CAR T cell research solutions

  • Isolation of donor T cells
  • Efficient T cell activation, gene transfer, and expansion
  • CAR T cell flow analytics 
  • Protocols and research hacks 

Webinars

Tips and tricks to get your CAR T cell research moving.

Webinar
Improve your CAR T cell research

Introduction into Miltenyi Biotec’s workflow for CAR T cell manufacturing for research use, as well as possibilities to easily translate the workflow into the clinic.

Webinar
Our latest solutions to improve your CAR T cell research 

Following on from our renowned webinar “Improve your CAR T cell research”, this webinar will cover our latest product solutions and strategies to efficiently move your CAR T cell research forward. Tune in and learn how to: Isolate fully functional T cells directly from whole blood and blood products, boost lentiviral T cell transduction efficiency, easily activate and expand your T cells without hassle, and advance your IPC and QC with our latest flow analysis solutions.

Generate and analyze CAR T cells – step by step  

Standardized workflow for successful CAR T cell engineering 

Take advantage of our experience and expertise in CAR T cell research and clinical manufacturing and benefit from our straightforward, translational workflow. 
Start with our tailored solutions for research and development of your engineered T cells. Once your small-scale workflow has been successfully developed, you can transfer your process to the CliniMACS Prodigy® Platform using the corresponding MACS® GMP Portfolio for larger scale production. 
Seamless, quick, and easy from bench to bedside. 

1. T cell enrichment
 

Starting your workflow with a well-defined, enriched T cell population is key to obtain robust, standardized, and reproducible results. Depending on the starting material, PBMCs or blood products, you choose either standard MACS® MicroBeads and Isolation Kits or the innovative StraightFrom® Technology. But independent of the approach you decide for, the basis is always the proven and most cited MACS MicroBead Technology. In addition, to enable a seamless transition of your research workflow to the CliniMACS Prodigy® Platform, our MACS MicroBeads are available in MACS GMP Quality. 

Separation of T cells from PBMCs 

Positive selection enables straightforward isolation of CD4+ and CD8+ T cells from PBMCs using MACS MicroBeads and MicroBead Kits. 

Purity of CD4+ and CD8+ T cells before and after enrichment with MACS Technology.
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T cell enrichment from PBMCs increases reproducibility

Simultaneous enrichment of CD4+ and CD8+ T cells makes for a homogeneous starting cell population. T cell enrichment minimizes donor variability, improves transduction efficiency, and avoids vector consumption by unwanted cells, allowing for robust, standardized, and reproducible results. 
 

If subsequent magnetic sorting steps are required, choose untouched cell isolation: MACS T Cell Isolation Kits allow for isolation of unlabeled target cells via depletion of non-target cells.

Separation of human T cells directly from blood products

Increase throughput and productivity with StraightFrom® Technology for T cell isolation directly from whole blood and blood products (buffy coat, LRSC, and Leukopak®) without density gradient centrifugation or RBC lysis.

Immune cell composition before and after enrichment.
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Fast and easy T cell enrichment without density-gradient centrifugation 

Using the innovative StraightFrom MicroBead Technology facilitates the omission of the density-gradient centrifugation step for PBMC generation, saving processing time and reducing user variability. 

A comparison of standard MACS MicroBeads and StraightFrom MicroBead Kits reveal that regardless of the isolation approach, magnetic cell separation using MACS Technology ensures preservation of T cell functionality enabling full downstream compatibility for CAR T cell generation. This novel approach is automatable using the MultiMACS™ Cell24 Separator Plus to further increase throughput and productivity.

CAR T cell enrichment from patient blood

The MACSprep™ CD19 CAR MicroBead Kit has been developed for fast and convenient CAR T cell isolation from fresh blood samples.  

Enrichment of CD19 CAR T cells using the MACSprep CD19 CAR MicroBead Kit. 
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Enrichment of CAR T cells directly from whole blood samples 

Low frequency CAR T cells were enriched directly from whole blood samples without density gradient centrifugation using the MACSprep CD19 CAR MicroBead Kit for further flow cytometric, functional, and molecular analysis. 
 

