Publications Using the CellRaft™ Technology
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Human induced pluripotent stem cells (hiPSCs) are widely used for disease modeling, tissue engineering, and clinical applications. Although the development of new disease-relevant or customized hiPSC lines is of high importance, current automated hiPSC isolation technologies rely largely on the fluorescent labeling of cells, thus limiting the cell line development from many applications. The objective of this research was to develop a platform for high-throughput hiPSC cytometry and splitting that utilized a label-free cell sensing approach. An image analysis pipeline utilizing background subtraction and standard deviation projections was implemented to detect hiPSC colonies from bright-field microscopy data. The pipeline was incorporated into an automated microscopy system coupling quad microraft cell-isolation arrays, computer-based vision, and algorithms for smart decision making and cell sorting. The pipeline exhibited a hiPSC detection specificity of 98% and a sensitivity of 88%, allowing for the successful tracking of growth for hundreds of microcolonies over 7 days. The automated platform split 170 mother colonies from a microarray within 80 min, and the harvested daughter biopsies were expanded into viable hiPSC colonies suitable for downstream assays, such as polymerase chain reaction (PCR) or continued culture. Transmitted light microscopy offers an alternative, label-free modality for isolating hiPSCs, yet its low contrast and specificity for adherent cells remain a challenge for automation. This novel approach to label-free sensing and microcolony subsampling with the preservation of the mother colony holds the potential for hiPSC colony screening based on a wide range of properties including those measurable only by a cell destructive assay.
Large-scale in vitro screening techniques have greatly advanced basic research and helped to identify drug candidates for treating human diseases, but current methods are optimized for dividing cells that can be expanded to generate large sample sizes, not for post-mitotic cells such as neurons. As such, the effort and cost of obtaining neurons for large-scale screens have limited drug discovery efforts in neuroscience. The authors used the CellRaft® Technology to generate thousands of neuronal mini-cultures for both mouse neurons and human-induced pluripotent stem cell-derived neurons. Furthermore, they were able to successfully detect disease-related defects in synaptic transmission and identify candidate small molecule therapeutics. CytoSort™ Arrays were seeded to create sufficient neuronal densities for network maturation while maintaining effective numbers of neurons for compound screening. This new approach was shown to have advantages in terms of evaporation issues that plague traditional long-term micro-cultures, and favorable viability was demonstrated when the CytoSort™ Arrays were compared to 384-well plates.
In order to determine the underlying mechanisms of a broad range of issues related to human health and disease, it is important to first understand how somatic stem cells self-renew and differentiate to produce the functional cells of the resident tissue, as stem cells reside in niches where support cells provide signaling critical for tissue renewal. Magness and colleagues demonstrated the use of the CellRaft® Technology to prove that Paneth cells (PC), a known intestinal stem cell (ISC) niche component, enhance organoid formation in a contact-dependent manner. CellRafts® were used to facilitate retrieval of early enteroids for qPCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. This platform enabled the study of a large number of single ISCs simultaneously, either at the clonal level or in the presence of niche cells, with multi-day, three-dimensional culture in extracellular matrices applied directly to the CytoSort™ Arrays. The authors found that direct cell-to-cell contact between ISCs and PCs is required for enhanced ISC growth.