Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro.

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.

Vascular prostheses: performance related to cell-shear responses.

BACKGROUND: This work concerned the endothelialization of vascular prostheses and subsequent improvement of functionality with respect to tissue engineering. The aim of the study was to investigate the initial, pre-shear stress cellular behavior with respect to three vascular biomaterials to explain subsequent cellular responses to physiological shear stresses. MATERIALS AND METHODS: Expanded polytetrafluoroethylene (ePTFE), polyethyleneterephthalate (polyester; Dacron; PET), and electrostatically spun polyurethane (PU) (all pre-impregnated with collagen I/III) were cell-seeded with L929 immortalized murine fibroblasts or human umbilical vein endothelial cells (HUVECs). Cytoskeletal involvement, cell height profiles, and immunohistochemistry were examined after 7 d static culture. RESULTS: All three vascular biomaterials demonstrated different structures. Cell behavior varied both between the materials and the two cell types: cytoskeletal involvement was greater for the HUVECs and the more fibrous surfaces; height profiles were greater for the L929 and PET, and lowest on PU. Immunohistochemistry of HUVEC samples also showed differences: PU revealed the greatest expression of intercellular adhesion molecule-1 and E-selectin (PET and ePTFE the lowest, respectively); ePTFE produced the greatest for vascular cell adhesion molecule-1 (PET the lowest). CONCLUSIONS: Material substrate influenced the cellular response. Cells demonstrating firm adhesion increased their cytoskeletal processes and expression of cell-substratum and inter-cellular adhesion markers, which may explain their ability to adapt more readily to shear stress. The fibrous PU structure appeared to be most suited to further shear stress exposure. This study demonstrated the potential of the underlying vascular material to affect the long-term cellular functionality of the prosthesis.

Effects of sterilisation method on surface topography and in-vitro cell behaviour of electrostatically spun scaffolds.

Electrostatic spinning is a potentially significant technique for scaffold production within the field of tissue engineering; however, the effect of sterilisation upon these structures is not known. This research investigated the extent of any topographical alteration to electrostatically spun scaffolds post-production through sterilisation, and examined any subsequent effect on contacting cells. Scaffolds made from Tecoflex SG-80A polyurethane were sterilised using ethylene oxide and UV-ozone. Scaffold topography was characterized in terms of inter-fibre separation (ifs), fibre diameter (f.dia) and surface roughness. Cell culture was performed over 7 days with both mouse L929 and human embryonic lung fibroblasts, the results of which were assessed using SEM, image analysis and confocal microscopy. Sterilisation by UV-ozone and ethylene oxide decreased ifs and increased f.dia; surface roughness was decreased by UV-ozone but increased by ethylene oxide. Possible mechanisms to explain these observations are discussed, namely photo-oxidative degradation in the case of UV-ozone and process-induced changes in surface roughness. UV-ozone sterilised scaffolds showed greater cell coverage than those treated with ethylene oxide, but lower coverage than all the controls. Changes in cell attachment and morphology were thought to be due to the changes in topography brought about by the sterilisation process. We conclude that surface modification by sterilisation could prove to be a useful tool at the final stage of scaffold production to enhance cell contact, phenotype or function.