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These cell lines are good models for studying in vitro osteoblast differentiation, particularly ECM signaling. They have behavior similar to primary calvarial osteoblasts.

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 Subclone 24 (ATCC CRL-2595) and the MC3T3 Subclone 30 (ATCC CRL-2596) lines exhibit poor osteoblast differentiation after growth in ascorbic acid. 

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. [51540] 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. RefWang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097

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. RefWang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097

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. RefWang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097

After implantation into immunodeficient mice, highly differentiating subclones form bone-like ossicles resembling woven bone, while poorly differentiating cells only produce fibrous tissue. RefWang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-053mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Wang D, et al. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J. Bone Miner. Res. 14: 893-903, 1999. PubMed: 10352097