237 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment Preparation of lung ECM-derived hydrogels Decellularized lung ECM powders were pooled (n = 7 each for both control and IPF lung ECM samples) with equal amounts of powder dry weight per donor in order to minimize the patient-to-patient variation. Using this powder, pepsin digestion was performed using 2 mL of 2 mg/mL pepsin (Sigma-Aldrich) solution in 0.01 M HCl for 40 mg dry ECM powder in a 7.5 mL glass vial as previously described [34]. The digestion was performed for 72 hours with constant stirring at room temperature. After the incubation, the pH was brought back to physiological conditions (pH = 7.4) using 0.1 M NaOH (Sigma-Aldrich) solution before being mixed with 10X PBS (Gibco) solution to supplement the solution (will be referred to as pre-gel from here on) with physiological salts and ions. Resulting control and IPF pre-gels were cast into the wells of a 48-well plate or in a 1.5 mL Eppendorf tube and incubated for 1-2 hours to allow gelation and after the incubation, the hydrogel formation was checked. Primary lung fibroblast isolation and cell culture Control and IPF primary human lung fibroblasts were isolated from lung tissue of patients undergoing surgery for tumor resection or lung transplantation at the University Medical Center Groningen (UMCG) as previously described [35]. Control tissue was obtained from macroscopically normal looking tissue from tumor excision surgery, as far as possible from the tumor. IPF primary human lung fibroblasts were isolated from the peripheral lung tissue of explanted lung tissue of patients who underwent lung transplantation as previously described. This study protocol was consistent with the Research Code of the University Medical Center Groningen ((https://www.umcg.nl/documents/770534/2183586/umcg-research-code-2018en.pdf/9680a460-3feb-543d-7d58-bc9d4f7277de?t=1614951313016, last accessed 12/01/2023) and the national ethical and professional guidelines (“Code of conduct for Health Research (only in Dutch):Gedragscode-Gezondheidsonderzoek-2022. pdf (https://www.coreon.org/gedragscode-gezondheidsonderzoek, last accessed 12/01/2023). Lung tissues used in this study were derived from leftover lung material after lung surgery from archival materials that are exempt from consent in compliance with applicable laws and regulations (Dutch laws: Medical Treatment Agreement Act (WGBO) art 458 / GDPR art 9/ UAVG art 24). This material was not subject to the Medical Research Human Subjects Act in the Netherlands, and, therefore, an ethics waiver was provided by the Medical Ethical Committee of the University Medical Center Groningen. The characteristics of the patients are presented in Table 1. The fibroblast cultures were maintained in complete growth media (DMEM Low Glucose (Gibco), supplemented with 10% FBS (Sigma-Aldrich), 1% Penicillin-Streptomycin (Gibco) and 1% GlutaMAX (Gibco)) and only used when tested negative against mycoplasma infection using a PCR screening. Fibroblasts were used at passage 5. 9
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