CTS Xenon Electroporation System Sample Data

Chimeric antigen receptor (CAR) T cells were generated from three different donors by using Cas9/gDNA to knock out the endogenous T-cell receptor (TCR) and knock in an FMC CAR. As a benchmark, lentiviral transduction would be expected to achieve 20–40% success for this process.

Knock-out and knock-in performance with the CTS Xenon system was strong. Across all three donors, successful transfection percentages (cells both knocked out and knocked in) ranged from about 22% to more than 45%, depending on the electroporation chamber used. Transfection efficiency on the Xenon instrument surpassed even the Neon Transfection System on which the process was developed. Cell viability—which must be balanced against transfection efficiency—exceeded 70% in all but one case and was usually within 10% of the untransfected controls. Percentages of CD4 and CD8 T cells remained relatively consistent before and after electroporation.

Figure 1. High transfection efficiency (knock-out and knock-in performance) in CAR T cells. T cells from three donors were transfected with an FMC CAR construct using Cas9/gRNA on the Neon Transfection System (100 µL) or the CTS Xenon Electroporation System with the SingleShot (1 mL) or MultiShot (9 mL) electroporation chamber or were left untransfected (0). Cells were characterized after 72 hours as untransfected (TCRαβ⁺, gray), knocked out but not knocked in (TCRαβ^–CAR^–, light blue), or successfully knocked out and knocked in (TCRαβ^–CAR⁺, dark blue). Across all donors, successful knock-in percentages on the Xenon system ranged from 21.9% to 45.6%, exceeding even the Neon system.

Figure 2. High cell viability in CAR T cells. In the same experiment (explained in Figure 1 above), cells were assessed for viability after 72 hours using trypan blue exclusion. For the three donors, cell viability on the Xenon system ranged from 64.6% to 83.6%.

Figure 3. Preservation of CD4 vs CD8 T cell ratio. In the same experiment, CD4 and CD8 T cells were identified by flow cytometry. Non-electroporated cells (condition 0) were gated on live, single, and untransfected (TCRαβ⁺), while electroporated cells were gated on live, single, and knocked out (TCRαβ^–). Proportions of CD4 (light blue) and CD8 (dark blue) T cells remained largely consistent from non-electroporated cells to cells electroporated with the Neon system (100 µL) and with the Xenon system (1 and 9 mL).

Figure 4. CAR T cell analysis by flow cytometry. In the same experiment, electroporated cells were analyzed by flow cytometry. Plots and analysis are representative of all three donors. (A) Gating on live single cells, 89.5% lost TCR function (TCRαβ–) and thus were successfully knocked out, while 9.59% retained TCR expression (TCRαβ⁺) and thus were not successfully transfected. (B) Analysis of untransfected cells shows the percentages of CD4 (43.1%) and CD8 (51.7%) single-positive (SP) cells. Double-positive (DP), double-negative (DN), and V5⁺ (successfully knocked-in) populations are small and reflect noise. (C) Analysis of knocked-out cells similarly shows the percentages of CD4 (38.0%) and CD8 (49.5%) SP cells. 31.2% of the knocked-out cells were successfully knocked in (V5⁺), including 36.2% of CD4 and 30.2% of CD8.

The CTS Xenon system can help generate and scale up manufacturing of CAR NK cells, and also enable efficient electroporation at high NK cell densities.

Figure 5. CAR NK cell generation studies. A combination of viral and nonviral strategies were used to genetically modify NK cells efficiently. These cells were able to (A) expand and (B) maintain a CD56⁺ phenotype and (C) high viability.

Scale up of smaller volume gene modification from the Invitrogen Neon system to the CTS Xenon system facilitates flexible transition from research to larger volume commercial manufacturing for nonviral delivery for cGMP cell therapy processing.

Figure 6. Gene editing efficiency. The light blue bars represent total cells for the no-electroporation control, and the dark blue bars represent total edited cells for the two electroporation volumes (100 µL and 1 mL). Data were collected 3 days post-electroporation (day 6).

The CTS Xenon system can be physically integrated with other cell therapy manufacturing instrumentation to create a closed workflow that can be controlled by process automation software to help minimize drawbacks associated with manual processes. Here we demonstrate the closed and integrated use of the CTS Rotea system with the CTS Xenon system to edit T cells isolated with the CTS DynaCellect system.

Figure 7. Physical integration of the CTS Xenon system with the CTS Rotea system. Schematic representation of the physical integration via a welded PVC line between the CTS Rotea system and CTS Xenon system used for T cell electroporation on day 3.