Loading of Acute Myeloid Leukemia Cells with Poly(I:C) by Electroporation

Abstract

In this chapter, we describe the technique of electroporation as an efficient method to load primary leukemic cells with the double-stranded RNA (dsRNA) analogue, polyriboinosinic polyribocytidylic acid (poly(I:C)), and detail on the delicate freezing and thawing procedure of primary leukemic cells.Electroporation is a non-viral gene transfer method by which short-term pores in the membrane of cells are generated by an electrical pulse, allowing molecules to enter the cell. RNA electroporation, a technique developed in our laboratory, is a widely used and versatile transfection method for efficient introduction of both coding RNA (messenger RNA) and non-coding RNA, e.g., dsRNA and small interfering (siRNA), into mammalian cells. Accurate cell processing and storage of patient material is essential for optimal recovery and quality of the cell product for downstream applications.

Intracellular Delivery of Synthetic dsRNA to Leukemic Cells Induces Apoptotic and Necrotic Cell Death

“…In the late stages of apoptosis, nuclei fragment and condense to form distinct apoptotic bodies (Gregory and Devitt, 2004). It is possible that the reported increase in the immunogenicity of poly I:C-electroporated leuke mic cells (Lion et al, 2009(Lion et al, , 2011(Lion et al, , 2014Smits et al, 2007) is not solely due to recognition of apoptotic tu mour cells, but could potentially be a response to ne crotic tumour cells. The decision of antigenpresenting cells, such as DCs, to mount an immunogenic or tolero genic adaptive immune response against internalized material has strong implications in devising the most ap propriate immunotherapeutic treatment for leukemic diseases such as AML.…”