A series of subclones were isolated from the cloned but phenotypically heterogeneous MC3T3-E1 cell line.
The subclones were selected for high or low osteoblast differentiation and mineralization after growth in medium containing ascorbic acid.
The MC3T3-E1 Subclone 4 (ATCC CRL-2593
) and the MC3T3 Subclone 14 (ATCC CRL-2594
) lines exhibit high levels of osteoblast differentiation after growth in ascorbic acid and 3 to 4 mM inorganic phosphate. 
They form a well mineralized extracellular matrix (ECM) after 10 days.
The MC3T3 Subclone 24 (ATCC CRL-2595
) and the MC3T3 Subclone 30 (ATCC CRL-2596
) lines exhibit poor osteoblast differentiation after growth in ascorbic acid. They do not form ECM. They can be used as negative controls for Subclones 4 and 14. 
Mineralizing subclones selectively express mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. 
Subclones with both high and low differentiation potential produce similar amounts of collagen in culture and express comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. 
After implantation into immunodeficient mice, highly differentiating subclones form bone-like ossicles resembling woven bone, while poorly differentiating cells only produce fibrous tissue. 
These cell lines are good models for studying in vitro osteoblast differentiation, particularly ECM signaling. They have behavior similar to primary calvarial osteoblasts.