Decellularized Rat Lung Research Articles

Fibroblast engraftment in the decellularized mouse lung occurs via a β1-integrin-dependent, FAK-dependent pathway that is mediated by ERK and opposed by AKT

Creation of bioartificial organs has been enhanced by the development of strategies involving decellularized mammalian lung. Because fibroblasts critically support lung function through a number of mechanisms, study of these cells in the context of the decellularized lung has the potential to improve the structure and function of tissue-engineered lungs. We characterized the engraftment and survival of a mouse fibroblast cell line in decellularized rat lung slices and found a time-dependent increase in cell numbers assessed by hematoxylin and eosin staining, cell proliferation assessed by Ki67 staining, and minimal cell death assessed by TUNEL staining. We developed a repopulation index to allow quantification of cell survival that accounts for variation in cell density throughout the seeded scaffold. We then applied this method to the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a β1-integrin-neutralizing antibody significantly reduced the repopulation index after 24 h of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation scores while treatment with AKT inhibitor increased initial scores. Rho-associated kinase inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occur in a β1-integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT.

Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy

Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n=11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ∼7μm thick slices. The complex elastic modulus (G∗) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G′, real part of G∗) of alveolar ECM was ∼6kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G′ of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (G″, imaginary part of G∗) was 10-fold lower and showed a frequency dependence similar to that of G′ at low frequencies (0.1–1Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.

Abstract 345: Circulating tumor cells from perfusable tumor nodules grown on decellularized rat lung scaffold has mesenchymal characteristics

Abstract Circulating tumor cells are thought to be responsible for metastasis. These cells leave the primary site of cancer and enter the circulation after undergoing epithelial to mesenchymal transition. The tumor cells lose the epithelial features, such as expression of E-cadherin, and acquire mesenchymal characteristics, such as expression of Twist1, Zeb and N-cadherin. In this study, we developed a novel ex vivo model that utilizes a decellularized rat lung scaffold to grow tumor nodules and study circulating tumor cells. We hypothesized that circulating tumor cells in our ex vivo model will have mesenchymal characteristics. We created the decellularized rat lung scaffold using SDS and Triton-X. We then placed 344SQ or 393P mice lung cancer cell lines in the decellularized rat lung scaffold and perfused them in a customized bioreactor. We measured tumor growth on the scaffold and the number of circulating tumor cells for 14 days. We then analyzed the functional characteristics of the circulating tumor cells using the Boyden chamber and determined gene expression of E-cadherin, N-cadherin, Zeb1 and Twist. Both the 344SQ and 393P lung cancer cell lines formed tumor nodules that gradually increased in size with an increase in the number of circulating tumor cells over the 14-day period in our model. The circulating tumor cells had greater ability to migrate than the monolayer cell cultures in the Boyden chamber assay, with 100% of the circulating tumor cells migrating in contrast to 69% and 71% of the monolayer cell cultures of 344SQ and 393P, respectively. The circulating 393P tumor cells had a significant increase in Twist (58-fold), Zeb (20-fold) and N-cadherin (14-fold) and decrease in E-cadherin compared to the 393P cells from the monolayer cell culture. This difference was even more dramatic for the circulating 344SQ tumor cells with an increase in Twist (>6000-fold), Zeb (14-fold) and N-cadherin (88-fold) compared to the 344SQ cells from the monolayer. There was small 4-fold increase in E-cadherin expression in the circulating 344SQ tumor cells compare to the 344SQ cells from the monolayer. The circulating tumor cells in our model have both phenotypic and genotypic characteristics of mesenchymal cells. The cancer cells that are more mesenchymal at the baseline have a greater difference in these characteristics. The circulating tumor cells in our model may be the sub-population of cells responsible for metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 345. doi:1538-7445.AM2012-345

Human Lung Cancer Cells Grown on Acellular Rat Lung Matrix Create Perfusable Tumor Nodules

Extracellular matrix allows lung cancer to form its shape and grow. Recent studies on organ reengineering for orthotopic transplantation have provided a new avenue for isolating purified native matrix to use for growing cells. Whether human lung cancer cells grown in a decellularized rat lung matrix would create perfusable human lung cancer nodules was tested. Rat lungs were harvested and native cells were removed using sodium dodecyl sulfate and Triton X-100 in a decellularization chamber to create a decellularized rat lung matrix. Human A549, H460, or H1299 lung cancer cells were placed into the decellularized rat lung matrix and grown in a customized bioreactor with perfusion of oxygenated media for 7 to 14 days. Decellularized rat lung matrix showed preservation of matrix architecture devoid of all rat cells. All three human lung cancer cell lines grown in the bioreactor developed tumor nodules with intact vasculature. Moreover, the lung cancer cells developed a pattern of growth similar to the original human lung cancer. Overall, this study shows that human lung cancer cells form perfusable tumor nodules in a customized bioreactor on a decellularized rat lung matrix created by a customized decellularization chamber. The lung cancer cells grown in the matrix had features similar to the original human lung cancer. This ex vivo model can be used potentially to gain a deeper understanding of the biologic processes involved in human lung cancer.