Nanostraw- and Nanopore-Based Transfection of Pancreatic Islet-Like β-Cell Spheroids In Vitro

Sammanfattning

Diabetes, characterized by high blood glucose due to β-cell dysfunction, remains one of the most common chronic diseases in the world. A promising method for diabetes research is nanoelectroporation using nanostraws and nanopores, which enables controlled delivery of molecular cargo into β-cells for genetic and epigenetic manipulation. However, most studies rely on 2D β-cell cultures, which limits physiological relevance. This thesis focuses on producing β-cell spheroids using the hanging droplet method and evaluating whether nanostraw- and nanopore-based electroporation can deliver small molecules (PI) and nucleic acids (pMax-GFP plasmid) into these spheroids. Additional experiments assessed spheroid handling during Ki-67 staining and explored fluorescent bead incorporation to examine particle distribution and potential release over time. Physiologically relevant homotypic β-cell spheroids were successfully generated in vitro using the hanging droplet method, with the mean diameter comparable to human pancreatic islets. Results showed that nanoelectroporation can successfully deliver small cargos, such as propidium iodide, into the spheroids. Transfection of the larger pMax-GFP plasmids was much less successful, as only a few spheroids were transfected using nanostraws. Additional parameter optimization may improve the transfection efficiency. Furthermore, a β-cell spheroid fixation and staining method was developed using the adherent properties of the β-cells to prevent spheroid loss. Lastly, 2.1 µm fluorescent beads were randomly distributed within the spheroids, but incorporation was inconsistent and experiments to assess whether the spheroids could release the incorporated beads were not carried out.