Texmacs gmp medium

Manufactured by Miltenyi Biotec

Sourced in Germany

TexMACS GMP medium is a cell culture medium designed for the expansion and differentiation of T cells. It is manufactured under GMP (Good Manufacturing Practice) conditions to ensure consistent quality and safety for clinical applications.

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TexMACS medium (120 citations) TexMACS (54 citations)

23 protocols using texmacs gmp medium

SARS-CoV-2-specific T-cell Generation

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For preclinical studies SARS-CoV-2-VST were generated by culturing PBMC (1.25x10⁷) in a G-Rex5 (Wilson Wolf Manufacturing Corporation, St. Paul, MN, USA) with 50 mL of VST medium (90% TexMACS™ GMP medium [Miltenyi Biotec, GmbH], 2 mM GlutaMAX, and 10% human AB serum supplemented with IL7 [20 ng/mL], IL4 [800 U/mL] [R&D Systems, Minneapolis, MN, USA]) and pepmixes (2 ng/peptide/mL) and cultured for 10-16 days at 37°C in 5% CO₂. For clinical production, VST received a second stimulation with irradiated, autologous, pepmix-pulsed PBMC as antigen-presenting cells (4:1 APC:VST) and were cultured in IL-2-supplemented medium (100 U/mL). The VST lines were checked for identity, phenotype and sterility, and cryopreserved prior to administration. All cell culture manipulations were carried out in the Center for Cell and Gene Therapy GMP facility using current standard operating procedures. Products that met study-specific release criteria were released for clinical use.
Full details on VST phenotypic and functional characterization can be found in the Online Supplementary Materials.

Vasileiou S., Hill L., Kuvalekar M., Workineh A.G., Watanabe A., Velazquez Y., Lulla S., Mooney K., Lapteva N., Grilley B.J., Heslop H.E., Rooney C.M., Brenner M.K., Eagar T.N., Carrum G., Grimes K.A., Leen A.M, & Lulla P. (2022). Allogeneic, off-the-shelf, SARS-CoV-2-specific T cells (ALVR109) for the treatment of COVID-19 in high-risk patients. Haematologica, 108(7), 1840-1850.

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Automated Expansion of Human Regulatory T Cells

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Freshly isolated cells were seeded at 0.3–0.5 × 10⁶ cells/ml in G-Rex® 100 flasks (Wilson Wolf Manufacturing) containing TexMACS GMP Medium (Miltenyi Biotec) supplemented with 100 ng/ml MACS GMP Rapamycin (Miltenyi Biotec), 1,000 U/ml IL-2 (Proleukin S, Novartis Pharma), 5% cell therapy-grade pooled human AB serum (Blutspendedienst Tübingen, Germany) and MACS GMP ExpAct Treg beads (Miltenyi Biotec) at a cell to bead ratio of 1:4. Cells were cultured at 37°C and 5%CO₂. Cells were counted on days 5, 8, 10, and 12 and adjusted to 0.3–0.5 × 10⁶ cells/ml (day 5, 8), 0.3–1.0 × 10⁶ cells/ml (day 10) or 0.5–1.0 × 10⁶ cells/ml (day 12) by partial (50%) media replacement. Fresh medium contained all additives except ExpAct Treg beads. Cells were re-stimulated with fresh beads at a 1:1 ratio on day 8. For direct comparison to the automated culture, manual G-Rex® 10 flask cultures containing the same isolated Treg pool at 10-fold lower cell number and volume as compared to the automated culture were performed.

Marín Morales J.M., Münch N., Peter K., Freund D., Oelschlägel U., Hölig K., Böhm T., Flach A.C., Keßler J., Bonifacio E., Bornhäuser M, & Fuchs A. (2019). Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System. Frontiers in Immunology, 10, 38.

