Rpmi8226

Manufactured by American Type Culture Collection

Sourced in United States, Germany, France

RPMI8226 is a human myeloma cell line derived from the peripheral blood of a patient with multiple myeloma. The cell line is widely used in research for studying multiple myeloma and related blood disorders.

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RPMI8226 cell line RPMI8226/s

335 protocols using rpmi8226

Teriflunomide Cytotoxicity in Multiple Myeloma

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The study was conducted on multiple myeloma cell line- RPMI 8226 (catalog no. CCL-155, ATCC, Manassas, VA, USA) and skin fibroblast cell line- BJ (catalog no. CRL-2522, ATCC, USA). RPMI 8226 cells were grown on RPMI-1640 Medium (USA, ATCC) and BJ cells on EMEM Medium (USA, ATCC). The culture media were supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA). Cells were cultured at 37 °C in 5% CO₂-air. The media contained antibiotics such as penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA). In all assays, the cells were seeded into plates at concentration of 1.5 × 10⁵ cells/mL.
The cell cultures were treated with teriflunomide (A77 1726) (Selleckchem, München, Germany) in a wide range of concentrations (5–500 µM) or DMSO as vehicle in control cultures (maximal DMSO concentration < 0.5%). After the addition of compound, the cells were incubated for 24 h. The concentrations (100, 200, and 500 µM) of teriflunomide used in the further research had been selected after MTT assay and the assessment of cells morphology under the microscope.

Adamczuk G., Humeniuk E., Iwan M., Natorska-Chomicka D., Adamczuk K, & Korga-Plewko A. (2021). The Mitochondria-Independent Cytotoxic Effect of Leflunomide on RPMI-8226 Multiple Myeloma Cell Line. Molecules, 26(18), 5653.

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Wnt Signaling Pathway Assay Protocol

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RPMI8226, HEK293, and Wnt3a-L cells were purchased from the American Type Culture Collection and cultured in RPMI1640 (RPMI8226 and MM.1S) and Dulbecco’s modified Eagle’s media (HEK293 and Wnt3a-L) supplemented with 10% fetal bovine serum, 120 μg/ml penicillin, and 200 μg/ml streptomycin. HEK293-FL (TOPFlash) reporter cells were established as previously described [13 (link)]. Wnt3a-CM was prepared as previously described [14 (link)]. Luciferase assays were conducted using the Dual-Luciferase Assay Kit (Promega, USA), following the manufacturer’s instructions. Lithium chloride (LiCl), MG-132, and ursolic acid were purchased from Sigma-Aldrich (USA).

Song G.R., Choi Y.J., Park S.J., Shin S., Lee G., Choi H.J., Lee D.Y., Song G.Y, & Oh S. (2021). Root Bark of Morus alba L. and Its Bioactive Ingredient, Ursolic Acid, Suppress the Proliferation of Multiple Myeloma Cells by Inhibiting Wnt/β-Catenin Pathway. Journal of Microbiology and Biotechnology, 31(11), 1559-1567.

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Isolation and Culture of Human Myeloma Cells

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Human myeloma cell lines RPMI-8226, U266 and OPM2 were purchased from the American Type Culture Collection (Manassas, VA, USA); OCI-MY5 and the bortezomib-resistant cell line RPMI-8226/R5 were kindly provided by Fenghuang Zhan (Department of Internal Medicine, University of Iowa, Iowa City, USA). Primary cells are CD138⁺ mononuclear cells isolated from the bone marrow of patients with multiple myeloma by Ficoll-Hypaque density gradient centrifugation. Written informed consent was signed by multiple myeloma patients in accordance with the Helsinki Declaration. The institutional review board of the Shanghai Tenth People's Hospital, Tongji University approved this protocol for collection of clinical samples. Human myeloma cell lines and CD138⁺ myeloma cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, BRL, USA) and 1% penicillin-streptomycin (PS; Gibco, Carlsbad, CA, USA). All cells were incubated in artificial incubator containing 5% CO₂ at 37°C.

Feng Q., Yao Q., Li B., Xie Y., Zhang H., Xu Z., Lu K., Hu K., Cheng Y., Shi B., Huang C., Li L., Wu X., You S., Shi J, & Zhu W. (2020). Glycolysis is suppressed by DCZ0801-induced inactivation of the Akt/mTOR pathway in Multiple Myeloma. Journal of Cancer, 11(16), 4907-4916.

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Culturing Multiple Myeloma Cell Lines

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Cell lines MM1.S (Dexamethasone sensitive), MM1.R (Dexamethasone resistant) and RPMI 8226 were kindly provided by Dr. Jonathan Keats (TGen, Phoenix, AZ, USA), DOX 40 (Doxorubicin resistant) was kindly provided by Dr. William Dalton (Moffitt Cancer Center, Tampa, FL, USA), and NC-H929 was purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI 1640 media (Mediatech Inc., Manassas, VA, USA) containing 10% fetal bovine serum (Mediatech Inc.), 100U/mL penicillin, 100uG/ml streptomycin, and 2mM glutamine (Invitrogen, Grand Island, NY, USA). Freshly obtained bone marrow aspirates from MM patients were obtained after informed consent and processed to collect either myeloma cells or bone marrow stromal cells (BMSCs) as described earlier [49 (link)–51 (link)]. Patient cells were cultured as MM cell lines except that 20% fetal bovine serum was used instead of 10% fetal bovine serum.

