Macs gmp t cell transact

Manufactured by Miltenyi Biotec

Sourced in Germany

The MACS GMP T Cell TransAct is a lab equipment product from Miltenyi Biotec. It is designed to activate and expand T cells ex vivo.

Automatically generated - may contain errors

Lab products found in correlation

7 protocols using macs gmp t cell transact

T Cell Activation and Poxvirus Infection

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were first plated at 2 × 10⁶ cells mL^–1 of TexMACS medium (Miltenyi Biotec) supplemented with 20 U mL⁻¹ human IL‐2 in 6‐well plates, and T cells were stimulated for 2 days by adding MACS GMP T Cell TransAct (final dilution 1/17.5; Miltenyi Biotec). PBMCs were then seeded at 2 × 10⁵ and 1 × 10⁵ cells per well in 96‐well plates for 1‐ and 5‐day cultures, respectively, and incubated with recombinant PCPV‐GFP (crude SN), MVA‐GFP or VV‐GFP at a MOI of 1 in TexMACS medium without cytokine. Half the medium was replaced at day 1 and day 4 post‐infection in 5‐day cultures. Cells were stained and analysed by flow cytometry as described for experiments studying poxvirus tropism. The percentage of GFP⁺ cells and the total cell count were measured for CD8 (CD3⁺CD4⁻CD19⁻) and CD4 (CD3⁺CD4⁺CD19⁻) T subsets.

Ramos R.N., Tosch C., Kotsias F., Claudepierre M., Schmitt D., Remy‐Ziller C., Hoffmann C., Ricordel M., Nourtier V., Farine I., Laruelle L., Hortelano J., Spring‐Giusti C., Sedlik C., Le Tourneau C., Hoffmann C., Silvestre N., Erbs P., Bendjama K., Thioudellet C., Quemeneur E., Piaggio E, & Rittner K. (2022). Pseudocowpox virus, a novel vector to enhance the therapeutic efficacy of antitumor vaccination. Clinical & Translational Immunology, 11(5), e1392.

+ Open protocol

+ Expand

Isolation and Expansion of Human T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Buffy coats and non-mobilized leukapheresis were obtained from healthy volunteer donors from the DRK Dortmund and Ulm. Human PBMCs were purified by density gradient centrifugation using Pancoll solution (Pan-Biotech, Aidenbach, Germany). Primary human T cells were isolated from PBMCs by negative bead selection according to manufacturer’s recommendations (Pan T cell isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and activated using MACS GMP T Cell TransAct (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured in TexMACS GMP medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL of recombinant human IL-7 and 12.5 ng/mL of recombinant human IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were transduced using lentiviral vectors 24 h after stimulation using an MOI of 5 to maintain the vector copy number of the drug product below 5. T cells were washed 3 days after stimulation to remove MACS GMP T Cell TransAct and lentiviral particles. T cells were expanded for 13–14 days with the addition of fresh culture medium every 2–3 days, before the experiments were started. Timing of T cell generation, including activation, transduction, and addition of medium was kept consistent for experiments comparing multiple donors and CAR constructs.

Kotter B., Engert F., Krueger W., Roy A., Rawashdeh W.A., Cordes N., Drees B., Webster B., Werchau N., Lock D., Dapa S., Schneider D., Ludwig S., Rossig C., Assenmacher M., Mittelstaet J, & Kaiser A.D. (2021). Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors. Cancers, 13(19), 4741.

+ Open protocol

+ Expand

CAR T Cell Production Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lentiviral supernatant was produced after triple transfection of the HEK293T cell line. Experimental CAR T cells were produced from healthy donors as previously described [11 (link)]. Briefly, cells were transduced after an initial activation and CD3+ MACS-selection step using CD3/CD28 beads following an expansion period in the presence of IL-2. Preclinical-grade IL-1RAP CAR T cells were also produced from healthy donors in a Prodigy device (Miltenyi Biotech) according to the manufacturer’s protocol. Briefly, selected CD4+ and CD8+ cells were activated with CD3/CD28+ IL-7/IL-15 (MACS GMP T Cell TransAct, Miltenyi Biotech) before being transduced and expanded for 9 days in a closed tubing set and TexMACS medium (Miltenyi Biotech). In both cases, genetically modified T cells were purified to increase purity to > 99%.

Haderbache R., Warda W., Hervouet E., da Rocha M.N., Trad R., Allain V., Nicod C., Thieblemeont C., Boissel N., Varlet P., Agha I.Y., Bouquet L., Guiot M., Venet F., Sujobert P., Roussel X., Rouzaire P.O., Caillot D., Casasnovas O., Bories J.C., Bachy E., Caillat-Zucman S., Deschamps M, & Ferrand C. (2021). Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients. Journal of Translational Medicine, 19, 265.

+ Open protocol

+ Expand

TALEN mRNA Transfer for T Cell Modification

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs-derived T cells or CD4/8 selected T cells were thawed and recovered for 24 h with TexMACS GMP medium supplemented with 12.5 ng/mL of recombinant hIL-7 and 12.5 ng/mL of recombinant hIL-15. On the same day or 24 h later, T cells were activated with MACS GMP T cell TransAct (Miltenyi Biotec), according to the manufacturer’s instructions, for 3 days prior to electroporation. For TALEN mRNA transfer, 1 × 10⁶ activated T cells were electroporated with 7.5 μg of each TALEN mRNA, unless stated otherwise, in a final volume of 50 μL CliniMACS electroporation buffer (Miltenyi Biotec) in the test cuvette adaptor using the electroporation settings listed in Table 1.

