Roswell park memorial institute (rpmi)

Manufactured by GE Healthcare

Sourced in Germany, Austria, United States

RPMI is a cell culture medium used for the in vitro cultivation of various cell types, including mammalian cells, primary cells, and cell lines. It is a complex mixture of inorganic salts, amino acids, vitamins, and other components that provide the necessary nutrients and support for cell growth and maintenance.

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Lab products found in correlation

Fetal bovine serum (fbs), by GE Healthcare (7 mentions) Glutamax, by Thermo Fisher Scientific (6 mentions) Dulbecco modified eagle medium (dmem), by GE Healthcare (6 mentions) Penicillin streptomycin, by GE Healthcare (5 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (5 mentions) Click s medium, by Irvine Scientific (4 mentions) Streptomycin, by Thermo Fisher Scientific (4 mentions) Penicillin, by Thermo Fisher Scientific (4 mentions) Glutamax, by GE Healthcare (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Phytohemagglutinin (pha), by Merck Group (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Interleukin 4 (il 4), by R&D Systems (2 mentions) Interleukin 6 (il 6), by R&D Systems (2 mentions) Human ab serum, by Gemini Bio (2 mentions) L glutamine, by GE Healthcare (2 mentions) Hyclone, by GE Healthcare (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Hepes, by GE Healthcare (2 mentions)

34 protocols using roswell park memorial institute (rpmi)

Dendritic Cells, Lymphoblasts, and LCLs Generation

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For dendritic cells (DCs) generation, PBMCs or CBMCs were plated at 1 × 10⁷ cells/well on 6-well plates and incubated for 2 hours in DC media (CellGenix, Freiburg, Germany) supplemented with 2 mmol/l GlutaMax (Gibco, Grand Island, NY). Non-adherent cells were harvested and cryopreserved. Adherent cells were cultured in DC media with IL-4 (1000 U/ml; R&D Systems, Minneapolis, MN) and GM-CSF (800 U/ml, R&D Systems). On day 5, immature DCs were matured using IL-4 (1000 U/ml), GM-CSF (800 U/ml), TNF-α (10 ng/ml), IL-6 (100 ng/ml), IL-1β (10 ng/ml; all R&D Systems), PGE-2 (1 μg/ml; Sigma-Aldrich, St. Louis, MO), and LPS (10 ng/ml, Sigma-Aldrich) and were harvested after 24 hours of maturation to use as APCs. To generate phytohemagglutinin (PHA)-treated lymphoblasts (PHA-blasts), PBMCs or CBMCs were stimulated with PHA (5 μg/ml, Sigma-Aldrich) in the presence of IL-2 (100 U/ml; Prometheus, San Diego, CA) in CTL media consisting of 45% RPMI (GE Healthcare, Logan, UT), 45% Click’s medium (Irvine Scientific, Santa Ana, CA), 10% human AB serum (Gemini BioProducts, West Sacramento, CA) and supplemented with 2 mmol/l GlutaMax. For lymphoblastoid cell lines (LCLs) generation, PBMCs or CBMCs were infected with live B95–8 EBV in complete media consisting of RPMI, 10% FBS (GE Healthcare), and 2 mmol/l GlutaMax containing cyclosporine A (1 μg/ml).

Hanajiri R., Sani G.M., Hanley P.J., Silveira C.G., Kallas E.G., Keller M.D, & Bollard C.M. (2019). Generation of Zika Virus-Specific T-Cells from Seropositive and Virus Naïve Donors for Potential Use as an Autologous or “Off the Shelf” Immunotherapeutic. Cytotherapy, 21(8), 840-855.

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Generation of PRAME-specific T-cell Products

Cited 1 time

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PRAME-specific T-cell products were generated from total PBMCs by previously established protocol [28 (link)]. Briefly, mature dendritic cells (DC), generated by adherence, were used as antigen-presenting cells. Non-adherent cells were stimulated with PRAME peptide-pulsed (200 ng/200 uL/5 x 10⁶ cells), irradiated (25 Gy) DC at an effector-to-target ratio of 10:1 and cultured in complete media consisting of 50% Click’s medium (Irvine Scientific, Santa Ana, CA), 40% RPMI (GE Healthcare, Logan, UT), 10% human AB serum (Gemini BioProducts, West Sacramento, CA), and supplemented with 2 mM GlutaMax (Gibco, Grand Island, NY). Second and third restimulations of T-cells were carried out weekly with PRAME peptide-pulsed, irradiated DC at an effector-to-target ratio of 20:1. On day 28, cells were harvested and evaluated for antigen specificity and functionality.

