Modelos de cultura celular para rastreio de fármacos

Two-dimensional (2D) cell culture is the prime methodology used, for screening anticancer therapeutics. However, when 2D cellular models are used, the architecture of native tumors is not fully represented, leading in some cases to an unsuccessful prediction of cancer cells response to drugs. On the other hand, there is a need to reduce the use of animal research models once they have economical and ethical problems associated. To overcome the limitations associated to 2D cell culture models and in vivo assays, the researchers started to perform cell growth in three-dimensions (3D), for reproducing in vitro the 3D structure of solid tumors. One of the most applied techniques to produce these 3D cellular aggregates, also known as spheroids, is Liquid Overlay Technique (LOT), in which cells are forced to aggregate due to their limited adhesion to certain biomaterials, usually agarose or agar. However, these biopolymers cannot interact with cancer cells, neither establish interactions that are similar to those occurring between cells and extracellular matrix (ECM) in solid tumors, which are responsible for the activation of cellular signaling pathways that regulate cancer cells behavior. In order to mimic not only the 3D structure but also the cell-ECM interactions that occur in tumors, it has been proposed the production of 3D models in which cells can interact with tumor ECM components. One of the biomaterial that has been used with this objective is the hyaluronic acid (HA). This compound is one of the main constituents of tumor ECM, it avoids cell adhesion and it has an essential role in cancer progression. In this work it was optimized, for the first time, the coating of surfaces with HA that were used for the production of reproducible heterotypic breast cancer spheroids. The obtained results revealed that the HA coated surfaces allow the production of spheroids that reproduce the 3D structure and the cellular heterogeneity presented by breast solid tumors. Furthermore, it was possible to control the size, shape and number of spheroids produced by changing the HA concentration and the number of cells initially seeded. Overall, these breast cancer spheroids assembled on HA-coated surfaces represent a huge improvement for the future development of anticancer therapies.A cultura de células em duas dimensões (2D) é a principal metodologia utilizada para o rastreio de agentes terapêuticos anticancerígenos. No entanto, quando os modelos celulares em 2D são utilizados, a arquitetura dos tumores nativos não é reproduzida na sua plenitude, levando, em alguns casos, a uma previsão pouco precisa da resposta das células aos fármacos. Por outro lado, existe a necessidade de reduzir a utilização de modelos animais em laboratório, uma vez que estes têm associados problemas económicos e éticos. Para superar as limitações associadas aos modelos celulares produzidos em 2D e aos ensaios in vivo, os investigadores começaram a efetuar o crescimento de células em três dimensões (3D) com o objetivo de reproduzir in vitro a estrutura 3D dos tumores sólidos. Uma das técnicas mais utilizadas para a produção destes agregados celulares 3D, também conhecidos como esferoides, é a técnica de sobreposição líquida (Liquid Overlay Technique – LOT), na qual as células são forçadas a agregar devido à sua limitada adesão a certos biomateriais, geralmente agarose ou agar. No entanto, estes biopolímeros não têm a capacidade de interagir com as células cancerígenas nem de estabelecer interações semelhantes às que ocorrem entre as células e a matriz extracelular (MEC) nos tumores sólidos, que ativam as vias de sinalização celular reguladoras do comportamento das células tumorais. Com o intuito de mimetizar não só a estrutura 3D mas também as interações MEC que ocorrem nos tumores, têm sido produzidos modelos 3D nos quais as células interagem com componentes da MEC. Um dos biomateriais que tem sido usado com este objetivo é o ácido hialurónico (AH). Este composto é um dos principais componentes da MEC dos tumores, evita a adesão celular e tem um papel essencial na progressão do cancro. No presente estudo foi pela primeira vez otimizado o revestimento de superfícies com AH para a produção de forma reprodutível de esferóides heterotípicos do cancro da mama. Os resultados obtidos revelaram que as superfícies revestidas com AH permitem a produção de esferóides que reproduzem a estrutura 3D e a heterogeneidade celular encontrada nos tumores sólidos. Por outro lado, é possível controlar o tamanho, forma e número de esferóides produzidos alterando a concentração de AH e o número inicial de células semeadas. Em suma, os esferóides aqui produzidos em superfícies revestidas com AH representam uma grande melhoria para o futuro desenvolvimento de terapias anticancerígenas

Assembly of breast cancer heterotypic spheroids on hyaluronic acid coated surfaces

Drug screening is currently demanding for realistic models that are able to reproduce the structural features of solid tumors. 3D cell culture systems, namely spheroids, emerged as a promising approach to provide reliable results during drug development. So far, liquid overlay technique (LOT) is one of the most used methods for spheroids assembly. It comprises cellular aggregation due to their limited adhesion to certain biomaterials, like agarose. However, researchers are currently improving this technique in order to obtain spheroids on surfaces that mimic cancer extracellular matrix (ECM), since cell–ECM interactions modulate cells behavior and their drug resistance profile. Herein, hyaluronic acid (HA) coated surfaces were used, for the first time, for the production of reproducible heterotypic breast cancer spheroids. The obtained results revealed that it is possible to control the size, shape, and number of spheroids gotten per well by changing the HA concentration and the number of cells initially seeded in each well.info:eu-repo/semantics/publishedVersio

