|
Three-Dimensional Printing for High-Throughput Liquid Suspension Spheroids
Christen Boyer, Postdoctoral Fellow, LSU Health Sciences Center
Three-dimensional (3D) cell cultures have recently gained tremendous attention due to their superiority over 2D cell cultures which have less translational potential. 3D scaffold supports, cell aggregate systems and hydrogels have been shown to more accurately mimic native tissues and support more relevant cell-cell interactions for studying actions of drugs and bioactive agents. [1-5] 3D printing continues to democratize manufacturing, offering economical assay solutions for laboratories. Here, consumer grade 3D printing was examined as a fabrication method for creation of high-throughput scaffold-free spheroid microtissues. ‘Hanging drop’ 3D printed polylactic acid inserts were designed to fit standard 6, 12, 24, and 96 well tissue culture plates. These inserts were seeded with human glioblastoma, placental-derived mesenchymal stem cells and intestinal smooth muscle cells. Spheroid microtissue formation was monitored by optical and fluorescent microscopy over 72 hours and microtissue topographies examined with Calcein AM, ethidium homodimer 1, and Hoechst 33342 fluorescence staining. Modular 3D printed inserts supported 3D spheroids which were cell density ‘tunable’ to allow reproducible drug screening. 3D printed microtissue inserts represent a highly cost-effective approach with many potential applications in pharmaceuticals and tissue-engineering.
Antibody-Based Platform for High-Content Analysis of 3D iPSC-Derived Human Neural Cultures and Tissue Constructs
Martin Tomov, Postdoctoral Associate, Broad Institute of MIT and Harvard
Moving personalized medicine and drug discovery from the lab to the clinic will require addressing the current hurdles impeding high-throughput generation of experimental and clinical data. Traditional immunofluorescence caps at up to five channels before having to rely on specialized and expensive equipment. Recent advances in multiplexed imaging techniques now allow tracking large numbers of markers at once, which creates exciting possibilities for teasing out pathways and generating refined cell phenotypes. One such technique, optimized in our lab, uses DNA-conjugated antibodies (DNA-PRISM) to acquire highly multiplexed images and provides comparable results to traditional immunofluorescence, while eliminating some of its more frustrating limitations, such as long secondary antibody incubation and the need to match animal hosts to avoid cross-reactivity. It is readily adaptable using standard imaging microscopes and without the need for specialized equipment or extensive training. Here we use PRISM to stain for multiple markers in 3D spinner flask dissociated cortical neurons, derived from human iPSCs, looking at cell subtypes within the complex culture. In conjunction with automation, DNA-PRISM can establish an effective high-throughput platform for drug and compound screening, as well as neurodevelopmental disease phenotyping of complex model systems by eliminating variability due to culture conditions or staining efficacy.
|
