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RATIONALE: Analysis of the stable isotope ratios of carbon and nitrogen (δ13 C and δ15 N values) is increasingly being used to gain insight into predator trophic ecology, which requires accurate diet-tissue discrimination factors (DTDFs), or the isotopic difference between prey and predator. Accurate DTDFs must be calculated from predators consuming an isotopically constant diet over time in controlled feeding experiments, but these studies have received little attention to date, especially among seabird species. METHODS: In this study, aquarium-housed Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins were fed a single-prey source diet (capelin Mallotus villosus) for eight weeks. Stable isotope ratios (δ13 C and δ15 N values) of penguin blood (cellular component and plasma) and capelin were measured using mass spectrometry and then used to calculate DTDFs for both components of penguin blood by comparison with prey values. RESULTS: The DTDFs for plasma were -0.63 ± 0.49 (mean ± SD) and -0.27 ± 0.22 for δ13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ15 N values for Magellanic and southern rockhopper penguins, respectively, while the DTDFs for the cellular component were 1.22 ± 0.03 and 1.26 ± 0.03 for δ13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ15 N values. CONCLUSIONS: We compare our DTDFs with published values from blood components of penguins and discuss the effects that lipid extraction, sample storage, and diet have on the DTDFs of penguin blood components. This study provides accurate DTDFs of blood components for two seabird species of conservation concern, and is one of the first to provide plasma DTDFs for penguins, which are underrepresented in the seabird literature.
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Alimentación Animal , Isótopos de Carbono/sangre , Isótopos de Nitrógeno/sangre , Spheniscidae/sangre , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Isótopos de Carbono/análisis , Dieta , Conducta Alimentaria , Peces/fisiología , Cadena Alimentaria , Masculino , Isótopos de Nitrógeno/análisis , Conducta Predatoria , Spheniscidae/fisiologíaRESUMEN
Biomimetic 3D models emerged some decades ago to address 2D cell culture limitations in the field of replicating biological phenomena, structures or functions found in nature. The fabrication of hydrogels for cancer disease research enables the study of cell processes including growth, proliferation and migration and their 3D design is based on the encapsulation of tumoral cells within a tunable matrix. In this work, a platform of gelatin methacrylamide (GelMA)-based photocrosslinked scaffolds with embedded colorectal (HCT-116) or pancreatic (MIA PaCa-2) cancer cells is presented. Prior to cell culture, the mechanical characterization of hydrogels was assessed in terms of stiffness and swelling behavior. Modifications of the UV curing time enabled a fine tuning of the mechanical properties, which at the same time, showed susceptibility to the chemical composition and crosslinking mechanism. All scaffolds displayed excellent cytocompatibility with both tumoral cells while eliciting various cell responses depending on the microenvironment features. Individual and collective cell migration were observed for HCT-116 and MIA PaCa-2 cell lines, highlighting the ability of the colorectal cancer cells to cluster into aggregates of different sizes governed by the surrounding matrix. Additionally, metabolic activity results pointed out to the development of a more proliferative phenotype within stiffer networks. These findings confirm the suitability of the presented platform of GelMA-based hydrogels to conduct 3D cell culture experiments and explore biological processes associated with colorectal and pancreatic cancer.
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Neoplasias Colorrectales , Gelatina , Humanos , Gelatina/química , Hidrogeles/química , Técnicas de Cultivo de Célula , Páncreas , Técnicas de Cultivo Tridimensional de Células , Microambiente TumoralRESUMEN
A combination of human-induced pluripotent stem cells (hiPSCs) and 3D microtissue culture techniques allows the generation of models that recapitulate the cardiac microenvironment for preclinical research of new treatments. In particular, spheroids represent the simplest approach to culture cells in 3D and generate gradients of cellular access to the media, mimicking the effects of an ischemic event. However, previous models required incubation under low oxygen conditions or deprived nutrient media to recreate ischemia. Here, we describe the generation of large spheroids (i.e., larger than 500 µm diameter) that self-induce an ischemic core. Spheroids were generated by coculture of cardiomyocytes derived from hiPSCs (hiPSC-CMs) and primary human cardiac fibroblast (hCF). In the proper medium, cells formed aggregates that generated an ischemic core 2 days after seeding. Spheroids also showed spontaneous cellular reorganization after 10 days, with hiPSC-CMs located at the center and surrounded by hCFs. This led to an increase in microtissue stiffness, characterized by the implementation of a constriction assay. All in all, these phenomena are hints of the fibrotic tissue remodeling secondary to a cardiac ischemic event, thus demonstrating the suitability of these spheroids for the modeling of human cardiac ischemia and its potential application for new treatments and drug research.
