This work was funded by the Scientific and Technological Research Council of Turkey (T&#220;B&#304;TAK) (Grant No. 118C238). The entire responsibility of the publication/paper belongs to the owner of the publication. The financial support received from TU&#776;BI&#775;TAK does not mean that the content of the publication is approved in a scientific sense by TU&#776;BI&#775;TAK. The authors gratefully acknowledge the use of services and facilities of Koc&#807; University Research Center for Translational Medicine (KUTTAM). Figure 1 and Figure 2a were created using Biorender.com.

Fresh native lungs from young (1-2 years old) bovine donors were obtained from a local slaughterhouse and transported in a sealed plastic container on ice to the laboratory. Animal sacrifice is performed for general meat consumption (lungs discarded as waste) and is not related to or due to the study. We confirm that the slaughterhouse complies with the national laws and regulations of animal sacrifice. Furthermore, we confirm that we only used waste material and the research project did not have an effect on the number of animals sacrificed.
1. Harvest of organs and tissue preparation
<ol>
	<li>Store freshly obtained bovine lung tissues at -80 &#176;C until the experiment.</li>
	<li>On the day of the experiment, wait for frozen lung tissues to thaw at room temperature.</li>
	<li>Dissect the tissues with sterile scalpels and scissors to remove trachea and cartilaginous airways, and further mince into small pieces (5 mm3).</li>
	<li>Wash thoroughly with ultrapure water containing 2% penicillin/streptomycin (P/S) at least 3x.</li>
</ol>
2. Decellularization of tissues
NOTE: Native bovine lung tissues were decellularized using two distinct protocols.
<ol>
	<li>Freeze-thaw method
	<ol>
		<li>Prepare tissue samples according to step 1. Immerse minced tissues in 2% iodine solution in sterile distilled water (dH2O) for 1 min. Perform two consecutive washes in sterile dH2O.</li>
		<li>Transfer minced tissue pieces into a 50 mL tube up to the 15 mL level and implement five manual freeze-thaw cycles using a portable liquid nitrogen container. Fill tubes to the top with sterile dH2O and freeze tubes for 2 min in liquid nitrogen. After 2 min, immediately transfer them to a 37 &#176;C water bath for 10 min. This constitutes 1 cycle of freeze-thaw.</li>
		<li>Incubate the samples with 30 mL of DNase solution (10 U/mL) in 10 mM MgCl2 buffer (pH 7.5) for 1 h at 37 &#176;C under constant shaking at 100 rpm.</li>
		<li>Continue with an extensive wash with sterile dH2O for 3 days under constant shaking at 100 rpm, with solution replenishment every 24 h.</li>
		<li>Perform lyophilization and cryomilling as follows20: Freeze the wet dECM samples at -80 &#176;C for 24 h. Transfer frozen samples into a lyophilizer and operate in drying mode under vacuum for 3 to 4 days until fully dried. Upon completion of freeze-drying, perform milling of the samples into a fine powder form using a grinding apparatus and dry ice. 
		NOTE: This fine powder state is deemed necessary due to its enhanced solubility properties during subsequent stages of the digestion process.</li>
	</ol>
	</li>
	<li>Triton-X-100 method
	<ol>
		<li>Prepare tissue samples according to step 1. Treat lung tissues with 1% Triton-X-100 for 3 days under gentle rotation at 4 &#176;C. Exchange solution every 24 h.</li>
		<li>Incubate the samples with DNase solution (10U/mL) in 10 mM MgCl2&#160;buffer (pH 7.5) for 1 h at 37 &#176;C under constant shake.</li>
		<li>Continue with an extensive wash with sterile dH2O for 3 days under gentle rotation, with solution replenishment every 24 h.</li>
		<li>Perform lyophilization followed by cryo-milling as described in step 2.1.</li>
	</ol>
	</li>
</ol>
3. Pepsin digestion
<ol>
	<li>Digest powdered dECM samples at a concentration of 15 mg/mL (w/v) in pepsin solution (1 mg/mL pepsin in 0.01 M HCl, pH 2). Carry out the sample digestion process at room temperature under constant stirring for 48 h and maintain the pH of the solution frequently for effective digestion.</li>