Ramakrishnan V., Gomez M., Prasad V., Kimlinger T., Painuly U., Mukhopadhyay B., Haug J., Bi L., Rajkumar S.V, & Kumar S. (2016). Smac mimetic LCL161 overcomes protective ER stress induced by obatoclax, synergistically causing cell death in multiple myeloma. Oncotarget, 7(35), 56253-56265.

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RPMI-8226 Cell Lysis and Western Blot

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RPMI-8226 (ATCC, Manassas, VA) cells were incubated (37 °C and 5% CO₂) with test compounds for 48 hrs in RPMI-1640 media containing 10% fetal bovine serum and penicillin-streptomycin. Whole cell lysate was obtained using RIPA buffer (0.15 M NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton (v/v) X-100, 0.05 M Tris HCl) containing protease and phosphatase inhibitors. Protein content was determined using the bicinchoninic acid (BCA) method (Pierce Chemical, Rockford, IL). Equivalent amounts of cell lysate were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed with the appropriate primary antibodies, and detected using HRP-linked secondary antibodies and Amersham Pharmacia Biotech ECL Western blotting reagents per manufacturer's protocols.

Foust B.J., Allen C., Holstein S.A, & Wiemer D.F. (2016). A New Motif for Inhibitors of Geranylgeranyl Diphosphate Synthase. Bioorganic & medicinal chemistry, 24(16), 3734-3741.

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Myeloma Cell Line Culturing Protocol

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A panel of 11 myeloma cell lines was used. Four of the cell lines were in-house: OH-2, IH-1, URVIN and KJON, whereas 7 were from other sources: INA-6, CAG, JJN3 and ANBL-6 were kind gifts from Dr M. Gramatzki (University of Erlangen-Nurnberg, Erlangen, Germany), Dr J. Epstein (University of Arkansas for Medical Sciences, Little Rock, AR, USA), Dr J. Ball (University of Birmingham, UK), and Dr D. Jelinek (Mayo Clinic, Rochester, MN, USA), respectively, KMS-12-BM was obtained from DSMZ (Braunschweig Germany), and RPMI-8226 and U266 were from ATCC (Rockville, MD, USA). URVIN, INA-6 and ANBL-6 cells were grown in 10% heat inactivated fetal calf serum (FCS) in RPMI-1640 (RPMI) supplemented with interleukin (IL)-6 (1 ng/mL) (Biosource, Camarillo, CA, USA). CAG, JJN3, KMS-12-BM, RPMI-8226 and U266 were grown in RPMI with 10, 10, 20, 20 or 15% FCS, respectively. OH-2 and IH-1 were maintained in 10% heat-inactivated human serum (HS) (Department of Immunology and Transfusion Medicine, St. Olav's University Hospital, Trondheim, Norway) whereas KJON was maintained in 5% HS, all in RPMI and IL-6 (2 ng/mL). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. For experiments 2% HS in RPMI was used as medium, with IL-6 (1 ng/mL) added for all IL-6 dependent cell lines.

Holien T., Misund K., Olsen O.E., Baranowska K.A., Buene G., Børset M., Waage A, & Sundan A. (2015). MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy. Oncotarget, 6(26), 22698-22705.

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Expansion and Transduction of T Cells and Cell Lines

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PBMCS were isolated by Ficoll-Paque (GE Healthcare, cat no. Cytvia 17-1440-03)
from buffy coats obtained from Sanquin Blood Bank). αβT cells were expanded from PBMCs using CD3/CD28 dynabeads (Thermo Fisher scientific, cat no. 40203D) and (1.7 × 10³ IU/ml of MACS GMP Recombinant Human interleukin (IL)-7 (Miltenyi Biotec, cat no. 130-095-361), and 1.5 × 10² IU/ml MACS GMP Recombinant Human IL-15 (Milteny Biotec, cat no. 130-095-762). HL60, RPMI 8226, and SSC9 stably expressing GFP-luciferase was generated by a previously described retroviral transduction protocol (30 (link)). The plasmid containing the GFP and luciferase transgenes was kindly provided by Jeanette Leusen (UMC Utrecht, Utrecht, Netherlands). The following cell lines were obtained from ATCC between 2010 and 2018, HL60 (CCL-240), RPMI 8226 (CCL-155), SCC9 (CRL-1629) and Daudi (CCL-213). Freestyle 293-F cells (R790-07) were obtained from Invitrogen. ML-1, HL60, RPMI 8226 and Daudi were cultured in RPMI (Gibco, cat no. 12017599), 10% FCS (Bodinco), 1% Pen/Strep (Invitrogen, cat no. 11548876). Freestyle 293-F in Freestyle expression medium (Gibco, cat no. 10319322). SCC9 in DMEM (Gibco, cat no. 31966047) 10% FCS, 1% Pen/Strep.

van Diest E., Nicolasen M.J., Kramer L., Zheng J., Hernández-López P., Beringer D.X, & Kuball J. (2023). The making of multivalent gamma delta TCR anti-CD3 bispecific T cell engagers. Frontiers in Immunology, 13, 1052090.

