Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate

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Abstract

Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (RL) and elastance (EL) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

Introduction

Lung transplantation is the main therapeutic indication for diseases in which the organ function is irreversibly damaged, such as chronic obstructive pulmonary disease and lung fibrosis. However, the scarcity of available organs and the lack of compatibility between donor and recipient human antigens reduce the success rate of this intervention. Therefore, intensive research has been conducted in an attempt to achieve a higher organ viability for transplantation and to improve the survival rate, which is currently only 50% in these patients within the first 5 years of lung transplantation (Yusen et al., 2010).

An alternative method recently proposed to overcome the shortage of viable organs from donors is the use of extracellular matrix (ECM) scaffolds as a platform for subsequent recellularization (Badylak, 2002, He and Callanan, 2013). The ECM of an organ preserves the 3D structure and the original architecture of components such as collagen, elastin, and laminin, allowing the cells to proliferate and differentiate into the specific phenotype for lung bioengineering (Crapo et al., 2012).

Several procedures to decellularize the kidney (Ross et al., 2009, Sullivan et al., 2012), liver (Uygun et al., 2010), heart (Ott et al., 2008), and lungs (Cortiella et al., 2010, Ott et al., 2010, Petersen et al., 2010, Petersen et al., 2012) have been described in the literature, each one using different variants of physical, chemical, or enzymatic methods. Basically, these methods consist of a first treatment to break down the integrity of the cells and a subsequent treatment to extract the cellular material by using different types of detergents to ultimately obtain the decellularized lung scaffold.

Studies comparing different methods of lung decellularization have been performed by using temperature changes, alternative perfusion/infusion pathways and different chemical agents (Cortiella et al., 2010, Ott et al., 2010, Petersen et al., 2010, Price et al., 2010, Daly et al., 2012, Wallis et al., 2012). However, which is the most efficient protocol for obtaining an optimal scaffold remains unclear. The aim of this study was to assess whether lung mechanical properties of the lung scaffold vary when subjected to artificial ventilation during the different stages of the decellularization process based on a sodium dodecyl sulfate detergent protocol.

Section snippets

Methods

Fifteen 7–8-week-old female C57BL/6 mice were intraperitoneally anesthetized with urethane (1 mg/kg) and euthanized by exsanguination according to a protocol previously approved by the Ethical Committee for Animal Research of the University of Barcelona. The lungs and trachea were excised and cleaned to remove any attached esophageal, lymphatic, and connective tissues. Then, lungs were placed in a 50 mL polystyrene conical tube with 5 mL of PBS, frozen and kept in a −80 °C freezer until

Results

As shown in a representative image of DAPI staining (Fig. 4) and in agreement with previous reports (Nonaka et al., 2013, Melo et al., 2014), the decellularization protocol applied in this study resulted in acellular lungs.

From fifteen lungs at the beginning of the process, only four of them maintained the structural integrity with no air leaks and intact trachea until the end of the last decellularization step. The mechanical evaluation of these four lungs revealed minor differences between

Discussion

Lung decellularization achieved by a combination of the freeze/thaw method and SDS detergent efficiently eliminated cellular material and preserved the mechanical properties of the scaffold. We have shown that the resistance and elastance values of the lungs did not exhibit significant changes between the initial and final values when measured during maneuvers of conventional mechanical ventilation.

The significant increase in the resistance and elastance parameters observed after the first SDS

Acknowledgments

The authors wish to thank Miguel A. Rodríguez for his excellent technical support.

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    Sources of support: This work was supported in part by the Spanish Ministry of Economy and Competitiveness (SAF2011-22576, FIS-PI11/00089) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Research Productivity modality 307618/2010-2). Paula Naomi Nonaka has a fellowship (2012/04052-2) from Fundação de Amparo à Pesquisa do Estado de São Paulo.

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