Automated procedure for biomimetic de-cellularized lung scaffold supporting alveolar epithelial transdifferentiation

Abstract

The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.

Pressure- and flow-controlled media perfusion differently modify vascular mechanics in lung decellularization

“…As expected from previous data (Girard et al, 2013), pressurevs. flow-controlled decellularization procedures performed satisfactorily and did not result in apparent differences in terms of maintenance of scaffold structure and composition of main extracellular matrix components.…”

“…Indeed, recent studies have compared perfusion methods (e.g. pressure-vs flow-controlled and manual vs automated) in terms of elimination of donor cell material in the scaffold, preservation of the extracellular matrix composition and structure, and suitability of the acellular scaffold as substrate for culturing cells (Girard et al, 2013;Wagner et al, 2014aWagner et al, , 2014bGuyette et al, 2014). Nevertheless, none of these studies has investigated lung vascular resistance in detail.…”

“…deoinized water, detergents, enzymes) through the vascular circuit of the organ is a conventional procedure for eliminating donor cell material since the widespread distribution of the capillary network within the organ allows both to distribute the media to short diffusion distance from any cell and to washout the resulting debris. In the specific case of the lung, different experimental studies in animal and human organs have shown that perfusing media through the pulmonary artery allows to adequately decellularize not only the vessel walls but also to eliminate the cells in the lung parenchyma and in the airway walls (Girard et al, 2013;Price et al, 2014;Gilpin et al, 2014aGilpin et al, , 2014bWagner et al, 2014aWagner et al, , 2014b.…”

An Official American Thoracic Society Workshop Report: Stem Cells and Cell Therapies in Lung Biology and Diseases

“…Some of the progress has been more incremental (e.g., the optimization of protocols for using decellularized and recellualrized lung scaffolds, both in rodents and more recently in larger animal models and in human lungs (7,(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90) (91). In presenting more recent studies on the use of organ-specific decellularized extracellular matrix (ECM) scaffolds for engineering a variety of whole visceral organs, (e.g., lung, liver, heart, and kidney) (92), Dr. Badylak stressed the importance of fine tuning harvesting methods, including decellularization techniques.…”

Enhanced Re-Endothelialization of Decellularized Rat Lungs

“…Decellularization of whole rat lungs was carried out essentially according to the protocol developed by Girard et al 21 As previously described by Price et al 13 upon decellularization the color of the organ turned from red to opaque/white (Fig. 1A, B, respectively).…”