Publications Using the CellRaft® Technology
CRISPR nucleases can introduce damages to DNA strands that when repaired by the cell, result in introduction or deletion of nucleotides to the sequence. Presented here is a strategy that utilizes a ‘sequence-ascertained favorable editing’ (SAFE) donor approach that has the ability to make such edits to the sequence of DNA using combinations of substitutions unlikely to have any effect on the target site. To test and evaluate the success of the SAFE donor editing approach, two target sites in the genes TEX2 and TTF1 were chosen as these are known to display frequent deletions affecting the editing site. Human stem cells were electroporated, seeded in 12-well plates to recover before being passaged for use with the CellRaft Technology. Cells were seeded on a CellRaft Array and scanned for 5 days before being isolated into 96-well collection plates. A total of 437 and 435 clones from each target site TEX2 and TTF1 pools, respectively were isolated and evaluated downstream. DNA was extracted from the clones, amplified, and the sequences were analyzed. The use of the SAFE donor approach easily enabled the detection of unintended effects by sequencing the target site.
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.
Sirtuin 6 (SIRT6) is a deacylase and mono-ADP-ribosyltransferase enzyme implicated in multiple aging-related pathways including DNA repair, telomere maintenance, and metabolism regulation. A strong correlation between SIRT6 activity and longevity has been observed across species, and genetic polymorphisms in the SIRT6 gene correlate with human longevity in SNP analyses. To demonstrate that the centSIRT6 allele improves DNA repair efficiency, the authors used the CellRaft AIR system to isolate monoclonal mesenchymal stem cells (MSCs) with a centSIRT6 knock-in. MSCs with the centSIRT6 allele demonstrated a higher efficiency of DNA repair and increased survival upon treatment with methyl methanesul-fonate, a potent DNA-damaging agent, supporting the hypothesis that the centSIRT6 allele contributes to human longevity by improving genome maintenance.
A major limitation with pooled screening platforms is the inability to identify phenotypic characteristics associated with cells of interest. Most platforms generate simple readouts such as viability and fluorescence. Using a method termed “Raft-Seq” this paper demonstrates how the CellRaft AIR System was able to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusion 2. The CellRaft AIR System was used to generate clones for monitoring phenotypes as well as isolating cells of interest for downstream sequencing. The CellRaft Technology was compared against 4 other cell screening-imaging platforms (Arrayed screening, In situ sequencing, photo-activation, and CRaft-ID) and among 7 comparison criteria (Imaging constraints, how cells are collected, genotype resolution, live cell collection, genotyping method, pooled scalability, and bottleneck). Overall, the platforms and comparison criteria, the CellRaft Technology dominated the competition by making it possible to examine phenotypic characteristics of live cells on a single cell genotype resolution with an ability to isolate out cells of interest for further downstream NGS.
Cohesin regulates three-dimensional gene organization and gene expression contributing an important role in the structure integrity of many proteins. PDS5A and PDS5B are two subunits that make up HEAT repeat proteins that interact with cohesin proteins. To understand more about the role these two subunits play in cohesin and gene expression, two stable knockout lines were constructed using the CellRaft AIR System. Cells were transfected with two plasmids, allowed to settle for a day and then seeded onto a CellRaft Array to expand. Once clones were generated and measured with fluorescence markers to determine successful edit, CellRafts containing colonies of interest were isolated using the CellRaft AIR System. Downstream, cells were sequenced and analyzed for further investigation against the role the two subunits play in gene expression when involved with the cohesin proteins.
Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses, simulate compound diffusion through complex structures, and assess cellular heterogeneity of tissues, making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell cul- ture that are grown from stem cells, are self-organizing, and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development, model diseases, test drug sensitivity and toxicity, and advance regenerative medicine. However, traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging, phenotypic assessment, and isolation from heterogenous organoid populations. To address these bottlenecks, we have adapted our tissue culture consumable and instrumentation to enable automated imaging, identification, and isolation of individual organoids. Organoids grown on the 3D CellRaft Array can be reliably tracked, imaged, and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time, then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids, we have demonstrated the use of this technology for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single- organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR® System to facilitate efficient, user-friendly, and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment, 2) establishment of single cell-derived organoids, and 3) isolation and retrieval of single organoids for downstream applications.
