Figure 1. Optimizing Pressure Parameters for CellPore™ FITC-Dextran Delivery to Isolated Human Primary Cells

The graphs show the results of pressure sweeps to determine the best delivery parameters for CellPore™ FITC-Dextran in human (A) T and (B) NK cells from a leukopak and (C) cryopreserved human cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs). Delivery efficiency (orange bars) and cell viability (black circles) were assessed via flow cytometry on the day of delivery. Optimal delivery conditions were defined as those in which delivery efficiency is maximized while impact on cell viability is minimal. Control groups include untreated samples and endocytosis controls for comparison. The endocytosis control represents the natural uptake of CellPore™ FITC-Dextran in undelivered samples without pressure application. Data are presented as mean ± SD, with n = 3 - 18 replicates.

Figure 2. mCherry mRNA Delivery to Isolated Human Peripheral Blood NK Cells Using CellPore™

mCherry mRNA was delivered to freshly isolated human NK cells (2 x 106 cells in 20 µL reactions) from a leukopak using the CellPore™ Transfection System at 70 psi. Doses ranged from 0.2 to 4.0 µg of mRNA. mCherry expression was measured via flow cytometry 2 days after delivery. Control conditions included untreated NK cells and an endocytosis control (4.0 µg mRNA added without pressure application). Data shown as mean ± SD, n = 2.

Figure 3. The CellPore™ Transfection System Can Efficiently Knock Down Gene Expression in Unactivated Pan T Cells at a Wide Range of Cell Concentrations

Cas9 RNPs targeting B2M (A, B) or TRAC (C, D) genes were delivered to 0.5 - 25 x 106 isolated unactivated pan T cells in 50 µL reactions using the CellPore™ Transfection System at 90 psi. Flow cytometry was used to assess B2M (MHC-I) after 2 days or TRAC (TCRαβ) knockout efficiency after 6 days, alongside viability (using DRAQ7) in Pan T, CD4+, and CD8+ T cell subsets. Comparable viability and editing performance were observed across all tested cell numbers. Control groups included untreated T cells and scramble controls delivering non-targeting sgRNA Cas9 RNPs. Data presented as mean ± SD; n = 3.

Figure 4. The CellPore™ Transfection System Preserves Unactivated State of Pan T Cells

Isolated unactivated pan T cells were manipulated using the CellPore™ Transfection System (delivery pressure 90 psi) or an optimized electroporation protocol. One and two days post-delivery, flow cytometry was used to assess CD69 expression, an early T cell activation marker. Electroporation increased CD69 expression in both (A) CD4+ and (B) CD8+ subsets of pan T cells. (C) Electroporation also elevated secretion of pro-inflammatory cytokines, measured using a bead-based multiplex cytokine assay 24 hours post-delivery. In contrast, the CellPore™ Transfection System maintained CD69 expression levels comparable to untreated control pan T cells. These findings underscore the gentle impact of the CellPore™ Transfection System on cellular function following intracellular cargo delivery. Data presented as mean ± SD; n = 3.

Figure 5. The CellPore™ Transfection System Enables Efficient Gene Knock-Out in CD34+ HSPCs

Cryopreserved cord blood-derived CD34+ HSPCs were cultured in complete StemSpan™ SFEM II medium for 24 hours. RNP complexes targeting the B2M or PTPRC gene were delivered to 5 x 10⁴ HSPCs. After a culture period of 4 days, flow cytometry was used to determine viability and (A) B2M knock-out (assessed via MHC-I surface expression, representative histogram shown) or (B) PTPRC knock-out (assessed via CD45 surface expression, representative histogram shown). (C) A pressure sweep was performed to identify the optimal delivery pressure for achieving robust gene knock out in CD34+ HSPCs, which was determined to be 30 psi. (D) When utilizing the optimal 30 psi delivery pressure, the average editing efficiency for MHC-I knock-out was 91.8% ± 1.4, and 84.2% ± 2.7 for CD45 knock-out, while retaining high viability. Data are presented as mean ± SD, n = 2 - 7.