Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens

Bailey, Steve N; Sabatini, David M.; Stockwell, Brent R.

doi:10.1073/pnas.0404425101

Cited by 131 publications

(99 citation statements)

References 52 publications

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“…More recently, to emulate native microenvironments, 3D cell cultures have been used extensively, particularly in tissue engineering applications, e.g., cell-seeded scaffolds (17) and patterned cocultures (18) as well as in directing cell fate and differentiation (19). Although miniaturization of 3D platforms has been performed for high-throughput applications (20, 21), relatively little effort has been directed toward using 3D cell cultures as screening tools for microscale toxicology assays (12,22,23). Herein, we address this technology gap by developing a miniaturized 3D cell-culture array (the Data Analysis Toxicology Assay Chip or DataChip) for high-throughput toxicity screening of drug candidates and their cytochrome P450-generated metabolites.…”

mentioning

confidence: 99%

Three-dimensional cellular microarray for high-throughput toxicology assays

Lee

Kumar

Sukumaran

et al. 2008

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

286

236

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cytochromes P450 ͉ in situ drug metabolism ͉ in vitro cytotoxicity ͉ on-chip cell encapsulation R ecent advances in genomics and proteomics coupled with sequencing of the human genome have led to a dramatic increase in the number of screenable drug targets (1). Combinatorial (2) and diversity-oriented synthesis (3) programs along with increased access to natural products and their structural scaffolds (4) have provided vast numbers of compounds to screen against these targets. However, there has not been a commensurate increase in the number of approved drugs (5). A major reason for this is the high failure rate of drug candidates because of factors that are not typically considered in the early stages of drug discovery, including poor ADME/tox (absorption, distribution, metabolism, excretion, and toxicology) profiles (6, 7). Thus, pharmaceutical companies are beginning to evaluate toxicity of drug candidates early in the discovery process to reduce the chances of late-stage failure (8).Such early-stage toxicity information requires the development of accurate, reproducible, and predictive in vitro assays. In some cases, validation of in vitro assays has been achieved, such as in percutaneous absorption, skin corrosivity, and phototoxicity (9); however, these tests are limited and are not effective for acute organ-specific toxicity or metabolite toxicity. Moreover, in some European industries (e.g., cosmetics and chemicals), animal testing is being phased out entirely, thereby forcing companies to adopt new in vitro screens that effectively predict human toxicity (10-12). Thus, the need for concordance between in vitro assays and in vivo responses is becoming greater and more pressing, particularly in high throughput that would enable prioritization of compounds for further development involving animal testing of pharmaceutical candidates (13) or direct human testing of cosmetic ingredients.High-throughput screening (HTS) assays for toxicity routinely use 96-or 384-well plates with 2D cell monolayer cultures (14, 15). The multiwell plate format, however, suffers from several limitations, including inefficient removal of reagents from the wells and the difficulty of subsequent washing of cell monolayers (16). These limitations are further compounded when highthroughput screening of cellular targets is coupled with metabolite synthesis, which requires addition of multiple reagents. More recently, to emulate native microenvironments, 3D cell cultures have been used extensively, particularly in tissue engineering applications, e.g., cell-seeded scaffolds (17) and patterned cocultures (18) as well as in directing cell fate and differentiation (19). Although miniaturization of 3D platforms has been performed for high-throughput applications (20, 21), relatively little effort has been directed toward using 3D cell cultures as screening tools for microscale toxicology assays (12,22,23). Herein, we address this technology gap by developing a miniaturized 3D cell-culture array (the Data Analysis Toxicology Assay Chip or D...

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mentioning

confidence: 99%

Three-dimensional cellular microarray for high-throughput toxicology assays

Lee

Kumar

Sukumaran

et al. 2008

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

286

236

View full text Add to dashboard Cite

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“…Previous work on a related type of microarray configuration has suggested that a 1.5-mm spacing between islands is sufficient to isolate cell populations from agents released from neighboring polymer spots (13). To determine whether paracrine signaling or diffusion of drugs from adjacent polymer islands was a factor in our drug-eluting microarray using this 1.5-mm (center to center) island spacing, we analyzed camptothecin-loaded arrays in various configurations ( Fig.…”

Section: -H)mentioning

confidence: 99%

“…Array platforms capable of capturing single cells have been established (11,12), but determination of chemotherapeutic efficacy is better investigated through methods using greater cell numbers, which better capture variability in cellular responses. Furthermore, arrays of drug-loaded polymer films with an overlying cell monolayer have been developed (13), but monolayers of cells are susceptible to juxtacrine and paracrine signaling, which are particularly important for multipotent cells. In the present work, the provision of differential cell adhesion to promote seeding onto spotted drug-loaded films against a surrounding nonfouling background (i.e., a surface that resists protein adsorption and thus cell adhesion) can separate drugeluting polymer films to create isolated culture environments.…”

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confidence: 99%

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

Carstens

Fisher

Acharya

et al. 2015

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

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A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.personalized medicine | chemopredictive | cancer stem cell | microarray | combination therapy

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“…Although effective, this method remains unavailable to many laboratories due to the requirement for expensive robotics. A second approach that can be used for high-throughput cell studies is reverse transfection [4][5][6] . Here, cells are seeded onto an array of DNA, RNA, or small molecules.…”

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confidence: 99%

Live mammalian cell arrays

et al. 2013

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High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We have developed a simple, yet powerful method that uses contact spotting to generate highdensity nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries. High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We have developed a simple, yet powerful method that uses contact spotting to generate highdensity nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.Cell-based assays and the tools used to perform them are constantly undergoing improvements toward higher experimental throughput, reduced reagent consumption, and advanced control of the cell microenvironment 1,2 . There are currently two main approaches for conducting highcontent cell culture experiments. In the first, cells are cultured in microtiter plates and robotic equipment is used to perform all necessary fluidic operations 3 . Although effective, this method remains unavailable to many laboratories due to the requirement for expensive robotics. A second approach that can be used for high-throughput cell studies is reverse transfection [4][5][6] . Here, cells are seeded onto an array of DNA, RNA, or small molecules. Large gene expression and silencing studies can be conducted in this manner, but complex cell libraries cannot be investigated since each array is limited to a single cell type.As an alternative to these approaches, contact spotting offers an affordable yet high-throughput platform. This technique is extensively used to generate high-density DNA and protein arrays and has been adapted to a variety of other purposes. For example, Hart et al. created a high-content immunoassay by spotting fixed mammalian cells that were cultured under different conditions 7 . To date, contact spotting has not been used to array live mammalian cells due to rapid spot evaporation and consequent cell death. Therefore, arraying of live mammalian cells has been limited to inkjet printing, which lacks the ability to handle a large number of different samples 8,9 .Large collections of mammalian cell lines have recently become available, including primary tumor and cancer cell lines 10 , stably transfected expression cell lines, and GFP-fusion libraries 11 . Novel approaches are required to efficiently assemble complex arrays of hundreds to thousands of genetically diverse cells. To address this arising need, we developed a simple, fast, and scalable method that uses standard microarray printing tools to generate high-density nanowell arrays. A minimal sample requirement of 500 cells enables the interrogation of cells that are availab...

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Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens

Cited by 131 publications

References 52 publications

Three-dimensional cellular microarray for high-throughput toxicology assays

Three-dimensional cellular microarray for high-throughput toxicology assays

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

Live mammalian cell arrays

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