Alzubi J., Lock D., Rhiel M., Schmitz S., Wild S., Mussolino C., Hildenbeutel M., Brandes C., Rositzka J., Lennartz S., Haas S.A., Chmielewski K.O., Schaser T., Kaiser A., Cathomen T, & Cornu T.I. (2020). Automated generation of gene-edited CAR T cells at clinical scale. Molecular Therapy. Methods & Clinical Development, 20, 379-388.

+ Open protocol

+ Expand

Isolation and Transduction of CD4 and CD8 T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Lymphoprep, Fresenius) gradient separation. T cells were isolated using the Pan T-cell isolation kit (Miltenyi), CD4 and CD8 fractions were selected with CD4 and CD8 Microbeads (Miltenyi) and stimulated with MACS-GMP T-Cell TransAct (Miltenyi). Both subsets were transduced with a bidirectional LV encoding for either a CD19.CAR.28z or a CD19.CAR.BBz in sense and the truncated LNGFR (ΔLNGFR marker gene in antisense and kept in culture in TeXmacs medium (Miltenyi) supplemented with interleukin (IL)-7 and IL-15 (Miltenyi). Untransduced (UT) CD4 and CD8 T cells were employed as control and separately kept in culture. CAR+T cells were enriched by sorting through magnetic labeling of ΔLNGFR. Phenotypic and functional analyses were performed at the end of manufacturing.

Bove C., Arcangeli S., Falcone L., Camisa B., El Khoury R., Greco B., De Lucia A., Bergamini A., Bondanza A., Ciceri F., Bonini C, & Casucci M. (2023). CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses. Journal for Immunotherapy of Cancer, 11(1), e005878.

+ Open protocol

+ Expand

Manufacturing and Characterization of CAR T Cells from Healthy and Leukemia Donors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Buffy coats from healthy donors were obtained after written informed consent and IRB approval. CD45RA⁺CD62L⁺ T_N/SCM were isolated by FACS. B-ALL samples were selected on the basis of the disease classification (type B) and all patients received chemotherapeutic treatment. Patient-derived CD4⁺CD8⁺ T_BULK and CD4⁺CD8⁺CD62L⁺CD45RA⁺ T_N/SCM were isolated by FACS from peripheral blood mononuclear cells. T_BULK and T_N/SCM, derived from either healthy donors or patients, were stimulated through MACS-GMP T Cell TransAct (Miltenyi Biotec) and transduced with a bidirectional lentiviral vector encoding either CD19.CAR.28z or CD19.CAR.BBz and the LNGFR marker gene. Bidirectional lentiviral backbones were provided by Luigi Naldini (San Raffaele-Telethon Institute for Gene Therapy, Milan, Italy). Cells were maintained in culture in TexMacs medium (Miltenyi Biotec), supplemented with low-dose IL-7/IL-15 (Miltenyi Biotec) for 15 days. Healthy donor CAR⁺ cells were enriched by sorting through magnetic labeling of the LNGFR marker gene. Phenotypic and functional analyses of each CAR T cell product were performed at the end of manufacturing.

Arcangeli S., Bove C., Mezzanotte C., Camisa B., Falcone L., Manfredi F., Bezzecchi E., El Khoury R., Norata R., Sanvito F., Ponzoni M., Greco B., Moresco M.A., Carrabba M.G., Ciceri F., Bonini C., Bondanza A, & Casucci M. (2022). CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. The Journal of Clinical Investigation, 132(12), e150807.

+ Open protocol

+ Expand

Expansion and Transduction of CD19-CAR T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

On day 0, CD45RA⁻CD14⁻ T cells (2×10⁶ cells/mL) were cultured in X-VIVO15 media (Lonza) containing 5% Human AB serum (Valley Biomedicals), 10 ng/mL each of rhIL-7 (Miltenyi Biotec) and rhIL-15 (Miltenyi Biotec) and activated with 1:17.5 v/v of MACS GMP T Cell TransAct (Miltenyi Biotec) reagent for 18 to 24 hours in 6 well plates. On day 1, activated T cells were transduced with clinical-grade CD19-CAR vector in the presence of 4 μg/mL protamine sulfate for 18 to 24 hours. LentiBOOST (Sirion Biotech) is also added at this step in some groups. On day 2, cells were resuspended in X-VIVO15 medium containing 5% Human AB serum, 10 ng/mL each of rhIL-7 and rhIL-15 (complete media), and transferred to G-Rex-6M culture plates (Wilson Wolf) with a total volume of 100 mL for each well. On day 5, rhIL-7 (10 ng/mL) and rhIL-15 (10 ng/mL) were added again to the medium. On day 7 or 8, expanded T cells were harvested, washed with plasmaLyte A (Baxter) containing 4% HSA, and cryopreserved in plasmaLyte A containing 6% pentastarch (Preservation Solutions), 5.75% HSA, and 5% DMSO using a controlled-rate freezer.

Gronowski A.M., Copper S., Baorto D, & Murray P.R. (2000). Reproducibility Problems with the Abbott Laboratories LCx Assay for Chlamydia trachomatis and Neisseria gonorrhoeae. Journal of Clinical Microbiology, 38(6), 2416-2418.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!