Acellular biologic scaffolds have been created from a variety of different tissues, including skin, esophagus, trachea, heart, and lung (Keane and Badylak 2014). Creation of biologic scaffolds involves removing the endogenous cell population while maintaining the gross structure of the ECM and its composition (Fig. 8.2). While a variety of methods have been utilized to generate acellular scaffolds, those that minimize destruction to ECM components and loss of mechanical properties are viewed as most desirable, as these are critical inputs to regulating cellular behavior. The most common techniques utilize a series or combination of chemical agents (e.g., detergents, solvents, acids/bases, and hypotonic/hypertonic solutions), biological agents (e.g., enzymes or chelating agents), and physical methods (e.g., freeze/thaw cycles, sonication) and vary in duration based on the species, organ, and method of decellularization [reviewed in (Crapo et al. 2011)] (see Table 8.1 for a list of commonly utilized reagents). In general, most protocols last from 1 to 7 days.

In the lung, maintenance of both large and small airways and vessels, as well as the more delicate alveolar, capillary, and lymphatic systems, is vital. Thus far, several techniques have been reported for decellularization of healthy mouse, rat, porcine, non-human primate, and human lungs (Tables 8.2 and 8.3). Perfusion decellularization has been most commonly utilized for whole organ decellularization, but there have also been reports of excising segments from native lung and decellularizing smaller segments (O’Neill et al. 2013; Nakayama et al. 2013; Zhou et al. 2014; Parker et al. 2014). Detergents are most commonly utilized for perfusion-based lung decellularization and several works have compared differences in the proteomic composition and mechanical properties of the final acellular scaffold and potency for recellularization in the different detergent-based protocols (Wallis et al. 2012; Gilpin et al. 2014; O’Neill et al. 2013). The most commonly utilized detergents for lung are either the ionic detergents sodium deoxycholate (SDC) or sodium dodecyl sulfate (SDS) in combination with the nonionic detergent Triton X-100 (Bonenfant et al. 2013; Bonvillain et al. 2012; Booth et al. 2012; Daly et al. 2012a; Jensen et al. 2012; Longmire et al. 2012; Sokocevic et al. 2013; Wagner et al. 2014a, b; Wallis et al. 2012; Gilpin et al. 2014; Ott et al. 2010; Song et al. 2011; Parker et al. 2014). Alternatively, zwitterionic detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have also been used (Petersen et al. 2010, 2011, 2012; O’Neill et al. 2013). Further, most protocols incorporate hypertonic lysis of cells with sodium chloride (NaCl) as well as a DNase and/or RNase step to clear residual DNA and RNA which is difficult to remove. There is, as yet, no consensus on the best route of administration and removal of decellularization agents (i.e., vascular versus airway perfusion or a combination). While both vascular-only perfusion and a combination of vascular and airway perfusion have produced acellular scaffolds capable of supporting recellularization, how differences in protocols and routes of administration for decellularization reagents might affect recellularization protocols or potential immunogenicity of implanted scaffolds is not yet known.

While standards do not yet exist for generating decellularized scaffolds, Crapo et al. proposed three minimal criteria: (1) <50 ng dsDNA per 1 mg ECM dry weight; (2) <200 bp DNA fragment length; (3) absence of visible nuclear content in histological sections by 4′,6-diamidino-2-phenylindole (DAPI) or hematoxylin–eosin (H&E) staining (Crapo et al. 2011). However, these criteria do not set forth additional characteristics which acellular scaffolds should have, such as preservation of specific ECM components, mechanical properties, or biological activity.

Scaling up decellularization protocols from rodent models to potential large animal xenogeneic sources (e.g., porcine) or human scaffolds presents additional challenges. In addition to anatomical differences, the larger size is a significant consideration for the actual decellularization technique. While rodent and macaque lungs can be decellularized by hand, higher pressures and volumes must be utilized for sufficient inflation of perfusion pathways (e.g., vasculature, airways, etc.) in larger organs to ensure that perfused solutions and the ensuing cellular debris are cleared from the lungs. All published protocols to date for decellularizing whole large animal or human lungs utilize perfusion pumps to generate acellular scaffolds which can support recellularization (Wagner et al. 2014a, b; Nichols et al. 2013; Booth et al. 2012; Petersen et al. 2010; Price et al. 2015; Gilpin et al. 2014). While not clinically translational for transplantation, human and porcine lung segments have also been decellularized using small segments (Zhou et al. 2014; O’Neill et al. 2013; Nakayama et al. 2013; Parker et al. 2014) (Table 8.3).

