biotin  (Miltenyi Biotec)

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Workflow for lentiviral vector (LV) production and results with the control vector LV/CAR-GFP. ( a ) Schematic of the vector production process. Packaging cells were expanded in progressively larger culture vessels (100 mm dishes, T-175 flasks, 5-layer stacks) before calcium phosphate transfection with the LV packaging system (transfer vector, Gag/Pol helper, and VSV-G envelope plasmid). Conditioned medium was harvested 48 h post-transfection, clarified, and filtered. Viral particles from ~600 mL supernatant underwent concentration through tangential flow filtration (TFF), followed by ultracentrifugation, yielding 1 mL of high-titer preparation. These procedures provided a sufficient viral stock for multiple CAR-T cell manufacturing runs. ( b ) Comparison of the transgene in the control vector LV/CAR-GFP and the experimental vector NCB.LV.CD19-CAR. In LV/CAR-GFP, the CAR gene is fused to GFP via an uncleavable linker. In similar experiments, amounts of GFP-fluorescent cells served as an indicator of transfection efficiency. ( c – e ) Results of preliminary experiments with the CAR-GFP-expressing vector. The photographs on panels ( c ) and ( d ) show typical results from transfection for LV packaging. Calcium phosphate precipitation method allows the efficient transfection of high-density cultures (100,000 cells/cm 2 ). The photographs were taken at 48 h post-transfection. Magnification = 50×. Scale bar = 100 µm. ( e ) Determining the optimal acceleration for centrifugal concentration of LV particles. The average functional titers are shown as means ± SDs, expressed as a percentage of the maximum titer across all experiments. The titer obtained at 20,000× g was set to 100%.

Article Snippet: For CAR detection, cells were stained with 2 μL of biotinylated CD19 CAR Detection Reagent (Miltenyi Biotec 130-129-550) per 1 × 10 6 cells.

Techniques: Plasmid Preparation, Control, Transfection, Concentration Assay, Filtration, Comparison, Expressing, Functional Assay

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Functional characterization of CAR-T cells via cytokine secretion and cytotoxicity assays. ( a ) Key Th1 cytokine release profile. CAR + T-cells were cocultured with CD19 + targets (B cells) (Exp) and compared to controls: non-transduced T-cells (NT) cultured with targets, and CAR + T-cells without targets (baseline, BL). Coculture of CAR + effectors with targets induced significant production of IFN-γ, TNF-α, and IL-2, indicating antigen-specific activation. Data shown as means ± SDs (V1: n = 6; V2: n = 6); ( b ) cytotoxic activity of CAR + T-cells was assessed by measuring lysis of labeled target cells at effector-to-target (E:T) ratios of 10:1 and 5:1. Control cocultures contained non-transduced (NT) T-cells with labeled targets. Both V1- and V2-derived CAR + cells exhibited dose-dependent target cell lysis (increasing with higher E:T ratios). Each data point represents the mean of triplicate measurements per sample; ( c ) violin plots with connected medians show a trend toward enhanced cytotoxicity in V2- vs. V1-derived CAR + T-cells; ns = not significant, * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: For CAR detection, cells were stained with 2 μL of biotinylated CD19 CAR Detection Reagent (Miltenyi Biotec 130-129-550) per 1 × 10 6 cells.

Techniques: Functional Assay, Cell Culture, Activation Assay, Activity Assay, Lysis, Labeling, Control, Derivative Assay