190 Chapter 8 where j varies from 0 to 100 s, Ej is the experimentally measured value at time j, E(tj) is the fit value at time j calculated with Eq. 1, and σj is the standard error that the LLCT makes because of inherent errors in position, time, and load measurements. Histological characterisation of ECM hydrogel fibre structure LdECM and Ru-LdECM hydrogels were prepared and washed as described above and fixed with 2% paraformaldehyde in PBS (PFA; Sigma-Aldrich) at RT for 20 min. The gels were then embedded in 1% Ultrapure agarose (Invitrogen, Waltham, MA, USA) before using a graded alcohol series to dehydrate followed by paraffin embedding. Sections (4 µm) were deparaffinised, and stained with 0.1 % Picrosirius Red (PSR) (Sigma-Aldrich) in 1.3% aqueous solution of picric acid to visualize collagens and their network. Slides were mounted with Neo-Mount® Mounting Medium (Merck, Darmstadt, Germany). Cell culture MRC-5 foetal lung fibroblasts (n=5) were cultured in complete growth medium. The MRC-5s were washed with Hank’s Balanced Salt Solution (HBSS; Gibco), harvested using 0.25% Trypsin-EDTA (Gibco) and centrifuged at 500 x g for 5 minutes. Cells were resuspended in 1 mL complete growth media and counted with a NucleoCounter NC-200™ (Chemometec, Allerod, Denmark). Fibroblasts were seeded on top of preprepared and washed LdECM and Ru-LdECM hydrogels in complete growth media with the seeding density 10.000 cells/gel. The cells were cultured on the gels for 1 or 7 days. Gels used for live/dead staining were stained with the live/dead stain and were subsequently harvested. Gels intended for immunofluorescent imaging were fixed in 2% PFA in PBS for 30 minutes. After fixation, hydrogels were washed three times with PBS and stored in PBS containing 1% penicillin-streptomycin at 4°C until analyses. Live/dead staining Cell viability of the MRC-5 cells cultured on LdECM and Ru-LdECM hydrogels was assessed after 1 and 7 days using Calcein AM (Thermo Scientific, Breda, the Netherlands) to stain live cells and propidium iodide (PI; Sigma-Aldrich) for staining dead cells, as previously described [31]. The hydrogels were first washed with HBSS and then incubated with serum free media containing 5 µM Calcein AM and 2 µM PI, for 1 hour at 37°C. After incubation, fluorescent images were captured using a EVOS Cell Imaging System (Thermo Scientific) with GFP (509 nm) and Texas Red (615 nm) channels.
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