Flow electroporation with pulsed electric fields for purging tumor cells.

Electroporation has been used in biological laboratories for many years to transiently porate cell membranes and permit plasmid or protein transfection. It has been shown that the application of pulsed electric fields (PEFs) of defined strength will kill off larger cells and select for viable small cells, in samples containing heterogeneous cells. This permits the selective killing of several blood and bone marrow-resident tumor cells. PEF technology is being applied to tumor purging of progenitor-cell transfusions, in support of high-dose chemotherapy, for the treatment of cancers such as lymphoma and multiple myeloma. Autologous stem-cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Progenitor cells are preserved or enriched. To overcome issues of electrical resistance, purging fidelity, and large sample volume, a flowing chamber PEF apparatus was designed and constructed for large-scale purging of clinical quantities of progenitor-cell transfusions. The specifics of this technique are described here. Treatment of greater than 10(9) cells is achieved in 30 min, under optimized flow conditions designed to overcome surface area or resistance issues and to optimize exposure of cells to electric fields. Efficient, large volume tumor purging of greater than 3 logs, for mixtures of tumor cells and mononuclear cells, is routinely achieved under defined conditions.

Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem cell function.

Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.