In vitro models最新文献

Purpose: In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems.

Methods: Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period.

Results: Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro.

Conclusion: This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00032-x.

Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal condition. Ulcerative colitis and Crohn's disease are types of inflammatory bowel disease. Over many decades, the disease has been a topic of study, with experts still trying to figure out its cause and pathology. Researchers have established many in vivo animal models, in vitro cell lines, and ex vivo systems to understand its cause ultimately and adequately identify a therapy. However, in vivo animal models cannot be regarded as good models for studying IBD since they cannot completely simulate the disease. Furthermore, because species differences are a crucial subject of concern, in vitro cell lines and ex vivo systems can be employed to recreate the condition properly. In vitro models serve as the starting point for biological and medical research. Ex vivo and in vitro models for replicating gut physiology have been developed. This review aims to present a clear understanding of several in vitro and ex vivo models of IBD and provide insights into their benefits and limits and their value in understanding intestinal physiology.

3D in vitro culture models of cancer cells in extracellular matrix (ECM) have been developed to investigate drug targeting and resistance or, alternatively, mechanisms of invasion; however, models allowing analysis of shared pathways mediating invasion and therapy resistance are lacking. To evaluate therapy response associated with cancer cell invasion, we here used 3D invasion culture of tumor spheroids in 3D fibrillar collagen and applied Ethanol-Ethyl cinnamate (EtOH-ECi) based optical clearing to detect both spheroid core and invasion zone by subcellular-resolved 3D microscopy. When subjected to a single dose of irradiation (4 Gy), we detected significant cell survival in the invasion zone. By physical separation of the core and invasion zone, we identified differentially regulated genes preferentially engaged in invading cells controlling cell division, repair, and survival. This imaging-based 3D invasion culture may be useful for the analysis of complex therapy-response patterns in cancer cells in drug discovery and invasion-associated resistance development.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00040-x.

The drug development process is a lengthy and expensive challenge for all involved players. Experience with the COVID-19 pandemic underlines the need for a rapid and effective approval for treatment options. As essential prerequisites for successful drug approval, a combination of high-quality studies and reliable research must be included. To this day, mainly in vivo data are requested and collected for assessing safety and efficacy and are therefore decisive for the pre-clinical evaluation of the respective drug. This review aims to summarize the current state of the art for safety and efficacy studies in pharmaceutical research and industry to address the relevant regulatory challenges and to provide an outlook on implementing more in vitro methods as alternative to animal testing. While the public demand for alternative methods is becoming louder, first examples have meanwhile found acceptance in relevant guidelines, e.g. the OECD guidelines for skin sensitizer. Besides ethically driven developments, also the rather low throughput and relatively high costs of animal experiments are forcing the industry towards the implementation of alternative methods. In this context, the development of orally inhaled drug products is particularly challenging due to the complexity of the lung as biological barrier and route of administration. The replacement of animal experiments with focus on the lungs requires special designed tools to achieve predictive data. New in vitro test systems of increasing complexity are presented in this review. Limits and advantages are discussed to provide some perspective for a future in vitro testing strategy for orally inhaled drug products.

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SARS-CoV-2 is a pandemic coronavirus that causes severe respiratory disease (COVID-19) in humans and is responsible for millions of deaths around the world since early 2020. The virus affects the human respiratory cells through its spike (S) proteins located at the outer shell. To monitor the rapid spreading of SARS-CoV-2 and to reduce the deaths from the COVID-19, early detection of SARS-CoV-2 is of utmost necessity. This report describes a flexible colorimetric biosensor capable of detecting the S protein of SARS-CoV-2. The colorimetric biosensor is made of polyurethane (PU)-polydiacetylene (PDA) nanofiber composite that was chemically functionalized to create a binding site for the receptor molecule-nucleocapsid antibody (anti-N) protein of SARS-CoV-2. After the anti-N protein conjugation to the functionalized PDA fibers, the PU-PDA-NHS-anti fiber was able to detect the S protein of SARS-CoV-2 at room temperature via a colorimetric transition from blue to red. The PU-PDA nanofiber-based biosensors are flexible and lightweight and do not require a power supply such as a battery when the colorimetric detection to S protein occurs, suggesting a sensing platform of wearable devices and personal protective equipment such as face masks and medical gowns for real-time monitoring of virus contraction and contamination. The wearable biosensors could significantly power mass surveillance technologies to fight against the COVID-19 pandemic.

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Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00022-z.

