Chemotherapy Assessment in Advanced Multicellular 3D Models of Pancreatic Cancer: Unravelling the Importance of Spatiotemporal Mimicry of the Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a challenge for global health with very low survival rate and high therapeutic resistance. Hence, advanced preclinical models for treatment screening are of paramount importance. Herein, chemotherapeutic (gemcitabine) assessment on novel (polyurethane) scaffold-based spatially advanced 3D multicellular PDAC models is carried out. Through comprehensive image-based analysis at the protein level, and expression analysis at the mRNA level, the importance of stromal cells is confirmed, primarily activated stellate cells in the chemoresistance of PDAC cells within the models. Furthermore, it is demonstrated that, in addition to the presence of activated stellate cells, the spatial architecture of the scaffolds, i.e., segregation/compartmentalization of the cancer and stromal zones, affect the cellular evolution and is necessary for the development of chemoresistance. These results highlight that, further to multicellularity, mapping the tumor structure/architecture and zonal complexity in 3D cancer models is important for better mimicry of the in vivo therapeutic response.

Unravelling the impact of fat content on the microbial dynamics and spatial distribution of foodborne bacteria in tri-phasic viscoelastic 3D models.

The aim of the current study is to develop and characterise novel complex multi-phase in vitro 3D models, for advanced microbiological studies. More specifically, we enriched our previously developed bi-phasic polysaccharide (Xanthan Gum)/protein (Whey Protein) 3D model with a fat phase (Sunflower Oil) at various concentrations, i.e., 10%, 20%, 40% and 60% (v/v), for better mimicry of the structural and biochemical composition of real food products. Rheological, textural, and physicochemical analysis as well as advanced microscopy imaging (including spatial mapping of the fat droplet distribution) of the new tri-phasic 3D models revealed their similarity to industrial food products (especially cheese products). Furthermore, microbial growth experiments of foodborne bacteria, i.e., Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis on the surface of the 3D models revealed very interesting results, regarding the growth dynamics and distribution of cells at colony level. More specifically, the size of the colonies formed on the surface of the 3D models, increased substantially for increasing fat concentrations, especially in mid- and late-exponential growth phases. Furthermore, colonies formed in proximity to fat were substantially larger as compared to the ones that were located far from the fat phase of the models. In terms of growth location, the majority of colonies were located on the protein/polysaccharide phase of the 3D models. All those differences at microscopic level, that can directly affect the bacterial response to decontamination treatments, were not captured by the macroscopic kinetics (growth dynamics), which were unaffected from changes in fat concentration. Our findings demonstrate the importance of developing structurally and biochemically complex 3D in vitro models (for closer proximity to industrial products), as well as the necessity of conducting multi-level microbial analyses, to better understand and predict the bacterial behaviour in relation to their biochemical and structural environment. Such studies in advanced 3D environments can assist a better/more accurate design of industrial antimicrobial processes, ultimately, improving food safety.

A Step-by-Step Methodological Guide for Developing Zonal Multicellular Scaffold-Based Pancreatic Cancer Models.

The tumor microenvironment (TME), a complex heterogeneous mixture of various cellular, physical, and biochemical components and signals, is a major player in the process of tumor growth and its response to therapeutic methods. In vitro 2D monocellular cancer models are unable to mimic the complex in vivo characteristics of cancer TME involving cellular heterogeneity, presence of extracellular matrix (ECM) proteins, as well as spatial orientation and organization of different cell types forming the TME. In vivo animal-based studies have ethical concerns, are expensive and time-consuming, and involve models of non-human species. In vitro 3D models are capable of tiding over several issues associated with both 2D in vitro and in vivo animal models. We have recently developed a novel zonal multicellular 3D in vitro model for pancreatic cancer involving cancer cells, endothelial cells, and pancreatic stellate cells. Our model (i) can provide long-term culture (up to 4 weeks), (ii) can control the ECM biochemical configuration in a cell specific manner, (iii) shows large amounts of collagen secretion by the stellate cells mimicking desmoplasia, and (iv) expresses cell-specific markers throughout the whole culture period. This chapter describes the experimental methodology to form our hybrid multicellular 3D model for pancreatic ductal adenocarcinoma, including the immunofluorescence staining on the cell culture.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes' viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells' rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

The antimicrobial efficacy of remote cold atmospheric plasma effluent against single and mixed bacterial biofilms of varying age.

