Publications Using the CellRaft™ Technology
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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®.
The widespread use of lab-on-a-chip devices containing microcavities in the form of microwells, traps, dead ends, and corners, is often precluded by the trapping of air bubbles. Allbritton and colleagues present a straightforward and simple approach to mitigating bubble formation that can be applied to the CytoSort™ Arrays that are the cornerstone of the CellRaft® Technology. Air bubbles are eliminated in a two-step process, whereby microarrays are first hydrophilized by plasma treatment, and subsequently coated with a monosaccharide such as D-glucose or D-sorbitol. Following this treatment, microwell arrays can be stored for as long as 6 months in air, and complete rewetting of the microwells is demonstrated by the dissolution of the monosaccharide with an aqueous solution.
Selection and isolation of single cells from a mixed population is a common procedure, for example, during the development of clonal cell lines that are genetically engineered, derived from stem cells, or grown from patient samples, single cells must be isolated and then cloned to form a homogeneous population. While myriad methods exist, they commonly rely on enzymatic or mechanical release, which imposes drawbacks in terms of loss of cell morphology, removal of cell surface markers, damage to cell membranes, alterations in cellular physiology, and loss of viability. In this seminal work describing the CellRaft® Technology, the authors demonstrate the fabrication of a microwell array that possesses detachable concave elements, termed CellRafts®, using a PDMS mold combined with standard dip-coating procedures. The resulting microarrays have low auto-fluorescence and are easily removable, in a targeted fashion, allowing for isolation and clonal expansion of single cells. Furthermore, the CytoSort™ Arrays allow for the assessment of cells based on morphology in a time-resolved manner and can be tailored to workflows requiring few to hundreds of thousands of cells.
Genomic mosaicism is prevalent throughout human somatic tissues and is much more common than previously thought. Here, we describe step-by-step methods to isolate neuronal nuclei from human brain and identify megabase-scale copy number variants (CNVs) in single nuclei. The approach detailed herein includes use of CellRaft technology for single-nucleus isolation, the PicoPLEX approach to whole-genome amplification and library preparation, and a pooled library purification protocol, termed Gel2Gel, which has been developed in our laboratory. These methods are focused toward neuroscience research, but are adaptable to many biomedical fields.
Mutations are the driving molecular causes of various biological processes such as developmental and cancer. Recent findings of genomic heterogeneity among ostensibly homogeneous cell populations such as cancer cells demand genomic characterization of mutations at the individual cell level to better understand the underlying biology. Additionally, single cell technologies make genomic analysis feasible for the characterization of rare cells such as circulating tumor cells. Due to its high sensitivity, next generation sequencing (NGS) represents the ideal technology to analyze a collection of mutations in single cells. The challenge, however, is that single cells yield limited amounts of DNA, that need to be amplified prior to NGS. To overcome this challenge, whole genome amplification (WGA), coupled with multiplex PCR-based targeted enrichment, were tested for mutation detection in single cells isolated from two colon cancer cell lines, Lovo and HT29.