Stanojevic M., Hont A.B., Geiger A., O’Brien S., Ulrey R., Grant M., Datar A., Lee P.H., Lang H., Cruz C.R., Hanley P.J., Barrett A.J., Keller M.D, & Bollard C.M. (2021). Identification of Novel HLA-Restricted PRAME Peptides to Facilitate off-the-shelf Tumor- Associated Antigen-specific T-cell Therapies. Cytotherapy, 23(8), 694-703.

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Small Cell Lung Cancer Cell Line Viability Assay

Cited 2 times

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Human small cell lung cancer cell lines (NCI-H417, NCI-H82, NCI-H2171, NCI-H847, NCI-H1048, NCI-H146, NCI-H510A, NCI-H1963, and NCI-H1876) were kindly provided by Dr. Adi Gazdar at the University of Texas Southwestern. Cells were cultured in RPMI (GE Lifesciences) supplemented with 10% heat-inactivated FBS. Cell lines were tested for and free of mycoplasma, and cell line identities were verified using short tandem repeat genotyping as compared with the original primary sample material within the CCcells database: www.CCcells.org. Small cell lung cancer cells (2 × 10⁶ cells) were plated in 96-well plates for 24 h before treatment with inhibitors (1 nM–10 μM in 3× increment). Six replicates of each drug concentration were used. After 96 h of drug incubation, cellular viability was measured using the DIMSCAN assay, as outlined in previous studies (Kang et al., 2011 (link); Zhang et al., 2012 (link)).

Mohiuddin I.S., Wei S.J., Yang I.H., Martinez G.M., Yang S., Cho E.J., Dalby K.N, & Kang M.H. (2021). Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4–MK2 interactions. Biotechnology and bioengineering, 118(5), 1987-2000.

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Candida-Induced Macrophage Cytotoxicity

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Exponential-phase Candida in PBS was diluted into serum-free, phenol red-free RPMI (GE Healthcare) and added to macrophages at an MOI of 3 for 6 h before supernatants were taken and lactate dehydrogenase release was measured using the CytoTox nonradioactive cytotoxicity assay kit (Promega).

Pountain A.W., Collette J.R., Farrell W.M, & Lorenz M.C. (2021). Interactions of Both Pathogenic and Nonpathogenic CUG Clade Candida Species with Macrophages Share a Conserved Transcriptional Landscape. mBio, 12(6), e03317-21.

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Cultivation of Cell Lines for Research

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K562 cells expressing either CD19 or NGFR were kindly provided by Dr Steven Feldman (from the National Cancer Institute, Bethesda). EBV-transformed B cells were established from HDs’ PBMCs using B95–8 cell supernatant. The cell lines were maintained in culture in RPMI (GE Healthcare Life Sciences, HyClone) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific, HyClone), and 100 U/mL of Penicillin and 100 μg/mL Streptomycin (ThermoFisher Scientific, HyClone).

Magalhaes I., Kalland I., Kochenderfer J.N., Österborg A., Uhlin M, & Mattsson J. (2018). CD19 Chimeric Antigen Receptor T Cells From Patients With Chronic Lymphocytic Leukemia Display an Elevated IFN-γ Production Profile. Journal of immunotherapy (Hagerstown, Md. : 1997), 41(2), 73-83.