A variety of post-decellularization techniques can be utilized to assess differences in potential decellularization techniques, including histologic, immunofluorescent staining, and DNA detection and quantification (Fig. 8.2). Further, proteomic analysis is a powerful tool which can be used to help delineate differences between protein loss and retention in protocols and can aid in the selection of optimal protocol parameters such as flow rates or pressures (Wagner et al. 2014b). For example, proteomic analysis can help delineate the impact of different steps and parameters in decellularization protocols (e.g., flow rate, pressure, rinse volumes, etc.) which preferentially retain certain ECM components or minimize retention of cellular-associated proteins (Wagner et al. 2014a, b; Booth et al. 2012).

The lung ECM itself has long been known to provide instructional cues during prenatal development as well as postnatally in maintaining tissue homeostasis and directing remodeling responses after injury (Fernandes et al. 2010; Lin et al. 2010; Mariani et al. 1997; Nguyen and Senior 2006; Rippon et al. 2006). While synthetic scaffolds offer the advantages of precise control and the ability to be more readily mass produced and stored, synthetic materials lack the biologically inductive capabilities observed in acellular scaffolds. Acellular scaffolds retain native integrin-binding sites in their correct spatial arrangement, in addition to preserving organ macro-architecture. A number of studies have shown their ability to induce phenotypic differentiation in the absence of the use of additional growth factors. While synthetic materials could be engineered to include specific integrin-binding sites to enhance cell adhesion [e.g., Arg-Gly-Asp (RGD) binding sites], it remains unknown what specific integrin-binding sites need to be included and in what spatial arrangement they need to be. Hybrid materials, consisting of synthetic and acellular matrix components, are also an attractive possibility and could be utilized to enhance cell adhesion and biological activity while taking advantage of the ability to more precisely manufacture scaffolds or scaffold components with synthetic materials (Ingenito et al. 2010, 2012; Cortiella et al. 2010; Nichols et al. 2013; Fischer et al. 2011). (Differences between acellular and synthetic scaffold approaches summarized in Table 8.4.)

Owing to the importance of ECM components, retention of key ECM components is a critical parameter to control and assess as an endpoint when evaluating potential decellularization protocols. The precise combination of ECM proteins that must be retained to preserve the ability of the acellular scaffold to give organotypic cues for cellular differentiation and functional tissue level assembly remains unknown. The major structural and functional molecules in the ECM include both proteins such as collagens, elastin, fibronectin, and laminins as well as a variety of glycoproteins including the glycosaminoglycans (GAGs). Collagens are important structural components of the lung and are responsible for overall mechanical strength, while elastin gives the lung its elastic properties of reversible distension and intrinsic recoil. GAGs help control macromolecular and cellular movement across the basal lamina and may play a role in the mechanical integrity of the lung. Matrix molecules are generally highly conserved proteins in eukaryotic organisms. This may theoretically explain the lack of an adverse immune response seen in xenotransplantation of other decellularized organs such as skin, trachea, and esophagus (Jungebluth et al. 2012a, b; Keane and Badylak 2014; Fishman et al. 2013).

There are a variety of techniques which have been used to evaluate ECM components, including histology, immunohistochemistry, Western Blotting, mass spectrometry-based proteomics, and component-specific assays such as Picrosirius, Fastin elastase, etc. (Table 8.2). The majority of decellularization techniques used for lung results in loss of elastin and sulfated GAGs in all species (Ott et al. 2010; Petersen et al. 2010; Price et al. 2010; Bonenfant et al. 2013; Bonvillain et al. 2012; Daly et al. 2012a; Jensen et al. 2012; Sokocevic et al. 2013; Wallis et al. 2012). In head to head comparison studies in perfused lungs, SDS and SDC have been found to retain more elastin as compared to CHAPS-based protocols (Petersen et al. 2012; Wallis et al. 2012). However, despite the differences in retention of ECM components, inoculated cells appear to behave similar in the repopulation assays currently used (including histological and immunofluorescence evaluation). Therefore, it remains unknown if there is an optimal decellularization protocol, and if so, which is best suited for translation to the clinic.

