A microparticle approach for non-viral gene delivery within 3D human mesenchymal stromal cell aggregates

Three-dimensional (3D) multicellular aggregates, in comparison to two-dimensional monolayer culture, can provide tissue culture models that better recapitulate the abundant cell-cell and cell-matrix interactions found in vivo. In addition, aggregates are potentially useful building blocks for tissue engineering. However, control over the interior aggregate microenvironment is challenging due to inherent barriers for diffusion of biological mediators (e.g. growth factors) throughout the multicellular aggregates. Previous studies have shown that incorporation of biomaterials into multicellular aggregates can support cell survival and control differentiation of stem cell aggregates by delivering morphogens from within the 3D construct. In this study, we developed a highly efficient microparticle-based gene delivery approach to uniformly transfect human mesenchymal stromal cells (hMSC) within multicellular aggregates and cell sheets. We hypothesized that release of plasmid DNA (pDNA) lipoplexes from mineral-coated microparticles (MCMs) within 3D hMSC constructs would improve transfection in comparison to standard free pDNA lipoplex delivery in the media. Our approach increased transfection efficiency 5-fold over delivery of free pDNA lipoplexes in the media and resulted in homogenous distribution of transfected cells throughout the 3D constructs. Additionally, we found that MCMs improved hMSC transfection by specifically increasing macropinocytosis-mediated uptake of pDNA. Finally, we showed up to a three-fold increase of bone morphogenetic protein-2 (BMP-2) expression and enhanced calcium deposition within 3D hMSC constructs following MCM-mediated delivery of a BMP-2 encoding plasmid and culture in osteogenic medium. The technology described here provides a valuable tool for achieving efficient and homogenous transfection of 3D cell constructs and is therefore of particular value in tissue engineering and regenerative medicine applications. STATEMENT OF SIGNIFICANCE: This original research describes a materials-based approach, whereby use of mineral-coated microparticles improves the efficiency of non-viral gene delivery in three-dimensional human mesenchymal stromal cell constructs. Specifically, it demonstrates the use of mineral-coated microparticles to enable highly efficient transfection of human mesenchymal stromal cells in large, 3D culture formats. The manuscript also identifies specific endocytosis pathways that interact with the mineral coating to afford the improved transfection efficiency, as well as demonstrates the utility of this approach toward improving differentiation of large cell constructs. We feel that this manuscript will impact the current understanding and near-term development of materials for non-viral gene delivery in broad tissue engineering and biofabrication applications, and therefore be of interest to a diverse biomaterials audience.