Robust Serum-Free Expansion Of Human B Cells With An Animal Component-Free Cell Culture Supplement

Abstract Human B cells are utilized in workflows for discovering therapeutic antibodies and in the developing field of cellular therapy. The ability to expand the B cell population in blood samples from pathogen-exposed or naive patients offers the opportunity to improve the recovery of antibody sequences, increase the diversity of the B cell repertoire, or achieve de novo immunization in vitro. It can be difficult, however, to obtain robust expansion of B cells in cultures. Addition of serum improves performance, albeit with high lot-to-lot variability, and introduces the risk of contamination by adventitious agents. We have developed an animal component-free (ACF) supplement for culturing human B cells that does not require the use of serum, feeder cells, or specialized culture plates to achieve robust in vitro expansion. The ACF supplement is a 50X concentrate comprising non-animal-derived recombinant proteins and factors that can be added to a suitable base medium of choice to promote B cell activation and expansion. Human pan-B cells isolated from peripheral blood mononuclear cells (leukopaks) by immunomagnetic enrichment could be cultured and expanded in standard 24-well tissue culture plates for over 30 days, starting with seeding densities of 1 × 105 cells/well and passaging every few days from day 7 (± 1 day) onwards. Though pronounced inter-donor cell variability was observed in the rate of proliferation, an average ~55-fold, ~110-fold and ~160-fold expansion of viable B cells was obtained after 7, 11 and 14 days of culture, respectively (n = 21 donors; range 27- to 1090-fold at day 14 ± 1 day). Differentiation to plasma cells was apparent by flow cytometric analysis of CD138 and CD20.