Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration
Introduction
Rapid developments in tissue engineering utilizing decellularized whole organs as biologic scaffolds coupled with advances in stem and endogenous lung progenitor cell biology have offered the potential of using acellular suboptimal donor or cadaveric lungs recellularized with appropriate cell types for ex vivo lung regeneration [1], [2], [3], [4], [5], [6]. Acellular lungs are also powerful in vitro platforms for studying cell–extracellular matrix (ECM) interactions in lung diseases such as emphysema and fibrosis, and in aging [7], [8], [9]. The resulting acellular scaffold retains characteristic disease architecture and recellularization attempts have shown differential responses, consistent with disease phenotypes [7], [8].
As acellular scaffolds derived from rodent lungs are readily available and easily handled, high throughput approaches can be utilized to study cell–matrix interaction or to assess the multiple conditions needed for effective recellularization and development of functional lung tissue [5], [7], [10], [11]. However, there is a more limited supply of larger lungs, including those from humans and potential xenogenic sources (e.g. porcine). Further, approaches utilized in rodent models may be insufficient for adequate decellularization of lungs obtained from larger animals and humans. As such, a limited number of reports have examined the feasibility of decellularization in large organs [1], [8], [12], [13].
Technical difficulties in handling larger tissue represent a major challenge in attempting to scale up rodent model decellularization techniques to porcine and human lungs or lobes. To address this issue, we assessed several methods of decellularization in porcine and human lungs using peristaltic pump-driven flow of decellularization reagents into both the airways and vasculature, with or without physical agitation, and compared these to manual instillations with static incubations, a technique commonly utilized for decellularizing rodent lungs [5], [7], [10], [11]. Optimization of fluid volumes was assessed for each step of the decellularization protocol.
Attempts to recellularize acellular scaffolds of larger lungs present additional hurdles. Recellularization of an entire acellular lobe or lung will require extremely large cell numbers. Supply of adequate nutrients and oxygen to maintain tissue vitality will be particularly challenging. Additionally, study of entire lungs or lobes does not readily lend itself to high throughput studies. Prior approaches have utilized thin slices of acellular human or porcine lungs onto which cells were non-specifically layered or thicker acellular tissue slices into which cells were non-specifically injected [1], [8], [12], [14]. While these studies demonstrated cell survival and even phenotypic changes following inoculation or seeding, these methods do not permit the selected study of cells with their respective ECM components (i.e. endothelial cells introduced into the vasculature or epithelial cells in airspaces). Rather, cells were heterogeneously introduced throughout the acellular scaffold and study of specific cell–ECM interactions is limited.
Methods to introduce cells in a biologically relevant fashion, as done in acellular rodent lungs [1], [2], [3], [4], [7], [10], [15], [16], [17] (e.g. airway or vasculature instillation), and which allow high throughput study approaches from larger lungs would be a significant step forward. We have developed a method of excising small (∼1–3 cm3) segments of acellular human and porcine lung scaffolds that maintain 3-dimensional structure and into which cells can be selectively inoculated via the preserved vascular and airway conduits. Excised segments can be obtained from select regions of the lung to study specific regional cell–matrix interactions. However, as excision of individual lung segments damages the integrity and function of the lung pleura, we have also developed a synthetic pleural coating that encapsulates the recellularized segments to provide an isolated lung unit for culture and analysis. Recellularized segments can then be sliced for high throughput studies or left as 3-dimensional segments.
Section snippets
Cell culture
Human bronchial epithelial cells (HBEs) (courtesy of Albert van der Vliet, University of Vermont, originally from Drs. J. Yankaskas and R. Wu) [18], human lung fibroblasts (HLFs) (ATCC, CCL 171), and human bone marrow-derived mesenchymal stromal cells (hMSCs) (obtained from the Texas A&M Stem Cell Core facility) [19] were cultured and expanded on tissue culture treated plastic at 37 °C and 5% CO2. Human vascular endothelial cells CBF12 (courtesy of Mervin Yoder, Indiana University – Purdue
Decellularization of cadaveric human and porcine lungs
Normal cadaveric human lungs, obtained from patients without a history of smoking or significant pulmonary disease, were used in the current study (19 patients, 29 lobes) and assigned to one of five different decellularization methods: M, VP, CP1, CP2, or CP3 (detailed in Methods and Table 1; Clinical characteristics of cadaveric human lungs are listed in Supplementary Table 1). The order of decellularization reagents is based on our previous work in rodent and non-human primate models [5], [7]
Discussion
One of the primary goals in developing techniques to derive acellular scaffolds from large animal and human lungs is as a potential tissue engineering platform for development of functional lung tissue that can be used in clinical transplantation. Several approaches have been tested in large animal and human lungs, such as continuous whole lung perfusion in a bioreactor chamber [12], manual instillation of fluids [1], [8], and exposing excised sheets of lung tissue to decellularization fluids
Conclusions
Acellular whole porcine lungs or human lobes can be decellularized using a combined perfusion and physical agitation approach that retains key ECM proteins, minimizes residual cellular debris, and retains gross airway and vascular architecture. To maximize use of the larger acellular lungs, we developed a technique to excise small pieces with identifiable bronchovascular bundles. By coating the excised segments with calcium alginate prior to cell inoculations, we are able to inflate individual
Support
Studies were supported by NIH ARRA RC4HL106625 (DJW), NHLBI R21HL094611 (DJW), R21HL108689 (DJW), and the UVM Lung Biology Training grant T32 HL076122 from the NHLBI. The Proteomics Core Facility is supported by the Vermont Genetics Network through NIH grant 8P20GM103449 from the INBRE program of the National Institute of General Medical Sciences (NIGMS) and the National Center for Research Resources (NCRR).
Acknowledgments
The authors are grateful to Helene Langevin MD, University of Vermont, and the Langevin lab for their assistance in obtaining porcine lungs and the autopsy staff at Fletcher Allen Hospital; Meagan Goodwin, Roberto Loi and Melissa Lathrop for helpful discussions; John Wallis and Amanda Daly for performing the first manual decellularizations; Elliot Marks for assistance with calcium alginate hydrogels and critical reading of the manuscript; Michael Bula and Alex Trick for compiling the FLIR
References (39)
- et al.
The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs
Biomaterials
(2013) - et al.
Human lung cancer cells grown on acellular rat lung matrix create perfusable tumor nodules
Ann Thorac Surg
(2012) - et al.
The effects of storage and sterilization on de-cellularized and re-cellularized whole lung
Biomaterials
(2013) - et al.
Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies
Biomaterials
(2013) - et al.
Decellularization of human and porcine lung tissues for pulmonary tissue engineering
Ann Thorac Surg
(2013) - et al.
Enhanced in vivo function of bioartificial lungs in rats
Ann Thorac Surg
(2011) - et al.
Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells
Cell Stem Cell
(2012) - et al.
An overview of tissue and whole organ decellularization processes
Biomaterials
(2011) - et al.
Engineered whole organs and complex tissues
Lancet
(2012) - et al.
Texas 3-step decellularization protocol: looking at the cardiac extracellular matrix
J Proteomics
(2013)
Alginate: properties and biomedical applications
Prog Polym Sci
Real-time in vivo detection of biomaterial-induced reactive oxygen species
Biomaterials
Tissue-engineered lungs for in vivo implantation
Science
Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded
Tissue Eng Part A
Regeneration and orthotopic transplantation of a bioartificial lung
Nat Med
Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation
Tissue Eng Part A
Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells
Tissue Eng Part A
Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized whole lung scaffolds
Respirology
Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation
Am J Respir Crit Care Med
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