by Keyword: Cell culture
Hortelão, Ana C., Carrascosa, Rafael, Murillo-Cremaes, Nerea, Patiño, Tania, Sánchez, Samuel, (2019). Targeting 3D bladder cancer spheroids with urease-powered nanomotors ACS Nano 13, (1), 429-439
Cancer is one of the main causes of death around the world, lacking efficient clinical treatments that generally present severe side effects. In recent years, various nanosystems have been explored to specifically target tumor tissues, enhancing the efficacy of cancer treatment and minimizing the side effects. In particular, bladder cancer is the ninth most common cancer worldwide and presents a high survival rate but serious recurrence levels, demanding an improvement in the existent therapies. Here, we present urease-powered nanomotors based on mesoporous silica nanoparticles that contain both polyethylene glycol and anti-FGFR3 antibody on their outer surface to target bladder cancer cells in the form of 3D spheroids. The autonomous motion is promoted by urea, which acts as fuel and is inherently present at high concentrations in the bladder. Antibody-modified nanomotors were able to swim in both simulated and real urine, showing a substrate-dependent enhanced diffusion. The internalization efficiency of the antibody-modified nanomotors into the spheroids in the presence of urea was significantly higher compared with antibody-modified passive particles or bare nanomotors. Furthermore, targeted nanomotors resulted in a higher suppression of spheroid proliferation compared with bare nanomotors, which could arise from the local ammonia production and the therapeutic effect of anti-FGFR3. These results hold significant potential for the development of improved targeted cancer therapy and diagnostics using biocompatible nanomotors.
Keywords: 3D cell culture, Bladder cancer, Enzymatic catalysis, Nanomachines, Nanomotors, Self-propulsion, Targeting
Torras, N., García-Díaz, M., Fernández-Majada, V., Martínez, Elena, (2018). Mimicking epithelial tissues in three-dimensional cell culture models Frontiers in Bioengineering and Biotechnology 6, Article 197
Epithelial tissues are composed of layers of tightly connected cells shaped into complex three-dimensional (3D) structures such as cysts, tubules, or invaginations. These complex 3D structures are important for organ-specific functions and often create biochemical gradients that guide cell positioning and compartmentalization within the organ. One of the main functions of epithelia is to act as physical barriers that protect the underlying tissues from external insults. In vitro, epithelial barriers are usually mimicked by oversimplified models based on cell lines grown as monolayers on flat surfaces. While useful to answer certain questions, these models cannot fully capture the in vivo organ physiology and often yield poor predictions. In order to progress further in basic and translational research, disease modeling, drug discovery, and regenerative medicine, it is essential to advance the development of new in vitro predictive models of epithelial tissues that are capable of representing the in vivo-like structures and organ functionality more accurately. Here, we review current strategies for obtaining biomimetic systems in the form of advanced in vitro models that allow for more reliable and safer preclinical tests. The current state of the art and potential applications of self-organized cell-based systems, organ-on-a-chip devices that incorporate sensors and monitoring capabilities, as well as microfabrication techniques including bioprinting and photolithography, are discussed. These techniques could be combined to help provide highly predictive drug tests for patient-specific conditions in the near future.
Keywords: 3D cell culture models, Biofabrication, Disease modeling, Drug screening, Epithelial barriers, Microengineered tissues, Organ-on-a-chip, Organoids
Rajzer, I., Menaszek, E., Kwiatkowski, R., Planell, J. A., Castaño, O., (2014). Electrospun gelatin/poly(ε-caprolactone) fibrous scaffold modified with calcium phosphate for bone tissue engineering Materials Science and Engineering: C 44, 183-190
In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.
Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning
Rigat, L., Elizalde, A., Del Portillo, H. A., Homs-Corbera, A., Samitier, J., (2014). Selective cell culturing step using laminar co-flow to enhance cell culture in splenon-on-a-chip biomimetic platform MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences , CBMS (San Antonio, USA) , 769-771
Constant evolution and improvements on areas such as tissue engineering, microfluidics and nanotechnology have made it possible to partially close the gap between conventional in vitro cell cultures and animal model-based studies. A step forward in this field concerns organ-on-chip technologies, capable of reproducing the most relevant physiological features of an organ in a microfluidic platform. In this work we have exploited the capabilities of laminar co-flow inside our biomimetic platform, the splenon-on-a-chip, in order to enhance cell culture inside its channels to better mimic the spleen's environment. Â© 14CBMS.
