Culture conditions induce the appearance of immortalized C₃H mouse cell lines

Mouse fibroblasts were cultured by three different procedures: (a) changing the 0.2 ml/cm² of growth medium every 2nd d and seeding 1×10⁵ cells/cm² after confluency; (b) changing the 0.4 ml/cm² of growth medium only at subculture performed at confluency by a 1∶2 split and keeping the bottles incubated on a rocking platform; (c) the same as Method b but keeping the bottles stationary throughout culture. By Method a no lines were immortalized over 36 experiments whereas Method b gave 1/4 immortalized lines and Method c gave 10∶12 immortalized lines. Cells always went into crisis at the 9th to 11th doubling. Immortalized lines had a tetraploid DNA content.