Disease:

Osteoblast; SV40 large T antigen transfected

Origin:

This line was established by transfection of limb tissue obtained from a spontaneous miscarriage with the temperature sensitive expression vector pUCSVtsA58 and the neomycin resistance expression vector pSV2-neo. Clones were selected in the presence of 0.6 mg/mL G418.

Species:

Homo sapiens, human

Tissue:

Bone

Morphology:

Osteoblast

Properties:

Adherent

Cytogenic data:

diploid, 43%; tetraploid, 57%.

Patient:

Fetus.

Medium:

1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's Medium,with 2.5 mM L-glutamine (without phenol red) + 10% FBS + G418 (0.3 mg/mL)

Subculture:

Remove medium, and rinse with 0.05% trypsin, 0.03 mM EDTA solution. Remove the solution and add an additional 1 to 2 mL of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 34°C) until the cells detach. Add fresh culture medium, aspirate and dispense into new culture flasks. Culture at 5% CO2, 34°C. A subcultivation ratio of 1:4 is recommended. Renew medium once every 2 to 3 days depending on cell density. Cells grown at a permissive temperature of 33.5°C exhibit rapid cell division (Doubling time of 36 hrs), whereas little cell division occurs at a restrictive temperature of 39.5°C (Doubling time of 96 hrs). The cells have the ability to differentiate into mature osteoblasts expressing the normal osteoblast phenotype. At the restrictive temperatures, cell division is slowed, differentiation increases, and a more mature osteoblast phenotype is produced.

Freezing Medium:

Culture medium, 72%; additional fetal bovine serum, 20%; DMSO, 8%