While T lymphocytes have been employed as a cancer immunotherapy, the development of effective and specific T-cell-based therapeutics remains challenging. A key obstacle is the difficulty in identifying T cells reactive to cancer-associated antigens. The objective of this research was to develop a versatile platform for single cell analysis and isolation that can be applied in immunology research and clinical therapy development. Methods: An automated microscopy and cell sorting system was developed to track the proliferative behavior of single-cell human primary CD4+ lymphocytes in response to stimulation using allogeneic lymphoblastoid feeder cells. Results: The system identified single human T lymphocytes with a sensitivity of 98% and specificity of 99% and possessed a cell collection efficiency of 86%. Time-lapse imaging simultaneously tracked 4,534 alloreactive T cells on a single array; 19% of the arrayed cells formed colonies of ≥2 cells. From the array, 130 clonal colonies were isolated and 7 grew to colony sizes of >10,000 cells, consistent with the known proliferative capacity of T cells in vitro and their tendency to become exhausted with prolonged stimulation. The isolated colonies underwent ELISA assay to detect interferon-γ secretion and Sanger sequencing to determine T cell receptor β sequences with a 100% success rate. Conclusion: The platform is capable of both identification and isolation of proliferative T cells in an automated manner. Significance: This novel technology enables the identification of TCR sequences based on T cell proliferation which is expected to speed the development of future cancer immunotherapies.
Motility and invasion are key steps in the metastatic cascade, enabling cells to move through normal tissue borders into the surrounding stroma. Most available in vitro assays track cell motility or cell invasion but lack the ability to measure both simultaneously and then separate single cells with unique behaviors. In this work, we developed a cell-separation platform capable of tracking cell movement (chemokinesis) and invasion through an extracellular matrix in space and time. The platform utilized a collagen scaffold with embedded tumor cells overlaid onto a microraft array. Confocal microscopy enabled high resolution (0.4 × 0.4 × 3.5 µm voxel) monitoring of cell movement within the scaffolds. Two pancreatic cancer cell lines with known differing invasiveness were characterized on this platform, with median motilities of 14 ± 6 μm and 10 ± 4 μm over 48 h. Within the same cell line, cells demonstrated highly variable motility, with XYZ movement ranging from 144 μm to 2 μm over 24 h. The ten lowest and highest motility cells, with median movements of 33 ± 11 μm and 3 ± 1 μm, respectively, were separated and sub-cultured. After 6 weeks of culture, the cell populations were assayed on a Transwell invasion assay and 227 ± 56 cells were invasive in the high motility population while only 48 ± 10 cells were invasive in the low motility population, indicating that the resulting offspring possessed a motility phenotype reflective of the parental cells. This work demonstrates the feasibility of sorting single cells based on complex phenotypes along with the capability to further probe those cells and explore biological phenomena.
The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was developed to perform semi-automated cell sampling, bioassay, and banking on ultra-small sample sizes. The system biopsies and collects colony fragments, quantifies intracellular protein levels via immunostaining, and then retrieves the living mother colonies based on the fragments’ immunoassay outcome. To accomplish this, a magnetic, microwell-based plate was developed to mate directly above the microraft array and capture colony fragments with a one-to-one spatial correspondence to their mother colonies. Using the Signal Transducer and Activator of Transcription 3 (STAT3) model pathway in basophilic leukemia cells, the system was used to sort cells based on the amount of intracellular STAT3 protein phosphorylation (pSTAT3). Colonies were detected on quad arrays using bright field microscopy with 96 ± 20% accuracy (true-positive rate), 49 ± 3% of the colonies were identified as originating from a single cell, and the majority (95 ± 3%) of biopsied clonal fragments were successfully collected into the microwell plate for immunostaining. After assay, biopsied fragments were matched back to their mother colonies and mother colonies with fragments possessing the greatest and least pSTAT3/STAT3 were resampled for expansion and downstream biological assays for pSTAT3/STAT3 and immune granule exocytosis. This approach has the potential to enable colony screening and sorting based on assays not compatible with cell viability, greatly expanding the cell selection criteria available to identify cells with unique phenotypes for subsequent biomedical research.
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.
