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

Abstract

Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five 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 two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second 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 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.

Cortisol Increases Interstitial Collagenase Expression in Osteoblasts by Post-transcriptional Mechanisms

“…Nuclear Run-off Assay-Subconfluent cultures of Ob cells were treated with trypsin, harvested, subcultured at a 1:6 dilution, and allowed to grow to confluence as described previously (20). These first passage Ob cells retain the osteoblastic phenotype and respond to cortisol in a manner similar to that of primary cultures (7).…”

Observations on the Mechanism of Bone Resorption Induced by Multiple Myeloma Marrow Culture Fluids and Partially Purified Osteoclast-activating Factor

“…With the mouse calvaria only IBMX was used. cAMP responses were also tested in bone cell populations isolated from neonatal rat calvaria by sequential enzyme digestions (19). Digestions were carried out for three consecutive 10-min periods, then two 20-min periods.…”

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent

“…Fetal RC cell populations were obtained by sequential enzymatic digestion of calvariae from 21-day-old fetuses of timed pregnant Wistar rats as previously described [36]. Cells from the last four digest steps (pop II-V) were plated for 24 hours in separate T-75 flasks containing α-minimum essential medium (α-MEM) from the Tissue Culture Media Prep (TCMP) core at the University of Toronto, 15% heatinactivated fetal bovine serum (FBS) purchased from CanSera (Toronto, Ontario; http://www.cansera.ca), and the following antibiotics: 0.25 µg/ml fungizone and 50 µg/ml gentamycin sulfate (both from GIBCO/BRL; Burlington, Ontario; http://www.invitrogen.com), and 0.1 mg/ml penicillin G (Sigma Chemical Co.; St. Louis, MO; http://www.sigmaaldrich.com).…”