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The MCA-3D cell line is derived from primary mouse epidermal cultures that exhibit resistance to calcium-induced terminal differentiation. These cells were initially treated with the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA), and subsequently exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The resistance to terminal differentiation was assessed by elevating calcium levels in the culture medium to 1.2 mM, which selectively allows for the growth of transformed cells while normal cells typically undergo terminal differentiation and death.
The MCA-3D cell line displays an epithelial morphology and forms well-defined colonies in culture. Ultrastructural analysis reveals that MCA-3D cells contain keratin filaments and desmosomes, which are indicative of their epithelial origin and suggest maintenance of some degree of normal keratinocyte differentiation. However, the exact abundance of these structures can vary among subpopulations within the line.
MCA-3D cells have been tested for tumorigenicity by subcutaneous injection into syngeneic Balb/c neonates, with results indicating that this line is non-tumorigenic, even after prolonged culture in high calcium conditions. Additionally, the MCA-3D cells do not grow in soft agar, further supporting their non-malignant phenotype. Biochemical assays for gamma glutamyl transpeptidase (GGT) activity and transglutaminase activity have shown that MCA-3D cells are negative for GGT, and their transglutaminase activity does not correlate with tumorigenic potential, aligning with their non-tumorigenic classification.
Overall, the MCA-3D cell line serves as a model for studying the early stages of carcinogenesis and the factors that influence the progression from preneoplastic lesions to fully malignant tumors.
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- cultureMedium: Ham's F12, w: 1.0 mM stable Glutamine, w: 1.0 mM Sodium pyruvate, w: 1.1 g/L NaHCO3 (Cytion article number 820600a)
- supplements: Supplement the medium with 10% FBS
- dissociationReagent: Accutase
- subculturing: Remove medium and rinse the adherent cells using PBS without calcium and magnesium (3-5 ml PBS for T25, 5-10ml for T75 cell culture flasks). Add TrypleExpress(1-2ml per T25, 2.5ml per T75 cell culture flask), the cell sheet must be covered completely. Incubate at 37 degree Celsius for 15-20 minutes. Carefully resuspend the cells with medium (10 ml), centrifuge for 5 min at 300xg, resuspend cells in fresh medium and dispense into new flasks which contain fresh medium.
- seedingDensity: 0.5 to 1 x 104 cells/cm2
- fluidRenewal: 2 to 3 times per week
- postThawRecovery: After thawing, plate the cells at 5 x 104 cells/cm2 and allow the cells to recover from the freezing process and to adhere for at least 24 hours.
- freezeMedium: As a cryopreservation medium, use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.
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