PEF selectively purges myeloma cell lines from mixtures with blood or bone marrow cells. (A) RPMI8226 myeloma cells were mixed 1:1 with PBMCs, and were PEF-treated in standard pulsing buffer (90% isotonic [282 mM] dextrose/10% PBS, pH 7.2) at 0 kV/cm or 1.4 kV/cm, using the flowing PEF apparatus. After ficolling, cells were photographed at ×50 magnification. The distinct large myeloma cells seen at 0 kV/cm (top) are not present at 1.4 kV/cm (bottom). Photographs were taken with a Nikon Diaphot 300 microscope, ×50 magnification, and a Nikon 6006 camera and using IP Labview Macintosh software (National Instruments, Austin, TX). (B) Raw flow cytometry data for PEF-treated and control mixed cells. Flow cytometry was performed on a Becton Dickinson FACSVantage flow cytometer (San Diego, CA), and data were analyzed using CellQuest software. Mixtures of PBMCs and CFDA-SE–labeled RPMI8226 myeloma cells were PEF-treated at 0 kV/cm or 1.4 kV/cm. PBMC suspensions from whole blood or cord blood were seeded with a CFDA-SE–labeled myeloma cell line, RPMI8226 or U266. Mixed cells were resuspended in the high resistivity pulsing buffer (500 Ω-cm) at between 10⁶ and 2 × 10⁶/mL. A quantity of 5 mL to 10 mL of cells was run through the flowing apparatus at a 4 mL/min flow rate, exposing cells to 250 20-μs pulses. Cell samples were treated at different electric field strengths, or pumped through the flowing apparatus without pulsing (0 kV/cm, control group). Cells were stained with anti-CD3/CD19–phycoerythrin (PE), anti-CD14–allophycocyanin (APC), and the nuclear exclusion dye 7-aminoactinomycin (7-AAD). Dot plots represent cells gated as 7-AAD–negative. Dot plots for forward and side scatter (left), CD3/CD19 and CFDA-SE (center), and CD14 and CFDA-SE (right) are shown for stained cells that were PEF-treated at 0 kV/cm (top row) or 1.35 kV/cm (bottom row). The results of this experiment are representative of more than 20 experiments using several different tumor cell lines. (C) Percent survival of RPMI8226 myeloma cells (▴), CD3⁺/CD19⁺ lymphocytes (), and CD14⁺ monocytes (▪) for mixtures of tumor cells and PBMCs PEF-treated at the indicated electric field strengths. Percent survival for tumor cells and other blood cells was determined as follows: (number of immunostained viable cells in the PEF-treated group)/(number of immunostained viable cells in the control [0 kV/cm] group) × 100. Results are representative of more than 5 experiments using RPMI8226, U266, and other tumor cell lines. (D) Survival of viable PEF-treated myeloma cells, as assayed by serial dilution tumor regrowth. Tumor regrowth assays were performed using triplicate serial dilutions of PEF-treated and 0 kV/cm aliquots of cells in tumor-conditioned medium, then assessing tumor colony formation after 2 weeks at 37°C. Percent survival is determined by comparing regrowth of PEF-treated and 0 kV/cm groups. Results from 1 of 5 representative experiments performed using RPMI8226 tumor cells is shown. (E) Average RPMI8226 tumor purging and standard errors, assessed by different methodologies, for 6 independent experiments is shown in the bar chart. (F) Human bone marrow mononuclear cells were treated with PEF at 0 kV/cm (control) or at 1.4 kV/cm. Cells (2 × 10⁶) from each group were injected into the tail veins of NOD/SCID/β₂m^-/- mice. After 7 weeks, mice were killed and bone marrow was removed. Mononuclear cells were isolated and stained with antihuman CD45 and antimurine CD45, then analyzed by flow cytometry (BD FACSVantage). Summary of percent human CD45 cells in mice that did not undergo transplantation (control), mice that received a transplant of 0 kV/cm human bone marrow (n = 5), and mice that received a transplant of PEF-treated (1.4 kV/cm) human bone marrow cells. (G) Representative data from CFC and LTC-IC assays performed after PEF at indicated electric field strengths.²⁴ Blue bars indicate LTC-IC; red bars, erythroid blast-forming units/colony-forming units (BFU/CFU-E); and cream bars, granulocyte/macrophage colony-forming units (CFU-GM) + granulocyte/erythroid/monocyte/megakaryocyte colony-forming units (CFU-GEMM). Error bars indicate standard error for means.