Dynamic Single-Cell Analysis for Quantitative Biology

Carlo, Dino Di; Lee, Luke P.

doi:10.1021/ac069490p

Cited by 346 publications

(221 citation statements)

References 46 publications

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“…Spatial registration of cells in a defined array obviates these problems and allows spatial encoding of multiple experimental conditions on the same slide, and cell spacing can be tuned to maximize the number of conditions per slide. Several methods currently exist for patterning cells, including dielectrophoresis (27), hydrostatic trapping (28), and microwell cell traps (29)(30)(31). Of these methods, only microwells capture cells passively, eliminating the need for external energy sources and introducing minimal external stress on cells.…”

Section: Resultsmentioning

confidence: 99%

Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

242

284

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With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24-and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.base excision repair | comet assay | DNA repair D NA damage is a critical risk factor for cancer, aging, and heritable diseases (1-3), and it is the underlying basis for most frontline cancer therapies (4). Increased knowledge about DNA damage and repair would facilitate disease prevention, identification of individuals at increased risk of cancer, discovery of novel therapeutics, and better drug safety testing. Despite their obvious translational impact, most DNA damage assays have not changed significantly in more than two decades, and thus are not compatible with modern high throughput screening technologies. To better assess the biological impact of damage, we need data that reflect the integrated effect of multiple DNA repair pathways and subpathways, in response to a range of DNA lesions, measured in an accurate and high throughput fashion.The single cell gel electrophoresis or "comet" assay is based on the principle that relaxed loops (induced by single strand breaks) and DNA fragments migrate farther in an agarose gel than undamaged DNA (5-8). The alkali comet assay sensitively detects a range of DNA lesions, including strand breaks (single and double), as well as alkali sensitive sites. Although most base lesions are not directly detected, base adducts can be detected when converted to abasic sites or single strand breaks with the addition of purified DNA repair enzymes (7-11). The comet assay has been used in a variety of applications, including genotoxicity testing, human biomonitoring and epidemiology, environmental healt...

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Section: Resultsmentioning

confidence: 99%

Single cell trapping and DNA damage analysis using microwell arrays

Wood

Weingeist

Bhatia

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

242

284

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“…The topics that have been covered in this article represent a small group of tools that we feel are fundamental to obtaining accurate single-cell analyses. In addition to the research highlighted here, more extensive reviews on the topic of cellular analyses and other cell-related procedures on microfabricated devices show the breadth of work being conducted in this area (28)(29)(30).…”

Section: Separation Of Analytesmentioning

confidence: 97%

Chemical Analysis of Single Mammalian Cells with Microfluidics

Price¹,

Culbertson²

2007

Anal. Chem.

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of Single Mammalian Cells with Microfluidics C ells are the fundamental building blocks of life. All basic physiological functions of multicellular organisms reside ultimately in the cell. The misregulation of cellular physiology results in disease at the organism level. Thus, comprehending cell physiology is key to understanding and curing diseases. Many physiological processes can be studied using populations of cells. Others occur either on short timescales (e.g., kinase signaling cascades) or nonsynchronously (e.g., response to an external chemical gradient), so that taking a population average will not lead to an understanding of how the cellular chemistry occurs. These types of processes require single-cell analysis, and thus discretion must be exercised when deciding under which circumstances bulk versus singlecell analyses are more appropriate (1). In addition, many diseases like cancer start with a single cell; therefore, if one would like to find the rare mutations in populations of cells that herald the inception of a disease, then cells must be examined individually.Probing behavior at the single-cell level, however, is a very challenging task, primarily because of the small sample volume, the low abundance of material, and the fragile nature of the cell itself. Analyzing the contents of a single cell requires sensitive detection techniques and handling procedures that do not stress or damage it. Additionally, no proper blank exists that can be used, so truly quantitative studies are difficult. Intense interest in single-cell physiology, however, is driving the analytical and biomedical engineering fields to improve the technology for examining cells. One of the most popular and promising areas is lab-on-a-chip devices to manipulate and analyze single cells.Since Jorgenson's groundbreaking work in 1989, CE has been used to examine the contents of single cells (2-4). However, many procedures for cell injection and lysis are time-consuming, and accuracy and reproducibility rely heavily on the skill of the researcher. Furthermore, the limited number of architectures provided by microbore tubing and its relatively large volume restrict the types of processes that can be investigated. Conversely, microfluidics, or lab-on-a-chip technology, offers a versatile format in which biological cells can be analyzed.These miniaturized devices provide several analytical and operational advantages over conventional macroscale systems (5, 6). Microfluidic architectures provide precise spatial control over reagents and samples, are capable of fast analysis times, can be automated, and can precisely manipulate picoliter volumes of material without dilution. In addition, microfluidic systems are amenable to many different detection schemes, can be manufactured from many different materials at relatively low cost, allow flexibility of design, and provide the capability of integrating a series of multiple tasks (sample preparation, mixing, separation, etc.) in both serial and parallel schemes. Portable versions of these sys...

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“…These methods, however, employ static testing conditions with low measurement throughput and restricted sample sizes of $100 cells (20). Because of the inherent heterogeneity of biological samples (21), populations on the order of 10 3 210 4 , similar to those generated by techniques such as fluorescence-activated cell sorting (FACS) (22,23), are required to make confident diagnoses based on deformability as a clinical biomarker.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput linear optical stretcher for mechanical characterization of blood cells

et al. 2015

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This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. V C 2015 International Society for Advancement of Cytometry

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Dynamic Single-Cell Analysis for Quantitative Biology

Cited by 346 publications

References 46 publications

Single cell trapping and DNA damage analysis using microwell arrays

Single cell trapping and DNA damage analysis using microwell arrays

Chemical Analysis of Single Mammalian Cells with Microfluidics

High‐throughput linear optical stretcher for mechanical characterization of blood cells

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