Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

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' 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 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.

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 ECM^1,2,3,4,5. ECM-based carriers have been used in many applications spanning tissue regeneration to disease model development. They are used as injectable or implantable biomaterial scaffolds^4,5, in drug screening applications^6,7, in the development of materials that induce cell growth^8,9,10, as bio-inks^11,12,13, in microfluidics¹⁴, and in cancer tissue models^{15,16,17,18,19}.

Decellularization of tissues and organs is a popular approach for 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 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' biochemical and mechanical properties, including viscoelasticity and stiffness, is crucial for 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 setting^20,22,23. Previous studies have demonstrated decellularization of 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.

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

1. Store freshly obtained bovine lung tissues at -80 °C until the experiment.
2. On the day of the experiment, wait for frozen lung tissues to thaw at room temperature.
3. Dissect the tissues with sterile scalpels and scissors to remove trachea and cartilaginous airways, and further mince into small pieces (5 mm³).
4. Wash thoroughly with ultrapure water containing 2% penicillin/streptomycin (P/S) at least 3x.

2. Decellularization of tissues

NOTE: Native bovine lung tissues were decellularized using two distinct protocols.

1. Freeze-thaw method
   1. 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.
   2. 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 °C water bath for 10 min. This constitutes 1 cycle of freeze-thaw.
   3. 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 °C under constant shaking at 100 rpm.
   4. Continue with an extensive wash with sterile dH₂O for 3 days under constant shaking at 100 rpm, with solution replenishment every 24 h.
   5. Perform lyophilization and cryomilling as follows²⁰: Freeze the wet dECM samples at -80 °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.
2. Triton-X-100 method
   1. Prepare tissue samples according to step 1. Treat lung tissues with 1% Triton-X-100 for 3 days under gentle rotation at 4 °C. Exchange solution every 24 h.
   2. Incubate the samples with DNase solution (10U/mL) in 10 mM MgCl₂ buffer (pH 7.5) for 1 h at 37 °C under constant shake.
   3. Continue with an extensive wash with sterile dH₂O for 3 days under gentle rotation, with solution replenishment every 24 h.
   4. Perform lyophilization followed by cryo-milling as described in step 2.1.

3. Pepsin digestion

1. 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.
2. Transfer the digests into a tube and centrifuge at 5000 x g for 10 min.
3. 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 detrimentally impact the gelation in the subsequent steps.
4. Store the pre-gel digests at −20 °C for further studies.

4. Histological staining

1. Fix a small portion (5 mm³) of decellularized tissue sample in 1 mL of 3.7% formaldehyde solution at 4 °C overnight.
2. Incubate tissues in tubes containing 1 ml of 30% sucrose solution in PBS for 12 h at 4 °C on a steady rock.
3. 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.
4. 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 °C until use.
5. Obtain 10 µm sections in the cryostat at -25 °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 °C until use.
6. In order to confirm the absence of nuclei after decellularization, perform a Hematoxylin and Eosin (H&E) staining as described below.
   1. 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.
   2. Immerse the slides in 95% ethanol and stain with 50 mL of 0.5% alcoholic Eosin solution in a staining jar for 45 s.
7. Perform Sirius red staining to show the retaining of collagen content after decellularization.
   1. 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.
   2. 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.
8. To analyze sGAG content in tissue samples, perform Alcian blue staining as described below.
   1. 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.
   2. Dehydrate all slides by soaking them in 70%, 95%, and 100% ethanol sequentially for 1 min each. 
9. Add 0.1 mL of mounting medium on top of the samples, and visualize using light microscopy using 10x magnification.

5. Mechanical characterization

1. Implement oscillatory rheology measurements using a rheometer with parallel plate geometry.
2. Pour 250 µL of pre-gel solution kept on ice onto a pre-cooled (4 °C) lower plate to avoid rapid gelation.
3. Lower the 20 mm parallel plate until the pre-gel solution forms a disk with a 1 mm gap width between the two plates.
4. Start the measurement immediately. Measure storage and loss moduli over time with a fixed frequency and strain while heating the lower plate to 37 °C for 30 min to observe gelation kinetics. Use a fixed frequency of 0.5 Hz and 0.1% strain.
5. Perform a creep-recovery test after the storage modulus value stops increasing and reaches a plateau.
6. 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.
7. Draw a strain versus time graph to show the stress-relaxing behavior. Repeat measurements for three different dECM digests as replicates.