	<li>Transfer the digests into a tube and centrifuge at 5000 x g for 10 min.</li>
	<li>Collect the supernatant and neutralize and buffer them to physiological conditions (pH 7, 1x phosphate buffer saline (PBS)) through the addition of 5M NaOH and 10x PBS. 
	NOTE: It is suggested to use concentrated alkaline solutions for pH adjustment of the acidic digest, as this approach avoids undesired volume expansion that could&#160;detrimentally impact the gelation in the subsequent steps.</li>
	<li>Store the pre-gel digests at &#8722;20 &#176;C for further studies.</li>
</ol>
4. Histological staining
<ol>
	<li>Fix a small portion (5 mm3) of decellularized tissue sample in 1 mL of 3.7% formaldehyde solution at 4 &#176;C overnight.</li>
	<li>Incubate tissues in tubes containing 1 ml of 30% sucrose solution in PBS for 12 h at 4 &#176;C on a steady rock.</li>
	<li>To embed tissues in optimal cutting temperature compound (OCT), pour 1 mL of OCT in a cryomold, place tissue in the middle of the cryomold, then pour 2 mL of OCT on top of the tissue.</li>
	<li>Place cryomolds containing tissues on dry ice and snap-freeze using liquid nitrogen. Samples are ready when OCT turns all white, which usually takes 3 min. Store OCT-embedded tissues at -20 &#176;C until use.</li>
	<li>Obtain 10 &#181;m sections in the cryostat at -25 &#176;C on a poly-L-lysine-coated glass slide and transfer the slide to room temperature to allow the tissue section to melt on the slide. Store the slides at -20 &#176;C until use.</li>
	<li>In order to confirm the absence of nuclei after decellularization, perform a Hematoxylin and Eosin (H&#38;E) staining as described below.
	<ol>
		<li>Incubate slides at room temperature for 10 min. Immerse slides in PBS and stain them with ready-to-use 50 mL of hematoxylin solution in a staining jar for 3 min, followed by a 3 min wash with tap water.</li>
		<li>Immerse the slides in 95% ethanol and stain with 50 mL of 0.5% alcoholic Eosin solution in a staining jar for 45 s.</li>
	</ol>
	</li>
	<li>Perform Sirius red staining to show the retaining of collagen content after decellularization.
	<ol>
		<li>Hydrate the slides with PBS and immerse them in 50 mL of 0.1% Sirius red in saturated aqueous solution of picric acid in a staining jar for 1 h.</li>
		<li>Rinse slides in 0.5% acetic acid solution for 5 s and dehydrate all slides by soaking them in 70%, 95%, and 100% ethanol sequentially for 1 min each.</li>
	</ol>
	</li>
	<li>To analyze sGAG content in tissue samples, perform Alcian blue staining as described below.
	<ol>
		<li>Immerse the slides in 50 mL of 1% Alcian blue in 3% acetic acid solution (pH 2.5) in a staining jar for 30 min. Wash the slides with running tap water for 2 min.</li>
		<li>Dehydrate all slides by soaking them in 70%, 95%, and 100% ethanol sequentially for 1 min each.&#160;</li>
	</ol>
	</li>
	<li>Add 0.1 mL of mounting medium on top of the samples, and visualize using light microscopy using 10x magnification.</li>
</ol>
5. Mechanical characterization
<ol>
	<li>Implement oscillatory rheology measurements using a rheometer with parallel plate geometry.</li>
	<li>Pour 250 &#181;L of pre-gel solution kept on ice onto a pre-cooled (4 &#176;C) lower plate to avoid rapid gelation.</li>
	<li>Lower the 20 mm parallel plate until the pre-gel solution forms a disk with a 1 mm gap width between the two plates.</li>
	<li>Start the measurement immediately. Measure storage and loss moduli over time with a fixed frequency and strain while heating the lower plate to 37 &#176;C for 30 min to observe gelation kinetics. Use a fixed frequency of 0.5 Hz and 0.1% strain.</li>
	<li>Perform a creep-recovery test after the storage modulus value stops increasing and reaches a plateau.</li>
	<li>Apply 1 Pa shear stress to the hydrogel for 15 min, measure the strain, unload the sample from the stress, and record the change in strain value for 15 min.</li>
	<li>Draw a strain versus time graph to show the stress-relaxing behavior. Repeat measurements for three different dECM digests as replicates.</li>
</ol>

