title: Prolonged Ex vivo expansion and differentiation of naïve murine CD43(-) B splenocytes.
abstract: Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell-independent or T-cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co-culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen-activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp-1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978-989, 2016.
authors.affiliation: Institute of Bioprocess Engineering, Friedrich-Alexander-University Erlangen Nürnberg, Paul-Gordan-Straße 3, Erlangen, D-91054, Germany.
authors.forename: Kenny
authors.initials: K
authors.lastname: Zambrano
authors.affiliation: Chair for Process Biotechnology, University of Bayreuth, Universitätsstraße 30, Bayreuth, D-95447, Germany.
authors.forename: Valérie
authors.initials: V
authors.lastname: Jérôme
authors.affiliation: Chair for Process Biotechnology, University of Bayreuth, Universitätsstraße 30, Bayreuth, D-95447, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Freitag
authors.affiliation: Institute of Bioprocess Engineering, Friedrich-Alexander-University Erlangen Nürnberg, Paul-Gordan-Straße 3, Erlangen, D-91054, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Buchholz
authors.affiliation: Division Molecular Immunology, Dept. of Internal Medicine III, Nikolaus Fiebiger Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstr. 6, Erlangen, D-91054, Germany.
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Jäck
authors.affiliation: Institute of Bioprocess Engineering, Friedrich-Alexander-University Erlangen Nürnberg, Paul-Gordan-Straße 3, Erlangen, D-91054, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Hübner
authors.affiliation: Division Molecular Immunology, Dept. of Internal Medicine III, Nikolaus Fiebiger Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstr. 6, Erlangen, D-91054, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schuh
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D051917:Leukosialin / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology* / Q000378:metabolism
chemical_list: D051917:Leukosialin; C497863:Spn protein, mouse
keywords: B lymphocytes; CD40 ligand; MACS; antibodies; feeder cells; in vitro B cell expansion