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

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#160;</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.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix 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

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

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

Harvesting, Decellularization, and Pepsin Digestion of Bovine Lung Tissues for Decellularized Lung Extracellular Matrix Hydrogel Formation

Assessment of Gelation Kinetics and Stress-Relaxation Properties in Decellularized Lung Extracellular Matrix Hydrogels Using Oscillatory Rheology

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.

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Summary