One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.
Electrostatic spinning was investigated as an alternative to electrospinning to establish the potential of the technique for the production of a range of microfibrous polyurethane scaffolds with a variety of structures and properties related to the fabrication conditions. Tecoflex® SG-80A polyurethane was spun, systematically altering the spinning parameters, and the resulting scaffolds were characterised using scanning electron microscopy. Inter-fibre separation was significantly affected by flow rate, spray distance and grid and mandrel voltages; fibre diameter by flow rate and mandrel voltage; void fraction by flow rate; fibre orientation by traverse speed and mandrel speed; and thickness by flow rate. Thus, scaffold (three-dimensional) architecture may be controlled through manipulation of the electric fields and the fibre deposition (spinning parameters of flow rate and grid and mandrel voltages); and by spray movement and direction (spinning parameters of relative spray height, spray distance, traverse speed and mandrel speed). There were significant differences between the internal and external scaffold surfaces, due in part to the manner in which the surface of the mandrels was prepared. We conclude that the process may be used to produce a range of polyurethane scaffolds for use in many tissue engineering applications
Electrostatic spinning is receiving increasing attention in the field of tissue engineering, due to its ability to produce 3-dimensional, multidirectional, microfibrous scaffolds. These structures are capable of supporting a wide range of cell growth; however, there is little knowledge relating material substrates with specific cellular interactions and responses. The aim of this research was to investigate if electrostatically spun scaffolds, with controlled topographical features, would affect the adhesion mechanisms of contacting cells. A range of electrostatically spun Tecoflex SG-80A polyurethane scaffolds was characterized in terms of inter-fibre separation, fibre diameter, surface roughness, void fraction and fibre orientation. Human embryonic lung fibroblasts and human vein endothelial cells were cultured on these scaffolds for 7, 14, 28 days, and analysed for their expression of extracellular matrix and adhesion molecules using image analysis and laser scanning confocal microscopy. There were significant differences in adhesion mechanisms between scaffolds, cell types and culture periods. Fibroblast-scaffolds were stimulated and oriented to a greater degree, and at earlier cultures, by the controlled topographical features than the endothelial cells. These conclusions confirm that cellular behaviour can be influenced by the induced scaffold topography at both molecular and cellular levels, with implications for optimum application specific tissue engineering constructs.
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