RESUMEN
A growing body of evidence suggests that studying cell biology in classical two-dimensional formats, such as cell culture plasticware, results in misleading, non-physiological findings. For example, some aspects of cancer biology cannot be observed in 2D, but require 3D culture methods to recapitulate observations in vivo. Therefore, we developed a microsphere-based model to permit 3D cell culture incorporating physiological extracellular matrix components. Bio-electrospraying was chosen as it is the most advanced method to produce microspheres, with THP-1 cells as a model cell line. Bio-electrospraying parameters, such as nozzle size, polymer flow rate, and voltage, were systematically optimized to allow stable production of size controlled microspheres containing extracellular matrix material and human cells. We investigated the effect of bio-electrospraying parameters, alginate type and cell concentration on cell viability using trypan blue and propidium iodide staining. Bio-electrospraying had no effect on cell viability nor the ability of cells to proliferate. Cell viability was similarly minimally affected by encapsulation in all types of alginate tested (MVM, MVG, chemical- and food-grade). Cell density of 5 × 106 cells ml-1 within microspheres was the optimum for cell survival and proliferation. The stable generation of microspheres incorporating cells and extracellular matrix for use in a 3D cell culture will benefit study of many diverse diseases and permit investigation of cellular biology within a 3D matrix.
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Currently, in vitro testing examines the cytotoxicity of biomaterials but fails to consider how materials respond to mechanical forces and the immune response to them; both are crucial for successful long-term implantation. A notable example of this failure is polypropylene mid-urethral mesh used in the treatment of stress urinary incontinence (SUI). The mesh was largely successful in abdominal hernia repair but produced significant complications when repurposed to treat SUI. Developing more physiologically relevant in vitro test models would allow more physiologically relevant data to be collected about how biomaterials will interact with the body. This study investigates the effects of mechanochemical distress (a combination of oxidation and mechanical distention) on polypropylene mesh surfaces and the effect this has on macrophage gene expression. Surface topology of the mesh was characterised using SEM and AFM; ATR-FTIR, EDX and Raman spectroscopy was applied to detect surface oxidation and structural molecular alterations. Uniaxial mechanical testing was performed to reveal any bulk mechanical changes. RT-qPCR of selected pro-fibrotic and pro-inflammatory genes was carried out on macrophages cultured on control and mechanochemically distressed PP mesh. Following exposure to mechanochemical distress the mesh surface was observed to crack and craze and helical defects were detected in the polymer backbone. Surface oxidation of the mesh was seen after macrophage attachment for 7 days. These changes in mesh surface triggered modified gene expression in macrophages. Pro-fibrotic and pro-inflammatory genes were upregulated after macrophages were cultured on mechanochemically distressed mesh, whereas the same genes were down-regulated in macrophages exposed to control mesh. This study highlights the relationship between macrophages and polypropylene surgical mesh, thus offering more insight into the fate of an implanted material than existing in vitro testing.
Asunto(s)
Mallas Quirúrgicas , Incontinencia Urinaria de Esfuerzo , Humanos , Ensayo de Materiales , Mallas Quirúrgicas/efectos adversos , Polipropilenos/química , Materiales Biocompatibles , Macrófagos , Incontinencia Urinaria de Esfuerzo/cirugíaRESUMEN
BACKGROUND AIMS: Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. METHODS: CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. RESULTS: The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. CONCLUSIONS: Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps.
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Antígenos CD34/metabolismo , Eritrocitos/fisiología , Células Madre Hematopoyéticas/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula/métodos , Muerte Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Eritrocitos/metabolismo , Sangre Fetal/metabolismo , Sangre Fetal/fisiología , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , LigandosRESUMEN
High internal phase emulsion (HIPE) templating is a well-established method for the generation of polymeric materials with high porosity (>74%) and degree of interconnectivity. The porosity and pore size can be altered by adjusting parameters during emulsification, which affects the properties of the resulting porous structure. However, there remain challenges for the fabrication of polyHIPEs, including typically small pore sizes (â¼20-50 µm) and the use of surfactants, which can limit their use in biological applications. Here, we present the use of gelatin, a natural polymer, during the formation of polyHIPE structures, through the use of two biodegradable polymers, polycaprolactone-methacrylate (PCL-M) and polyglycerol sebacate-methacrylate (PGS-M). When gelatin is used as the internal phase, it is capable of stabilising emulsions without the need for an additional surfactant. Furthermore, by changing the concentration of gelatin within the internal phase, the pore size of the resulting polyHIPE can be tuned. 5% gelatin solution resulted in the largest mean pore size, increasing from 53 µm to 80 µm and 28 µm to 94 µm for PCL-M and PGS-M respectively. In addition, the inclusion of gelatin further increased the mechanical properties of the polyHIPEs and increased the period an emulsion could be stored before polymerisation. Our results demonstrate the potential to use gelatin for the fabrication of surfactant-free polyHIPEs with macroporous structures, with potential applications in tissue engineering, environmental and agricultural industries.