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 °C water bath was applied for the freeze-thaw method to disrupt cellular structures. In the second decellularization method, native lung tissue pieces were treated with 1% Triton-X-100 solution for 3 days under constant stirring. In both methods, cutting the lung tissue into small pieces is critical to allow diffusion of solutions to core regions as well as the physical disruption of the nuclear content. As the first visual indicator of decellularization, the pink color of the tissue turned into a whitish color with repeated cycles in both methods. It is also crucial to remove the residual DNA, which was achieved with DNase treatment in this protocol. Furthermore, after decellularization with both methods, tissues were extensively washed for 3 days in dH₂O supplemented with antibiotics to preserve the sterility of the tissues. Another point to consider is that during the neutralization and buffering of dECM digests to physiological conditions, all solutions were filter-sterilized, again for sterility purposes, and kept on ice to avoid premature gel formation. Lung dECM hydrogel formation was then achieved with thermal gelation at the final step (Figure 1).

Cryo-sectioning and histological stainings
Fixation and cryo-sectioning of native and decellularized lung tissues followed by indicated histological stainings were performed to assess the elimination of nuclei and retention of ECM molecules after decellularization (Figure 2A). H&E staining demonstrated a significant reduction in nuclei content in decellularized samples with both freeze-thaw and Triton-X-100 methods compared to native tissue, implying that decellularization was achieved by both physical and chemical methods (Figure 2B). Sirius red staining showed that retained collagen in decellularized tissues was comparable with native tissue, indicating both decellularization methods had minimal impact on collagen content (Figure 2B). Additionally, Alcian blue staining demonstrated that sGAG content in decellularized tissues was efficiently preserved compared to native tissues (Figure 2B).

Mechanical characterization
Rheology measurements of decellularized lung hydrogels were carried out with a 20 mm parallel plate set-up to obtain a hydrogel disc of 1 mm thickness (Figure 3A). Frequency sweep was used to optimize the strain values used in the measurements. The average storage modulus (G') and loss modulus (G'') of hydrogels obtained with the freeze-thaw method were 204 Pa and 28.4 Pa, respectively, which were significantly stiffer than hydrogels obtained with the Triton-X-100 method (Figure 3B-C). Interestingly, creep-recovery tests showed that hydrogels obtained with freeze-thaw and Triton-X-100 methods had distinct responses to stress, implicating that hydrogels have different viscoelastic properties (Figure 3D).

Figure 1: Schematic representation of the decellularization process. Bovine lung tissue was dissected into small pieces and washed thoroughly. Removal of cellular content was achieved with two different methods. Physical method with repeating freeze-thaw cycles and chemical method with Triton-X-100 treatment. DNase treatment was performed in both methods to remove residual DNA; after lyophilization of the decellularized tissues, cryomilling, pepsin digestion, neutralization, and gelation steps were pursued. Please click here to view a larger version of this figure.

Figure 2: Cryo-sectioning and staining of native and decellularized bovine lung tissues. (A) Native and decellularized bovine lung tissue pieces were fixed with 3.7% formaldehyde solution, immersed in 30% sucrose solution in PBS, then embedded in optimal cutting temperature (OCT) compound and snap-frozen. For each sample, 10 µm sections were mounted on glass slides to proceed with staining. (B) Representative images of native and decellularized samples by freeze-thaw or Triton-X-100 methods. Hematoxylin & Eosin (H&E) staining for nuclei, Sirius red staining for collagen and Alcian blue staining for sGAGs were performed. Scale bar: 100 µm. Please click here to view a larger version of this figure.

Figure 3: Measurement of mechanical properties of decellularized lung ECM hydrogels. (A) Representative image of a thermally crosslinked decellularized lung hydrogel after mechanical characterization. Oscillatory rheology was performed using a rheometer with parallel plate geometry. Rheological properties of decellularized hydrogels, (B) storage modulus (G'), (C) loss modulus (G''), (D) creep-recovery test to assess stress relaxation behavior of hydrogels. Error bars represent s.d. (*p < 0.05), n=3. An unpaired t-test with Welch's correction was used for statistical analysis. Please click here to view a larger version of this figure.