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Tumor-Derived Biopsy Culture on FiSS

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Cell lines were purchased from the American type culture collection (ATCC) and passaged no more than 25 times. Cells were cultured in a humidified incubator at 37C in a 5% CO₂ (Carbon Dioxide) atmosphere. Cells were cultured in tissue culture treated plates in the appropriate complete cell culture media [DMEM or RPMI (GE Healthcare) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin/streptomycin (GE Healthcare)]. The Fiber Inspired Smart Scaffold (FiSS) was prepared and seeded with cells as previously described.35, 36, 37 Tumor derived biopsy cultures: mice were injected subcutaneously on flanks with 3 million H1650 parental (right) and DT (left) cells. The tumors were harvested when they reached about 10mm in diameter and digested using MACS Miltenyi Biotec Mouse Tumor Dissociation kit and gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer's protocol. Tumor cell suspensions were then plated on the FiSS just as cell line suspensions.

Howell M.C., Green R., Khalil R., Foran E., Quarni W., Nair R., Stevens S., Grinchuk A., Hanna A., Mohapatra S, & Mohapatra S. (2019). Lung cancer cells survive epidermal growth factor receptor tyrosine kinase inhibitor exposure through upregulation of cholesterol synthesis. FASEB bioAdvances, 2(2), 90-105.

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Directed Migration of Human Mesenchymal Stem Cells

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Studies on directed migration of hMSCs were performed using the µ-Slide Chemotaxis^3D assay (Ibidi, Martinsried, Germany) as previously described by us in detail^{27 (link)}. Briefly, 0.5 × 10⁶ hMSCs suspended in SFM were placed into the observation channel of a µ-Slide. One medium reservoir was filled with 65 µL RPMI (GE Healthcare) supplemented with 100 ng/mL SDF-1α (PeproTech) and the second reservoir with RPMI only. The µ-Slide was then incubated for 24 h at 37 °C in an IX70 microscope (Olympus, Tokyo, Japan) that was connected to a SensiCam camera (PCO Imaging, Kelheim, Germany). Serial images of cell movement from a total of 30 hMSCs in each observation channel were captured by taking pictures every 20 min. The resulting images were converted to a stack using the ImageJ software (NIH). To determine the migration path of hMSCs, the image stacks were analyzed by deploying the Manual Tracking plug-in as well as the Chemotaxis and Migration Tool (both from Ibidi) following the recommendations of the provider. To quantify and characterize effects on both chemotactic and migratory potential of hMSCs, the Rayleigh test, the moving direction, and the forward migration index (FMI) were determined. The migration distance displays the summary of all cell center movements between images.

Egea V., Kessenbrock K., Lawson D., Bartelt A., Weber C, & Ries C. (2021). Let-7f miRNA regulates SDF-1α- and hypoxia-promoted migration of mesenchymal stem cells and attenuates mammary tumor growth upon exosomal release. Cell Death & Disease, 12(6), 516.

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Standardized Cell Culture Conditions

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All cell lines were cultured at 5% CO₂ and 37 °C with HEK293T, Saos2, and Skov3 cells cultured in DMEM high glucose (GE Healthcare Life Sciences, Hyclone, Amersham. UK, Cat# SH30081), HCT116 and U2OS cells in McCoy’s 5a (ThermoFisher Scientific, Pittsburgh, MA, USA, Cat# SH30200), and PC3 cells in RPMI (GE Healthcare Life Sciences, Hyclone, Cat# SH3009602) and 2 mM l-glutamine (GE Healthcare Life Sciences, Hyclone, Cat# SH3003402). All growth mediums were supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific, Gibco, Cat# 12657029) and 50 μg/ml of streptomycin (ThermoFisher Scientific, Gibco, Cat# 15140122) and 50 μg/ml of penicillin (ThermoFisher Scientific, Gibco, Cat# 15140122). All cell lines were purchased from ATCC, tested negative for mycoplasma, and their identity was verified via STR profiling.

Serviss J.T., Andrews N., Van den Eynden J., Richter F.C., Houtman M., Vesterlund M., Schwarzmueller L., Johnsson P., Larsson E., Grandér † D, & Pokrovskaja Tamm K. (2018). An antisense RNA capable of modulating the expression of the tumor suppressor microRNA-34a. Cell Death & Disease, 9(7), 736.

