Massively parallel delivery of large cargo into mammalian cells with light pulses

Wu, Yi-Chien; Wu, Ting-Hsiang; Clemens, Daniel L.; Lee, Bai‐Yu; Wen, Ximiao; Horwitz, Marcus A.; Teitell, Michael A.; Chiou, Pei-Yu

doi:10.1038/nmeth.3357

Cited by 158 publications

(174 citation statements)

References 34 publications

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“…Collectively, these distinctions between virulent strains and LVS imply that the biological relevance of our current findings for F. tularensis strains in general will ultimately require that the work be validated using virulent strains. Further, it was recently shown that the ⌬iglC mutant of F. novicida U112 was unable to replicate upon delivery into the cytosol of mammalian cells (79), in contrast to our findings after microinjection. Thus, phenotypic differences appear to exist between the different subspecies of F. tularensis that affect their intracellular survival.…”

Section: Discussioncontrasting

confidence: 99%

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

et al. 2015

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bCertain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ⌬iglA, ⌬iglC¸⌬iglG, ⌬iglI, and ⌬pdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC ؊/؊ BMDM, MyD88 ؊/؊ BMDM, J774 cells, or HeLa cells, LVS, ⌬pdpE and ⌬iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ⌬iglA and ⌬iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC ؊/؊ BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88 ؊/؊ BMDM and LVS showed no replication in either BMDM or MyD88 ؊/؊ BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection. Bacteria and other microbes have developed an ability to invade host cells and use them as a principal habitat for replication. These so-called intracellular pathogens are able to trigger their uptake by mammalian cells, by phagocytosis when the host cells are professional phagocytes, e.g., monocytes or macrophages, or by triggered phagocytosis in the case of nonprofessional phagocytic host cells, such as epithelial or endothelial cells, hepatocytes, and fibroblasts (1, 2). After internalization, virulence factors produced by the intracellular pathogen modulate the intracellular environment to facilitate microbial survival (3, 4). For protection against intracellularly located microorganisms, the immune system is dependent on pattern recognition receptors (PRR) that identify conserved microbial components (5). The best-characterized family of PRR is the one of Toll-like receptors (TLR), a group of integral membrane proteins that recognize microbial components, such as lipopolysaccharide, bacterial lipoprotein, and CpG DNA (6, 7). Triggering of TLR leads to rapid initiation of an antimicrobial proinflammatory response (8, 9). These innate defense mechanisms are normally sufficient to mediate the eradication of phagocytosed extracellular pathogens but not to control those capable of intracellular replication.Many intracellular bacteria, e.g., Mycobacterium,...

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Section: Discussioncontrasting

confidence: 99%

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

et al. 2015

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“…Spatial control can be achieved by laser excitation of an absorbent particle or substrate. Recently, this concept was scaled up for deployment with cell monolayers 93 . Substrates arrayed with pores lined by metallic absorbers were irradiated underneath adherent cells.…”

Section: Towards Precision Membrane Disruptionmentioning

confidence: 99%

In vitro and ex vivo strategies for intracellular delivery

Stewart¹,

Sharei²,

Ding³

et al. 2016

Nature

738

742

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Although carrier-mediated delivery systems offer promise for nucleic acid transfection in vivo 1,2 , membrane-disruption-based modalities are attractive candidates for universal delivery systems in vitro and ex vivo. In this review, we begin with motivations driving next-generation intracellular delivery strategies and suggest relevant requirements for future systems. Next, a broad overview of current delivery concepts covering salient strengths, challenges and opportunities is presented. Following that, our focus shifts to prevalent mechanisms of membrane disruption and recovery in the context of intracellular delivery. Finally,

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“…This work represents a shift in microfluidic design that should allow microfluidic devices to be incorporated in a wider range of commercial applications. High-Throughput Photothermal BLAST Platform for Large-Cargo Intracellular Delivery 8 While there are a number of well-established methods for delivery of small-scale cargo, such as kilobit-sized nucleic acids, reliably delivering large cargo into cells has remained elusive. Developing a method for large-cargo delivery in a high-throughput manner is an even more difficult hurdle to overcome.…”

Section: A 3d Platform For Modular Microfluidic Circuitsmentioning

confidence: 99%

“…[8][9][10] This year's honorees represent the best in research that advances translational science and technology, and we are proud to honor these researchers for their amazing work. JALA and SLAS would like to thank all the nominees as well as those who nominated them.…”

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confidence: 99%

Congratulations to the 2016 JALA Ten!

Chow

2016

SLAS Technology

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EditorialOn behalf of the JALA scientific advisors and JALA editorial board, I am happy to present this year's honorees of the prestigious JALA Ten. Each year, JALA seeks to highlight and honor the very best work of the year that will have a deep impact on how technology is used across a wide range of disciplines, including automation, life sciences and biomedical research, diagnostics, drug delivery, and regenerative medicine. Implementing the latest advances in microfluidics, nanotechnology, materials science, and other fields of research, this year's JALA Ten honorees have and will continue to change the way research is performed and the way diseases are diagnosed and treated. As such, the work highlighted here should have far-reaching impact in our everyday lives. It demonstrates the promise that science brings toward a better future.While a number of areas of research will feel the impact of this year's JALA Ten, one highlight of the collection is the diverse ways in which the honorees have advanced biological molecule detection and established foundations for tomorrow's biosensors in life sciences research and medical diagnostics.1-3 For example, Peter Lillehoj at Michigan State University (USA) and his collaborators have developed a microfluidic biosensor using immobilized antimicrobial peptides for highly specific, multiplexed detection of bacterial pathogens.1 Showing both high pathogen detection specificity and accurate pathogen quantification, this work is an example of how microfluidic technology is changing how medical professionals will track and diagnose infectious diseases.On the other end of the spectrum, Somin Eunice Lee at the University of Michigan (USA) has harnessed the power of nanotechnology to develop a gold nanoparticle-based plasmon ruler that is capable of single-molecule measurements.2 Even more impressive is the application of this plasmon ruler toward the accurate detection and measurement of secreted single molecules in the cellular microenvironment. Understanding how cells communicate with their cellular microenvironment remains a challenge to study. Tools such as Lee's plasmon ruler vastly improve life sciences researchers' abilities to learn more about the interplay between cells and their microenvironments.Another area of research highlighted this year is the advancement of technology toward customizable platforms for increased personalized clinical and research applications. [4][5][6][7] The development of tunable injectable microporous gel scaffolds by researchers at the University of California, Los Angeles (USA) not only brings truly personalized medicine to regenerative medicine, but also allows researchers to quickly optimize culture conditions in research and drug development applications that require complex three-dimensional (3D) cell cultures. 4 The development of integrated platforms composed of modular technologies increases the diversity of clinical and research applications to which technology such as microfluidics can be applied and is highlighted here by work f...

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Massively parallel delivery of large cargo into mammalian cells with light pulses

Cited by 158 publications

References 34 publications

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

In vitro and ex vivo strategies for intracellular delivery

Congratulations to the 2016 JALA Ten!

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