CAR T cell research solutions

Enrichment of CD4⁺ and CD8⁺ T cells by positive selection using a mixture of CD4 and CD8 MicroBeads (1:1) in combination with MACS Technology, either manually or automatically using the autoMACS Pro. Cells were fluorescently stained with CD45-VioBlue®, CD4-VioGreen™, and CD8-APC-Vio® 770 and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and 7-AAD fluorescence.

Immune cell composition of independent healthy donors (n=3) was determined before and after T cell enrichment with StraightFrom Buffy Coat CD4/CD8 MicroBeads using the MACSQuant Analyzer 10 and applying the CAR T Cell Express Mode Package (Immune_Cell_Composition_human). After T cell enrichment using StraightFrom Buffy Coat CD4/CD8 MicroBeads, isolated CD4⁺ and CD8⁺ T cells exhibited a purity of 95%, 96%, and 94% for the three donors, whereas the percentage of other immune cell subsets (NK, NKT, monocytes, and B cells) amounted to 5%, 4%, and 6%, respectively.

CD19 CAR T cells were spiked into a whole blood sample from a healthy donor and isolated using the MACSprep CD19 CAR MicroBead Kit, one LS Column, one MS Column, a MACSmix™ Tube Rotator, and a QuadroMACS™ Separator. Cells were fluorescently stained with CD19 CAR Detection Reagent, human, Biotin, Biotin Antibody-PE, CD45-VioBlue®, 7-AAD Staining Solution, CD3-FITC, and analyzed by flow cytometry using the MACSQuant Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and 7-AAD fluorescence. The purity of CD19 CAR T cells after separation is defined by the percentage of CD19 CAR T cells (viable CD45^dim/+, 7-AAD^– cells). The purity of CD19 CAR T cells varies due to the variation of human samples, experimental set-up, and starting frequency of CD19 CAR T cells.

Human T cells were isolated using the Pan T Cell Isolation Kit, human. Purified T cells were stimulated with T Cell TransAct, human and cultured in TexMACS Medium containing IL-2. CD4, CD8, CD25, and CD69 expressions were analyzed via flow cytometry on a MACSQuant Analyzer. Proliferation was measured using the cell division tracker CFSE. T cells activated with T Cell TransAct, human showed high expression of activation markers CD25 and CD69 after 48 h, as well as strong proliferation after 7 days of culture. 

Human T cells were isolated using the Pan T cell Isolation Kit, human. Subsequently, cells were stimulated either with T Cell TransAct, human (research grade), with activation beads (bead-based competitor product), or left untreated and cultured for 14 days either in RPMI (10% FBS) or serum-free TexMACS Medium. Both media were supplemented with 50 U/mL Human IL-2 IS, premium grade. Cell proliferation (fold expansion) was analyzed on the MACSQuant Analyzer 10. Culturing in serum-free TexMACS Medium resulted in over 100-fold T cell expansion comparable to the expansion rates in RPMI + 10% FBS.

Human T cells were transduced with gamma-retroviral vectors encoding GFP in the presence of Vectofusin-1®. Transduction was performed either at 37 °C in an incubator (static), or the T cells were centrifuged at 400×g for 2 hours at 32 °C (spin) before transfer to 37 °C. GFP expression was analyzed on day 7 of cultivation using a MACSQuant Analyzer. T cell viability was assessed by flow cytometry using Viobility™ Fixable Dye.

The CD19 CAR T Cell Express Mode Package contains 12 optimized application-specific Express Modes.

Flow cytometry is the perfect tool for in-process and quality control of CAR T cell products, as well as for monitoring CAR T cell persistence in patients under treatment. CD19 CAR Detection Reagent (Biotin) detects CD19 CAR T cells via recognition of the CD19 CAR molecule. 

CD19 CAR T cells, generated using the CliniMACS Prodigy TCT Process within 12 days, were stained with CD19 CAR Detection Reagent, Anti-Biotin-PE, CD3-FITC, CD4-VioGreen™, CD8-APC-Vio® 770, and 7-AAD Staining Solution and analyzed by flow cytometry using the MACSQuant Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and 7-AAD fluorescence. Shown are viable CD4⁺ and CD8⁺ T cells as well as viable CD3⁺ T cells.

CAR T cells were labelled with CD19 CAR Detection Reagent, human, Biotin before co-culture. The cells were either co-cultured with antigen-positive cells (lower row) or with antigen-negative cells (lower row) for 4 hours. Cells were fluorescently stained using the CD19 CAR Detection Reagent, human, Biotin, Biotin Antibody-PE, CD3-VioBlue®, CD8-PE-Vio® 615, IFN-γ-APC-Vio 770, TNF-α-APC, IL-2-PE-Vio 770, Fixable Viobility™ Dye VioGreen™ following the “Intracellular flow cytometry staining protocol”, and analyzed by flow cytometry using the MACSQuant Analyzer 10. 
Cell debris and dead cells were excluded from the analysis based on scatter signals and Viobility VioGreen. CD3⁺ and CAR T cells were further analyzed regarding their effector cytokine expression (IFN-γ, TNF-α, or IL-2 ). 

Automatically manufactured CD20 CAR T cells are fully functional in terms of cytokine secretion. CD20 CAR T cells, derived from healthy donor (A, n = 5) or patient (B, n = 4) samples, were co-cultured with JeKo-1 as the target cell line (effector:target = 1:1), and cytokine secretion was analyzed using the MACSPlex Cytokine 12 Kit, human.