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Establishing Multiple Myeloma Cell Line Database

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The human multiple myeloma cell lines LP-1, OPM2, H929, KMS28BM, KMS11, RPMI8226, U266, KMS12 and Burkitt lymphoma cell lines Daudi and Raji were obtained from the ATCC. All of the cell lines were grown in RPMI-1640 medium (BioWest) supplemented with 10% FBS and 1% penicillin–streptomycin, and kept at 37°C with 5% CO₂. The identity of all cell lines was recently authenticated by short tandem repeat analysis. Plasma cells were isolated by CD138 immunomagnetic bead selection from bone marrow aspirates of newly diagnosed patients with multiple myeloma, obtained from the National University Hospital. The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration, and was approved by the Human Ethics Committee of National University of Singapore. Written informed consent was obtained from individual or guardian participants. DMSO was purchased from Sigma. THZ1 (HY-80013) and JQ1 (HY-13030) were obtained from Cayman Chemical and Medchemexpress, respectively. All antibodies used are shown in Supplementary materials and methods.

Jia Y., Zhou J., Tan T.K., Chung T.H., Chen Y., Chooi J.Y., Sanda T., Fullwood M.J., Xiong S., Toh S.H., Balan K., Wong R.W., Lim J.S., Zhang E., Cai Z., Shen P, & Chng W.J. (2021). Super Enhancer-Mediated Upregulation of HJURP Promotes Growth and Survival of t(4;14)-Positive Multiple Myeloma. Cancer Research, 82(3), 406-418.

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Cell Line Origin, Culture, and Consent

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The following cell lines (Kasumi-1, NB4, NALM-6, Jurkat, K562, Daudi, IM-9, RPMI8226, NCI-H929, HeLa, and RK13) were obtained from ATCC between 2004 and 2013. FL-218 was obtained from the Japanese Collection of Research Bioresources Cell Bank in 2011. Normal human skin fibroblasts (NB1RGB) were obtained from the RIKEN BioResource Center in 2014. All cell lines were passaged for less than 6 months. No further authentication was done for these cell lines in the past 6 months. Bone marrow samples were collected from patients admitted to the Japanese Red Cross Medical Center between February 2014 and December 2014; written informed consent was obtained from all patients prior to collection. All relevant study-related protocols were approved by the Institutional Review Board of the Japanese Red Cross Medical Center and the Institute of Medical Science, The University of Tokyo. Cells were grown in appropriate culture medium supplemented with 10% fetal bovine serum (JRH Biosciences), 100 U/mL penicillin (Wako), and 100 μg/mL streptomycin (Wako). Cell lines derived from hematological malignancies and patient bone marrow mononuclear cells were grown in RPMI 1640 medium (Wako). HeLa cells and NB1RGB cells were grown in minimum essential medium (MEM), alpha modification (Wako). RK13 cells were grown in standard MEM (Wako).

Futami M., Sato K., Miyazaki K., Suzuki K., Nakamura T, & Tojo A. (2017). Efficacy and Safety of Doubly-Regulated Vaccinia Virus in a Mouse Xenograft Model of Multiple Myeloma. Molecular Therapy Oncolytics, 6, 57-68.

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Inhibition of Aminopyrimidine Kinase in Multiple Myeloma

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The aminopyrimidine kinase inhibitor JP11646 and other members of its family of inhibitors (Table 1), LGB321 (Novartis, Berkeley, CA) and AZD1208 (Astra Zeneca, London, UK) were synthesized and obtained from Jasco Pharmaceuticals (Woburn, MA) and were used as 10 mM stocks in DMSO. The MM cell lines MM1.S (gift from Dr. Stephen Rosen, Chicago, IL), RPMI8226, U266 and KMS11 (ATCC, Manassas, VA), were maintained in RPMI 1640 media (Mediatech Inc, Manassas, VA) containing 10% FBS (Hyclone Inc., Logan, UT), 1000 U/ml penicillin/streptomycin, 4 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate. Recombinant IL-6 was purchased from R&D Systems (Minneapolis, MN).

Nair J.R., Caserta J., Belko K., Howell T., Fetterley G., Baldino C, & Lee K.P. (2016). Novel inhibition of PIM2 kinase has significant anti-tumor efficacy in multiple myeloma. Leukemia, 31(8), 1715-1726.

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