3.2.2 Cell Culture Good Practice

Rigorous aseptic technique underlies every reliable in-vitro result.

Rigorous aseptic technique underlies every reliable in-vitro result. Sterility must be maintained using a Class II biological safety cabinet, 70% isopropyl alcohol surface disinfection, and appropriate personal protective equipment. Standard growth media are selected according to cell type — DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for most adherent lines, RPMI for lymphocyte culture, and L-15 medium for suspension or CO2-independent culture. Cells are typically maintained at 37°C in a humidified incubator with 5% CO2 and passaged every two to three days once they reach 70–80% confluence. Test compounds are conventionally dissolved in dimethyl sulfoxide (DMSO), with the final assay concentration of vehicle kept below 0.1% v/v, since DMSO itself becomes cytotoxic above approximately 0.5%. Every assay plate must include a negative control (vehicle alone), a positive control (a known active reference compound), and a blank (medium only, no cells), enabling background subtraction and confirming assay validity.