scholarly journals Using a micro-physiological system to prolong the preservation of ex vivo lung tissue

2021 ◽  
Vol 7 (2) ◽  
pp. 207-210
Author(s):  
Sebastian Böhlen ◽  
Sebastian Konzok ◽  
Jennifer Labisch ◽  
Susann Dehmel ◽  
Dirk Schaudien ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Lactate Dehydrogenase Release ◽  
Repeated Dose Toxicity ◽  
Prolonged Culture ◽  
Static Conditions ◽  
Precision Cut Lung Slices

Abstract Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.

Biaxial distension of precision-cut lung slices

2010 ◽  
Vol 108 (3) ◽  
pp. 713-721 ◽  
Author(s):  
C. Dassow ◽  
L. Wiechert ◽  
C. Martin ◽  
S. Schumann ◽  
G. Müller-Newen ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Cell Injury ◽  
Mechanical Stretch ◽  
Longitudinal Direction ◽  
Lung Parenchyma ◽  
Cell Types ◽  
Static Conditions ◽  
Precision Cut Lung Slices

The mechanical forces acting on lung parenchyma during (mechanical) ventilation and its (patho)physiological consequences are currently under intense scrutiny. Several in vivo and cell culture models have been developed to study the pulmonary responses to mechanical stretch. While providing extremely useful information, these models do also suffer from limitations in being either too complex for detailed mechanical or mechanistic studies, or in being devoid of the full complexity present in vivo (e.g., different cell types and interstitial matrix). Therefore in the present study it was our aim to develop a new model, based on the biaxial stretching of precision-cut lung slices (PCLS). Single PCLS were mounted on a thin and flexible carrier membrane of polydimethylsiloxane (PDMS) in a bioreactor, and the membrane was stretched by applying varying pressures under static conditions. Distension of the membrane-PCLS construct was modeled via finite element simulation. According to this analysis, lung tissue was stretched by up to 38% in the latitudinal and by up to 44% in the longitudinal direction, resulting in alveolar distension similar to what has been described in intact lungs. Stretch for 5 min led to increased cellular calcium levels. Lung slices were stretched dynamically with a frequency of 15/min for 4 h without causing cell injury {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test; live/dead straining}. These findings suggest that stretching of PCLS on PDMS-membranes may represent a useful model to investigate lung stretch in intact lung tissue in vitro for several hours.

scholarly journals Distinct niches within the extracellular matrix dictate fibroblast function in (cell free) 3D lung tissue cultures

2018 ◽  
Vol 314 (5) ◽  
pp. L708-L723 ◽  
Author(s):  
Gerald Burgstaller ◽  
Arunima Sengupta ◽  
Sarah Vierkotten ◽  
Gerhard Preissler ◽  
Michael Lindner ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Lung Tissue ◽  
Ex Vivo ◽  
3D Culture ◽  
Focal Adhesions ◽  
3D Cell Culture ◽  
Small Gtpases ◽  
Cellular Migration

Cues from the extracellular matrix (ECM) and their functional interplay with cells play pivotal roles for development, tissue repair, and disease. However, the precise nature of this interplay remains elusive. We used an innovative 3D cell culture ECM model by decellularizing 300-µm-thick ex vivo lung tissue scaffolds (d3D-LTCs) derived from diseased and healthy mouse lungs, which widely mimics the native (patho)physiological in vivo ECM microenvironment. We successfully repopulated all d3D-LTCs with primary human and murine fibroblasts, and moreover, we demonstrated that the cells also populated the innermost core regions of the d3D-LTCs in a real 3D fashion. The engrafted fibroblasts revealed a striking functional plasticity, depending on their localization in distinct ECM niches of the d3D-LTCs, affecting the cells’ tissue engraftment, cellular migration rates, cell morphologies, and protein expression and phosphorylation levels. Surprisingly, we also observed fibroblasts that were homing to the lung scaffold’s interstitium as well as fibroblasts that were invading fibrotic areas. To date, the functional nature and even the existence of 3D cell matrix adhesions in vivo as well as in 3D culture models is still unclear and controversial. Here, we show that attachment of fibroblasts to the d3D-LTCs evidently occurred via focal adhesions, thus advocating for a relevant functional role in vivo. Furthermore, we found that protein levels of talin, paxillin, and zyxin and phosphorylation levels of paxillin Y118, as well as the migration-relevant small GTPases RhoA, Rac, and CDC42, were significantly reduced compared with their attachment to 2D plastic dishes. In summary, our results strikingly indicate that inherent physical or compositional characteristics of the ECM act as instructive cues altering the functional behavior of engrafted cells. Thus, d3D-LTCs might aid to obtain more realistic data in vitro, with a high relevance for drug discovery and mechanistic studies alike.

scholarly journals Pegylation of Antimicrobial Peptides Maintains the Active Peptide Conformation, Model Membrane Interactions, and Antimicrobial Activity while Improving Lung Tissue Biocompatibility following Airway Delivery

