Elsevier

Biomaterials

Volume 34, Issue 13, April 2013, Pages 3231-3245
Biomaterials

The effects of storage and sterilization on de-cellularized and re-cellularized whole lung

https://doi.org/10.1016/j.biomaterials.2013.01.031Get rights and content

Abstract

Despite growing interest on the potential use of de-cellularized whole lungs as 3-dimensional scaffolds for ex vivo lung tissue generation, optimal processing including sterilization and storage conditions, are not well defined. Further, it is unclear whether lungs need to be obtained immediately or may be usable even if harvested several days post-mortem, a situation mimicking potential procurement of human lungs from autopsy. We therefore assessed effects of delayed necropsy, prolonged storage (3 and 6 months), and of two commonly utilized sterilization approaches: irradiation or final rinse with peracetic acid, on architecture and extracellular matrix (ECM) protein characteristics of de-cellularized mouse lungs. These different approaches resulted in significant differences in both histologic appearance and in retention of ECM and intracellular proteins as assessed by immunohistochemistry and mass spectrometry. Despite these differences, binding and proliferation of bone marrow-derived mesenchymal stromal cells (MSCs) over a one month period following intratracheal inoculation was similar between experimental conditions. In contrast, significant differences occurred with C10 mouse lung epithelial cells between the different conditions. Therefore, delayed necropsy, duration of scaffold storage, sterilization approach, and cell type used for re-cellularization may significantly impact the usefulness of this biological scaffold-based model of ex vivo lung tissue regeneration.

Introduction

Increasing interest in the use of de-cellularized complex whole organ scaffolds for ex vivo tissue engineering has provided both opportunity and also unique challenges. Among the unresolved issues which require clarification include defining optimal, organ specific approaches for de-cellularization and for sterilization and storage of de-cellularized organs prior to re-cellularization [1], [2], [3], [4]. With respect to trachea and lung, a number of recent publications have comparatively assessed different de-cellularization protocols. Notably, the resulting architecture and extracellular matrix (ECM) protein composition of either trachea or lungs may differ substantially between the different regimens utilized [5], [6], [7]. Whether this will subsequently affect re-cellularization of scaffolds and therefore the generation of functional tissue suitable for transplantation, remains unresolved [4], [5]. Methods of optimal sterilization and storage have been suggested for trachea [7], [8] but not yet clearly delineated for de-cellularized lungs. One further consideration is that of post-mortem time prior to lung harvest and de-cellularization, a practical issue for procurement of human lungs. A number of hours or even days may pass prior to post-mortem tissue harvest. It is unknown at present whether this will affect the suitability of the donor lung for de-cellularization and subsequent re-cellularization.

To address these questions, we assessed architecture and ECM protein content and distribution in mouse lungs obtained following a prolonged post-mortem period prior to harvest compared to freshly procured lungs. We also assessed lungs obtained immediately after euthanasia and then subsequently stored after de-cellularization for prolonged periods (3 and 6 months). We further evaluated effects of sterilization using either irradiation or a final rinse with peracetic acid, a commonly used protocol in storage of other biologic scaffolds [9], [10], [11], [12], [13]. We then assessed growth of two different cell types, murine bone marrow-derived mesenchymal stromal cells (MSCs) and C10 mouse lung type 2 alveolar epithelial cells, following intratracheal inoculation into the different de-cellularized lungs.

Section snippets

Mice

Adult male C57BL/6J mice (8–24 wks, Jackson Laboratories) were maintained at UVM in accordance with institutional and American Association for Accreditation of Laboratory Animal Care standards and review. There was a total of 34 mice used for this experiment.

Lung de-cellularization

Mice were euthanized by lethal intraperitoneal injection of sodium pentobarbital in accordance with accepted AAALAC standards. After opening the chest, the trachea was cannulated with a blunted 18 gauge Luer-lock syringe, the thymus was

Comparison of de-cellularized mouse lungs: architecture and ECM composition

Histologic evaluation with H&E, Verhoeff's Van Gieson (EVG), and Masson's trichrome stains demonstrates, as we and others have previously shown [5], [6], [14], [15], [16], [20], [21], [22], [23], [24], that freshly de-cellularized lungs maintain the architecture of the extracellular matrix compared to native lung (Fig. 1A). Glycosaminoglycans (GAGs) were less evident by Alcian Blue staining in freshly de-cellularized lungs, likely representing, in large part, loss of cell-associated GAGs during

Discussion

Use of de-cellularized whole lung scaffolds for ex vivo generation of functional lung tissue is a rapidly growing area that may provide a viable option for clinical lung transplantation [4], [5], [6], [14], [15], [16], [20], [21], [22], [23], [24], [25]. As demonstrated in other tissue types such as skin, muscle, bladder, and others, successful use of biologic scaffolds has already entered clinical practice [1], [2], [3], [4]. However, the complicated 3-dimensional structure–functional biology

Conclusions

In summary, the current results demonstrate that conditions of storage or of sterilization of de-cellularized lung scaffolds can significantly impact both the structure and residual protein content as well as the ability of different cell types to survive and proliferate following inoculation. While excellent progress is being made in developing techniques for utilizing cadaveric de-cellularized lungs as scaffolds for ex vivo generation of functional lung tissue suitable for clinical use, these

Disclosure statement

No competing financial interests exist.

Acknowledgments

The authors gratefully acknowledge the staffs of the Offices of Animal Care Management at the University of Vermont, Bruce Bunnell, Christine Finck, and Andrew Hoffman for critical reads of the manuscript and Tyler Bittner and Ian Johnson for valuable contributions to the experimental studies. Studies were supported by NIH ARRA RC4HL106625 (DJW), NHLBI R21HL094611 (DJW), UVM Lung Biology Training grant T32 HL076122 from the NHLBI, and UVM Environmental Pathology Training grant T32 ES007122 from

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