A Bioassay for Optimization of Macrophage-Conditioned Medium as a Culture Supplement to Promote Hybridoma Cell Survival and Growth

Macrophage-conditioned medium (MCM) is an important cell culture supplement used to support the survival and growth of newly fused hybridoma cells. The use of macrophage cells, as a part of hybridoma technology, has proven to be an effective and inexpensive source of growth factors that promote the early survival and growth of hybridoma cells. Despite the widespread use of MCM as a hybridoma culture supplement, there is limited guidance and standardization for MCM production to achieve optimal hybridoma support. As an undefined supplement, significant variations in production of MCM may negatively impact hybridoma cell survival and growth. The lack of an available method for standardization of MCM bioactivity has limited validation, optimization, and commercial production. Consequently, variations in batch production of MCM may result in low-quality MCM that limits hybridoma viability and negatively impacts monoclonal antibody production. In this report, we describe a novel bioassay based on the newly generated, MCM-dependent RMH359 hybridoma cell line that can be used to validate MCM bioactivity and standardize production. We demonstrate the utility of the RMH359 bioassay (1) for evaluating MCM hybridoma bioactivity, (2) to define optimal conditions for production of MCM, and (3) as a method for MCM validation and standardization. In conclusion, the RMH359 cell bioassay provides a specific and sensitive assessment of MCM bioactivity in support of hybridoma cell survival and growth.