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Isquemia Miocárdica , Miocitos Cardíacos , Humanos , Constricción , Células Cultivadas , IsquemiaRESUMEN
From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization has evolved extensively. Several properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, the influence of the reaction media (RM) and swelling media (SM)-phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations-is described in gelatin methacrylamide (GelMA) hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco-2 and HCT-116). All scaffolds are UV-photocrosslinked under identical conditions and evaluated for mass swelling ratio and stiffness. The results indicate that stiffness is highly susceptible to the RM, but not to the SM. Additionally, PBS-prepared hydrogels exhibited a higher photopolymerization degree according to high resolution magic-angle spinning (HR-MAS) NMR. These findings correlate with the biological response of Caco-2 and HCT-116 cells seeded on the substrates, which demonstrated flatter morphologies on stiffer hydrogels. Overall, cell viability and proliferation are excellent for both cell lines, and Caco-2 cells displayed a characteristic apical-basal polarization based on F-actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture.
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Hidrogeles , Ingeniería de Tejidos , Humanos , Hidrogeles/farmacología , Hidrogeles/química , Ingeniería de Tejidos/métodos , Gelatina/química , Células CACO-2 , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Andamios del Tejido/químicaRESUMEN
The mechanical microenvironment plays a crucial role in the evolution of colorectal cancer, a complex disease characterized by heterogeneous tumors with varying elasticity. Toward setting up distinct scenarios, herein, we describe the preparation and characterization of gelatin methacrylamide (GelMA)-based hydrogels via two different mechanisms: free-radical photopolymerization and photo-induced thiol-ene reaction. A precise stiffness modulation of covalently crosslinked scaffolds was achieved through the application of well-defined irradiation times while keeping the intensity constant. Besides, the incorporation of thiol chemistry strongly increased stiffness with low to moderate curing times. This wide range of finely tuned mechanical properties successfully covered from healthy tissue to colorectal cancer stages. Hydrogels prepared in phosphate-buffered saline or Dulbecco's modified Eagle's medium resulted in different mechanical and swelling properties, although a similar trend was observed for both conditions: thiol-ene systems exhibited higher stiffness and, at the same time, higher swelling capacity than free-radical photopolymerized networks. In terms of biological behavior, three of the substrates showed good cell proliferation rates according to the formation of a confluent monolayer of Caco-2 cells after 14 days of cell culture. Likewise, a characteristic apical-basal polarization of cells was observed for these three hydrogels. These results demonstrate the versatility of the presented platform of biomimetic materials as in vitro cell culture scaffolds.
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The tissue microenvironment plays a crucial role in tissue homeostasis and disease progression. However, the in vitro simulation has been limited by the lack of adequate biomimetic models in the last decades. Thanks to the advent of microfluidic technology for cell culture applications, these complex microenvironments can be recreated by combining hydrogels, cells and microfluidic devices. Nevertheless, this advance has several limitations. When cultured in three-dimensional (3D) hydrogels inside microfluidic devices, contractile cells may exert forces that eventually collapse the 3D structure. Disrupting the compartmentalisation creates an obstacle to long-term or highly cell-concentrated assays, which are extremely relevant for multiple applications such as fibrosis or ischaemia. Therefore, we tested surface treatments on cyclic-olefin polymer-based microfluidic devices (COP-MD) to promote the immobilisation of collagen as a 3D matrix protein. Thus, we compared three surface treatments in COP devices for culturing human cardiac fibroblasts (HCF) embedded in collagen hydrogels. We determined the immobilisation efficiency of collagen hydrogel by quantifying the hydrogel transversal area within the devices at the studied time points. Altogether, our results indicated that surface modification with polyacrylic acid photografting (PAA-PG) of COP-MD is the most effective treatment to avoid the quick collapse of collagen hydrogels. As a proof-of-concept experiment, and taking advantage of the low-gas permeability properties of COP-MD, we studied the application of PAA-PG pre-treatment to generate a self-induced ischaemia model. Different necrotic core sizes were developed depending on initial HCF density seeding with no noticeable gel collapse. We conclude that PAA-PG allows long-term culture, gradient generation and necrotic core formation of contractile cell types such as myofibroblasts. This novel approach will pave the way for new relevant in vitro co-culture models where fibroblasts play a key role such as wound healing, tumour microenvironment and ischaemia within microfluidic devices.
