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.

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

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

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 ...

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 ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...