Publications Using the CellRaft® Technology
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.
