Transient Transfection of Porcine Granulosa Cells after 3D Culture in Barium Alginate Capsules

Abstract

Three-dimensional culture systems in barium alginate capsules can be employed to maintain primary granulosa cells in an undifferentiated state for almost 6 days. This is due to a self-organization of cells in a pseudofollicular structure. The transfection of primary granulosa cells is a necessary condition when employing these culture systems for several purposes, for example as an in vitro toxicity test or the development of oocytes or zygotes. In this work, the feasibility of two transient transfection techniques (liposome-mediated and electroporation) was assessed in primary porcine granulosa cells after a 6-day culture in an artificial extracellular matrix (barium alginate membrane). Human recombinant green fluorescent protein was chosen as a molecular readout, and protein expression was assessed after 48 hours from transfection. Liposome-mediated transfection gave low transfection levels, with increasing yields from 2 to 12 μgDNA/ml of medium; the maximum percentage (85.7%) was reached at 12 μgDNA/ml of medium. Electroporation-mediated transfection yields were higher: the best results (81.7% of transfected cells) were achieved with two 50V pulses and 12 μg/ml DNA. The application of a single or double pulse (50V) at 4 mgDNA/ml gave negligible results. These results indicate that primary granulosa cell cultured in barium alginate capsules can be transfected by electroporation with high transfection yields.