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Treg Cell Isolation and Activation Assay

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CD25⁺ Treg cells were isolated from the leukapheresis product of end stage renal patients as previously described [16 (link),17 (link),18 (link)]. Briefly, the leukapheresis product was conjugated for 10 min at 4 °C with the CliniMACS CD25 MicroBeads reagent (Miltenyi Biotec). CD25⁺ cells were isolated in the Clini MACS Plus system (Miltenyi Biotec, software program Enrichment 3.2), following the manufacturer’s instructions. Treg cells were identified as CD4⁺/CD25⁺/FOXP3⁺/CD127⁻ and isolated with a purity above 85% throughout the study. Tconv were sorted from PBMCs with the human CD4⁺ T cell isolation kit (StemCell Technologies, Cambridge, MA, USA). For initial stimulation of cells, Treg and/or Tconv cells were placed in culture at a concentration of 10⁶ cells/mL in Treg cell culture medium: TexMACS GMP medium (Miltenyi) supplemented with recombinant human Interleukin-2 (IL2, MACS GMP, Miltenyi, at 500 IU/mL) and MACS GMP ExpAct Treg (beads conjugated to CD28, Anti-Biotin, and CD3-Biotin monoclonal antibodies, Miltenyi) at a bead-to-cell ratio of 2:1. Cells were exposed to treatment conditions of vehicle control (DMSO 0.1%), RAPA (100 nM) or AZD8055 (20 nM) for 60 min prior addition of cell activation supplements. Additionally, half of cell culture media was routinely replaced every three days.

Gedaly R., Orozco G., Ancheta A.P., Donoho M., Desai S.N., Chapelin F., Khurana A., Lewis L.J., Zhang C, & Marti F. (2023). Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application. Cells, 12(16), 2066.

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Generation of Research-Grade MultiVSTs

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To generate research-grade multiVSTs for characterization studies, PBMCs (1.25x10⁷) were transferred in a G-Rex5M (Wilson Wolf Manufacturing Corporation, St. Paul, MN) with 50 ml of VST medium [90% TexMACS™ GMP medium (Miltenyi Biotec, GmbH), 2 mM GlutaMAX, and 10% human AB serum] supplemented with IL7 (20 ng/ml), IL4 (800 U/ml) (R&D Systems, Minneapolis, MN) and pepmixes (2 ng/peptide/ml) and cultured for 14 ± 2 days at 37°C, 5% CO₂ prior to cryopreservation. MultiVSTs were cryopreserved in freeze medium [45% RPMI + 45% Fetal Bovine Serum (FBS; Gibco) + 10% DMSO] at a concentration of 1.5-2x10⁷ cells/ml/cryovial. After overnight storage at -80°C in a Mr. Frosty freezing container (Thermo Fisher Scientific), cells were transferred to a liquid nitrogen tank for long-term storage in the vapor phase.

Koukoulias K., Papayanni P.G., Jones J., Kuvalekar M., Watanabe A., Velazquez Y., Gilmore S., Papadopoulou A., Leen A.M, & Vasileiou S. (2023). Assessment of the cytolytic potential of a multivirus-targeted T cell therapy using a vital dye-based, flow cytometric assay. Frontiers in Immunology, 14, 1299512.

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Rapid Expansion and Transduction of T Cells

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A leukapheresis product (STEMCELL Technologies) from a healthy donor was positively selected for CD4⁺ and CD8⁺ cells on the CliniMACS Prodigy (Miltenyi). The selected cells were cultured on the Prodigy in TexMACS GMP Medium (Miltenyi) with 3% (v/v) human AB serum (Valley Biomedical) and 200 IU/mL rhIL-2 (Prometheus Therapeutics and Diagnostics) using the Miltenyi T Cell Transduction Process for the CliniMACS Prodigy. Cells were activated on day 0 using GMP T cell TransAct (Miltenyi). Cells were transduced on day 1 with the MSCV-CAR1922-WPRE lentivirus (Lentigen) at an MOI of 40. Cells received a full culture wash of fresh medium on day 5, culture feed by medium addition on day 6, and culture feed by medium demi-depletion on day 7. Samples were removed from the culture on day 9 for RCL analysis prior to harvest on the same day.