Objective: Imaging endothelial cell behaviour under physiological conditions, particularly those associated with chronic fibrotic pathologies, is an incredibly challenging endeavour. While short-term assessments (hours) can be achieved with techniques such as intravital microscopy, vascular changes often occur over days and weeks which is unfeasible with current imaging techniques. These challenges are exemplified within the liver where liver sinusoidal endothelial cells (LSECs) are known to undergo dramatic changes termed endothelial-to-mesenchymal transition (EndMT) during fibrotic liver disease. Despite the established presence of EndMT in liver disease, the inaccessibility of viable liver tissue, and simplicity of 2D culture techniques has meant, the role of EndMT during disease progression remains largely undetermined. This study describes the development of novel fluorescent EndMT reporters to identify, track, and characterise the migratory behaviour of EndMT cells. We show that liver-on-a-chip (LOAC) platforms provide a flexible, optically accessible, and physiologically relevant microenvironment to study the vascular dynamics of EndMT during liver disease.

Methods: Identification, creation, and application of an EndMT-specific fluorescent reporter construct (EndMT-Rep). Transduction of EC using lentiviral packaged CNN1-eGFP construct as an inducible EndMT-Rep (CNN1-Rep) to 2D, 3D, and 4D imaging techniques for fixed and live cell imaging. Combined application of live and fixed imaging technologies to measure EndMT using CNN1-Rep on LOAC platform under physiological conditions. Demonstration of the high-resolution single-cell EndMT tracking by live cell time-lapse microscopy and with post-acquisition processing to perform a comparative study of CNN1-Rep and healthy LSECs within a NASH-like LOAC microenvironment.

Conclusions: LOAC enables prolonged, multi-platform imaging of endothelial cell sub-populations such as those undergoing EndMT in 2D and 3D cultures. Our study highlights the application of EndMT reporters, such as CNN1-Rep, to provide high-resolution imaging of EndMT behaviour for the first time under physiologically relevant liver microenvironment. Overall, these methods reveal the adaptability and impact of live-cell imaging on uncovering vascular behaviours, such as EndMT, that are unattainable in viable tissue or conventional 2D in vitro experiments.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00034-9.

Inflammatory bowel disease (IBD) is a widespread disease, affecting a growing demographic. The treatment of chronic inflammation located in the GI-tract is dependent on the severity; therefore, the IBD treatment pyramid is commonly applied. Animal experimentation plays a key role for novel IBD drug development; nevertheless, it is ethically questionable and limited in its throughput. Reliable and valid in vitro assays offer the opportunity to overcome these limitations. We combined Caco-2 with monocyte-derived macrophages and exposed them to known drugs, targeting an in vitro-in vivo correlation (IVIVC) with a focus on the severity level and its related drug candidate. This co-culture assay addresses namely the intestinal barrier and the immune response in IBD. The drug efficacy was analyzed by an LPS-inflammation of the co-culture and drug exposure according to the IBD treatment pyramid. Efficacy was defined as the range between LPS control (0%) and untreated co-culture (100%) independent of the investigated read-out (TEER, P_app, cytokine release: IL-6, IL-8, IL-10, TNF-α). The release of IL-6, IL-8, and TNF-α was identified as an appropriate readout for a fast drug screening ("yes-no response"). TEER showed a remarkable IVIVC correlation to the human treatment pyramid (5-ASA, Prednisolone, 6-mercaptopurine, and infliximab) with an R² of 0.68. Similar to the description of an adverse outcome pathway (AOP) framework, we advocate establishing an "Efficacy Outcome Pathways (EOPs)" framework for drug efficacy assays. The in vitro assay offers an easy and scalable method for IBD drug screening with a focus on human data, which requires further validation.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-022-00035-8.

Purpose: Current air-liquid interface (ALI) models of bovine proximal airways have their limitations. They do not simulate blood flow necessary to mimic systemic drug administration, and repeated sampling requires multiple, independent cultures. A bovine lung-on-chip (bLOC) would overcome these limitations, providing a convenient and cost-effective model for pharmacokinetic or pathogenicity studies.

Methods: Bovine pulmonary arterial endothelial cells seeded into the endothelial channel of an Emulate Lung-Chip were interfaced with bovine bronchial epithelial cells in the epithelial channel. Cells were cultured at ALI for up to 21 days. Differentiation was assessed by mucin quantification, phase-contrast light microscopy and immunofluorescence of cell-specific markers in fixed cultures. Barrier integrity was determined by FITC-labelled dextran 3-5 kDa permeability. To evaluate the model, endothelial-epithelial transport of the antibiotic drug, danofloxacin, was followed using liquid chromatography-mass spectrometry, with the aim of replicating data previously determined in vivo.

Results: bLOC cultures secreted quantifiable mucins, whilst cilia formation was evident in the epithelial channel. Barrier integrity of the model was demonstrated by resistance to FITC-Dextran 3-5 kDa permeation. Bronchial epithelial and endothelial cell-specific markers were observed. Close to plasma, representative PK data for danofloxacin was observed in the endothelial channel; however, danofloxacin in the epithelial channel was mostly below the limit of quantification.

Conclusion: A co-culture model of the bovine proximal airway was successfully generated, with potential to replace in vivo experimentation. With further optimisation and characterisation, the bLOC may be suitable to perform drug pharmacokinetic studies for bovine respiratory disease (BRD), and other applications.