Publications Using the CellRaft® Technology
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.
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.
The self-renewal potential of hematopoietic stem cells (HSCs) is essential for maintaining tissue and blood homeostasis after stress. Previous studies have identified RNA Binding Proteins (RBPs) as central regulators of HSC fate and promotion of self-renewal, with SYNCRIP being one such RBP upregulated in HSPCs. Using a conditional Cre-lox system to deplete SYNCRIP in HSCs, the authors observed that knocking out SYNCRIP resulted in decreased in vitro colony formation and in vivo engraftment activity, suggesting that SYNCRIP is necessary for HSC self-renewal potential. To test the hypothesis that loss of SYNCRIP alters HSC proliferation, the researchers utilized Cell Microsystem’s CellRaft AIR system to directly image wild type and SYNCRIP KO cells and monitor single cell division in vitro. As the CellRaft arrays allow for single cell segregation in flask-like culture conditions, single HSCs were identified, and their division was tracked with CellRaft Cytometry software. While there was a slight increase in time to first cell division in the SYNCRIP KO cells compared to WT, there was no significant difference in the percent of cumulative dividing cells over 60hrs, indicating that the loss of self-renewing potential of SYNCRIP KO HSCs is not due to a deficit in HSC proliferation. Further investigation found that loss of SYNCRIP resulted in increased unfolded protein response and Endoplasmic Reticulum stress leading to dysregulated proteostasis in KO HSCs. Interestingly, as SYNCRIP is an RBP, it is required for translating CDC42 and a decrease in this RHO-GTPase’s expression resulted in dysregulated HSC polarity and asymmetric segregation with a loss of self-renewal. These studies highlight the role of SYNCRIP in HSC maintenance and repopulation which is necessary for blood homeostasis as well as engraftment following transplantation.
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.