2. T cell activation and expansion 
 

Physiological T cell activation is vital to start the proliferation and expansion process and facilitate an efficient T cell transduction. Optimal T cell expansion relies on the synergistic effect of T Cell TransAct™, TexMACS™ Medium, and MACS Cytokines.  

Activation and expansion of human CD4+ and CD8+ T cells using T Cell TransAct and TexMACS Medium.
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Robust activation of human T cells

T Cell TransAct enables biologically appropriate, robust activation of resting T cells via humanized CD3 and CD28 antagonists conjugated to a polymeric nanomatrix. T cell activation was verified by expression of T Cell activation marker CD25 and CD69. 

Moreover, with its unique features, T Cell TransAct is superior to conventional methods and has been developed to be used in an automated closed system.

  • Ready-to-use reagent quick and easy protocol.
  • No magnet needed, no bead removal.
  • Available in research grade and MACS GMP Grade.
     
Optimal T cell expansion relies on the synergistic effect of T Cell TransAct, TexMACS Medium, and MACS Cytokines.
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Highly efficient serum-free expansion of human T cells 

T cell expansion rates after activation with T Cell TransAct and culture in serum-free TexMACS Medium were compared with stimulation using a bead-based competitor product and RPMI medium. Analyses revealed identical expansion rates, even under serum-free conditions. Especially in clinical manufacturing, a serum-free T cell expansion is advantageous as human serum can be the limiting factor. Both media were supplemented with Human IL-2 IS, premium grade (50 U/mL). 
 

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Video:
T Cell activation and expansion made simple and convenient 

Watch the video and learn how T Cell TransAct can optimize your T cell research.
 

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Application protocol:  
Engineering of CAR T cells for research use 

Our application protocol describes a complete workflow for engineering CAR T cells for research. Including T cell isolation, activation, gene-transfer of the CAR construct, CAR T cell expansion, as well as phenotyping and functional analysis. 

3. Gene delivery
 

Transgene expression depends on gene transfer efficiency. However, viral transduction of suspension cells with retroviral vectors is often inefficient and requires the use of transduction enhancers.  

Using the synthetic, non-toxic soluble Vectofusin-1® with a simple and easy-to-follow protocol enhances the transduction efficiency of viral vectors.  

Viability and transduction efficiency of T cells in the presence and absence of Vectofusin-1®.
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Enhanced retroviral transduction of primary T cells 

Applying Vectofusin-1® directly to the cell culture medium increases the transduction efficiency and thus reduces the amount and cost of viral vector needed per experiment. Applicable for both, static and spinoculation protocols. 
 

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Scientific poster:  
Effective retroviral transduction of primary T cells and hematopoietic
stem cells using the soluble transduction enhancer: Vectofusin-1®

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4. CAR T cell flow analysis tools
 

As the success of CAR T cell therapies has been associated with the phenotype, activation status, and functional profiling of CAR T cells, the reliable and standardized characterization of those parameter is crucial. 

Our CAR T cell analytics portfolio provides all you need for reliable, reproducible, and standardized analysis. Use our high-quality reagents, instruments, and software to streamline your CAR T cell analysis – from basic research and discovery to IPC/QC in clinical manufacturing.

Tune into the recordings of Miltenyi Biotec´s Cell Therapy Analytics Day 2021 to discover lessons learned, master best practices, and overcome regulatory hurdles in order to simplify your cell manufacturing analytics

Related videos:

Standardizing flow cytometry assays in cell manufacturing 
Watch our three-part webinar series on automated tools and learn how to take your standardization efforts to the next level.

Phenotypic characterization

Our optimized and validated panels of fluorochrome-conjugated REAfinity™ Recombinant Antibodies and CAR Detection Reagents help to streamline the phenotypic characterization of CAR T cells. For maximum reproducibility and standardization of in-process-control (IPC) and quality control (QC) for your clinical manufacturing process, combine the StainExpress™ Dried Antibody Cocktails with the MACSQuant® Analyzer and the Express Modes. 
 

