Use of Precision-Cut Lung Slices as an Ex Vivo Tool for Evaluating Viruses and Viral Vectors for Gene and Oncolytic Therapy

Gerpe, María Carla Rosales; Vloten, Jacob P. van; Santry, Lisa A.; Jong, Jondavid de; Mould, Robert C.; Pelin, Adrian; Bell, John C.; Bridle, Byram W.; Wootton, Sarah K.

doi:10.1016/j.omtm.2018.07.010

Cited by 43 publications

(41 citation statements)

References 57 publications

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“…Ovine lung slices were transduced with GFP-expressing LVs since we previously found that lung tissue from lambs <6 months of age expresses a heatresistant AP. 26 Forty-eight hours post-transduction, the lung tissue slices were sandwiched between a coverslip and glass slide and imaged with an inverted fluorescence microscope. Faint GFP-positive foci observed in ovine lung tissue slices transduced by EBOV GP LV are pinpointed by yellow arrows.…”

Section: Discussionmentioning

confidence: 99%

“…Here, we generated two novel LVs pseudotyped with DGP Jenv N592T and DGP Jenv M593E, mutant versions of the envelope glycoprotein from a lung tropic betaretrovirus, which can produce vector titers equivalent to VSVg LV, and optimized a method for their large-scale purification. Using a lung slice method we previously optimized, 26 we developed a novel murine lung tissue slice model that allowed us to test these pseudotyped LVs ex vivo. We showed that TFF, followed by gradient ultracentrifugation, dialysis, and PEG-20,000 concentration, is a scalable method for producing high-titer DGP Jenv-pseudotyped LVs for in vivo use.…”

Section: Discussionmentioning

confidence: 99%

“…Three weeks later, lungs were harvested and used to generate hHyal2-expressing lung tissue slices as previously described. 26 Immunohistochemical staining for FLAG revealed robust AAVmediated expression of FT-hHyal2 throughout the lung parenchyma (Fig. 3B, bottom panel).…”

Section: Development Of a Mouse Model To Evaluate Jenv-pseudotyped LVmentioning

confidence: 95%

“…Lung slices were prepared and maintained as previously described. 26 Briefly, mouse and lamb lungs were excised immediately after euthanasia. Each murine lung lobe and intact lamb lungs were perfused via the trachea with 2% low melting point agarose.…”

Section: Lung Tissue Slicesmentioning

confidence: 99%

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Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery

et al. 2020

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Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Development Of a Mouse Model To Evaluate Jenv-pseudotyped LVmentioning

confidence: 95%

Section: Lung Tissue Slicesmentioning

confidence: 99%

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Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery

et al. 2020

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“…Among these technologies, organotypic slice cultures have demonstrated the ability to closely resemble the TME architecture and allow for drug metabolism and toxicity studies in brain, 33 liver, [34][35][36] lung, 36,37 kidney, 36,38 and intestine 34 tumor slices. Patient-derived clinical samples provide a unique tool to explore the susceptibility of individual tumors to specific therapies, but the small amount of tumor tissue extracted from any given patient limits the utility of this approach.…”

Section: Introductionmentioning

confidence: 99%

A Microfluidic Platform for Functional Testing of Cancer Drugs on Intact Tumor Slices

Rodriguez

Horowitz

Castro

et al. 2020

Preprint

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AbstractPresent approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.

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An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision‐Cut Lung Slices to Understand Lung Injury and Repair

et al. 2020

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Recent advances in cell culture models like air-liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics.

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Use of Precision-Cut Lung Slices as an Ex Vivo Tool for Evaluating Viruses and Viral Vectors for Gene and Oncolytic Therapy

Cited by 43 publications

References 57 publications

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery

A Microfluidic Platform for Functional Testing of Cancer Drugs on Intact Tumor Slices

An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision‐Cut Lung Slices to Understand Lung Injury and Repair

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