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

<ol>
	<li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+micropattern+arrays+fabricated+by+lung-derived+dECM+hydrogels+for+chemotherapeutic+drug+screening.">Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.</a> Mater Today Bio. 15, 100274 (2022).</li><li>Ulldemolins, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+extracellular+matrix+hydrogels-derived+vesicles+contribute+to+epithelial+lung+repair.">Lung extracellular matrix hydrogels-derived vesicles contribute to epithelial lung repair.</a> Polymers. 14 (22), 4907 (2022).</li><li>Biehl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+standardized+multi-tissue+decellularization+protocol+for+the+derivation+of+extracellular+matrix+materials.">Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.</a> Biomater Sci. 11 (2), 641-654 (2023).</li><li>Pak, J., Lee, J. H., Park, K. S., Jeong, B. C., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+cartilage+in+human+knee+osteoarthritis+with+autologous+adipose+tissue-derived+stem+cells+and+autologous+extracellular+matrix.">Regeneration of cartilage in human knee osteoarthritis with autologous adipose tissue-derived stem cells and autologous extracellular matrix.</a> Biores Open Access. 5 (1), 192-200 (2016).</li><li>Spang, M. T., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogel+therapies.">Extracellular matrix hydrogel therapies.</a> In vivo applications and development.Acta Biomater. 68, 1-14 (2018).</li><li>Schwartz, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomaterial+screening+approach+reveals+microenvironmental+mechanisms+of+drug+resistance.">A biomaterial screening approach reveals microenvironmental mechanisms of drug resistance.</a> Integr Biol. 9 (12), 912-924 (2017).</li><li>Monteiro, M. V., Gaspar, V. M., Ferreira, L. P., Mano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+3D+in+vitro+tumor+models+for+screening+cell+aggregation+mediated+drug+response.">Hydrogel 3D in vitro tumor models for screening cell aggregation mediated drug response.</a> Biomater Sci. 8 (7), 1855-1864 (2020).</li><li>Nakamura, R., Nakamura, F., Fukunaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+composition+of+the+extracellular+matrix+accumulated+by+mesenchymal+stem+cells+during+in+vitro+expansion.">Changes in the composition of the extracellular matrix accumulated by mesenchymal stem cells during in vitro expansion.</a> Anim Sci J. 85 (6), 706-713 (2014).</li><li>Bual, R. P., Ijima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+extracellular+matrix+component+promotes+maintenance+of+liver-specific+functions+and+larger+aggregates+formation+of+primary+rat+hepatocytes.">Intact extracellular matrix component promotes maintenance of liver-specific functions and larger aggregates formation of primary rat hepatocytes.</a> Regen Ther. 11, 258-268 (2019).</li><li>Jhala, D., Vasita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+extracellular+matrix+mimicking+strategies+for+an+artificial+stem+cell+niche.">A review on extracellular matrix mimicking strategies for an artificial stem cell niche.</a> Poly Rev. 55 (4), 561-595 (2015).</li><li>Jeong, W., Kim, M. K., Kang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+detergent+type+on+the+performance+of+liver+decellularized+extracellular+matrix-based+bio-inks.">Effect of detergent type on the performance of liver decellularized extracellular matrix-based bio-inks.</a> J Tissue Eng. 12, 2041731421997091 (2021).</li><li>Lee, S. J., Lee, J. H., Park, J., Kim, W. D., Park, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+3D+printing+scaffold+with+porcine+skin+decellularized+bio-ink+for+soft+tissue+engineering.">Fabrication of 3D printing scaffold with porcine skin decellularized bio-ink for soft tissue engineering.</a> Materials. 13 (16), 3522 (2020).</li><li>Won, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+dermal+substitute+using+decellularized+dermis+extracellular+matrix+derived+bio-ink.">A potential dermal substitute using decellularized dermis extracellular matrix derived bio-ink.</a> Artif Cells Nanomed Biotechnol. 47 (1), 644-649 (2019).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+decellularized+extracellular+matrix+hydrogel+microspheres+fabricated+using+a+temperature-controlling+microfluidic+system.">Bioactive decellularized extracellular matrix hydrogel microspheres fabricated using a temperature-controlling microfluidic system.