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Figure 1. EasySep? Human T Cell Isolation Kit

Starting with human peripheral blood mononuclear cells (PBMCs), the T cell content (CD3+) of the isolated fraction is typically 96.7 ¡À 1.5% (mean ¡À SD).

Figure 2. ImmunoCult?-XF T Cell Expansion Medium Supports Faster T Cell Expansion Than Other Serum-Free and Serum-Supplemented Media

T cells were isolated from human peripheral blood samples using the EasySep? Human T Cell Isolation Kit (Catalog #17951), stimulated with ImmunoCult? Human CD3/CD28/CD2 T Cell Activator (Catalog #10970), and cultured in ImmunoCult?-XF T Cell Expansion Medium supplemented with rhIL-2. T cells were stimulated with ImmunoCult? Human CD3/CD28/CD2 T Cell Activator on Day 0 and every 7 to 8 days for the duration of the culture. T cells were analyzed on Days 4, 7, 8, 10, 11, 14, 18, and 21 for fold expansion relative to the initial cell seeding density. Compared to all competitor media tested, ImmunoCult?-XF T Cell Expansion Medium showed significantly higher expansion of total T cells. Commercial alternatives 1 to 4 include, in no particular order, X-VIVO? 15 (Lonza), AIM V? Medium (Life Tech), CellGro? DC Medium (CellGenix), and RPMI 1640 + serum. Each data point represents the mean fold expansion ¡À S.E.M. at the specified time points (p<0.05 for ImmunoCult?-XF versus all media for Days 8, 11, 14, 18, and 21, tested using two-tailed, paired t-test with unequal variance, n = 6 to 19 donors). The average fold expansion of T cells in ImmunoCult?-XF T Cell Expansion Medium were 15-fold on Day 7, 80-fold on Day 10, 450-fold on Day 14, and 4,000-fold on Day 21.

Figure 3. T Cell Expansion and Activation Ability Is Better Preserved When Leukopaks are Stored at Fridge Temperature

Using EasySep? Human T Cell Isolation Kit (Catalog #17951), T cells were isolated from 1 leukopak fraction (Catalog # 70500) of each storage condition daily for 5 days, and 1 x 10? isolated cells were cultured in ImmunoCult?-XF T Cell Expansion Medium (Catalog #10981) supplemented with ImmunoCult? Human CD3/CD28/CD2 T Cell Activator (Catalog # 10970) and 10 ng/mL IL-2 for 10 days with assessment of CD25 activation marker expression on day 3 of culture. (A) Representative flow cytometry data from leukopaks stored 1 day at fridge temperature (FT), showing that both CD4+ and CD8+ cells are CD25-negative at the start of culture (Day 0), and upregulate CD25 expression by Day 3 of culture. (B) Cellular expansion and corresponding cell yield over 7 days of culture decreases in correlation with storage duration of leukopak fractions. Leukopaks that were stored for 1 - 2 days at either room temperature (RT) or FT had high expansion potential yielding 1.5 - 2 x 10? cells, and this 15 - 20-fold expansion potential is maintained in T cells from leukopaks stored at FT for up to 5 days. In contrast, little or no expansion is observed in T cell cultures from leukopaks stored at RT for 3 or more days, indicating a loss of proliferative capacity. Moreover, T cells show a gradual reduction in their ability to become activated by ImmunoCult? T Cell activator, as shown by a reduction in Day 3 CD25 expression in gated CD4+ (bottom left) or CD8+ (bottom right) cells over time, and the effect is most pronounced with storage at RT for 3 or more days. All data points represent average ¡À standard deviation values from leukopak fractions of n = 3 unique donors.

Figure 4. ImmunoCult? Human T Cell Activators Can Be Used to Optimize Culture Conditions for High-Efficiency TRAC Knockout from Human Primary T Cells

The TRAC locus of human primary T cells was edited with an RNP-based-CRISPR-Cas9 system using multiple T cell activation reagents and dynamics then evaluated to identify a condition with the highest knockout efficiency. (A) TRAC knockout efficiency in human T cells isolated using EasySep? Human T Cell Isolation Kit (Catalog #17951) and activated with either ImmunoCult? Human CD3/CD28 or CD3/CD28/CD2 T Cell Activator (Catalog #10971/10970) for 2 or 3 days was assessed by binding the TCR¦Á¦Â and CD3 receptors with antibodies and performing flow cytometry analysis. Each data point per condition represents an individual donor; n = 4 - 8 donors. Error bars represent standard error of the mean. (B) Genome editing (cleavage) efficiency was assessed at 48 hours post electroporation in human T cells activated with ImmunoCult? Human CD3/CD28 T Cell Activator for 3 days using the ArciTect? T7 Endonuclease I Kit (Catalog #76021). Mock electroporated: - RNP; RNP electroporated: + RNP. (C - D) Representative dot plots of TCR¦Á¦Â and CD3 flow cytometry analysis from (C) mock electroporated and (D) RNP electroporated human T cells activated with ImmunoCult? Human CD3/CD28 T Cell Activator for 3 days. (E) Representative dot plot of CD4 and CD8 flow cytometry analysis of human T cells activated with ImmunoCult? Human CD3/CD28 T Cell Activator for 3 days.