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 °C and can be used immediately following neutralization or stored at 4 °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.

Electrospun Decellularized Lung Matrix Scaffold for Airway Smooth Muscle Culture.

Chronic respiratory disease affects many people worldwide with little known about the intricate mechanisms driving the pathology, making it difficult to develop novel therapies. Improving the understanding of airway smooth muscle and extracellular matrix (ECM) interactions is key to developing treatments for this leading cause of death. With currently no relevant or controllable in vivo or in vitro models to investigate cell-ECM interactions in the small airways, the development of a biomimetic in vitro model with cell attachment, signaling, and organization is needed. The goal of this study was to create a biologically and structurally relevant in vitro model of small airway smooth muscle. In order to achieve this goal, a scaffold was engineered from synthetic poly-l-lactic acid (PLLA) and decellularized pig lung ECM (PLECM). PLECM scaffolds have improved physical characteristics over synthetic scaffolds, by exhibiting a significant decrease in the elastic modulus and an increase in hydrophilicity. Histological staining and SDS-PAGE showed that essential proteins or protein fragments found in natural ECM were present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM 3D scaffolds formed confluent layers and maintained a contractile phenotype, as demonstrated by the organized arrangement of actin filaments within the cell and expected contractile protein expression of calponin 1. HBSMCs cultured on electrospun PLECM scaffold also increased alpha-1 type 1 collagen compared to those cultured on PLLA scaffolds. In summary, this research demonstrates that a PLLA/PLECM composite electrospun mat is a promising tool to produce an in vitro model of the airway with the potential for a better understanding of bronchiole smooth muscle behavior in diseased or normal states.

Development and characterization of a naturally derived lung extracellular matrix hydrogel.

The complexity and rapid clearance mechanisms of lung tissue make it difficult to develop effective treatments for many chronic pathologies. We are investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. The main objectives of this study include effective decellularization of porcine lung tissue, development of a hydrogel from the porcine ECM, and characterization of the material's composition, mechanical properties, and ability to support cellular growth. Our evaluation of the decellularized tissue indicated successful removal of cellular material and immunogenic remnants in the ECM. The self-assembly of the lung ECM hydrogel was rapid, reaching maximum modulus values within 3 min at 37°C. Rheological characterization showed the lung ECM hydrogel to have a concentration dependent storage modulus between 15 and 60 Pa. The purpose of this study was to evaluate our novel ECM derived hydrogel and measure its ability to support 3D culture of MSCs in vitro and in vivo delivery of MSCs. Our in vitro experiments using human mesenchymal stem cells demonstrated our novel ECM hydrogel's ability to enhance cellular attachment and viability. Our in vivo experiments demonstrated that rat MSC delivery in pre-gel solution significantly increased cell retention in the lung over 24 h in an emphysema rat model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1922-1935, 2016.