</a> ACS Biomater Sci Eng. 8 (4), 1644-1655 (2022).</li><li>Kaushik, N., Kim, S., Suh, Y., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinvasive+extracellular+matrix+remodeling+for+tumor+progression.">Proinvasive extracellular matrix remodeling for tumor progression.</a> Arch Pharm Res. 42 (1), 40-47 (2019).</li><li>Lu, P., Weaver, V. M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+a+dynamic+niche+in+cancer+progression.">The extracellular matrix: a dynamic niche in cancer progression.</a> J Cell Biol. 196 (4), 395-406 (2012).</li><li>Poltavets, V., Kochetkova, M., Pitson, S. M., Samuel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+extracellular+matrix+and+its+molecular+and+cellular+regulators+in+cancer+cell+plasticity.">The role of the extracellular matrix and its molecular and cellular regulators in cancer cell plasticity.</a> Front Oncol. 8, 431 (2018).</li><li>Takeda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+drug+screening+system+using+three-dimensional+cardiac+tissues+containing+multiple+cell+types.">Development of a drug screening system using three-dimensional cardiac tissues containing multiple cell types.</a> Sci Rep. 11 (1), 5654 (2021).</li><li>Jensen, A. R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific,+fibroblast-derived+matrix+as+a+tool+for+studying+breast+cancer+metastasis.">Organ-specific, fibroblast-derived matrix as a tool for studying breast cancer metastasis.</a> Cancers (Basel). 13 (13), 3331 (2021).</li><li>Ku&#351;o&#287;lu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+decellularization+methods+in+bovine+lung+tissue+reveals+distinct+biochemical+composition,+stiffness,+and+viscoelasticity+in+reconstituted+hydrogels.">Different decellularization methods in bovine lung tissue reveals distinct biochemical composition, stiffness, and viscoelasticity in reconstituted hydrogels.</a> ACS Appl Bio Mater. 6 (2), 793-805 (2023).</li><li>Fern&#225;ndez-P&#233;rez, J., Ahearne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+decellularization+methods+on+extracellular+matrix+derived+hydrogels.">The impact of decellularization methods on extracellular matrix derived hydrogels.</a> Sci Rep. 9 (1), 14933 (2019).</li><li>Park, S., Kim, T. H., Kim, S. H., You, S., Jung, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+vascularized+lung+cancer-on-a-chip+with+lung+extracellular+matrix+hydrogels+for+in+vitro+screening.">Three-dimensional vascularized lung cancer-on-a-chip with lung extracellular matrix hydrogels for in vitro screening.</a> Cancers (Basel). 13 (16), 3930 (2021).</li><li>Uriarte, J. J., Uhl, F. E., Rolandsson Enes, S. E., Pouliot, R. A., Weiss, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+bioengineering:+advances+and+challenges+in+lung+decellularization+and+recellularization.">Lung bioengineering: advances and challenges in lung decellularization and recellularization.</a> Curr Opin Organ Transplant. 23 (6), 673-678 (2018).</li><li>Roth, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+freeze-thaw+cycles+for+decellularization+of+tendon+tissue+-+a+pilot+study.">Automated freeze-thaw cycles for decellularization of tendon tissue - a pilot study.</a> BMC Biotechnol. 17 (1), 13 (2017).</li><li>Gilbert, T. W., Freund, J. M., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+DNA+in+biologic+scaffold+materials.">Quantification of DNA in biologic scaffold materials.</a> J Surg Res. 152 (1), 135-139 (2009).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Cox, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+matrix+in+cancer.">The matrix in cancer.</a> Nat Rev Cancer. 21 (4), 217-238 (2021).</li><li>Vining, K. H., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+direct+stem+cell+behaviour+in+development+and+regeneration.">Mechanical forces direct stem cell behaviour in development and regeneration.</a> Nat Rev Mol Cell Biol. 18 (12), 728-742 (2017).</li><li>Chaudhuri, O., Cooper-White, J., Janmey, P. A., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+extracellular+matrix+viscoelasticity+on+cellular+behaviour.">Effects of extracellular matrix viscoelasticity on cellular behaviour.</a> Nature. 584 (7822), 535-546 (2020).</li><li>Chaudhuri, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+with+tunable+stress+relaxation+regulate+stem+cell+fate+and+activity.">Hydrogels with tunable stress relaxation regulate stem cell fate and activity.</a> Nat Mater. 15 (3), 326-334 (2016).</li></ol>

