BioLegend Cell-Vive™ GMP Cell Activation and Expansion Tools are manufactured and tested in accordance to USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products and Ph. Eur. Chapter 5.2.12 guidelines in a dedicated ISO 13485:2016 certified GMP facility.

Specifications and processes include:

Cell-Vive GMP CD3/CD28 Human T Cell Activation Beads are suitable for in vitro or ex vivo activation and expansion of human T cells for ex vivo cell processing. Superparamagnetic microbeads are coated with anti-CD3 (clone OKT3) and anti-CD28 (clone S20013F) antibodies, providing both the primary and co-stimulatory signals required for the activation and expansion of human T cells without the need for antigen-presenting or feeder cells.

Downstream applications include use in ex vivo cell manufacturing workflows.

Here is an overview of the protocol. After washing beads, simply add the beads directly to isolated T cells or to PBMCs at a 1:1 bead-to-cell ratio for robust T cell activation. This is followed by incubation of cells in a humidified CO₂ incubator for precise stimulation and expansion of T cells. The antibody-coated beads can be removed from the cells with a magnetic separator.

*This product was optimized on isolated CD3+ T cells and PBMCs. Alternate sources of T cells may require protocol optimization.

Human peripheral blood mononuclear cells were isolated from whole blood and plated at 5x10⁵ cells/well in complete IMDM culture medium supplemented with 5% human AB serum and 30 IU/mL recombinant human IL-2 (Cat. No. 791908). On Day 0, cells were unstimulated or incubated with a 1:1 ratio of Cell-Vive™ GMP CD3/CD28 Human T Cell Activation Beads (Cat. No. 422606), RUO Human CD3/CD28 T Cell Activation Beads (Cat. No. 422604), or competitor CD3/CD28 Activation Beads. Cells were cultured at 37°C, 5% CO₂ for 11 days and expanded with fresh media every 2-4 days. Total cell count and fold expansion were measured at indicated timepoints.

Human peripheral blood mononuclear cells were isolated from whole blood and plated at 5x10⁵cells/well in complete IMDM culture medium supplemented with 5% human AB serum and 30 IU/mL recombinant human IL-2 (Cat. No. 791908). On Day 0, cells were unstimulated or incubated with a 1:1 ratio of Cell-Vive™ GMP CD3/CD28 Human T Cell Activation Beads (Cat. No. 422606), RUO Human CD3/CD28 T Cell Activation Beads (Cat. No. 422604), or competitor CD3/CD28 Activation Beads. Cells were cultured at 37°C, 5% CO₂ for 11 days and expanded with fresh media every 2-4 days. On indicated timepoints, cells were collected and surface stained with APC anti-human CD3 clone UCHT1 (Cat. No. 300439), PE anti-human CD4 clone RPA-T4 (Cat. No. 300539), FITC anti-human CD8 clone SK1 (Cat. No. 344704), PE/Cyanine7 anti-human PD1 clone A17188B (Cat. No. 621616), APC/Fire™ 750 anti-human CD69 clone FN50 (Cat. No. 310946), and 7-AAD viability dye (Cat. No. 420404) and analyzed by flow cytometry. The frequency of CD69+ or PD1+ cells is shown gated on viable CD3+CD4+ or CD3+CD8+ T cells.