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Despite advances in microsurgical techniques, treatment options to restore prior function following peripheral nerve injury remain unavailable, and autologous nerve grafting remains the therapy of choice. Recent experimental work has focused on the development of artificial constructs incorporating smart biomaterials and stem cells, aspiring to match/improve the outcomes of nerve autografting. Chemically stimulated human adipose-derived stem cells (dhASC) can improve nerve regeneration outcomes; however, these properties are lost when chemical stimulation is withdrawn, and survival rate upon transplantation is low. It is hypothesized that interactions with synthetic hydrogel matrices could maintain and improve neurotrophic characteristics of dhASC. dhASC are cultured on PeptiGel-Alpha 1 and PeptiGel-Alpha 2 self-assembling peptide hydrogels, showing comparable viability to collagen I control gels. Culturing dhASC on Alpha 1 and Alpha 2 substrates allow the maintenance of neurotrophic features, such as the expression of growth factors and neuroglial markers. Both Alpha 1 and Alpha 2 substrates are suitable for the culture of peripheral sensory neurons, permitting sprouting of neuronal extensions without the need of biological extracellular matrices, and preserving neuronal function. PeptiGel substrates loaded with hdASC are proposed as promising candidates for the development of tissue engineering therapies for the repair of peripheral nerve injuries.
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Tejido Adiposo/citología , Hidrogeles/farmacología , Regeneración Nerviosa/efectos de los fármacos , Péptidos/farmacología , Células Madre/citología , Potenciales de Acción/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Nanofibras/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas Sprague-Dawley , Células Madre/efectos de los fármacos , Células Madre/ultraestructuraRESUMEN
Self-assembling peptide hydrogels (SAPHs) represent emerging cell cultures systems in several biomedical applications. The advantages of SAPHs are mainly ascribed to the absence of toxic chemical cross-linkers, the presence of ECM-like fibrillar structures and the possibility to produce hydrogels with a large range of different mechanical properties. We will present a two-component peptide system with tuneable mechanical properties, consisting of a small pentapeptide (SFFSF-NH2 , SA5N) that acts as a gelator and a larger 21-mer peptide (SFFSF-GVPGVGVPGVG-SFFSF, SA21) designed as a physical cross-linker. The hydrogels formed by different mixtures of the two peptides are made up mainly of antiparallel ß-sheet nanofibers entangling in an intricate network. The effect of the addition of SA21 on the morphology of the hydrogels was investigated by atomic force microscopy and transmission electron microscopy and correlated to the mechanical properties of the hydrogel. Finally, the biocompatibility of the hydrogels using 2D cell cultures was tested. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 535-544, 2019.
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Hidrogeles , Ensayo de Materiales , Nanofibras/química , Oligopéptidos , Animales , Hidrogeles/química , Hidrogeles/farmacología , Ratones , Células 3T3 NIH , Nanofibras/ultraestructura , Oligopéptidos/química , Oligopéptidos/farmacologíaRESUMEN
The molecular signalling pathways mediating sexual dimorphism have principally been investigated in the gonads, and to a lesser extent in other organs. The brain plays a central role in coordinating sexual function, including the regulation of reproductive development, maturation and sexual behaviour in both sexes. In this study, we investigated sex-related differences in gene expression in the brains of breeding zebrafish (Danio rerio) to establish a greater understanding of the sex-specific physiology of the brain in lower vertebrates. The brain transcriptomic profiles of males and females were interrogated to identify the genes showing sexually dimorphic gene expression. 42 genes were differentially expressed between the sexes, from which 18 genes were over-expressed in males and 24 genes were over-expressed in females. In males, these included deiodinase, iodothyronine, type II and ribosomal protein S8, and in females, superoxide dismutase [Cu-Zn], sprouty-4, frizzled 10 and testis enhanced gene transcript. Estrogen responsive elements were found in the regulatory regions for 3 genes over-expressed in males and 7 genes over-expressed in females. We have demonstrated the existence of dimorphic patterns of gene expression in the brain of a sexually mature, non-mammalian, vertebrate model, with implications for studies into reproduction and chemical disruption of brain function.
