Original Pre-Clinical Science
Perfusion decellularization of human and porcine lungs: Bringing the matrix to clinical scale

https://doi.org/10.1016/j.healun.2013.10.030Get rights and content

Background

Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale.

Methods

Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs.

Results

Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture.

Conclusions

SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture.

Section snippets

Whole-organ perfusion decellularization

Animal organs were harvested in accordance with approved animal protocols and institutional guidelines. Outbred Sprague-Dawley rats (150–200 g, Charles River Laboratories, Inc, Wilmington, MA) were used throughout. Porcine lungs were harvested in-house from Yorkshire swine (40–200 kg) being otherwise used for experiments not involving the chest cavity. Rat donors were pre-treated with a dose of heparin (2.0 U/g of body weight) into the inferior vena cava. Porcine donors were not pre-treated

Results

We first aimed to analyze 3 published decellularization protocols for rat lung4, 5, 6 to determine the most suitable methods for subsequent up-scaling. A perfusion-based decellularization method was adopted for each protocol. The specific protocol details are outlined in the Methods and Supplemental Figure 1 (available in the online version of this article at jhltonline.org).

Histologic analysis of rat lungs decellularized by each protocol revealed equivalent removal of intracellular and nuclear

Discussion

More than 1,700 Americans are in need of a donor lung,1 with limited or no available alternative therapies.19 Waiting list mortality rates for lung candidates continue to rise, reported at 15.7/100 waiting list-years in 2011.20 In addition, only 21.0% of potential donor lungs are used for transplantation,20 further highlighting the shortage of adequate organs. A 50% probability of graft failure and more than 40% probability of death at 5 years after transplant remain clinical realities lung

Disclosure statement

This study was supported by the United Therapeutics Corporation and the National Institutes of Health (NIH) Director’s New Innovator Award (DP2-OD008749-01).

This work was presented at the Thirty-third Annual Meeting and Scientific Sessions of the International Society for Heart and Lung Transplantation, Montreal, Québec, Canada, April 24–27, 2013.

HC Ott is founder and stockholder of IVIVA Medical Inc. This relationship did not influence design, execution, and/or interpretation of the present

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