Cold atmospheric plasma (CAP) is a minimal food processing technology of increasing interest in the food industry, as it is mild in nature compared to traditional methods (e.g. pasteurisation) and thus can maintain the food's desirable qualities. However, due to this mild nature, the potential exists for post-treatment microbial survival and/or stress adaptation. Furthermore, biofilm inactivation by CAP is underexplored and mostly studied on specific foods or on plastic/polymer surfaces. Co-culture effects, biofilm age, and innate biofilm-associated resistance could all impact CAP efficacy, while studies on real foods are limited to the food product investigated without accounting for structural complexity. The effect of a Remote and Enclosed CAP device (Fourth State Medicine Ltd) was investigated on Escherichia coli and Listeria innocua grown as planktonic cells and as single or mixed bacterial biofilms of variable age, on a biphasic viscoelastic food model of controlled rheological and structural complexity. Post-CAP viability was assessed by plate counts, cell sublethal injury was quantified using flow cytometry, and biofilms were characterised and assessed using total protein content and microscopy techniques. A greater impact of CAP on planktonic cells was observed at higher air flow rates, where the ReCAP device operates in a mode more favourable to reactive oxygen species than reactive nitrogen species. Although planktonic E. coli was more susceptible to CAP than planktonic L. innocua, the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore, E. coli produced much higher protein content in both single and mixed biofilms than L. innocua. Consequently, greater survival of L. innocua in mixed biofilms was attributed to a protective effect from E. coli. These results show that biofilm susceptibility to CAP is age and bacteria dependent, and that in mixed biofilms bacteria may become less susceptible to CAP. These findings are of significance to the food industry for the development of effective food decontamination methods using CAP.

The impact of food model system structure on the inactivation of Listeria innocua by cold atmospheric plasma and nisin combined treatments.

Novel processing methods such as cold atmospheric plasma (CAP) and natural antimicrobials like nisin, are of interest to replace traditional food decontamination approaches as, due to their mild nature, they can maintain desirable food characteristics, i.e., taste, texture, and nutritional content. However, the microbial growth characteristics (planktonic growth/surface colonies) and/or the food structure itself (liquid/solid surface) can impact the inactivation efficacy of these novel processing methods. More specifically, cells grown as colonies on a solid(like) surface experience a completely different growth environment to cells grown planktonically in liquid, and thus could display a different response to novel processing treatments through stress adaptation and/or cross protection mechanisms. The order in which combined treatments are applied could also impact their efficacy, especially if the mechanisms of action are complementary. This work presents a fundamental study on the efficacy of CAP and nisin, alone and combined, as affected by food system structure. More specifically, Listeria innocua was grown planktonically (liquid broth) or on a viscoelastic Xanthan gum gel system (1.5% w/v) and treated with CAP, nisin, or a combination of the two. Both the inactivation system, i.e., liquid versus solid(like) surface and the growth characteristics, i.e., planktonic versus colony growth, were shown to impact the treatment efficacy. The combination of nisin and CAP was more effective than individual treatments, but only when nisin was applied before the CAP treatment. This study provides insight into the environmental stress response/adaptation of L. innocua grown on structured systems in response to natural antimicrobials and novel processing technologies, and is a step towards the faster delivery of these food decontamination methods from the bench to the food industry.

A Novel Scaffold-Based Hybrid Multicellular Model for Pancreatic Ductal Adenocarcinoma-Toward a Better Mimicry of the in vivo Tumor Microenvironment.

