CAR-T
Relevant Instruments:
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Current immuno therapies leverage a patient’s system against cancer, lupus, or other autoimmune diseases. Chimeric antigen receptors (CARs) have been used successfully to genetically modify human immune cells to enhance detection of a person’s own cancer cells.
Monitoring the CAR process in cell culture has been accomplished by image-based or coulter-based cell counting followed by cellular analysis using fluorophore tagged antibodies and flow cytometry. Rather than using two different instruments for CAR analysis, only ORFLO’s Moxi GO II has the power to perform both accurate counting and fluorescent analysis with speed, precision, and high performance sample differentiation.
Initial PBMC Checks
Fig. 1: Immune cell counting and viability from a fresh PBMC sample using a Moxi GO II
These sorts of therapies are typically performed using T cells, and as such CAR-T is the most prevalent procedure used. The first step in any CAR-T workflow is assessing the sample extracted from the patient. Microfluidics-based Coulter counting technology, which is the core of all the Moxi systems, enables the highest quality data and better reproducibility with CVs < 5%. With the ability to characterize samples to 0.1µm, Moxi systems can readily distinguish between cell populations in the PBMC sample that other cell counters that have a typical resolution of ~4 µm can’t (Fig. 1). Coupled with dual viability staining capabilities, getting an accurate picture of the immune cell content is simple and reliable.
T Cell Activation Monitoring
Fig. 2: Active Dynabead interaction with T Cells using a Moxi GO II
CD3/CD28 Dynabeads are one of the most commonly used methods to activate T cells ex-vivo after they have been plated in cell culture. With FITC-conjugated goat anti-mouse IgG, the Moxi Go II can characterize the activated T cell population based on both size and Dynabead interaction using fluorescence via the 525/40nm green channel (Fig. 2). Because of this two dimensional analysis and data output, it is easy to distinguish which T cells are interacting with Dynabeads.
Fig. 3: Identification of active T cells using dual-color fluorescent profiling on a Moxi GO II
Once the Dynabeads have been cleared and the T cells have begun the activation process, monitoring changes in both physiology and gene expression profile is critical. With the Moxi Go II, using a dual fluorophore profiling of the cell population with both the 525/45nm and 561nm/LP channels quickly identifies T cells, and quantifies the subset that are expressing an activation marker like CD25 (Fig. 3).
CAR-T Cell Expansion
Fig. 4: Identification of active T cells using a combination of fluorescence labeling and cell sizing with a Moxi GO II.
Final QC Checks
Once the CAR-T cells have been activated for reintroduction to the patient, final counting and viability QC checks are critical. Moxi GO II provides a simultaneous count of thousands of cells with the use of either ORFLO’s single color or two color viability reagent. Moxi systems also have CFR Part 11 features if needed for a validated environment within the laboratory.
Summary
For CAR-T research and product development, Moxi GO II is the trusted instrument to achieve the highest quality data and sample differentiation with a faster time to results and with CVs < 5%. From initial PBMC counting to viability checks, including monitoring dynabead assays and quantifying cells as they activate and expand, Moxi GO II combines the power of a cell counter and a flow cytometer in less than 10 square inches. At the price of a flow cytometer’s one-year service contract, unlock higher performance and convenient cellular analysis with ORFLO’s Moxi systems.