harvesting, decellularization, and pepsin digestion of bovine lung tissues for decellularized lung extracellular matrix hydrogel formation

Comparative Study of Decellularization Techniques for Bovine Lung ECM 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.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

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

harvesting-decellularization-pepsin-digestion-bovine-lung-tissues-for

Research

JoVE Journal

Bioengineering

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.

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 ...