Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Modulating the Barrier Function of Human Alveolar Epithelial (hAELVi) Cell Monolayers as a Model of Inflammation.

The incidence of inflammatory lung diseases such as acute respiratory distress syndrome (ARDS) remains an important problem, particularly in the present time with the Covid-19 pandemic. However, an adequate in vitro test system to monitor the barrier function of the alveolar epithelium during inflammation and for assessing anti-inflammatory drugs is urgently needed. Therefore, we treated human Alveolar Epithelial Lentivirus-immortalised cells (hAELVi cells) with the pro-inflammatory cytokines TNF-α (25 ng/ml) and IFN-γ (30 ng/ml), in the presence or absence of hydrocortisone (HC). While TNF-α and IFN-γ are known to reduce epithelial barrier properties, HC could be expected to protect the barrier function and result in an anti-inflammatory effect. We investigated the impact of anti-inflammatory/inflammatory treatment on transepithelial electrical resistance (TEER) and the apparent permeability coefficient (Papp) of the low permeability marker sodium fluorescein (NaFlu). After incubating hAELVi cells for 48 hours with a combination of TNF-α and IFN-γ, there was a significant decrease in TEER and a significant increase in the Papp. The presence of HC maintained the TEER values and barrier properties, so that no significant Papp change was observed. By using hAELVi cells to study anti-inflammatory drugs in vitro, the need for animal experiments could be reduced and pulmonary drug development accelerated.

In vitro tools for orally inhaled drug products-state of the art for their application in pharmaceutical research and industry and regulatory challenges.

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.

Gelatin nanoparticles with tunable mechanical properties: effect of crosslinking time and loading.

Tuning the elastic properties of nanoparticles intended to be used in drug delivery is of great interest. To this end, different potential formulations are developed since the particle elasticity is affecting the in vitro and in vivo performance of the nanoparticles. Here we present a method to determine the elasticity of single gelatin nanoparticles (GNPs). Furthermore, we introduce the possibility of tuning the elastic properties of gelatin nanoparticles during their preparation through crosslinking time. Young's moduli from 5.48 to 14.26 MPa have been obtained. Additionally, the possibility to measure the elasticity of single nanoparticles revealed the influence of loading a macromolecular model drug (FITC-dextran) on the mechanical properties, which decreased with raising amounts of loaded drug. Loaded particles were significantly softer, with Young's moduli between 1.06 and 5.79 MPa for the same crosslinking time, than the blank GNPs. In contrast to this, lysozyme as a crosslinkable macromolecule did not influence the mechanical properties. A good in vitro cell compatibility was found investigating blank GNPs and FITC-dextran-loaded GNPs in viability assays with the cancer cell line A549 and the human primary cell-derived hAELVi cell line.

Inflammatory bowel disease addressed by Caco-2 and monocyte-derived macrophages: an opportunity for an in vitro drug screening assay.

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, Papp, 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.

Ten-year trends of the characteristics in patients with advanced Parkinson's disease at the time of intestinal gel therapy introduction / Az intestinalis gél bevezetésekor rögzített jellemzok tízéves változásai elorehaladott Parkinson-kóros betegekben

Introduction: Parkinson's disease is a neurodegenerative disease, the symptoms of which can be treated reasonably well; however, therapeutic recommendations need to be refined based on the observations from everyday practice. Objective: We aimed to analyze the extent by which published expert recommendations were reflected in the manage-ment of patients with advanced Parkinson's disease, prior to the introduction of the intestinal gel. Method: Data from patients treated with levodopa-carbidopa intestinal gel were retrospectively examined. The period from 2011 to 2021 was divided into two five-year periods, prior and after the usage of the 5-2-1 rule in clinical decision-making. Results: Levodopa-carbidopa intestinal gel treatment was initiated in 150 patients during the study period. In the second five-year period, the mean age of the patients was lower and the time from diagnosis was shorter. Also, there were significantly fewer patients with peak-dose dyskinesias (p = 0.02), biphasic dyskinesias (p<0.001), and early morning akinesias (p = 0.02). Furthermore, in the last five years of the study, fewer patients were affected by delayed on (p = 0.03), no on (p = 0.02), and freezing (p = 0.01). The mean score measured on the Hoehn-Yahr scale was also lower in the second period, while the mean MMSE score was higher (p<0.001). Daily doses of levodopa were higher (p<0.01) in the second period, but with similar dosing frequency. Conclusion: Our retrospective analysis of trends during a ten-year period revealed that, in the second five-year period, levodopa-carbidopa intestinal gel was started in advanced Parkinson's disease patients with a significantly better physical and cognitive state. Compared to expert recommendations, our patients still had a more severe clinical pic -ture at the start of device-aided therapy, but acceptance of this invasive method has improved both for patients and for general practitioners and neurologists.