All authors declare no competing financial interests.

Organ-derived hydrogels have become promising models that recapitulate the native tissue ECM and mimic organotypic cellular function. Although decellularized lung ECM has often been used in tissue engineering, a thorough characterization of biomaterial composition and mechanical properties will benefit a better understanding of how cell-ECM interactions can be modulated for modeling biological processes during homeostasis or disease. Particularly, assessment and control of mechanical properties of reconstituted hydrogels...

Conventional monolayer culture conditions do not offer a faithful representation of native tissue microenvironments and lack the ability to provide a three-dimensional (3D) scaffold with instructive ligands that enable cell-matrix and cell-cell interactions1. Extracellular matrix (ECM) composition and mechanical properties are highly tissue-specific, time-dependent, and undergo alterations in pathological conditions. Therefore, there is a need for biomimetic 3D tissue models that allow tunability of such characteristics, modulation of cellular behavior, and achieving desired tissue functionality. Native ECM-derived biomaterials draw much attention in tissue engineering with the ability to directly use tissue-specific ECM1,2,3,4,5. ECM-based carriers have been used in many applications spanning tissue regeneration to disease model development. They are used as injectable or implantable biomaterial scaffolds4,5, in drug screening applications6,7, in the development of materials that induce cell growth8,9,10, as bio-inks11,12,13, in microfluidics14, and in cancer tissue models15,16,17,18,19.
Decellularization of tissues and organs is a popular approach for&#160;generating scaffolds that mimic tissue-specific ECM. The reconstitution of decellularized tissues and organs into hydrogels allows embedding of cells into biomimetic 3D tissue models20. Decellularization techniques mainly focus on eliminating cellular components while retaining the ECM composition. Physical methods such as freeze-thaw cycles or chemical processes such as Triton-X-100 treatment are commonly&#160;applied to decellularize tissues. Furthermore, DNase treatment is preferred for removing residual DNA to minimize the immunological responses upon cell embedding. It is critical to achieve maximal cell removal and minimal ECM impairment to optimize decellularization procedures21. Besides these aspects, the characterization of reconstituted scaffolds&#39; biochemical and mechanical properties, including viscoelasticity and stiffness, is crucial for&#160;improving engineered 3D tissue models derived from native matrices20.
Organ-specific ECM in lung tissue engineering allows mimicking the pulmonary microenvironment to model developmental, homeostatic, or pathological processes in vitro and testing therapeutics in a physio-mimetic setting20,22,23. Previous studies have demonstrated decellularization of&#160;lung tissue from several species, such as rats, porcine, and humans, but these methods have yet to be adapted to less frequently used species such as bovine. A better understanding of the parameters of the decellularization process and how they affect the resulting reconstituted ECM scaffolds regarding biochemical composition and mechanical properties will allow for better tuning of such aspects and pave the way for more reliable tissue models in health and disease. In this study, bovine lung decellularization with two distinct methods, freeze-thaw cycles and Triton-X-100 treatment, is explicitly described and followed by biochemical and mechanical analyses of decellularized lung ECM (dECM) hydrogels. The findings reveal that both methods yield effective decellularization and retention of ECM ligands. Notably, the choice of method significantly alters the resulting stiffness and viscoelasticity of reconstituted hydrogels. Hydrogels derived from the bovine dECM demonstrate notable biochemical analogies with the extracellular matrix of the human lung, and they exhibit reliable thermal gelation characteristics20. As previously described, both methods are suitable for the 3D culture of lung cancer cells, healthy bronchial epithelial cells, and patient-derived lung organoids20.