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Colágeno , Hidrogeles , Humanos , Hidrogeles/química , Colágeno/química , Matriz Extracelular/química , Isquemia , Dispositivos Laboratorio en un ChipRESUMEN
Photoembossing is a powerful photolithographic technique to prepare surface relief structures relying on polymerization-induced diffusion in a solventless development step. Conveniently, surface patterns are formed by two or more interfering laser beams without the need for a lithographic mask. The use of nanosecond pulsed light-based interference lithography strengthens the pattern resolution through the absence of vibrational line pattern distortions. Typically, a conventional photoembossing protocol consists of an exposure step at room temperature that is followed by a thermal development step at high temperature. In this work, we explore the possibility to perform the pulsed holographic exposure directly at the development temperature. The surface relief structures generated using this modified photoembossing protocol are compared with those generated using the conventional one. Importantly, the enhancement of surface relief height has been observed by exposing the samples directly at the development temperature, reaching approximately double relief heights when compared to samples obtained using the conventional protocol. Advantageously, the light dose needed to reach the optimum height and the amount of photoinitiator can be substantially reduced in this modified protocol, demonstrating it to be a more efficient process for surface relief generation in photopolymers. Kidney epithelial cell alignment studies on substrates with relief-height optimized structures generated using the two described protocols demonstrate improved cell alignment in samples generated with exposure directly at the development temperature, highlighting the relevance of the height enhancement reached by this method. Although cell alignment is well-known to be enhanced by increasing the relief height of the polymeric grating, our work demonstrates nano-second laser interference photoembossing as a powerful tool to easily prepare polymeric gratings with tunable topography in the range of interest for fundamental cell alignment studies.
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El Programa de Detección Precoz del Cáncer de Mama (PDPCM) se implantó en el Distrito Sanitario Córdoba Sur finales del año 1999. El objetivo de este estudio es evaluar el PDPCM en nuestro distrito sanitario a lo largo de las primaras 4 ediciones del programa. Se calcularon las tasas de participación (mujeres exploradas/ población elegible), las tasas de captación (mujer que acuden/ población elegible) y las tasas de derivación hospitalaria en porcentaje, además de las tasas de detección (numero de mujeres diagnosticados de tumor maligno x 10?/ numero de mujeres mamografiadas) en el nuestro distrito sanitario. La población elegible fue de 17.058, 16.421, 18.098 y 20.478 para las 4 ediciones, respectivamente. Las tasas de participación del Distrito fueron de 73,9% (1ª edición), 77% (2ª edición), 76,3% (3ª edición) y 76,2% (4ª edición). Las tasas de captación del Distrito fueron de 82% (1ª edición), 78,8% (2ª edición), 78,2% (3ª edición) y 77,9% (4ª edición). Las tasas de derivación hospitalaria del Distrito fueron de 7,8% (1ª edición), 4,4% (2ª edición), 6,3% (3ª edición) y 6,57% (4ª edición). Las tasas de detección del Distrito fueron de 4,68 (1ª edición), 2,21 (2ª edición), 2,97 (3º edición) y 2 (4ª edición). Desde que comenzó el PDPCM se han detectado 162 tumores malignos con una tasa de detección global del 7,13 por 103 mujeres mamografiadas. En conclusión, el PDPCM en el Distrito Sanitario Córdoba Sur ha obtenido unas tasas de participación y de captación más altas de las marcadas por la Conserjería de Salud de Andalucía. Esto ha permitido el diagnóstico temprano de 162 mujeres con tumores malignos, lo que su pone una mejora en el pronostico y la calidad de vida de las pacientes (AU)
The Early Detection of Breast Cancer Program (EDBCP) was implanted at South Cordoba Health District by the end of 1999. The objective of this study is to evaluate the EDBCP in our health district over the last 4 rounds of the program. We calculated the participation rate (examined women/ target population), the inclusion rate (attended women/ target population) and the hospital derivation rate in percentage, together with the detection rate (number of women diagnosed with malignant tumors x 10?/ number of women examined with mammograms) in our health district. The target population was 17058, 16421, 18.098 and 20478 for the 4 rounds respectively. The participation rates in our district were 73.9% (1st round), 77% (2nd round), 76.3% (3rd round) and 76.2% (4th round). The inclusion rates of the district were 82% (1st round), 78.8% (2nd round), 78.2% (3rd round) and 77.9% (4th round). The hospital derivation rates of the district were 7.8% (1st round), 4.4% (2nd round), 6.3% (3rd round) and 6.57% (4th round). The detection rates of the district were 4.68 (1st round), 2.21 (2nd round), 2.97 (3rd round) and 2 (4th round). Since the EDBCP was implanted we have detected 162 malignant tumours, with global detection rate of 7.13 per 103 examined women. In conclusion, the EDBCP at South Cordoba Health District have obtained higher participation and inclusion rates than the target objective of the Andalusian Health Services. This has permitted the early diagnosis of 162 women with malignant tumours, which would improve the prognosis and the quality of life of these patients (AU)