With a very low survival rate, pancreatic ductal adenocarcinoma (PDAC) is a deadly disease. This has been primarily attributed to (i) its late diagnosis and (ii) its high resistance to current treatment methods. The latter specifically requires the development of robust, realistic in vitro models of PDAC, capable of accurately mimicking the in vivo tumor niche. Advancements in the field of tissue engineering (TE) have helped the development of such models for PDAC. Herein, we report for the first time a novel hybrid, polyurethane (PU) scaffold-based, long-term, multicellular (tri-culture) model of pancreatic cancer involving cancer cells, endothelial cells, and stellate cells. Recognizing the importance of ECM proteins for optimal growth of different cell types, the model consists of two different zones/compartments: an inner tumor compartment consisting of cancer cells [fibronectin (FN)-coated] and a surrounding stromal compartment consisting of stellate and endothelial cells [collagen I (COL)-coated]. Our developed novel hybrid, tri-culture model supports the proliferation of all different cell types for 35 days (5 weeks), which is the longest reported timeframe in vitro. Furthermore, the hybrid model showed extensive COL production by the cells, mimicking desmoplasia, one of PDAC's hallmark features. Fibril alignment of the stellate cells was observed, which attested to their activated state. All three cell types expressed various cell-specific markers within the scaffolds, throughout the culture period and showed cellular migration between the two zones of the hybrid scaffold. Our novel model has great potential as a low-cost tool for in vitro studies of PDAC, as well as for treatment screening.

Textile-based non-invasive lithium drug monitoring: A proof-of-concept study for wearable sensing.

Flexible wearable chemical sensors are emerging tools which target diagnosis and monitoring of medical conditions. One of the potential applications of wearable chemical sensors is therapeutic drug monitoring for drugs that have a narrow therapeutic range such as lithium. We have investigated the possibility of developing a fibre-based device for non-invasive lithium drug monitoring in interstitial fluid. A flexible cotton-based lithium sensor was coupled with a carbon fibre-based reference electrode to obtain a potentiometric device. In vitro reverse iontophoresis experiments were performed to extract Li+ from under porcine skin by applying a current density of 0.4 mA cm-2 via two electrodes. Carbon fibre-based reverse iontophoresis electrodes were fabricated and used instead of a conventional silver wire-based version and comparable results were obtained. The fibre-based Li+ sensor and reference electrodes were capable of determining the Li+ concentration in samples collected via reverse iontophoresis and the results compared well to those obtained by ion chromatography. Additionally, biocompatibility of the materials used have been tested. Promising results were obtained which confirm the possibility of monitoring lithium in interstitial fluid using a wearable sensor.

Chemoradiotherapy screening in a novel biomimetic polymer based pancreatic cancer model.

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly and aggressive disease with a very low survival rate. This is partly due to the resistance of the disease to currently available treatment options. Herein, we report for the first time the use of a novel polyurethane scaffold based PDAC model for screening the short and relatively long term (1 and 17 days post-treatment) responses of chemotherapy, radiotherapy and their combination. We show a dose dependent cell viability reduction and apoptosis induction for both chemotherapy and radiotherapy. Furthermore, we observe a change in the impact of the treatment depending on the time-frame, especially for radiation for which the PDAC scaffolds showed resistance after 1 day but responded more 17 days post-treatment. This is the first study to report a viable PDAC culture in a scaffold for more than 2 months and the first to perform long-term (17 days) post-treatment observations in vitro. This is particularly important as a longer time-frame is much closer to animal studies and to patient treatment regimes, highlighting that our scaffold system has great potential to be used as an animal free model for screening of PDAC.

Modelling the microbial dynamics and antimicrobial resistance development of Listeria in viscoelastic food model systems of various structural complexities.

