Cellxvivo human b cell expansion kit

To assess the effects of STRO-001 on human B cells, peripheral blood mononuclear cells (PBMC) from three different healthy human donors (Buffy Coats from Stanford Blood Center, Palo Alto, CA, USA) were isolated by density gradient centrifugation (Nycoprep 1.077, Cosmo Bio USA, Carlsbad, CA, USA). Untouched human B cells were purified by negative isolation (B Cell Isolation Kit II, Miltenyi Biotec, San Diego, CA, USA). B cells were cultured at 0.1 × 10⁶ cells per well in 96-well plates with or without in vitro expansion reagents (CellXVivo Human B-cell Expansion Kit, R&D Systems) in RPMI media containing 10% heat inactivated fetal bovine serum (SH30070.03HI, HyClone, GE Healthcare Life Sciences). Next day, serial dilution of STRO-001 and control unconjugated Ab were added to naïve and proliferating B cells and cultured for 6 days. Cell killing was assessed by flow cytometry using propidium iodide (PI) staining to identify dead B cells and absolute counting beads (flow cytometry absolute count standard, Bangs Laboratories, Fishers, IN, USA) to quantify viable B-cell numbers. In addition, surface expression of CD80, CD86, HLA-DR and CD74 on B-cell was confirmed after 4 to 5 days by antibodies from eBioscience.

This study used 6 HMCLs, NCI-H929, OPM-2, AMO-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Neddersassen, Germany), RPMI8226 (American Type Culture Collection, Manassas, VA, USA), KMS-12-BM and KMS-28-PE (kind gifts from Dr. Ohtsuki T (Kawasaki Medical School, Kurashiki, Okayama, Japan)), harboring various types of chromosomal abnormalities and gene mutations (Supplementary Information). HMCLs were grown in RPMI-1640 with 10% fetal calf serum, 2 mM L-glutamate, and penicillin/streptomycin at 37 °C in a fully humidified atmosphere of 5% CO₂ in the air. Patient-derived CD138-positive myeloma cells were isolated using MACSprep^TM Multiple Myeloma CD138 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Nordrhein-Westfalen, Germany) [5 (link),29 (link)], and were grown in RPMI-1640 with 10% fetal calf serum, 2 mM L-glutamate, penicillin/streptomycin, and CellXVivo Human B Cell Expansion Kit (R&D systems, Minneapolis, MN, USA). The agents used were BI-D1870, which selectively inhibits the phosphorylation of RSK2^Ser227 at NTKD in an ATP-competitive manner [64 (link)] (Cayman Chemical Company, Ann Arbor, MI, USA), and ipatasertib, which binds to AKT in an ATP-competitive manner and prevents substrate phosphorylation [65 (link)] (Selleck Biotech Ltd., Tokyo, Japan).