Production and Assessment of Decellularized Pig and Human Lung Scaffolds

Abstract

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

“…Lamin A/C appears to be efficiently depleted during the decellularization process, despite known challenges of completely removing nuclear matrix. In contrast, h H2A, appears enriched postdecellularization, despite reports of efficient DNA depletion (5,44,45). Similarly, cellular proteins such as cytoskeletal actins, tubulins, and mitochondrial ATP synthase subunit alpha-1 appear to be inefficiently removed by the decellularization process according to the peptide spectral match comparisons in Table I.…”

Coming to terms with tissue engineering and regenerative medicine in the lung

“…There is now considerable evidence that MSCs play a role in regeneration and repair (61,62,81,91,112,201) and in secretion of growth factors, immune modulators, and other factors that support the idea that MSCs may be better taken up in organ scaffolds (158,198,220), especially if scaffolds enhance their differentiation (86,219), presumably through cell-matrix interactions (64,161,174,232). Several studies have shown that MSCs can attach and proliferate in lung scaffolds (39,117,131,137). For example, MSCs derived from human adipose tissue and bone marrow on decellularized rat lungs express markers of pulmonary epithelial cells (117), although the source of the MSCs may play a role in their local behavior such as cell attachment, migration, and proliferation.…”

“…An important consideration (as with allogeneic transplants) is the immunological response of the transplant recipient to the bioengineered lung (102,131,189,190). Regardless of the materials used to engineer a lung, generalized immunosuppression will likely be the initial approach to minimizing rejection, although techniques to enhance acceptance of a bioengineered lung containing specific types of scaffold materials and cell types are obviously a major research focus [e.g., biocompatible materials, novel (ideally personalized) sources for stem cells, and cell differentiation techniques].…”

“…It is certainly much easier to acquire adult lung cells (e.g., from surgical resections, transplant recipients, or donor lungs deemed unfit for transplantation), and such adult cells have been explored for their potential for reseeding of scaffolds (alveolar epithelial cells, fibroblasts, and small airway epithelial cells) (131,137,194). However, as expected, these adult cells show restricted proliferative capacities and are therefore unlikely to be useful for reseeding large surface areas needed for a bioengineered human lung.…”

Respiratory Tissue Engineering: Current Status and Opportunities for the Future

“…Since then, parallel investigations have been conducted with murine (168,169), primate (170), and more recently human tissue (171)(172)(173), as well as analysis with different stem cell populations such as bone marrow-derived MSCs (174). This emergent field could have great implications for transplant medicine, where the possibility of using acellular cadaveric donor tissue could reduce numbers of patients waiting to receive a transplant from viable sources (175).…”