Minimal processing for microbial decontamination, such as the use of natural antimicrobials, is gaining interest in the food industry as these methods are generally milder than conventional processing, therefore better maintaining the nutritional content and sensory characteristics of food products. The aim of this study was to quantify the impact of (i) structural composition and complexity, (ii) growth location and morphology, and (iii) the natural antimicrobial nisin, on the microbial dynamics of Listeria innocua. More specifically, viscoelastic food model systems of various compositions and internal structure were developed and characterised, i.e. monophasic Xanthan gum-based and biphasic Xanthan gum/Whey protein-based viscoelastic systems. The microbial dynamics of L. innocua at 10 °C, 30 °C and 37 °C were monitored and compared for planktonic growth in liquid, or in/on (immersed or surface colony growth) the developed viscoelastic systems, with or without a sublethal concentration of nisin. Microscopy imaging was used to determine the bacterial colony size and spatial organisation in/on the viscoelastic systems. Selective growth of L. innocua on the protein phase of the developed biphasic system was observed for the first time. Additionally, significant differences were observed in the colony size and distribution in the monophasic Xanthan gum-based systems depending on (i) the type of growth (surface/immersed) and (ii) the Xanthan gum concentration. Furthermore, the system viscosity in monophasic Xanthan gum-based systems had a protective role against the effects of nisin for immersed growth, and a further inhibitory effect for surface growth at a suboptimal temperature (10 °C). These findings give a systematic quantitative insight on the impact of nisin as an environmental challenge on the growth and spatial organisation of L. innocua, in viscoelastic food model systems of various structural compositions/complexities. This study highlights the importance of accounting for system structural composition/complexity when designing minimal food processing methods with natural antimicrobials.

Biophysical interactions between pancreatic cancer cells and pristine carbon nanotube substrates: Potential application for pancreatic cancer tissue engineering.

Novel synthetic biomaterials able to support direct tissue growth and retain cellular phenotypical properties are promising building blocks for the development of tissue engineering platforms for accurate and fast therapy screening for cancer. The aim of this study is to validate an aligned, pristine multi-walled carbon nanotube (CNT) platform for in vitro studies of pancreatic cancer as a systematic understanding of interactions between cells and these CNT substrates is lacking. Our results demonstrate that our CNT scaffolds-which are easily tuneable to form sheets/fibers-support growth, proliferation, and spatial organization of pancreatic cancer cells, indicating their great potential in cancer tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1637-1644, 2018.

A 3D bioinspired highly porous polymeric scaffolding system for in vitro simulation of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma is an aggressive disease with an extremely low survival rate. This is due to the (i) poor prognosis and (ii) high resistance of the disease to current treatment options. The latter is partly due to the very complex and dense tissue/tumour microenvironment of pancreatic cancer, which contributes to the disease's progression and the inhibition of apoptotic pathways. Over the last years, advances in tissue engineering and the development of three-dimensional (3D) culture systems have shed more light into cancer research by enabling a more realistic recapitulation of the niches and structure of the tumour microenvironment. Herein, for the first time, 3D porous polyurethane scaffolds were fabricated and coated with fibronectin to mimic features of the structure and extracellular matrix present in the pancreatic cancer tumour microenvironment. The developed 3D scaffold could support the proliferation of the pancreatic tumour cells, which was enhanced with the presence of fibronectin, for a month, which is a significantly prolonged in vitro culturing duration. Furthermore, in situ imaging of cellular and biomarker distribution showed the formation of dense cellular masses, the production of collagen-I by the cells and the formation of environmental stress gradients (e.g. HIF-1α) with similar heterogeneity trends to the ones reported in in vivo studies. The results obtained in this study suggest that this bioinspired porous polyurethane based scaffold has great potential for in vitro high throughput studies of pancreatic cancer including drug and treatment screening.

Designing a bio-inspired biomimetic in vitro system for the optimization of ex vivo studies of pancreatic cancer.

Pancreatic cancer is one of the most aggressive and lethal human malignancies. Drug therapies and radiotherapy are used for treatment as adjuvants to surgery, but outcomes remain disappointing. Advances in tissue engineering suggest that 3D cultures can reflect the in vivo tumor microenvironment and can guarantee a physiological distribution of oxygen, nutrients, and drugs, making them promising low-cost tools for therapy development. Here, we review crucial structural and environmental elements that should be considered for an accurate design of an ex vivo platform for studies of pancreatic cancer. Furthermore, we propose environmental stress response biomarkers as platform readouts for the efficient control and further prediction of the pancreatic cancer response to the environmental and treatment input.

