Abstract
The interactions of post-culture treatments reagents used for fixing, lysing and cell quantification on poly(lactide-co-glycolide) (PLGA) flat sheet membrane scaffolds are presented. Lysing with Alkaline buffer solution/Triton X-100/MilliQ water (ATM) and fixing with 10% Neutral Buffered Formalin (10% NBF) had no affect on membrane structure while fixing with 95% ethanol caused smoothing of the surface, shrinkage and a reduction in surface area of 55, 48 and 33, for 100:0, 75:25 and 50:50 (PLA:PGA), respectively. PicoGreen assay was selected for cell (560pZIPv.neo) quantification since the background noise would not affect readings for cell numbers over 3,000 cells/cm2, while the background reading was too high for MTT and Methylene Blue (MB). MB at 0.5% (w/v) was, however, deemed suitable for visualising cell morphology on the membranes. Furthermore ATM buffer was suitable for the PicoGreen assay, which allows the same samples to be used for quantification of alkaline phosphatase activity.
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Acknowledgements
We would like to acknowledge the financial support of the Engineering and Physical Sciences Research Council, the Biotechnology and Biological Sciences Research Council, the technical support of Mr Fernando Acosta (HPLC) and Ms Ting Ting Yue, and Ursula Potter and Hugh Perrott in the Centre for Electron Optical Studies, University of Bath.
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Ellis, M.J., Forsey, R. & Chaudhuri, J.B. Post-culture treatment protocols for PLGA membrane scaffolds. Biotechnol Lett 32, 215–222 (2010). https://doi.org/10.1007/s10529-009-0136-9
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DOI: https://doi.org/10.1007/s10529-009-0136-9