Publications Using the CellRaft® Technology
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In order to establish a causal relationship between somatic mutations and aging, mutational events must be directly identified in primary human tissues. Single cell sequencing holds promise for the detection of a full complement of mutations in somatic cells, overcoming the challenges that arise due to the random nature and very low abundance of most somatic mutations; however, the genome amplification procedures required for single cell genomics have a high error rate. To address this problem, Vijg, et al. developed a highly accurate single cell multiple displacement amplification (SCMDA) to comprehensively determine the full spectrum of base substitutions in a single somatic cell, and thereby assess mutation accumulation as a function of age in human B lymphocytes from healthy individuals. To aid in this analysis, bulk B lymphocytes were isolated from PBMCs and subsequently plated on gelatin coated CytoSort™ Arrays. Using the CellRaft® Technology, single B lymphocytes were isolated and collected in PCR tubes for SCMDA and downstream analysis of somatic mutations.
Neocortical neurons are among the most diverse and longest-lived mammalian cells, and human-specific brain phenotypes are attributed to neocortical expansion during evolution. McConnell and coworkers assembled a brain copy number variation (CNV) atlas to reveal the frequency of neocortical neurons with complex karyotypes and the associated variability among individuals. These CNVs represent rare variants with strong contributions to genetic risks for schizophrenia, autism, and other neurological disorders. The CellRaft® Technology was used to isolate, and verify the integrity of, single nuclei following flow sorting to assess the quality of the whole genome amplification (WGA) method utilized. The authors provide evidence that a functional consequence of CNV neurons may be selective vulnerability to aging-associated atrophy.
Generation of cell lines with specific mutations is integral to the in vitro study of many diseases and the associated pathogenesis, and the CRISPR-Cas9 gene editing system has revolutionized the ability to efficiently generate disease models. Limiting dilution and FACS have traditionally been used to obtain clonal cell lines with specific genomic modifications introduced by the CRISPR-Cas9 system; however, they require large sample sizes and often yield low cell viability. The authors used the CellRaft® Technology to sort cells based on the temporal evolution of fluorescent protein expression (EGFP) to generate a CRISPR gene-edited cell line with a leukemia-associated mutation (S34F) in the U2AF1 protein, allowing for the further study of the consequences of this mutation on mRNA splicing in AML.
It has long been known that genetic material is mutable at a rate subject to natural selection, but multicellular organisms have a somatic genome with a mutation rate that differs from the germline mutation rate. However, a lack of reliable methods to measure somatic mutation frequencies in DNA have precluded a direct comparison in mutation rates between somatic and germline cells. Vijg and colleagues present the first direct comparison of mutation rates in human and mouse single somatic cells, both of which are further compared to human and mouse de novo germline mutation rates. The CellRaft® Technology is utilized here to isolate single cells for downstream single cell whole genome sequencing after amplification. The results presented suggest that somatic mutation frequencies are significantly higher than germline mutation frequencies, which may point toward somatic mutations as a conserved mechanism of aging.
The authors present a novel methodology to address the artifacts associated with cell lysis and whole genome amplification during genome-wide DNA mutation analysis, termed Single-Cell Multiple Displacement Amplification (SCMDA). SCMDA and SCcaller were validated by direct comparison of SNVs from amplified single cells and unamplified clones derived from cells in the same population of early passage human primary fibroblasts. The CellRaft® Technology was utilized for multiple steps in the validation workflow, including isolation of single cells and generation of single cell clones. Single cells were plated on CytoSort™ Arrays, isolated, and subjected to downstream SCMDA, library preparation, and sequencing. Empty CellRafts® were also isolated to serve as negative controls in WGA. To generate single cell clones, cells were plated on CytoSort™ Arrays and isolated once the clones reached confluency on the CellRaft®, approximately 10-16 cells per raft, for further expansion in 96-well plates. Lastly, to generate kindred single cells and clones, small clones were transferred from 96-well and seeded on a fresh CellRaft Arrayto isolate single cells. This method, and the corresponding single-cell variant caller SCcaller, provide a foundation for standardizing somatic mutation analysis in single-cell genomics.
Antigen-specific cytotoxic CD8+ T cell responses are an essential component in the ability of the adaptive immune system to control viral infections and cancer. There are several immune monitoring assays that have been developed to measure the cytotoxic activity of a T cell population; however, there are limitations in terms of sample size, sample processing, overall assay time, and cost. Furthermore, these methods do not directly measure individual T cell mediated killing, nor do they provide data regarding the temporal dependence of CD8+ T cells. Armistead and colleagues developed a methodology to identify, isolate, and clonally expand antigen-specific CD8+ T cells based upon the time course measurement of the killing of antigen expressing target cells. The CellRaft® Technology was utilized wherein each CellRaft® contained a population of fluorescently-labeled antigen-presenting target cells and one CD8+ T cell. Over the 6-hour time course of the experiment, a cytotoxicity dye was used in the media of the CytoSort™ Array to monitor target cell death over time. The CD8+ T cells that demonstrated a high rate of target cell death were isolated from the CytoSort™ Array to a 96-well plate for clonal expansion and further characterization, including measurement of TCR affinity to a peptide/MHC tetramer and sequencing of the TCRα and TCRβ CDR3 regions.
Identifying genetic and genomic characteristics that determine the functional or phenotypic properties of individual tissues and cells in multicellular organisms is a fundamental problem in modern biology. While high-throughput techniques, such as RNA-seq, are indispensable tools, they are most often performed on bulk tissue samples and inherently blur the properties of individual cells within a heterogeneous tissue. The authors present the CellRaft® Technology as a method that provides two key advantages over existing single cell isolation approaches for genomics: selective isolation without the requirement for a pre-selection step, and the ability to sort cells based on complex temporal or spatial phenotypes or cellular markers. To evaluate this approach, CFPAC-1 cells were plated on CytoSort™ Arrays and treated with 0, 2, or 5 nM gemcitabine and allowed to grow on the array for 4 days, with strongly restricted growth seen in the cells treated with 5 nM gemcitabine. Cells of interest, either proliferative or non-proliferative, were subsequently isolated and sequenced, thereby linking a proliferative pancreatic cancer cell phenotype, in the presence of a chemotherapeutic agent, to the altered transcriptome of the cells.
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.
Cell transplantation has wide-ranging applications in establishing xenograft tumor models, studying in vivo differentiation of stem cells, and repairing tissue for regenerative medicine. Biodegradable microcarriers have long been used to deliver anchorage-dependent cells, and prolonged survival and function of transplanted cells has been demonstrated. However, cells isolated from animal or human populations are generally heterogeneous, and must, therefore, be selectively isolated from a mixed population prior to implantation into another animal. To address this, the authors build upon the original description of the CellRaft® Technology. Biodegradable CellRafts® are micromolded out of poly(lactic-co-glycolic acid) (PLGA), rather than polystyrene, as originally described. Cells plated on the arrays attach to the biodegradable CellRafts® and can be identified, sorted, and implanted into animals. As a proof-of-concept study, Allbritton and colleagues subcutaneously injected mice with biodegradable CellRafts® carrying human pancreatic adenocarcinoma cells to establish a xenograft tumor model and demonstrate the in vivo degradation of the implanted CellRafts®.