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Encéfalo/metabolismo , Expresión Génica , Caracteres Sexuales , Pez Cebra/genética , Animales , Mapeo Cromosómico , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Conducta Sexual Animal/fisiologíaRESUMEN
Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four ß-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution-pronase-was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed.
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Hidrogeles/química , Péptidos/química , ARN/aislamiento & purificación , Ingeniería de Tejidos , Materiales Biocompatibles/química , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Humanos , Hidrogeles/farmacología , Nanofibras/química , Conformación Proteica en Lámina beta , ARN/químicaRESUMEN
Environmental oestrogens are widespread in the aquatic environment and cause alterations in sexual development and function in vertebrates. The molecular pathways underpinning these effects, however, remain poorly understood. In this study, we aimed at generating a mechanistic understanding of the disruptive effects of exposure to environmentally relevant concentrations of 17 alpha-ethinyloestradiol (EE(2)) on reproduction in zebrafish, by anchoring the transcriptomic alterations induced with the physiological consequences of exposure. Breeding colonies of zebrafish were exposed for a 21-day period to three concentrations of EE(2) (0.05, 0.5 and 5 ng/L) and the gonadal transcriptomic alterations induced (determined using a 17,000 oligonucleotide microarray) were analysed together with physiological effects seen on reproductive output of both males and females. Exposure to 5 ng EE(2)/L resulted in reproductive impairment characterised by a decrease in egg production, alterations in sperm quality and reduced fertilisation success. The effects seen were associated with altered expression of 114 and 131 genes in the gonads of males and females, respectively. The biological processes most affected by the exposure were protein metabolism in males and mitochondria organisation and biogenesis in females. Genes involved in the regulation of cell cycle progression, the ubiquitin system and glutathione peroxidase were affected by the EE(2) exposure and associated with the changes observed in gamete quality in both genders. In summary, we demonstrated that EE(2) exposure compromised the reproductive health of breeding zebrafish at environmentally relevant concentrations. The molecular mechanisms mediating some of these effects were identified and included those impacting processes central to gametogenesis in both males and females.
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Etinilestradiol/toxicidad , Expresión Génica/efectos de los fármacos , Gónadas/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/fisiología , Animales , Análisis por Conglomerados , Exposición a Riesgos Ambientales , Femenino , Fertilización/efectos de los fármacos , Genes/efectos de los fármacos , Genes/fisiología , Masculino , Oogénesis/efectos de los fármacos , Fenotipo , Análisis de Componente Principal , Espermatozoides/efectos de los fármacos , Agua/análisis , Pez Cebra/genéticaRESUMEN
Huntington's disease is caused by a single mutation on the HTT gene which produces an expansion in the number of glutamine repeats present in the huntingtin protein. This mutation results in an array of motor, cognitive and behavioural problems mediated by a progressive loss of striatal neurons and brain atrophy. The identification of behavioural phenotypes in mouse models of the disease provides a baseline of efficacy for therapeutic interventions. The R6/1 mouse line carries â¼115 CAG repeats and has an aggressive form of the disease. The aim of the present study was to undertake longitudinal behavioural characterisation of this mouse line in order to quantify the time course and severity of disease progression. In the present study, when compared to wildtype littermates, male R6/1 heterozygous mice demonstrated a progressive weight loss from 3 months of age. The R6/1 carriers also demonstrated a relatively stable motor coordination deficit on the rotarod, and progressive impairments on each aspect of the balance beam test: latency to orientate and traverse the beam; number of fore- and hind-limb footslips. The R6/1 carriers were less reactive to acoustic startle stimuli and displayed less inhibition to prepulse warning stimuli than their wildtype littermates. In the Morris water maze, the R6/1 carriers demonstrated a deficit on latency to find the platform and path length measures, which was apparent by 3 months of age but not further progressive. They also demonstrated fewer entries into the target zone during probe trials. The data from the present study demonstrate that the R6/1 mouse has a profound behavioural phenotype that includes motor and cognitive deficits, but that not all of these deficits were robustly progressive in nature.