**4. Materials and Methods**

#### *4.1. Decellularization of Lung Tissue Samples*

Tissue samples for healthy controls originated from healthy human donor lungs, unusable for transplantation, or from an una ffected area in resection material (Table 1). The diagnosis of IPF was confirmed by clinicians with histological examination of explanted lungs that had reached end stage of disease, consistent with ERS and ATS criteria. Sample HL 4 showed no signs of pathological changes in the parenchymal tissue used for sca ffold extraction.


**Table 1.** Patient and donor tissue information.

\* No COPD diagnosis. Tissue obtained from non-affected area.

Lung tissue was dissected for scaffold isolation within the first 24 h of surgical removal of explanted lungs. Adjacent to the pleura, cubic blocks (1 cm3) of parenchymal tissue were dissected from peripheral regions of the lung (Figure 1A). Tissue blocks were immediately frozen in 2-methylbutane chilled with liquid nitrogen. After storage in −80 ◦C lung tissue was cryosectioned into 350 μm tissue slices with a HM-560 cryostat (Microm, Heidelberg, Germany). Antifreeze cryoprotective solution (30% v/v glycerol and 30% v/v ethylene glycol in 0.1 M sodium phosphate buffer) was used to maintain the integrity of the tissue at sectioning. Tissue slices were thawed on chilled D-PBS (Dublecco's phosphated buffer solution) (Invitrogen, Waltham, MA, USA) and treated for decellularization. Samples for histology were fixated and prepared as described below.

Tissue slices were decellularized according to Rosmark et al. [13]. In short, tissue was incubated with mild agitation in decellularization solution (8 mM CHAPS, 1 M NaCl, 25 mM EDTA in D-PBS) (1 mL/slice), with the solution replaced five times during the first 4 h of incubation. Tissue was stored overnight at +4 ◦C in D-PBS. The following day, tissue slices were rinsed with benzonase working buffer (20 mM Tris-HCl, 2 mM Mg<sup>2</sup>+, 20 mM NaCl) prior to incubation with benzonase nuclease (Sigma-Aldrich, Saint Louis, MO, USA, cat. no. E1014) (90 U/mL, 1 mL/slice, 30 min at 37 ◦C). The decellularized tissue (scaffold) was rinsed and stored in D-PBS supplemented with amphotericin B (2.5 μg/mL), penicillin-streptomycin (1%) and gentamicin (50 μg/mL) at +4 ◦C. Randomly selected scaffolds from each tissue cube were used, which further introduced biological variability between the technical replicates.