Tumor spheroid assembly on hyaluronic acid-based structures: A review

Two-dimensional (2D) cell culture is the main methodology used for screening anticancer therapeutics. However, these 2D cellular models misrepresent the architecture of native tumors, leading, in some cases, to unsuccessful prediction of cancer cell response to drugs. To overcome such limitations, cell growth in three dimensions (3D) arises as an alternative to reproduce in vitro the cellular arrangement found in tumors. Among the 3D cancer models developed so far, spheroids are the most attractive since these are cellular aggregates that broadly mimic many features of solid tumors affecting humans, like cell–cell interactions. One of the most applied techniques for producing spheroids is the liquid overlay technique, in which cells aggregate due to their limited adhesion to certain biomaterials, usually agarose or agar. Recently, the suitability of hyaluronic acid (HA) for spheroids assembly and HA-cell surface receptor interactions has been investigated. Ergo, this review gathers a summary of different studies where HA-based structures were developed and used for tumor spheroids production in order to be used in vitro as reliable 3D tumor models for therapeutic screening purposes.info:eu-repo/semantics/publishedVersio

Spheroids formation on non‐adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre‐clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell–cell and cell‐extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold‐based and ‐free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high‐throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold‐free techniques used for the spheroids formation are also addressed.info:eu-repo/semantics/publishedVersio

3D tumor spheroids: an overview on the tools and techniques used for their analysis

In comparison with 2D cell culture models, 3D spheroids are able to accurately mimic some features of solid tumors, such as their spatial architecture, physiological responses, secretion of soluble mediators, gene expression patterns and drug resistance mechanisms. These unique characteristics highlight the potential of 3D cellular aggregates to be used as in vitro models for screening new anticancer therapeutics, both at a small and large scale. Nevertheless, few reports have focused on describing the tools and techniques currently available to extract significant biological data from these models. Such information will be fundamental to drug and therapeutic discovery process using 3D cell culture models. The present review provides an overview of the techniques that can be employed to characterize and evaluate the efficacy of anticancer therapeutics in 3D tumor spheroids.info:eu-repo/semantics/publishedVersio

Coaxial electrospun PCL/Gelatin-MA fibers as scaffolds for vascular tissue engineering

Coaxial electrospinning is a technique that allows the production of nanofibers with a core–shell structure. Such fibers present several advantages as materials for the preparation of scaffolds, namely due to the possibility of combining a core with the desired mechanical properties with a shell prepared from biocompatible materials that will establish proper interactions with the host. Herein, core-shell fibrous meshes, composed of a polycaprolactone (PCL) core and a functionalized gelatin shell, were prepared by coaxial electrospinning and then photocrosslinked under UV light aiming to be used in vascular tissue regeneration. The suitability of the meshes for the pretended biomedical application was evaluated by assessing their chemical/physical properties as well as their haemo and biocompatibility in vitro. The obtained results revealed that meshes’ shell prepared with a higher content of gelatin showed fibers with diameters presenting a unimodal distribution and a mean value of 600 nm. Moreover, those fibers with higher content of gelatin also displayed lower water contact angles, and therefore higher hydrophilicities. Such features are crucial for the good biologic performance displayed by these meshes, when in contact with blood and with Normal Human Dermal Fibroblasts cells.info:eu-repo/semantics/publishedVersio

Photocrosslinkable electrospun fiber meshes for tissue engineering applications

Electrospun polymeric meshes are known to exhibit promising properties for the regeneration of several soft tissues. Herein, electrospun polymeric meshes were prepared from blends of polycaprolactone and functionalized gelatin. The meshes were then photocrosslinked under UV light using Irgacure® 2959 as the photoinitiator, aiming to improve membranes’ stability in biological fluids. Moreover, meshes suitability to be used as vascular grafts was evaluated by characterizing their chemical/physical properties as well as their haemo and biocompatibility in vitro. The obtained results show that the blended polymeric meshes are biodegradable and those with a higher content of gelatin display a lower water contact angle. Blood compatibility studies showed that the photocrosslinked membranes are haemocompatible, i.e. they display low values of thrombogenicity and do not trigger any haemolytic effect. Also, Normal Human Dermal Fibroblasts cells were incubated in the presence of the produced membranes and they were able to adhere and proliferate, thus revealing the biocompatibility of the photocrosslinked meshes.info:eu-repo/semantics/publishedVersio

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Theia: Faint objects in motion or the new astrometry frontier

In the context of the ESA M5 (medium mission) call we proposed a new satellite mission, Theia, based on relative astrometry and extreme precision to study the motion of very faint objects in the Universe. Theia is primarily designed to study the local dark matter properties, the existence of Earth-like exoplanets in our nearest star systems and the physics of compact objects. Furthermore, about 15 $\%$ of the mission time was dedicated to an open observatory for the wider community to propose complementary science cases. With its unique metrology system and "point and stare" strategy, Theia's precision would have reached the sub micro-arcsecond level. This is about 1000 times better than ESA/Gaia's accuracy for the brightest objects and represents a factor 10-30 improvement for the faintest stars (depending on the exact observational program). In the version submitted to ESA, we proposed an optical (350-1000nm) on-axis TMA telescope. Due to ESA Technology readiness level, the camera's focal plane would have been made of CCD detectors but we anticipated an upgrade with CMOS detectors. Photometric measurements would have been performed during slew time and stabilisation phases needed for reaching the required astrometric precision