<table><tbody><tr><td>Absolute ethanol</td><td>ISOLAB</td><td>64-17-5</td><td></td></tr><tr><td>Acetic acid</td><td>ISOLAB</td><td>64-19-7</td><td></td></tr><tr><td>Alcian blue solution</td><td>Sigma-Aldrich</td><td>B8438</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Discovery HR-2 rheometer</td><td>TA Instruments</td><td></td><td></td></tr><tr><td>Entellan mounting medium</td><td>Merck</td><td>107960</td><td></td></tr><tr><td>Eosin solution</td><td>Bright-slide</td><td>2.BS01-105-1000</td><td></td></tr><tr><td>Formaldehyde</td><td>Electron Microscopy Sciences</td><td>50-980-485</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100317</td><td></td></tr><tr><td>Iodine</td><td>Sigma-Aldrich</td><td>3002</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>Mayer&#039;s haematoxylin staining solution</td><td>Merck</td><td>2.BS01-103-1000</td><td></td></tr><tr><td>O.C.T compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Biowest</td><td>L0018-100</td><td></td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma-Aldrich</td><td>P6887</td><td></td></tr><tr><td>Picric acid</td><td>Polysciences</td><td>88-89-1</td><td></td></tr><tr><td>Sirius Red</td><td>Polysciences</td><td>09400-25</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Triton-X-100</td><td>Merck</td><td>112298</td><td></td></tr></tbody></table>

development and characterization of decellularized lung extracellular matrix hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Decellularization 
Decellularization of bovine lung tissue to produce dECM hydrogels that would recapitulate the native lung microenvironment has been achieved by both physical (freeze-thaw) and chemical (Triton-X-100) methods. After dissection, tissue pieces were washed in dH2O-containing antibiotics to remove pathogens that can later affect the sterility of the dECM hydrogels. A total of five cycles alternating between liquid nitrogen to 37 &#176;C water bath was applied for the freeze-...

Watch this Scientific Journal Video about Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels at JoVE.com

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

development-characterization-decellularized-lung-extracellular-matrix

Research

JoVE Journal

Bioengineering

1.3K Views.  Koç University. The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

Video: Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Cancer Research

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation. Ascertaining novel bioengineering approaches for the replacement of diseased lungs is imperative for improving patient survival and avoiding complications associated with current transplantation methodologies. An alternative approach involves the use of decellularized whole lungs lacking cellular constituents that are typically the cause of acute and chronic rejection. Since the lung is such a complex organ, it is of interest to examine the extracellular matrix components of specific regions, including the vasculature, airways, and alveolar tissue. The purpose of this approach is to establish simple and reproducible methods by which researchers may dissect and isolate region-specific tissue from fully decellularized lungs. The current protocol has been devised for pig and human lungs, but may be applied to other species as well. For this protocol, four regions of the tissue were specified: airway, vasculature, alveoli, and bulk lung tissue. This procedure allows for the procurement of samples of tissue that more accurately represent the contents of the decellularized lung tissue as opposed to traditional bulk analysis methods.

Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue  

Presented here is a protocol for the isolation of regional decellularized lung tissue. This protocol provides a powerful tool for studying complexities in the extracellular matrix and cell-matrix interactions.

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Summary

Explore More Videos

Chapters in this video

Procedure for Lung Engineering

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Engineered Lung Tissues Prepared from Decellularized Lung Slices

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Dissection and Isolation of Region-Specific Decellularized Lung Tissue