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 ...

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

1 Our aim is to develop an organotypic lung tissue model. 2 Therefore, we have optimized the decellularization 3 of bovine lung tissue 4 and reconstitution of lung extracellular matrix hydrogels. 5 In this study, 6 we seek to understand the effect 7 of different decellularization methods 8 on the biochemical and mechanical characteristics 9 of reconstituted lung hydrogels.10 One of the main challenges of decellularization protocols<v>11 is achieving mechanical stability 12 in reconstitute hydrogels, 13 which requires an understanding 14 of the effects of decellularization 15 on mechanical properties. 16 These properties, such as stiffness and viscoelasticity, 17 have crucial effects on cellular behaviors. 18 We have established methodologies<v>19 that effectively decellularize the bovine lung, 20 yielding reproducible lung hydrogels 21 that demonstrate noteworthy analogies 22 with the extracellular matrix of the human lung.23 Therefore, there is substantial promise 24 in utilizing native lung dECM hydrogels 25 for the purpose of disease modeling 26 in the context of the lung. 27 We engineered tissues<v>28 to model homeostatic and disease conditions 29 with a particular focus on cell matrix interactions. 30 Therefore, we're interested in understanding the role 31 of organotypic extracellular matrices, 32 both in terms of unique content and mechanical aspects 33 on cell behavior.34 Our current research focuses on building biomimetic, 35 patient-specific organoid models of cancer.

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

1 To begin, remove the frozen bovine lung tissues<v>2 from 80 degrees Celsius 3 and allow them to thaw at room temperature. 4 Dissect the tissue using sterile scalpels and scissors 5 to remove the trachea and cartilaginous airways, 6 then mint the tissue into small pieces. 7 Wash the tissue three times 8 with ultrapure water 9 containing 2%penicillin and streptomycin.10 Immerse the minced tissue sample 11 in 2%iodine solution for one minute, 12 then perform two consecutive washes 13 in sterile distilled water. 14 Transfer the tissue pieces into a 50 milliliter tube 15 up to the 15 milliliter mark. 16 Fill the tube to the top with distilled water 17 and freeze it in liquid nitrogen for two minutes.18 Then immediately transfer the tube to a beaker 19 containing 37 degrees Celsius water for 10 minutes. 20 After five freeze thaw cycles, 21 incubate the sample in 30 milliliters 22 of DNase solution for one hour 23 at 37 degrees Celsius with constant stirring. 24 Freeze the wet decellularized extracellular matrix samples 25 at 80 degrees Celsius for 24 hours.26 Transfer frozen samples into a lyophilizer 27 and operate in drying mode under a vacuum 28 for three to four days until fully dried. 29 Following freeze drying, 30 mill the samples into a fine powder 31 in a grinding apparatus containing dry ice. 32 Treat the lung tissues with 1%Triton-X-100 33 for three days under gentle rotation 34 at four degrees Celsius, 35 changing the solution every 24 hours.36 Incubate the sample in DNase solution 37 for one hour at 37 degrees Celsius with constant stirring. 38 Wash the tissue with distilled water 39 for three days under constant rolling, 40 replenishing the water every 24 hours. 41 After the last wash, perform lyophilization and cryo milling 42 as demonstrated previously.43 Digest 15 milligrams per milliliter 44 powdered decellularized extracellular matrix samples 45 in one milligram per milliliter pepsin solution 46 at room temperature under constant stirring for 48 hours. 47 Maintain the solution pH at two for effective digestion. 48 Centrifuge the tissue digest at 5, 000 g for 10 minutes 49 and remove the supernatant.

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

1 Turn on the Discovery HR-2 Rheometer<v>2 to implement oscillatory rheology measurements. 3 Pour 250 microliters of pre-gel digest solution 4 placed on ice onto a pre-cooled lower plate 5 to avoid rapid gelation. 6 Lower the 20 millimeter parallel plate 7 until the pre-gel solution forms a disc 8 with a one millimeter gap width between the two plates.9 Set the strain at 0.1%and frequency to 0.5 hertz. 10 Measure storage and loss moduli over time 11 while heating the lower plate 12 to 37 degrees Celsius for 30 minutes 13 to observe gelation kinetics. 14 After the storage modulus value stops increasing 15 and reaches a plateau, 16 perform a creep recovery test 17 to assess the stress relaxation behavior of hydrogels.18 Apply one Pascal shear stress to the hydrogel 19 for 15 minutes and measure the strain. 20 Then unload the sample from the stress 21 and record the change in strain values for 15 minutes. 22 Finally, draw a strain versus time graph 23 to show the stress relaxing behavior.24 The average storage modulus 25 and loss modulus of hydrogels 26 produced by the freeze-thaw method 27 demonstrated significantly higher stiffness 28 compared to hydrogels produced 29 using the Triton X-100 method. 30 Creep recovery tests revealed 31 that hydrogels obtained from both methods 32 exhibited distinct stress responses 33 indicating different viscoelastic properties.

920 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

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