A Facile, Transfection‐Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models

• DOI: 10.1002/cpz1.1121
• Publication Date: 2024-09-03T13:00:27Z
• Authors: Andrew S. Riching; Allyson Malloy; Emily M. Anderson; Jonathan Sheard; Piia Mikkonen; Anja van Brabant ...; Zaklina Strezoska; Josien Levenga
• Keywords: Druggability; Spheroid; 3D cell culture

Abstract Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two‐dimensional (2D) cell fails to faithfully model the complexity found in vivo, novel drug candidates that show promising results 2D models often do not translate clinic. More recently, three‐dimensional (3D) have gained popularity owing their greater physiological relevance vivo biology. In particular, 3D spheroid are becoming widely used due ability mimic solid tumors, both architecture gradation nutrients distributed from outer, proliferative layers into inner, quiescent cells. Similar lines grown tend be more resistant antitumor treatments than cultured counterparts, though distinct signaling pathways gene targets conferring this resistance yet fully explored. RNA interference (RNAi) is an effective tool elucidate function discover druggable models; however, only a few studies successfully performed RNAi complex date. Here, we demonstrate efficient RNAi‐mediated knockdown using “transfection‐free” Dharmacon Accell siRNAs three models, presence or absence extracellular matrix. This methodology potential scaled up arrayed screening experiments, which may aid identification with clinical those identified experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Generation spheroids matrix‐free ULA plates Alternate Matrigel matrix–embedded 2 GrowDex hydrogel–embedded Delivery siRNA collection 3 matrix‐embedded protein extraction characterization