Keywords: Cell culture, Co-flow, Laminar flow, Organ-on-a-chip, Spleen
Gil, V., Del Río, J. A., (2012). Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue Nature Protocols 7, (2), 268-280
This protocol uses rat tail-derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail-derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks.
Keywords: Cell biology, Cell culture, Developmental biology, Imaging, Model organisms, Neuroscience, Tissue culture
Gustavsson, J., Ginebra, M. P., Engel, E., Planell, J., (2011). Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media Acta Biomaterialia 7, (12), 4242-4252
Solution-mediated surface reactions occur for most calcium phosphate-based biomaterials and may influence cellular response. A reasonable extrapolation of such processes observed in vitro to in vivo performance requires a deep understanding of the underlying mechanisms. We therefore systematically investigated the nature of ion reactivity of calcium-deficient hydroxyapatite (CDHA) by exposing it for different periods of time to standard cell culture media of different chemical composition (DMEM and McCoy medium, with and without osteogenic supplements and serum proteins). Kinetic ion interaction studies of principal extracellular ions revealed non-linear sorption of Ca2+ (∼50% sorption) and K+ (∼8%) as well as acidification of all media during initial contact with CDHA (48 h). Interestingly, inorganic phosphorus (Pi) was sorbed from McCoy medium (∼50%) or when using osteogenic media containing β-glycerophosphate, but not from DMEM medium. Non-linear sorption data could be perfectly described by pseudo-first-order and pseudo-second-order sorption models. At longer contact time (21 days), and with frequent renewal of culture medium, sorption of Ca2+ remained constant throughout the experiment, while sorption of Pi gradually decreased in McCoy medium. In great contrast, CDHA began to release Pi slowly with time when using DMEM medium. Infrared spectra showed that CDHA exposed to culture media had a carbonated surface chemistry, suggesting that carbonate plays a key role in the ion reactivity of CDHA. Our data show that different compositions of the aqueous environment may provoke opposite ion reactivity of CDHA, and this must be carefully considered when evaluating the osteoinductive potential of the material.
Keywords: Hydroxyapatite, Bioactive materials, Cell culture medium, Ion exchange, Sorption models
Fernandez, Javier G., Mills, C. A., Samitier, J., (2009). Complex microstructured 3D surfaces using chitosan biopolymer Small 5, (5), 614-620
A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).
Keywords: Biocompatible Materials/ chemistry, Cell Adhesion, Cell Culture Techniques/ methods, Cell Proliferation, Cells, Cultured, Chitosan/ chemistry, Crystallization/methods, Endothelial Cells/ cytology/ physiology, Humans, Materials Testing, Nanostructures/ chemistry/ ultrastructure, Nanotechnology/methods, Particle Size, Surface Properties, Tissue Engineering/methods
Navarro, M., Engel, E., Planell, J. A., Amaral, I., Barbosa, M., Ginebra, M. P., (2008). Surface characterization and cell response of a PLA/CaP glass biodegradable composite material Journal of Biomedical Materials Research - Part A , 85A, (2), 477-486
Bioabsorbable materials are of great interest for bone regeneration applications, since they are able to degrade gradually as new tissue is formed. In this work, a fully biodegradable composite material containing polylactic acid (PLA) and calcium phosphate (CaP) soluble glass particles has been characterized in terms of surface properties and cell response. Cell cultures were performed in direct contact with the materials and also with their extracts, and were evaluated using the MTT assay, alkaline phosphatase activity, and osteocalcin measurements. The CaP glass and PLA were used as reference materials. No significant differences were observed in cell proliferation with the extracts containing the degradation by-products of the three materials studied. A relation between the materials wettability and the material-cell interactions at the initial stages of contact was observed. The most hydrophilic material (CaP glass) presented the highest cell adhesion values as well as an earlier differentiation, followed by the PLA/glass material. The incorporation of glass particles into the PLA matrix increased surface roughness. SEM images showed that the heterogeneity of the composite material induced morphological changes in the cells cytoskeleton.
Keywords: Glass, Polylactic acid, Surface analysis, Cell culture, In vitro test