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