Parathyroid Hormone- and Prostaglandin E1-Response in a Selected Population of Bone Cells After Repeated Subculture and Storage at ‒80 C

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Abstract
Five different cell populations, designated I to V, were isolated from minced newborn rat calvaria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all 5 populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in c[cyclic]AMP level. None of the 5 populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a 2- to 3-fold increase in cAMP concentration in response to PTH up to the 3rd subculture. No morphological differences could be observed among the 5 populations. The 3rd subculture of population IV cells that had been stored in 10% glycerol at -80.degree. C for 4 mo. was subsequently thawed and subcultured to the 6th subculture. These cells still responded to PTH with an increase in cAMP level. In a 2nd experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80.degree. C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the 6th subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U[unit]/ml to 5 U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 .mu.g/ml to 10 .mu.g/ml. The time course of action on population IV of PTH was different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.

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