| 1. |
- Alsafadi, Hani N, et al.
(författare)
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Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery
- 2020
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Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1044-1549. ; 62:6, s. 681-691
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Forskningsöversikt (refereegranskat)abstract
- Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.
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| 2. |
- Alsafadi, Hani N, et al.
(författare)
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Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:12, s. 2718-2731
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Tidskriftsartikel (refereegranskat)abstract
- The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.
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| 3. |
- Augusto Silva, Iran, et al.
(författare)
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A Semi-quantitative Scoring System for Green Histopathological Evaluation of Large Animal Models of Acute Lung Injury
- 2022
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Ingår i: Bio-protocol. - 2331-8325. ; 12:16
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Tidskriftsartikel (refereegranskat)abstract
- Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.
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| 4. |
- Augusto Silva, Iran, et al.
(författare)
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Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs
- 2023
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Ingår i: Frontiers in Biomaterials Science. - 2813-3749. ; 2-2023:1155919
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Tidskriftsartikel (refereegranskat)abstract
- Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable.
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| 5. |
- Broberg, Ellen, et al.
(författare)
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A new way of monitoring mechanical ventilation by measurement of particle flow from the airways using Pexa method in vivo and during ex vivo lung perfusion in DCD lung transplantation
- 2018
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Ingår i: Intensive Care Medicine Experimental. - : Springer Science and Business Media LLC. - 2197-425X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Different mechanical ventilation settings are known to affect lung preservation for lung transplantation. Measurement of particle flow in exhaled air may allow online assessment of the impact of ventilation before changes in the tissue can be observed. We hypothesized that by analyzing the particle flow, we could understand the impact of different ventilation parameters. Methods: Particle flow was monitored in vivo, post mortem, and in ex vivo lung perfusion (EVLP) in six porcines with the Pexa (particles in exhaled air) instrument. Volume-controlled ventilation (VCV) and pressure-controlled ventilation (PCV) were used to compare small versus large tidal volumes. The surfactant lipids dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC) were quantified by mass spectrometry. Results: In vivo the particle mass in VCV1 was significantly lower than in VCV2 (p= 0.0186), and the particle mass was significantly higher in PCV1 than in VCV1 (p= 0.0322). In EVLP, the particle mass in VCV1 was significantly higher than in PCV1 (p= 0.0371), and the particle mass was significantly higher in PCV2 than in PCV1 (p= 0.0127). DPPC was significantly higher in EVLP than in vivo. Conclusions: Here, we introduce a new method for measuring particle flow during mechanical ventilation and confirm that these particles can be collected and analyzed. VCV resulted in a lower particle flow in vivo but not in EVLP. In all settings, large tidal volumes resulted in increased particle flow. We found that DPPC was significantly increased comparing in vivo with EVLP. This technology may be useful for developing strategies to preserve the lung and has a high potential to detect biomarkers.
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| 6. |
- Broberg, Ellen, et al.
(författare)
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Mechanically ventilated patients exhibit decreased particle flow in exhaled breath as compared to normal breathing patients
- 2020
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Ingår i: Erj Open Research. - : European Respiratory Society (ERS). - 2312-0541. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: In this cohort study, we evaluated whether the particles in exhaled air (PExA) device can be used in conjunction with mechanical ventilation during surgery. The PExA device consists of an optical particle counter and an impactor that collects particles in exhaled air. Our aim was to establish the feasibility of the PExA device in combination with mechanical ventilation (MV) during surgery and if collected particles could be analysed. Patients with and without nonsmall cell lung cancer (NSCLC) undergoing lung surgery were compared to normal breathing (NB) patients with NSCLC. Methods: A total of 32 patients were included, 17 patients with NSCLC (MV-NSCLC), nine patients without NSCLC (MV-C) and six patients with NSCLC and not intubated (NB). The PEx samples were analysed for the most common phospholipids in surfactant using liquid-chromatography-mass-spectrometry (LCMS). Results: MV-NSCLC and MV-C had significantly lower numbers of particles exhaled per minute ( particle flow rate; PFR) compared to NB. MV-NSCLC and MV-C also had a siginificantly lower amount of phospholipids in PEx when compared to NB. MV-NSCLC had a significantly lower amount of surfactant A compared to NB. Conclusion: We have established the feasibility of the PExA device. Particles could be collected and analysed. We observed lower PFR from MV compared to NB. High PFR during MV may be due to more frequent opening and closing of the airways, known to be harmful to the lung. Online use of the PExA device might be used to monitor and personalise settings for mechanical ventilation to lower the risk of lung damage.
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| 7. |
- Broberg, Ellen, et al.
