title: IL-7 and immobilized Kit-ligand stimulate serum- and stromal cell-free cultures of precursor B-cell lines and clones.
abstract: Long-term proliferating, DHJH-rearranged mouse precursor B-cell lines have previously been established in serum- and IL-7-containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre-B cells to undefined factors, hampering accurate analyses of ligand-dependent signaling, which controls pre-B cell proliferation, survival, residence and migration. Here, we describe a novel serum-free, stromal cell-free culture system, which allows us to establish and maintain pre-B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit-ligand, also called stem cell factor, produced as a kit-ligand-Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre-B cells in an optimal density in plate-bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre-B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.
authors.affiliation: Department of Lymphocyte Development, Max-Planck Institute for Infection Biology, Berlin, Germany.|Deutsches Rheuma-Forschungszentrum Berlin (DRFZ), Berlin, Germany.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Kawano
authors.affiliation: Department of Lymphocyte Development, Max-Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Petkau
authors.affiliation: Department of Lymphocyte Development, Max-Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Wolf
authors.affiliation: Department of Lymphocyte Development, Max-Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Tornack
authors.affiliation: Department of Lymphocyte Development, Max-Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Fritz
authors.initials: F
authors.lastname: Melchers
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002999:Clone Cells; D003470:Culture Media; D015851:Interleukin-7 / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D019089:Stem Cell Factor / Q000276:immunology* / Q000494:pharmacology
chemical_list: D003470:Culture Media; D015851:Interleukin-7; D019089:Stem Cell Factor
keywords: IL-7; Kit-ligand; Pre-B cell; Proliferation; Serum-free; Stem cell factor; Stromal cell-free