Skrdlant L.M., Armstrong R.J., Keidaisch B.M., Lorente M.F, & DiGiusto D.L. (2017). Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G. Molecular Therapy. Methods & Clinical Development, 8, 1-7.

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Autologous CIK Cell Preparation

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Two complete aphereses of autologous peripheral blood mononuclear cells (PBMCs) were performed using a CS3000S cell separator (Baxter, Deerfield, IL, USA). The cell numbers apheresed were 1.28×10⁹ and 1.47×10⁹, respectively, and two aliquots were separated and cryopreserved from the second apheresis for later treatment. CIKs were prepared as previously described (8 (link)). Briefly, newly apheresed or thawed PBMCs were cultured in TexMACS™ GMP Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) with 1,000 U/ml IFN-γ (Beijing ShuangLu Pharmaceutical Co., Ltd., Beijing, China) added on day 0, 50 ng/ml antiCD3 (Hui Xin Qing Yuan Biotech Co., Ltd., Beijing, China) added on day 1, and 500 U/ml recombinant human IL2 included in the medium from day 1 onwards. From days 10 to 21, the cells were harvested on every other day for infusion.
Statistical analysis. Statistical analyses of the data were performed using GraphPad Prism software (San Diego, CA, USA).

Wang W., Wang W., Liu B., Suo Z., Zhou L, & Liu G. (2016). Effect of an immunomodulatory regimen for cancer prevention: A case report. Molecular and Clinical Oncology, 5(5), 540-544.

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Co-culture of naïve CD4+ T cells with NPC and NPE cells

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NPC and NPE cells were seeded into a 24-well plate with a density of 5×10⁴ cells/well in 1 mL complete RPMI-1640 24 hours prior to adding CD4+ naïve T cells. For the transwell-based co-culture system, 1 × 10⁵ naive CD4+ T cells were seeded into a 0.3 cm² transwell insert with 1 μm pore size, avoiding T cell migration but allowing cytokine exchange. For the direct co-culture system, 2 × 10⁵ naive CD4 + T cells were directly seeded into the 1.9 cm² 24-well plate together with C666 cells or NP69/NP460 cells. The negative control group has only 2 × 10⁵ CD4 + naïve T cells per well without any NPC and NPE cells seeded in the 24-well plate. Cells were cultured in 1.5 mL TexMACS™ GMP medium (Miltenyi) supplemented with 10% HI-FBS, 1% PS, 50 μM 2-mercaptoethanol (Gibco), 25 IU/mL IL-2 (Miltenyi) and 2 ng/mL TGF-β1 (PerproTech) for 3 days.

Gong L., Luo J., Zhang Y., Yang Y., Li S., Fang X., Zhang B., Huang J., Chow L.K., Chung D., Huang J., Huang C., Liu Q., Bai L., Tiu Y.C., Wu P., Wang Y., Tsao G.S., Kwong D.L., Lee A.W., Dai W, & Guan X.Y. (2023). Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction. Nature Communications, 14, 1912.

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Expansion of Regulatory T Cells

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In the BRC GMP Facility, cells were seeded in MACS GMP Cell Differentiation/Expansion Bags at 0.5 × 10⁶ cells/mL in TexMACS GMP Medium (Miltenyi Biotec, Germany), supplemented with 5% human serum containing 100 nM rapamycin (Rapamune) and activated with anti-CD3- and anti-CD28-coated beads (4:1 bead:cell ratio, MACS GMP ExpAct Treg Kit, Miltenyi Biotec, Germany). Human recombinant IL-2 (500 IU/mL; Proleukin) was added at day 4–6 and replenished every 2 to 3 days. The cells were rested for 4 days before restimulation. Stimulation occurred on days 12 and 24, during which time cells were pooled, fresh beads (1:1), rapamycin and IL-2 were added, and the suspension was re-seeded at 0.5 × 10⁶ cells/mL into new bags (250, 500, or 1,000 mL) For a schematic representation of the process see Figure 5. Expanded cells were harvested on day 36 and pooled. The ExpAct Treg expansion beads were depleted using the CliniMACS Plus System (Miltenyi Biotec) to form a bead-depleted cell population. A small aliquot of the cells was then taken for safety and functional analysis.

