187 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition 10 mM Tris pH8 (Sigma-Aldrich) solution each for 24 h at 4°C with constant shaking, except for the DNAse treatments, which were at 37°C with shaking. The volume ratio of tissue homogenate to decellularization/washing solution was always 1:10. Between treatments, the homogenate was washed three times with Milli-Q® water, with centrifugation at 3,000 x g between washes. After two cycles of decellularization, the tissue homogenate was sterilised by adding 0.18 % peracetic acid and 4.8 % ethanol, and left shaking at 4 °C for 24 h. After tissue sterilization the resultant decellularized ECM was washed three times with sterile Dulbecco’s phosphate-buffered saline (DPBS) and stored in sterile DPBS containing 1 % penicillin-streptomycin (Gibco Invitrogen, Carlsbad, CA, USA) at 4 °C (Figure 1A). Hydrogel preparation The decellularized lung ECM was snap-frozen in liquid nitrogen and lyophilized with a FreeZone Plus lyophilizer (Labconco, Kansas City, USA), before being ground into a powder with an A11 Analytical mill (IKA, Staufen, Germany). For solubilization, 20 mg/mL of ECM powder was digested with 2 mg/mL porcine pepsin (Sigma-Aldrich) in 0.01 M HCl under constant agitation at RT for 48 h [28]. Digestion was stopped by neutralising the pH with 0.1 M NaOH and the solution was brought to 1X PBS with one-tenth volume 10X PBS to generate the lung ECM pre-gel solution which was stored at 4°C indefinitely. A ruthenium Visible Light Photo initiator (400-450nm) kit (Advanced BioMatrix, San Diego, California, US) containing pentamethyl cyclopentadienyl bis(triphenylphosphine) ruthenium(II) chloride (CAS Number: 92361-49-4, hereafter referred as ruthenium) and sodium persulfate (CAS: 7775-27-1) was used to crosslink the LdECM hydrogels (Figure 1B). 20 µL of each ruthenium. (37.4 mg/mL) and sodium persulfate (119 mg/mL) solutions were added per 1 mL of ECM hydrogel. The control gel received the same volume of sterile ddH2O water. In the dark, both the rutheniumcontaining gel and the control gel without ruthenium were pipetted (200 µL) into a 48-well plate and incubated at 37°C for 1 h. After the hydrogels had settled, crosslinking of the ECM by photoinitiated ruthenium was triggered by exposing the samples to UV/Visible light from 4.5 cm distance using 2 x 9W UV lamps (405 nm) (20 mW/cm2 light intensity) for 5 min to generate Ru-LdECM hydrogel (Figure 1B). Finally, the gels were immersed in 400 µL of Dulbecco’s Modified Eagle Medium (DMEM) Low Glucose growth media (Lonza) supplemented with 10% foetal bovine serum (FBS), 1% penicillin-streptomycin and 1% GlutaMAX (Gibco) (hereafter referred as complete growth medium), and were washed 3X with media before cell seeding in order to remove excess (both reacted and unreacted) ruthenium and sodium persulfate. 8
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