*4.5. Mechanical Testing*

Native tissue slices and decellularized sca ffolds of equal size from healthy and IPF donor tissues (*n* = 4 biological replicates per group) were mounted in organ baths (emkaBATH4, emka Technologies, Paris, France) in D-PBS for mechanical testing. Tissue samples were mounted to triangular hooks with silk suture and original tissue length was measured (L0). Tissues/sca ffolds were then pre-loaded in tension with 350 mg by vertical elongation. After relaxation, the samples were loaded with a displacement corresponding to a strain of 5%, 10%, and 15% at a rate of 0.1 mm/s, and allowed to relax for 20 min between each sequential load step (sequential stress-relaxation). Finally, samples were tested in tension at a rate of 0.1 mm/s until they ruptured or reached a maximal additional 10 mm. The software iox 2.10.0.40 datanalystv2.6.1.18 was used for data acquisition. Tissue sti ffness (N/m) was calculated from the linear region of the force displacement curve during the final tensile test, *k* = *F* δ , where F is the load [mN] and δ is the displacement. Ultimate force was the maximum load [mN] at failure i.e., physical breakage of the tissue.

#### *4.6. Repopulation of Sca*ff*olds with Primary Lung Fibroblasts Labeled with Heavy Arginine and Lysine*

Human primary parenchymal lung fibroblasts were isolated from one healthy donor control lung as previously described [50]. Fibroblasts were expanded on regular culture flasks (Sarstedt, Nümbrecht, Germany, cat.no. 83.3910.002) in DMEM supplemented with amphotericin B (2.5 μg/mL), penicillin-streptomycin (1%), gentamicin (50 μg/mL), glutamine (1%), and 10% fetal clone serum (FCIII, Thermo Scientific) at 37 ◦C, 10% CO2. Cells in passage 7 were trypsinized and resuspended in complete SILAC DMEM Flex Media (Life Technologies, Carlsbad, CA, USA, cat.no. A2493901) supplemented with 10% dialyzed serum (Gibco, A3382001), glucose (4500 μg/mL), amphotericin B (2.5 μg/mL), penicillin-streptomycin (1%), gentamicin (50 μg/mL), 1% Glutamax along with "heavy" 13 C6 labeled l-Arginine-HCl (Thermo Fisher Scientific, 88210) and "heavy" 13 C6 15 N2-labeled l-lysine-2HCl (Thermo Fischer Scientific, 88209), as needed for optimal cell culture conditions. Fibroblasts were pre-cultured for 5 days on regular culture flasks in complete SILAC DMEM Media and sca ffolds were pre-conditioned for 1 h with SILAC DMEM Media prior to re-population of sca ffolds. In 24-well suspension culture plates (Sarstedt, cat.no. 83.3922.500) fibroblasts were seeded on sca ffolds with mild agitation for 24 h at 10% CO2, 37 ◦C. Culture media was analyzed for cellular content for the examination of cellular attachment to sca ffolds. The cell seeded sca ffolds were then mounted on sca ffold holders (8 mm inner diameter), composed of polyoxymethylene and incubated for up to 9 days, based on previous data [13]. Culture medium was changed after 24 h, 3 days, and 6 days of incubation. Schematics of the experimental layout is provided in Figure 1A. Repopulated sca ffolds were analyzed for cellular viability after 1, 3, and 9 days of incubation (biological replicates per group *n* = 4 with two technical replicates). Cell viability was analyzed with WST-1 (Roche, Basel, Switzerland) according to manufacturer's instructions. In brief, sca ffolds were incubated at 37 ◦C at 10% CO2 with WST-1 solution (diluted in 1:10 in cell culture medium). Color development in cell medium, corresponding to cellular metabolism, was measured at 450 nm.