Fraser H., Safinia N., Grageda N., Thirkell S., Lowe K., Fry L.J., Scottá C., Hope A., Fisher C., Hilton R., Game D., Harden P., Bushell A., Wood K., Lechler R.I, & Lombardi G. (2018). A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials. Molecular Therapy. Methods & Clinical Development, 8, 198-209.

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Isolation and Expansion of Human Thymic Regulatory T Cells

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Thymic tissue fragments were mechanically disaggregated in TexMACS GMP medium (Miltenyi Biotec) with the gentleMACS Dissociator (Miltenyi Biotec). Total thymocytes obtained were filtered through a 40 µm pore, and CD25⁺ cells were immunomagnetically selected using human CD25 Microbeads II and LS columns (Miltenyi Biotec). After isolation, CD25⁺ (thyTreg day 0) and CD25- (thyTconv day 0) were cultured in TexMACS GMP medium supplemented with 600 U/ml IL-2 (Miltenyi Biotec) at 10⁶ cells/ml at 37°C and 5% CO₂. Cells were stimulated with T Cell TransAct (Miltenyi Biotec), a polymeric nanomatrix, to activate and expand human T cells via CD3 and CD28 following the manufacturer’s instructions. On day 3, half of the medium was removed and replaced with fresh TexMACS GMP medium supplemented with 600 U/ml IL-2. Cells were monitored on days 4, 5 and 6 and passage was performed when required. On day 7, cells were harvested, and their phenotype, functionality and stability were analyzed (Figure 1A). Additionally, dried cell pellets and culture supernatants were stored at −80°C for further DNA methylation studies and cytokine quantification respectively.

Bernaldo-de-Quirós E., Cózar B., López-Esteban R., Clemente M., Gil-Jaurena J.M., Pardo C., Pita A., Pérez-Caballero R., Camino M., Gil N., Fernández-Santos M.E., Suarez S., Pion M., Martínez-Bonet M, & Correa-Rocha R. (2022). A Novel GMP Protocol to Produce High-Quality Treg Cells From the Pediatric Thymic Tissue to Be Employed as Cellular Therapy. Frontiers in Immunology, 13, 893576.

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Preparation of 19BBz CAR T cells

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Human 19BBz CAR T cells targeting human CD19 were prepared as previously described.^[²³
^] HEK293T cells were transfected with lentiviral 19BBz CAR expression plasmid together with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) using Lentifit (Hanbio, China) transfection reagents. Lentivirus‐containing supernatant was harvested 48 and 72 h after transfection, concentrated by ultracentrifugation, and filtered with a 0.22 µm filter. Primary human T cells from the PBMCs of healthy donors were stimulated with anti‐CD3/CD28 beads at a 1:1 ratio (Miltenyi Biotec, Bergisch Gladbach, Germany) in TexMACS GMP medium (Miltenyi Biotec) supplemented with 10% FBS and 50 IU ml⁻¹ IL‐2 for 2 days, followed by transduction with concentrated 19BBz CAR lentivirus containing supernatant through spin transduction for 2 h at 32 °C. Then, transduced cells were passaged daily with a fresh culture medium for another 5 days. Transfection efficiency was determined by GFP expression of the CAR plasmid using flow cytometry.

Ma S., Wu Q., Wu W., Tian Y., Zhang J., Chen C., Sheng X., Zhao F., Ding L., Wang T., Zhao L., Xie Y., Wang Y., Yue X., Wu Z., Wei J., Zhang K., Liang X., Gao L., Wang H., Wang G., Li C, & Ma C. (2024). Urolithin A Hijacks ERK1/2‐ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity. Advanced Science, 11(18), 2310065.

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