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® 3 High-Throughput Cytometry Platform
The iQue® 3 High-Throughput 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 High-Throughput Cytometry Explore Phenotype and Function Assays
Incucyte® Live-Cell Analysis Platform
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:
- Immune Cell Killing Assays
- Immune Cell Activation
- Live Cell Immunocytochemistry
- Cell Health and Proliferation
- Cell Culture QC
- Cell Morphology
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.
Benefits of Sartorius Function and Phenotypic Solutions:
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® 3 High-Throughput Cytometry Platform.
Assess multiple cell parameters faster, with fewer cells and less reagents.
- Immune cell killing
- Immune cell assessment
- Cytokine profiling
iQue® 3 High-Throughput Cytometry and Incucyte® Live-Cell Analysis platforms 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.
Sartorius’ quantitative functional and phenotypic cell-based assays provide insights that translate into clinical outcomes. The iQue® 3 High-Throughput Screening 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.
Frequently Asked Questions
The iQue® 3 High-Throughput 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.