(författare)
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Particle Flow Profiles From the Airways Measured by PExA Differ in Lung Transplant Recipients Who Develop Primary Graft Dysfunction
- 2019
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Ingår i: Experimental and clinical transplantation. - : Baskent University. - 1304-0855 .- 2146-8427. ; 17:6, s. 803-812
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Primary graft dysfunction is a severe form of acute lung injury and a major cause of early morbidity and mortality encountered after lung transplant.We used a customized PExA 2.0 instrument (PExA, Gothenburg, Sweden) to measure particle flow in exhaled air during mechanical ventilation in the intensive care unit. Our objective was to discover whether patients who developed primary graft dysfunction had different particle flow patterns from the airways. We used volume-controlled ventilation and pressure-controlled ventilation to see whether changes in particle patterns could be observed in both mechanical ventilation settings.MATERIALS AND METHODS: First, we investigated whether it was safe to use a customized PExA 2.0 in conjunction with mechanical ventilation. Next, 12 lung transplant patients were randomized to either daily volumecontrolled ventilation or pressure-controlled ventilation as the first mode of treatment until extubation.RESULTS: In our study group, 6 patients did not develop primary graft dysfunction and 6 developed primary graft dysfunction. Patients with primary graft dysfunction underwent mechanical ventilation significantly longer; they also showed a stepwise increase in particle count from day 0 until extubation. We observed no adverse events related to the PExA 2.0 device.CONCLUSIONS: This study suggests that the PExA 2.0 device is safe to use in conjunction with mechanical ventilation in the intensive care unit. Lung transplant patients who developed primary graft dysfunction showed a different particle profile from the airways before clinical signs of primary graft dysfunction developed. Online assessment of ventilation impact before presentation of tissue changes may allow realtime detection of primary graft dysfunction, thus preventing or reducing its effects.
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| 8. |
- Bölükbas, Deniz A., et al.
(författare)
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The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential
- 2019
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1940, s. 275-295
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Tidskriftsartikel (refereegranskat)abstract
- Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.
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| 9. |
- Bölükbas, Deniz, et al.
(författare)
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Organ-Restricted Vascular Delivery of Nanoparticles for Lung Cancer Therapy
- 2020
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Ingår i: Advanced Therapeutics. - : Wiley. - 2366-3987. ; 3:7
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Tidskriftsartikel (refereegranskat)abstract
- Nanoparticle-based targeted drug delivery holds promise for treatment of cancers. However, most approaches fail to be translated into clinical success due to ineffective tumor targeting in vivo. Here, the delivery potential of mesoporous silica nanoparticles (MSN) functionalized with targeting ligands for epidermal growth factor receptor and C─C chemokine receptor type 2 is explored in lung tumors. The addition of active targeting ligands on MSNs enhances their uptake in vitro but fails to promote specific delivery to tumors in vivo, when administered systemically via the blood or locally to the lung into immunocompetent murine lung cancer models. Ineffective tumor targeting is due to efficient clearance of the MSNs by the phagocytic cells of the liver, spleen, and lung. These limitations, however, are successfully overcome using a novel organ-restricted vascular delivery (ORVD) approach. ORVD in isolated and perfused mouse lungs of Kras-mutant mice enables effective nanoparticle extravasation from the tumor vasculature into the core of solid lung tumors. In this study, ORVD promotes tumor cell-specific uptake of nanoparticles at cellular resolution independent of their functionalization with targeting ligands. Organ-restricted vascular delivery thus opens new avenues for optimized nanoparticles for lung cancer therapy and may have broad applications for other vascularized tumor types.
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| 10. |
- Bölükbas, Deniz, et al.
(författare)
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Preclinical evidence for the role of stem/stromal cells in COPD
- 2019. - 1
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Ingår i: Stem Cell-Based Therapy for Lung Disease. - Cham : Springer International Publishing. - 9783030294021 - 9783030294038 ; , s. 73-96
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Bokkapitel (refereegranskat)abstract
- Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide and there are currently limited treatment options for these patients. The disease is characterized by a reduction in airflow due to chronic bronchitis, as well as airspace enlargement in the distal lung, resulting in a loss of surface area available for gas exchange. At end-stage disease, oxygen therapy and lung transplantation remain the only potential options. The disease is heterogeneous and both inflammatory cells as well as structural cells are thought to play a role in disease onset and progression. Pharmaceutical approaches are ineffective at reversing disease pathology and currently aim only to provide symptomatic relief. A recent area of investigation focuses on exogenous cell therapy, including stem cell administration, and its potential for directing lung regeneration. Cell therapies from a variety of sources, as well as cell-derived products such as extracellular vesicles, have recently shown efficacy in animal models of COPD, but early clinical trials have not yet shown efficacy. In this chapter, we discuss the different animal models of COPD as well as the studies which have been conducted to date with cell therapies. We conclude the chapter with a discussion regarding the limitations of current animal models and discuss potential areas for future study.
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