Cell Therapy CAR-T cell attacking cancer cell

Functional and Phenotypic Assessment of CAR-T Cell Functions

CAR-T Cells: Functional and Phenotype Screening

Once the design and assessment of the chimeric antigen receptor (CAR) construct is complete and the process is progressing towards the expansion phase, phenotype and function of the cells needs to be quantified to ensure required cell characteristics are maintained.

Sartorius offers in vitro, cell-based functional and phenotypic assay platforms which enable quantitative and predictive evaluation of key CAR-T cell functions facilitating the discovery of effective and novel CAR-T therapeutics.

  • Assessment of parameters that translate into in vivo function
  • Better understanding of T-Cell biology
  • Streamlining the discovery of effective CAR-T candidates

Functional and Phenotype Screening Solutions

iQue® Advanced Flow Cytometry Platform

iQue 3 and computer monitor

The iQue® advanced flow cytometry platform utilizes a fixed wide dynamic range allowing for the assessment of phenotype and functional analysis of CAR-T cells eliminating the discrepancy of different time points or the need to split samples for subsequent analysis. The high-throughput platform provides assays that:

  • Deliver reproducible and biologically relevant results
  • Assess the cell´s functional reactivity to the target of interest
  • Assess the phenotype of cells in combination with functional readouts, i.e., estimate effector cell activation and cytotoxic killing
  • Enable simultaneous quantification of secreted cytokine levels along with phenotype from the same sample
  • Quantify tumor expression of antigen and immunosuppressive molecules

Featured Assay: iQue® Human T Cell Activation Cell and Cytokine Profiling Kit

The iQue® Human T Cell Activation Cell and Cytokine Profiling Kit assesses the activation status of T cells while providing information about their health and role in cytokine secretion. This one-wash assay requires minimal hands-on time, and measures cells and beads to provide the following biological insights:

  • Live immune cells are distinguished from dead cells by staining with a fluorescent membrane integrity dye.
  • Cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+ T cells, CD3- non-T cells, CD4+ T helper cells, CD8+ T cytotoxic cells.
  • The T cell phenotypes are profiled for the expression of 3 activation markers, CD69 (early), CD25 (late), and HLA-DR (even later, with strong stimulation).

This optimized workflow enables the measurement of effector cytokines secreted by activated T cells, Th1 cytokine IFNγ/ multifunctional cytokine TNFα in the same assay well.

Explore iQue® Flow Cytometry Explore Phenotype and Function Assays

Incucyte® Live-Cell Analysis Platform

incucyte neural activitity

The Incucyte® Live-Cell Analysis System is designed to efficiently capture cellular changes where they happen - in the incubator. For functional and phenotypic assessment of T-Cells, it enables in-depth understanding of the underlying biology, such as the mechanisms of immune cell activation, proliferation and killing.

  • Improve biological insights using integrated image acquisition and analysis to identify state of cell activation
  • Perform non-invasive, non-disruptive and unbiased analysis of cell health, phenotype, subsets, and function
  • Discover new insights into the dynamic interactions of immune and cancer cells

Accelerate your next discovery with the Incucyte®  suite of live-cell applications:

Featured Live-Cell Analysis Assay: CD19-Targeted CAR-T Cell Killing of Antigen Positive Target Cells

Demonstrating the Specific Killing Profile of CD19-Targeted CAR-T Cells

Incucyte® immune cell killing assay was used for quantification of the functional activity of CAR-T cells. Quantification of images demonstrates a clear CAR-T cell density related decrease in Ramos target cells over time. 

A co-culture of Incucyte® Nuclight Green labeled Ramos or Jurkat cells with either anti-CD19 CAR-T or control T cells was set up at various T:E ratios in a 96-well plate. Cultures were imaged in the Incucyte® every 4 hours over 4 days and quantified for green fluorescent area. Images taken at 72 hours (A) show a clear- reduction in green area of Ramos cells in combination with CAR-T cells (1:2 T:E). Time course graphs (B and C) demonstrate increased killing of antigen positive Ramos cells compared to antigen negative Jurkat cells. Data shown as mean ± SEM of 3 wells.

Explore Live Cell Assays

Benefits of Sartorius Function and Phenotypic Solutions:

less water
less water
multiple data points
cell insights
Save Precious Samples

Avoid the need for multiple assays and get immunophenotyping data with measurements of cell health, and bead-based cytokine secretion, in a single experiment with iQue® Advanced Flow Cytometry Platfrom.

Assess multiple cell parameters faster, with fewer cells and less reagents.

  • Immune cell killing
  • Immune cell assessment
  • Cytokine profiling
Instant Access to Real-Time Data

iQue® Advanced Flow Cytometry and Incucyte® Live-Cell Analysis Systems integrate powerful software for instant access to real-time data, better visualization tools and dynamic gating capabilities, so it’s easy to interpret complex datasets. 

Translational Insights

Sartorius’ quantitative functional and phenotypic cell-based assays provide insights that translate into clinical outcomes. The iQue® high-throughput flow cytometer and Incucyte® live-cell analysis system can be combined to deliver visualization and quantification of immune cell driven tumor killing and detailed cell characteristics like biomarker phenotype, tri-lineage differentiation capacity, viability, exhaustion, and killing efficacy. 

Have Questions? Contact an Expert

Resources for Functional and Phenotype Screening

Phenotypic and Functional Characterization of CAR-T Cells white paper
White Paper

Phenotypic and Functional Characterization of CAR-T Cells

Learn about streamlined in vitro assays used for phenotyping and functional assessment of modified CAR-T cells, with three case studies.

Access White Paper
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Poster

Quantify T Cell Response in 3D Tumor Spheroids

Learn two 3D tumor spheroid-based solutions to study immune cell-tumor interactions including targeted activation and killing by anti-HER2 CAR-T cells...

Access Poster
eBook

iQue® Next-Gen CAR-T Development App Compendium

Discover how advanced flow cytometry enables you to expedite CAR-T discovery.

View eBook

Frequently Asked Questions

The iQue® Advanced Flow Cytometry Platform performs high-throughput assays with cells and beads in suspension, enabling a powerful multiplexing approach through encoding technology. Encoding technology allows screening against multiple target antigens in the same experiment because multiple populations of cells or beads bearing different antigens of interest can be combined into the wells of assay plates. For example, it allows measurement of T cell activation by proliferation, cytokine secretion, and changes in subtype populations (immunophenotyping) in the same analysis.

The Incucyte® Advanced Label-Free Classification Analysis Software Module provides a turnkey solution using an established label-free protocol for acquisition and advanced analysis protocols for simplified identification of target or immune cell populations based on morphologies.

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