Validated panels for CAR T cell flow analysis

Benefit from Miltenyi Biotec Tested Panels (MBTP) including antibody panels, step-by-step protocols, and gating strategies to accelerate your CAR T cell flow analysis.

The CD19 CAR T Cell Express Mode panels.
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Simplified flow cytometry analysis with Express Modes

Express Modes are a unique add-on for the MACSQuantify™ Software, developed to simplify flow cytometric analysis and maximize reproducibility of data analysis. They automate the measurement and analysis of flow experiments via predefined experiment settings as well as acquisition and automated analysis.

Reliable detection and enumeration of CAR-expressing T cells

Our ready-to-use biotinylated CAR Detection Reagents have been developed for the detection of T cells that are engineered to express a specific CAR on the cell surface, e.g., CD19 CAR or CD22 CAR. 

Mechanism of tumor cell recognition and CAR T cell detection.
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  • Reliable CAR T cell identification and enumeration by flow cytometry.
  • Binding of antigen-based fusion protein to CAR T cell simulates in vivo binding of CAR T cell to tumor antigen.
  • Background-free analysis, no need for additional FcR blocking steps.
  • Available for CD19, CD22, BCMA CAR T cells.
     
Flow cytometry analysis of transduced T cells after expansion. 
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Detection of viable CAR T cells via flow cytometry

CD19 CAR Detection Reagent (Biotin) detects CD19 CAR T cells via recognition of the CD19 CAR molecule, here shown on day 12 after expansion. 

Functional CAR T cell analysis 

We offer flow cytometry solutions to improve the quality of potency assay results. They can, for example, be used for high-throughput and automated in vitro potency assays to assess CAR T cells’ antigen-specific killing, T cell phenotype, intracellular cytokine detection, as well as effector cytokine secretion.
 

Rapid Cytokine Inspector Kits offer the fastest combination of surface marker and intracellular cytokine staining

  • High-throughput multiparameter analysis of antigen-specific T cells.
  • Rapid identification and enumeration of cytokine-producing activated antigen-specific CD4+ and CD8+ T cells upon in vitro stimulation with the respective antigen or upon polyclonal restimulation.
  • Immunomonitoring of antigen-specific T cells.

Conventional staining to detect effector cytokine secreting CAR T cells

Detection of CAR T cells secreting effector cytokines IFN-γ, TNF-α, or IL-2.
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Conventional intracellular staining procedure using REAfinity Antibodies and CAR Detection Reagents in combination with the Inside Stain Kit allow for successful detection of CAR T cells that secrete effector cytokines such as IFN-γ, TNF-α, or IL-2. 

MACSPlex Cytokine Kits for quatification of up to 12 analytes in one supernatant

Gene-engineered T cells are assessed via cytokine secretion. 
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The accurate quantification of cytokines facilitates detailed examination of newly engineered CAR T cells. MACSPlex Cytokine Kits were designed for easy quantification of secreted cytokines in cell culture supernatant, making it easy to assess CAR T cell functionality.

  • Up to 12 human cytokines in one sample.
  • True mulitplexing.
  • Analysis can be done with every standard multi-color flow cytometer.
  • Optimized for automated measurement using the Express Modes.
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Application note
Maximize the efficiency of CAR T cell functionality assessment via flow cytometry 

Phenotyping, cytokine profiling, and cytotoxicity testing: multiple readouts within one single co-culture assay.

5. Ultra high content imaging for CAR T cell research


Ultra high content imaging allows for CAR T cell target discovery, in-depth tumor infiltration analysis, preclinical CAR T cell target safety as well as CAR T cell biodistribution.
 

Discovery of CAR target candidates

A new publication in Nature Communications reveals target candidates for CAR T cell–based immunotherapy of pancreatic adenocarcinoma. 

Explore the scientific publication.

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