Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Summary

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

Abstract

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.

Introduction

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 attent....

Protocol

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 .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Absolute ethanol	ISOLAB	64-17-5
Acetic acid	ISOLAB	64-19-7
Alcian blue solution	Sigma-Aldrich	B8438
Deoxyribonuclease I from bovine pancreas	Sigma-Aldrich	DN25
Discovery HR-2 rheometer	TA Instruments
Entellan mounting medium	Merck	107960
Eosin solution	Bright-slide	2.BS01-105-1000
Formaldehyde	Electron Microscopy Sciences	50-980-485
Hydrochloric acid	Merck	100317
Iodine	Sigma-Aldrich	3002
Magnesium chloride	Sigma-Aldrich	7786-30-3
Mayer's haematoxylin staining solution	Merck	2.BS01-103-1000
O.C.T compound	Tissue-Tek	4583
Penicillin/Streptomycin	Biowest	L0018-100
Pepsin from porcine gastric mucosa	Sigma-Aldrich	P6887
Picric acid	Polysciences	88-89-1
Sirius Red	Polysciences	09400-25
Sodium hydroxide	Sigma-Aldrich	S5881
Sucrose	Sigma-Aldrich	S0389
Triton-X-100	Merck	112298