Protein-free culture medium improvement: testing additives and their interactive effects in 96-well plates

In order to achieve a rational screening of additives suspected to improve antibody production over basal Dulbecco's Modified Eagle Medium (DMEM), we have developed a 96-well plate method for simultaneously testing individual and interactive effects of multiple additives. Cell viabilities were determined directly in each well by a colorimetric assay (MTT assay) and antibody production was measured by an enzyme-linked immunosorbent assay (ELISA) performed on well supernatants. Such as supplemented culture medium might considerably reduce production costs since commercial serum- or protein-free media are sold at serum-enriched basal medium costs. The CBM-P22 mouse cell line used in this study was shown to be sensitive to key amino acids, oxalacetic acid, ethanolamine and selenium at low cell density (<1 × 10⁵ cells·ml^–1). When these cells were inoculated in 96-well plates their antibody productivity was improved (sevenfold) by adding these additives to the basal DMEM as evidenced by ELISA absorbance readings. This improved productivity, obtained with the supplemented DMEM, named DOWSENs, is comparable to the one obtained with the commercial, but costly, protein-free hybridoma medium (PFHM II), taken here as the positive control. Results were confirmed by growing these cells in different media in standard (25 cm²) T-flask static culture. In addition, specific amino acid consumption, as analysed by HPLC, showed that asparagine and tryptophan when added to DMEM may improve antibody production, even if these amino acids are not limiting or highly consumed in PFHM II. Using this 96-well plate assay allows the assessment of a large number of different assays with a few milligrams of the product(s) tested. This is a rapid technique that gives results for additives that are not easily quantified by analytical techniques or for the study of interactive effects, which is time consuming and labour-intensive when done in individual T-flasks. Correspondence to: J. Côté