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, such as stiffness and viscoelasticity, holds great importance as these have been implied in regulating several cellular phenomena. Therefore, it is crucial to compare and evaluate different decellularization methods for producing dECM hydrogels in terms of biochemical content and mechanical aspects^20,23. Here, bovine lung decellularization was demonstrated with two distinct approaches: Freeze-thaw cycles, which were mainly based on mechanical disruption and removal of DNA content, as well as chemical removal of nuclear content using a common detergent, Triton-X-100 (Figure 1). For the freeze-thaw method, it is essential to ensure that all tissue samples are frozen and appropriately thawed between liquid nitrogen and water bath in an automated and repetitive pathway²⁴. In order to achieve this, tissue samples could be divided into several tubes and sank into the nitrogen container for freezing and then a water bath for thawing. Changing the Triton-X-100 solution every 24 h is critical for the chemical method to disrupt the cellular content effectively²¹. DNase treatment was used in both methods to further enhance the breakdown of remaining DNA, which could reduce the viability of encapsulated cells. Both methods successfully eliminated cellular material during decellularization, with trace quantities of DNA remaining in both cases²⁰. Notably, commercially available extracellular matrix products have similarly been reported to contain small amounts of residual DNA with no adverse effect on cytocompatibility or immune responses in hosts upon implantation for their use in tissue engineering and translational studies^20,25.

ECM comprises many protein and polysaccharide macromolecules, such as collagens, elastins, and proteoglycans, which have critical regulatory roles in cellular signaling²⁶. Therefore, native ECM-derived scaffolds should demonstrate retention of such cell-instructive ECM ligands. For this purpose, collagen and sGAG stainings were performed to compare the ECM composition of decellularized matrices to that of native tissues. The results show that collagen and sGAG content were retained in decellularized tissues via freeze-thaw and Triton-X-100 decellularization methods, suggesting that they offer a native-like ECM microenvironment in reconstituted scaffolds^20,26. The structural organization of the ECM constituents showed differences according to the decellularization method despite slight differences in the ECM content. Even though representative images showed more similarity between detergent-based decellularization of lung tissue and native lung tissue stainings, both protocols successfully retained native ECM proteins overall (Figure 2).

Mechanical properties of 3D tissue models play a crucial role in cell culture studies since both the growth and behavior of cells are highly instructed by the mechanical characteristics of the native ECM^27,28,29,30. Various decellularization methods show distinct impacts on the gelation process and the final stiffness and viscoelasticity of dECM hydrogels. Those findings demonstrate that the freeze-thaw method produces more robust and stiffer hydrogels than the Triton-X-100 method. Furthermore, in our previous study, we exhibited that the stiffness of freeze-thaw dECM hydrogels can be adjusted by modifying the ligand concentration and can be lowered to match the stiffness of Triton-X-100-derived dECM hydrogels²⁰. Viscoelasticity is a feature of native tissue matrices that indicates viscous and elastic behavior in response to mechanical deformation. Recent studies revealed the role of ECM viscoelasticity in cell proliferation, morphology, and differentiation^29,30. This study shows lung dECM hydrogels as stress-relaxing materials similar to native fibrin, collagen, or reconstituted basement membrane. Moreover, the findings indicate that Triton-X-100 method-derived dECM hydrogels recover faster than freeze-thaw-derived hydrogels. Therefore, the choice of the decellularization method highly affects the creep response and relaxation kinetics of the resulting dECM gels.

Decellularization methods described here utilize bovine lung to reconstitute the biochemical and mechanical characteristics of the lung tissue ECM. We finely characterized the biochemical and physical composition of the ECM after the decellularization process. A major challenge in decellularization methods is the retention of ECM composition, which directly affects the gelation capability of dECM hydrogels between batches. Utilization of lung tissue from the same donor is an important parameter to overcome batch-to-batch variability. Even though healthy native tissues were used, variability between donors is inevitable. Overall, both freeze-thaw and Triton-X-100 methods show promising potential for using native dECM hydrogels in disease modeling of the lung.

All authors declare no competing financial interests.

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