2012 ◽  
Vol 56 (6) ◽  
pp. 3298-3308 ◽  
Author(s):  
Christopher J. Morris ◽  
Konrad Beck ◽  
Marc A. Fox ◽  
David Ulaeto ◽  
Graeme C. Clark ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Lung Tissue ◽  
Conformational Changes ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Pulmonary Infections ◽  
Cecropin A

ABSTRACTAntimicrobial peptides (AMPs) have therapeutic potential, particularly for localized infections such as those of the lung. Here we show that airway administration of a pegylated AMP minimizes lung tissue toxicity while nevertheless maintaining antimicrobial activity. CaLL, a potent synthetic AMP (KWKLFKKIFKRIVQRIKDFLR) comprising fragments of LL-37 and cecropin A peptides, was N-terminally pegylated (PEG-CaLL). PEG-CaLL derivatives retained significant antimicrobial activity (50% inhibitory concentrations [IC50s] 2- to 3-fold higher than those of CaLL) against bacterial lung pathogens even in the presence of lung lining fluid. Circular dichroism and fluorescence spectroscopy confirmed that conformational changes associated with the binding of CaLL to model microbial membranes were not disrupted by pegylation. Pegylation of CaLL reduced AMP-elicited cell toxicity as measured usingin vitrolung epithelial primary cell cultures. Further, in a fully intactex vivoisolated perfused rat lung (IPRL) model, airway-administered PEG-CaLL did not result in disruption of the pulmonary epithelial barrier, whereas CaLL caused an immediate loss of membrane integrity leading to pulmonary edema. All AMPs (CaLL, PEG-CaLL, LL-37, cecropin A) delivered to the lung by airway administration showed limited (<3%) pulmonary absorption in the IPRL with extensive AMP accumulation in lung tissue itself, a characteristic anticipated to be beneficial for the treatment of pulmonary infections. We conclude that pegylation may present a means of improving the lung biocompatibility of AMPs designed for the treatment of pulmonary infections.

scholarly journals Co-Spray Dried Nafamostat Mesylate with Lecithin and Mannitol as Respirable Microparticles for Targeted Pulmonary Delivery: Pharmacokinetics and Lung Distribution in Rats

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1519
Author(s):  
Ji-Hyun Kang ◽  
Young-Jin Kim ◽  
Min-Seok Yang ◽  
Dae Hwan Shin ◽  
Dong-Wook Kim ◽  
...  
Keyword(s):  
Lung Tissue ◽  
High Performance ◽  
Ex Vivo ◽  
Intratracheal Administration ◽  
Residual Time ◽  
Sites Of Action ◽  
Nafamostat Mesylate ◽  
Spray Dried

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution of NFM, administered via intravenous and intratracheal routes, were determined using high performance liquid chromatography analysis of blood plasma, lung lumen using bronchoalveolar lavage fluid, and lung tissue. Intratracheal administration had higher drug delivery and longer residual time in the lung lumen and tissue, which are the main sites of action, than intravenous administration. We confirmed the effect of lecithin as a stabilizer through an ex vivo stability test. Lecithin acts as an inhibitor of carboxylesterase and delays NFM decomposition. We prepared inhalable microparticles with NFM, lecithin, and mannitol via the co-spray method. The formulation prepared using an NFM:lecithin:mannitol ratio of 1:1:100 had a small particle size and excellent aerodynamic performance. Spray dried microparticles containing NFM, lecithin, and mannitol (1:1:100) had the longest residual time in the lung tissue. In conclusion, NFM-inhalable microparticles were prepared and confirmed to be delivered into the respiratory tract, such as lung lumen and lung tissue, through in vitro and in vivo evaluations.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons

2018 ◽  
Vol 41 (7) ◽  
pp. 400-412 ◽  
Author(s):  
Manuela Teresa Raimondi ◽  
Matteo Laganà ◽  
Claudio Conci ◽  
Michele Crestani ◽  
Alessia Di Giancamillo ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Biochemical Properties ◽  
Cyclic Stretch ◽  
Cell Alignment ◽  
Tendon Tissue ◽  
Medium Temperature ◽  
Prolonged Culture ◽  
Stimulation Of

Introduction: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. Methods: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. Results: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. Conclusion: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

scholarly journals Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Ali Kermanizadeh ◽  
Trine Berthing ◽  
Ewa Guzniczak ◽  
Melanie Wheeldon ◽  
Graeme Whyte ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Hepatic Function ◽  
Test System ◽  
Cell Membrane Integrity ◽  
Cytokine Analysis ◽  
Recovery Capacity

Abstract Background With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver’s recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. Results The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. Conclusion In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

scholarly journals Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue

2015 ◽  
Vol 309 (4) ◽  
pp. L323-L332 ◽  
Author(s):  
Gerald Burgstaller ◽  
Sarah Vierkotten ◽  
Michael Lindner ◽  
Melanie Königshoff ◽  
Oliver Eickelberg
Keyword(s):  
Smooth Muscle ◽  
Lung Tissue ◽  
Ex Vivo ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Cellular Tissue ◽  
Muscle Actin ◽  
Human Lung Tissue

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

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