magnetic resonance , biochemistry , biology , phosphate , cell , stereochemistry , membrane , energy metabolism , inorganic phosphate , ion , genetics , physics , organic chemistry , endocrinology
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 10 5 cells/cm of microcarrier (65% of the available area occupied). 31 P NMR spectra were typically recorded on 300 x 10 6 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 31 P 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.