Live-cell Microarray Surface Coatings Supporting Reverse Transduction by Adeno-Associated Viruses

McConnell, Kellie I.; Schweller, Ryan M.; Diehl, Michael R.; Suh, Junghae

doi:10.2144/000113748

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…Since the complete slide surface is covered with a cell carpet, a successful gene transfer into the cells could be easily observed by the additional expression of a reporter gene, e.g., green fluorescent protein [38]. This procedure results in fluorescent cells at the spots of oligonucleotides and non-fluorescent cells attached between the oligonucleotide spots [38,42,83,84,85]. Other research groups used GFP-transfected cells for creating a kind of grid between the non-fluorescent cells used in their studies [38,86].…”

Section: Literature Review Sectionmentioning

confidence: 99%

Living Cell Microarrays: An Overview of Concepts

et al. 2016

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Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.

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Section: Literature Review Sectionmentioning

confidence: 99%

Living Cell Microarrays: An Overview of Concepts

et al. 2016

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“…Using a similar strategy, reverse transduction has been developed where viruses are fixed on the glass slides to form transduction islands. In addition to the limitations associated with reverse transfection, the viruses have to be prepared before fixing in the conventional culture plates, which is laborious and low throughput [11,12].…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Platform for Efficient Chemical Transfection, Virus Packaging, and Transduction

Zhang

Wang

et al. 2019

Micromachines

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Intracellular gene delivery is normally required to study gene functions. A versatile platform able to perform both chemical transfection and viral transduction to achieve efficient gene modification in most cell types is needed. Here we demonstrated that high throughput chemical transfection, virus packaging, and transduction can be conducted efficiently on our previously developed superhydrophobic microwell array chip (SMAR-chip). A total of 169 chemical transfections were successfully performed on the chip in physically separated microwells through a few simple steps, contributing to the convenience of DNA delivery and media change on the SMAR-chip. Efficiencies comparable to the traditional transfection in multi-well plates (~65%) were achieved while the manual operations were largely reduced. Two transfection procedures, the dry method amenable for the long term storage of the transfection material and the wet method for higher efficiencies were developed. Multiple transfections in a scheduled manner were performed to further increase the transfection efficiencies or deliver multiple genes at different time points. In addition, high throughput virus packaging integrated with target cell transduction were also proved which resulted in a transgene expression efficiency of >70% in NIH 3T3 cells. In summary, the SMAR-chip based high throughput gene delivery is efficient and versatile, which can be used for large scale genetic modifications in a variety of cell types.

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“…The multiplicities of infection (MOIs)—number of viral genomes added per cell—necessary to produce optimal results were reported in the range of 20,000–40,000. Another study demonstrated the feasibility of developing live‐cell microarrays by depositing AAV2 onto nitrocellulose‐coated slides, resulting in effective reverse transduction when HeLa cells were seeded on top of these surfaces (McConnell, Schweller, Diehl, & Suh, ).…”

Section: Introductionmentioning

confidence: 99%

Reverse transduction can improve efficiency of AAV vectors in transduction‐resistant cells

Lee

Robinson

Tabor

et al. 2018

Biotech & Bioengineering

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Reverse transduction, also known as substrate-mediated gene delivery, is a strategy in which viral vectors are first coated onto a surface that subsequently comes into contact with mammalian cells. The cells internalize the surface-attached vectors, resulting in transgene expression. We hypothesized that forcing the interaction between cells and adeno-associated virus (AAV) vectors through a reverse transduction format would increase in vitro gene delivery efficiencies of the vectors in transduction-resistant cells. We tested this hypothesis by comparing the gene delivery efficiencies of three AAV serotypes using either standard or reverse transduction approaches. Our study reveals reverse transduction of AAV7 and AAV9 can significantly improve their delivery efficiencies. In contrast, AAV2 does not perform better under the reverse transduction format. Interestingly, increased vector uptake by cells does not provide a complete explanation for the increased transduction efficiency. Our findings offer a simple and practical method for improving transduction outcomes in vitro in cell types less permissive to a particular AAV vector.

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Live-cell Microarray Surface Coatings Supporting Reverse Transduction by Adeno-Associated Viruses

Cited by 6 publications

References 19 publications

Living Cell Microarrays: An Overview of Concepts

Living Cell Microarrays: An Overview of Concepts

High-Throughput Platform for Efficient Chemical Transfection, Virus Packaging, and Transduction

Reverse transduction can improve efficiency of AAV vectors in transduction‐resistant cells

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