Multifunctional, biocompatible and pH-responsive carbon nanotube- and graphene oxide/tectomer hybrid composites and coatings.

Here we present a route for non-covalent functionalization of carboxylated multi-walled carbon nanotubes and graphene oxide with novel two-dimensional peptide assemblies. We show that self-assembled amino-terminated biantennary and tetraantennary oligoglycine peptides (referred to as tectomers) effectively coat carboxylated multi-walled carbon nanotubes and also strongly interact with graphene oxide due to electrostatic interactions and hydrogen bonding as the driving force, respectively. The resulting hybrids can be made into free-standing conducting composites or applied in the form of thin, pH-switchable bioadhesive coatings onto graphene oxide fibers. Monitoring of cell viability of pancreatic cell lines, seeded on those CNT hybrids, show that they can be used as two- and three-dimensional scaffolds to tissue engineer tumour models for studying ex vivo the tumour development and response to treatment. This highly versatile method in producing pH-responsive hybrids and coatings offers an attractive platform for a variety of biomedical applications and for the development of functional materials such as smart textiles, sensors and bioelectronic devices.

Towards unravelling the kinetics of an acute myeloid leukaemia model system under oxidative and starvation stress: a comparison between two- and three-dimensional cultures.

A great challenge when conducting ex vivo studies of leukaemia is the construction of an appropriate experimental platform that would recapitulate the bone marrow (BM) environment. Such a 3D scaffold system has been previously developed in our group [1]. Additionally to the BM architectural characteristics, parameters such as oxygen and glucose concentration are crucial as their value could differ between patients as well as within the same patient at different stages of treatment, consequently affecting the resistance of leukaemia to chemotherapy. The effect of oxidative and glucose stress-at levels close to human physiologic ones-on the proliferation and metabolic evolution of an AML model system (K-562 cell line) in conventional 2D cultures as well as in 3D scaffolds were studied. We observed that the K-562 cell line can proliferate and remain alive for 2 weeks in medium with glucose close to physiological levels both in 20 and 5% O2. We report interesting differences on the cellular response to the environmental, i.e., oxidative and/or nutritional stress stimuli in 2D and 3D. Higher adaptation to oxidative stress under non-starving conditions is observed in the 3D system. The glucose level in the medium has more impact on the cellular proliferation in the 3D compared to the 2D system. These differences can be of significant importance both when applying chemotherapy in vitro and also when constructing mathematical tools for optimisation of disease treatment.

Role of growth morphology in the formulation of NaCl-based selective media for injury detection of Escherichia coli, Salmonella Typhimurium and Listeria innocua.

Sublethal injury (SI) poses major public health concerns since injured cells are responsible for serious limitations in food diagnostics and are susceptible to recovery, often developing adaptive stress responses. Detection of SI is based on the difference in plate counts between non-selective media, which represent the total cell population, and selective media, to which injured cells become sensitive. Selective media for detection of sublethal membrane damage are often based on NaCl supplement, although there is a lack of consensus in the literature about appropriate levels. Planktonic cells are generally used to investigate SI mechanisms, although they often exhibit different stress tolerance than cell colonies in/on solid food (model) systems. In this work, the effect of growth morphology, colony size and concentration of the gelling agent in the growth media, on the maximum non-inhibitory NaCl concentration in the plating medium was assessed for Escherichia coli, Salmonella Typhimurium and Listeria innocua. Stationary phase cultures of planktonic cells and large and small colonies grown in either 1.5% (w/v) xanthan gum-based system or 2.5% (w/v) xanthan gum-based system exhibited significantly different viable counts and osmotolerance. The effect of cell arrangement and xanthan gum percentage in the growth media depended on the microorganism under investigation. Additionally, differences in the maximum non-inhibitory concentration were evident, with 5.0% (w/v) NaCl for the Gram-negative bacteria and 6.5% (w/v), for L. innocua. Different extent of colony shrinkage and morphological damage was observed as NaCl concentration in the plating medium increased. This information will contribute to define NaCl-based selective media for accurate SI detection under realistic scenarios.