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Isolating and Characterizing Myeloid Leukemia Cells

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Steady-state apheresis units were purchased from AllCells and peripheral blood mononuclear cells (PBMCs) were isolated using lymphoprep density gradient. Before cryopreserving the PBMCs, expression of HLA-A2 was assessed by flow cytometry. In this study, T cells were cultivated in T cell medium composed of 50% RPMI (HyClone, GE Healthcare), 40% Click’s media (Millipore Sigma), 10% heat-inactivated fetal bovine serum (FBS; HyClone, GE Healthcare), 2 mM Glutamax (GIBCO, Thermo Fisher Scientific), and penicillin/streptomycin (P/S; GIBCO, Thermo Fisher Scientific). The AML cell lines KG1a and THP-1 and the large cell immunoblastic lymphoma cell line SR were purchased from American Type Culture Collection (ATCC). KG1a was cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; HyClone, GE Healthcare) base medium and RPMI base medium was utilized to culture the THP1 and SR cell lines, with both mediun containing 10% FBS plus P/S and 2mM Glutamax. For in vitro and in vivo experiments, KG1a and THP-1 cell lines were transduced with retroviral vector to express EGFP. The HLA-A∗0201 TAP-deficient cell line T2 was purchased from ATCC and maintained in growth culture media composed of IMDM plus 20% of FBS and 2 mM Glutamax.

Raskin S., Van Pelt S., Toner K., Balakrishnan P.B., Dave H., Bollard C.M, & Yvon E. (2021). Novel TCR-like CAR-T cells targeting an HLA∗0201-restricted SSX2 epitope display strong activity against acute myeloid leukemia. Molecular Therapy. Methods & Clinical Development, 23, 296-306.

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Cell Culture Conditions for Diverse Immune Cells

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EBV–B cell lines were cultured in RPMI (Invitrogen), and SV40-fibroblasts and 293T HEK cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Saimiri T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN-Biotech GmbH), supplemented with 10% FBS, 350 µg/ml glutamine, 100 µg/ml gentamycin, and 10 U/ml h IL-2 (Roche). PBMCs were isolated from blood by Ficoll-Hypaque density gradient centrifugation (GE Healthcare) and cultured at a density of 10⁶ cells/ml in RPMI supplemented with 10% FBS. Stimulations were performed, at the doses stated and for the indicated times, with IL-12 (R&D Systems), BCG (donated by C. Nathan, Weill Cornell Medical College, New York, NY), IL-18 (R&D Systems), PMA/ionomycin (Sigma-Aldrich), IL-2 (BD), IL-23 (R&D Systems), IFN-γ (Imukin; Boehringer Ingelheim), IL-27 (R&D Systems), IFNα-2b (Intron A; Schering Plough), IFNβ-1b (PeproTech), LPS (Sigma-Aldrich), IL-21 (R&D Systems), IL-29 (R&D Systems), IL-28B (R&D Systems), IL-10 (R&D Systems), IL-6 (R&D Systems), hyper–IL-6 (hIL-6; donated by S. Rose-John), and LIF (EMD Millipore). hIL-6 was prepared as described elsewhere (Fischer et al., 1997 (link)).

Kreins A.Y., Ciancanelli M.J., Okada S., Kong X.F., Ramírez-Alejo N., Kilic S.S., El Baghdadi J., Nonoyama S., Mahdaviani S.A., Ailal F., Bousfiha A., Mansouri D., Nievas E., Ma C.S., Rao G., Bernasconi A., Sun Kuehn H., Niemela J., Stoddard J., Deveau P., Cobat A., El Azbaoui S., Sabri A., Lim C.K., Sundin M., Avery D.T., Halwani R., Grant A.V., Boisson B., Bogunovic D., Itan Y., Moncada-Velez M., Martinez-Barricarte R., Migaud M., Deswarte C., Alsina L., Kotlarz D., Klein C., Muller-Fleckenstein I., Fleckenstein B., Cormier-Daire V., Rose-John S., Picard C., Hammarstrom L., Puel A., Al-Muhsen S., Abel L., Chaussabel D., Rosenzweig S.D., Minegishi Y., Tangye S.G., Bustamante J., Casanova J.L, & Boisson-Dupuis S. (2015). Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome. The Journal of Experimental Medicine, 212(10), 1641-1662.

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