In vitro models最新文献

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.

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.

Bone pain typically occurs immediately following skeletal damage with mechanical distortion or rupture of nociceptive fibres. The pain mechanism is also associated with chronic pain conditions where the healing process is impaired. Any load impacting on the area of the fractured bone will stimulate the nociceptive response, necessitating rapid clinical intervention to relieve pain associated with the bone damage and appropriate mitigation of any processes involved with the loss of bone mass, muscle, and mobility and to prevent death. The following review has examined the mechanisms of pain associated with trauma or cancer-related skeletal damage focusing on new approaches for the development of innovative therapeutic interventions. In particular, the review highlights tissue engineering approaches that offer considerable promise in the application of functional biomimetic fabrication of bone and nerve tissues. The strategic combination of bone and nerve tissue engineered models provides significant potential to develop a new class of in vitro platforms, capable of replacing in vivo models and testing the safety and efficacy of novel drug treatments aimed at the resolution of bone-associated pain. To date, the field of bone pain research has centred on animal models, with a paucity of data correlating to the human physiological response. This review explores the evident gap in pain drug development research and suggests a step change in approach to harness tissue engineering technologies to recapitulate the complex pathophysiological environment of the damaged bone tissue enabling evaluation of the associated pain-mimicking mechanism with significant therapeutic potential therein for improved patient quality of life.

Graphical abstract: Rationale underlying novel drug testing platform development. Pain detected by the central nervous system and following bone fracture cannot be treated or exclusively alleviated using standardised methods. The pain mechanism and specificity/efficacy of pain reduction drugs remain poorly understood. In vivo and ex vivo models are not yet able to recapitulate the various pain events associated with skeletal damage. In vitro models are currently limited by their inability to fully mimic the complex physiological mechanisms at play between nervous and skeletal tissue and any disruption in pathological states. Robust innovative tissue engineering models are needed to better understand pain events and to investigate therapeutic regimes.

Diabetes mellitus is a significant public health problem worldwide. It encompasses a group of chronic disorders characterized by hyperglycemia, resulting from pancreatic islet dysfunction or as a consequence of insulin-producing β-cell death. Organ-on-a-chip platforms have emerged as technological systems combining cell biology, engineering, and biomaterial technological advances with microfluidics to recapitulate a specific organ's physiological or pathophysiological environment. These devices offer a novel model for the screening of pharmaceutical agents and to study a particular disease. In the field of diabetes, a variety of microfluidic devices have been introduced to recreate native islet microenvironments and to understand pancreatic β-cell kinetics in vitro. This kind of platforms has been shown fundamental for the study of the islet function and to assess the quality of these islets for subsequent in vivo transplantation. However, islet physiological systems are still limited compared to other organs and tissues, evidencing the difficulty to study this "organ" and the need for further technological advances. In this review, we summarize the current state of islet-on-a-chip platforms that have been developed so far. We recapitulate the most relevant studies involving pancreatic islets and microfluidics, focusing on the molecular and cellular-scale activities that underlie pancreatic β-cell function.

Purpose: The aggressive nature of a tumor is presumably its inherent one, but different environmental cues can manipulate it in many ways. In this context, the influence of metabolic stresses on tumor behavior needs to be analyzed to understand their far-reaching implications on tumor aggression and dormancy. This work investigates different facets of the tumor, such as tumorigenic capacity, tumor phenotype, and migration, under multiple metabolic stress conditions.

Methods: Non-invasive and invasive multicellular spheroids (MTS) were created and subjected to multiple stress conditions, namely glucose, amino acid, and oxygen deprivation. Altered behavior of the MTS has been evaluated in the context of in vitro tumorigenesis, spheroid formation capacity, phenotype, mRNA profile, migration, and recruitment of mesenchymal stem cells.

Results: The metabolic stress conditions were observed to negatively impact the in vitro tumorigenesis and spheroid formation process of invasive and non-invasive breast cancer cells. While the stress seemingly influences the growth and phenotype of spheroids, it does not alter the organization of sub-cellular entities significantly. Metabolic stress conditions impact the transcriptomic landscape of hypoxic, angiogenic, ECM deformation, glycolysis shift, and protein starvation-related gene clusters. MTSs do not adhere or migrate under stress, but they exhibit different modalities of migration when rescued. Invasive spheroids, after the rescue, exhibit increased aggressiveness. Furthermore, stressed spheroid was observed to control the migration and recruitment of mesenchymal stem cells.

Conclusion: Multiplex metabolic stresses could control the tumorigenesis while influencing the physiology of invasive and non-invasive breast cancer spheroids along with their migration pattern and tumor-stromal crosstalk.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-021-00006-5.

Bone is the major connective tissue maintaining the structural integrity of the human body. However, fracture and many skeletal degenerative diseases can compromise this function. Thus, therapeutics related to bone degeneration are of significant research interest and require good in vitro models for such therapeutic evaluation. Bone is a highly vascularized tissue and incorporation of this feature is significantly important for mimicking the osteogenic microenvironment. In the current study, we developed a vascularized flat bone model via simultaneous mechanical actuation of mechanical strain and fluid shear. The mechanical strain was achieved by static magnetic field actuation of a magnetic nanocomposite scaffold. The fluid shear was generated by developing a micropattern on the magnetic nanocomposite via replica molding and laser-based microfabrication. From the live cell imaging window of the microdevice, both bone and vasculature like cellular morphology was observed. The SEM study showed thick ECM deposition in the dynamic culture. In the PCR study, both osteogenic (Col-1, osteocalcin) and angiogenic phenotypes (PECAM) were observed in the dynamic culture scaffolds while chondrogenic marker (Col-2) was downregulated.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-021-00004-7.

Purpose: Present blood-contacting materials have not yet demonstrated to be effective in reducing blood coagulation without causing additional side effects clinically. We have developed an endothelial glycocalyx-inspired biomimetic surface that combines nanotopography, heparin presentation, and nitric oxide (NO)-releasing features. The resulting modified surfaces have already shown promise in reducing unfavorable blood-material interactions using platelet-rich plasma. In this study, the efficacy of modified surfaces for reducing coagulation of human whole blood was measured. In addition, the effects of leached polysaccharides and chemical modification of the modified surfaces were evaluated.

Methods: Leached polysaccharides in the incubation solution were detected by a refractive index method to determine the potential influences of these modified surfaces on the blood coagulation observation. Chemical modifications by the nitrosation process on the polysaccharides in the modified surfaces were detected using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Clot formation parameters were measured using thromboelastography (TEG), a clinically relevant technique to evaluate whole blood coagulation.

Results: No polysaccharides were detected in the heparinized polyelectrolyte multilayer-coated titania nanotube array surface (TiO₂NT + PEM) incubation solution; however, polysaccharides were detected from NO-releasing TiO₂NT + PEM surface (TiO₂NT + PEM + NO) incubation solution both after the nitrosation process and after all NO was released. The structures of thiolated chitosan and heparin were altered by t-butyl nitrite. All heparin-containing surfaces were shown to slow or inhibit clot formation.

Conclusion: This study is the first to evaluate these endothelial glycocalyx-inspired surfaces using clinically relevant parameters, as well as proposing potential influences of these modified surfaces on the inhibition of clot formation.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-021-00001-w.