Pulmonary in vitro instruments for the replacement of animal experiments.

Advanced in vitro systems often combine a mechanical-physical instrument with a biological component e.g. cell culture models. For testing of aerosols, it is of advantage to consider aerosol behavior, particle deposition and lung region specific cell lines. Although there are many good reviews on the selection of cell cultures, articles on instruments are rare. This article focuses on the development of in vitro instruments targeting the exposure of aerosols on cell cultures. In this context, guidelines for toxicity investigation are taken into account as the aim of new methods must be the prediction of human relevant data and the replacement of existing animal experiments. We provide an overview on development history of research-based instruments from a pharmaceutical point of view. The standardized commercial devices resulting from the research-based instruments are presented and the future perspectives on pulmonary in vitro devices are discussed.

Safety assessment of excipients (SAFE) for orally inhaled drug products.

The development of new orally inhaled drug products requires their demonstration of safety, which must be proven in animal experiments. New in vitro methods may replace, or at least reduce, these animal experiments, provided they are able to correctly predict safety or possible toxicity in humans. However, the challenge is to link in vitro data obtained in human cells to human in vivo data. We here present a new approach to the safety assessment of excipients (SAFE) for pulmonary drug delivery. The SAFE model is based on a dose response curve of 23 excipients tested on the human pulmonary epithelial cell lines A549 and Calu-3. The resulting in vitro IC50 values were correlated with the FDA-approved concentrations in pharmaceutical products for either pulmonary (if available) or parenteral administration. Setting a threshold of 0.1% (1 mg/mL) for either value yielded four safety classes and allowed to link IC50 data as measured in human cell cultures in vitro with the concentrations of the same compounds in FDA-approved drug products. The necessary in vitro data for novel excipients can be easily generated, and the SAFE approach allows putting them into context for eventual use in human pulmonary drug products. Excipients that are most likely not safe for use in humans can be excluded early on from further pharmaceutical development. The SAFE approach thus helps to avoid unnecessary animal experiments.

Combining MucilAir™ and Vitrocell® Powder Chamber for the In Vitro Evaluation of Nasal Ointments in the Context of Aerosolized Pollen.

Hay fever is notoriously triggered when nasal mucosa is exposed to allergenic pollen. One possibility to overcome this pollen exposure may be the application of an ointment with physical protective effects. In this context, we have investigated Bepanthen® Eye and Nose Ointment and the ointment basis petrolatum as reference while using contemporary in vitro techniques. Pollen from false ragweed (Iva xanthiifolia) was used as an allergy-causing model deposited as aerosol using the Vitrocell® Powder Chamber (VPC) on Transwell® inserts, while being coated with either Bepanthen® Eye and Nose Ointment and petrolatum. No pollen penetration into ointments was observed upon confocal scanning laser microscopy during an incubation period of 2 h at 37 °C. The cellular response was further investigated by integrating the MucilAir™ cell system in the VPC and by applying pollen to Bepanthen® Eye and Nose Ointment covered cell cultures. For comparison, MucilAir™ were stimulated by lipopolysaccharides (LPS). No increased cytokine release of IL-6, TNF-α, or IL-8 was found after 4 h of pollen exposure, which demonstrates the safety of such ointments. Since nasal ointments act as a physical barrier against pollen, such preparations might support the prevention and management of hay fever.

Morphological identification of neuron types in the rat hippocampus.

The cerebral cortex ensures an optimal interaction of mammals, including humans, with their environment, by encoding, storing and combining information about the surrounding world and the internal milieu. Probably the simplest and the most popular region for studying the cortical network is the hippocampal CA1 area, because it has the least heterogeneous neuronal population, the somata and dendrites of principal neurons (pyramidal cells) are arranged into well defined layers and the extrinsic and intrinsic inputs are segregated. The relatively homogeneous pyramidal cell population is supported by a very heterogeneous GABAergic interneuron population, which provides not only general inhibition, but also regulates the precise timing of pyramidal cell activity. Interneurons usually innervate distinct domains of the surface of their target cell. The strategic placement of inhibitory synapses, indicate that GABAergic interneurons belonging to different classes serve distinct functions in the hippocampal network. Neuron types are usually defined according to various morphological, molecular and physiological features. Under typical experimental conditions only some of these parameters are available, therefore an important scientific question is: which partial measures are sufficient for correct recognition of a class of cell. By immunohistochemistry it is possible to stain all neurochemically identical neurons in a given brain region, therefore it is the most widely used method for identifying neuron classes. This review presents the neuron types identified so far in the area CA1 of the rat hippocampus with special emphasis on the immunocytochemical characterization of these cells.