In contrast to traditional protein detection methods, mass spectrometry-based proteomics is gaining traction as an invaluable tool for assessing acellular scaffolds. In addition to detecting ECM composition and residual proteins in acellular scaffolds, it has been used for distinguishing differences between decellularization methods or lung origin, including disease states or donor age (Wallis et al. 2012; Bonvillain et al. 2012; Sokocevic et al. 2013; Wagner et al. 2014a, b; Booth et al. 2012; Nakayama et al. 2013; Gilpin et al. 2014). One particularly striking and consistent result among the various groups utilizing this analysis approach is the amount and breadth of non-ECM proteins detected in the scaffold following decellularization. In particular, cytoskeletal elements and histones appear to be retained in the scaffolds, while lesser secreted proteins are detected. This suggests that transmembrane proteins and their associated cytoskeletal elements may remain anchored to the ECM with the currently used decellularization protocols. The impact of these residual proteins on recellularization, including potential immunogenicity, remains unknown.

A variety of in vitro assessments have been utilized to begin to establish metrics for assessing the potential functionality of acellular scaffolds. Investigators have explored both micro- (Melo et al. 2014a, b; Nonaka et al. 2014; Booth et al. 2012) and macro-scale (Daly et al. 2012a; Wallis et al. 2012; Nichols et al. 2013) mechanical measurements of acellular lungs as well as force tension relationships in linear strips of decellularized lungs (Petersen et al. 2012; O’Neill et al. 2013). While techniques such as atomic force microscopy (AFM) are useful in obtaining topographical information and initially assessing mechanical properties of the scaffolds (Melo et al. 2014a, b; Nonaka et al. 2014; Booth et al. 2012), these results do not necessarily translate to recellularization and functional performance. Traditional lung mechanics testing of acellular scaffolds has shown that in the absence of cells and surfactants, acellular scaffolds are stiffer than their naïve counterparts (Daly et al. 2012a). Introduction of exogenous surfactant into the acellular scaffolds can partially restore lung compliance (Daly et al. 2012a). This is an important finding and indicates that during recellularization strategies, serial measurements of lung mechanics could be used as a non-invasive and non-destructive means to assess functionality of the regenerating scaffold. For example, decreases in elastance could be used as a measurement of de novo surfactant production.

The lung is comprised of many different cell subtypes and all uniquely contribute to some critical aspect of lung function (Morrisey and Hogan 2010). This heterogeneous cell population is replenished by resident stem or progenitor cells following injury. Regeneration of lungs suitable for transplantation will require some minimal restoration of these subtypes for subsequent long-term functionality. While a variety of cell sources are being investigated for recellularizing acellular scaffolds, obtaining sufficient cell numbers with any source remains a significant open question. The ideal solution is to use an autologously derived source of cells to minimize post-transplantation immune complications, which are a significant cause of morbidity in transplanted patients. One potential source is the use of fully differentiated primary adult cells, which ideally would come from an autologous source. However, using this approach, the multiple cell types would need to be isolated from the eventual transplant recipient, grown to sufficient numbers ex vivo and then further be able to be used in a recellularization approach to restore functionality. While it has been shown that a strategy such as integrin blocking can be used to direct initial cell engraftment of a single cell population (Daly et al. 2012a; Sun et al. 2014), scaling this clinically and further adding the complex challenge of uniquely directing the right cell population to a specific architectural location would be challenging. Alternatively, autologous endogenous lung progenitor cells from the various compartments could be utilized (e.g., distal and proximal epithelial progenitor cells, endothelial progenitor cells, etc.) along with stromal cells to recellularize acellular scaffolds. However, the same challenges of obtaining sufficient cell numbers for an initial seeding strategy and directing cells to their correct compartment remain. In both instances, however, it remains unknown if normal cells could be obtained from a patient with a preexisting lung disease or if isolated diseased cells could be gene-corrected prior to subsequent recellularization. However, recent work indicates that the scaffold may more significantly contribute to phenotype than cell origin (Parker et al. 2014). An allogeneic cell source could also be used, but this re-introduces the potential for immune complications following transplantation. Furthermore, the identification of bona fide lung progenitor cells in the adult human lung remains controversial.

A potentially more appealing autologous approach is the use of induced pluripotent stem (iPS) cells, which are derived from reprogramming somatic cells to a stem-cell-like state. While iPS cells avoid the ethical controversies surrounding the use of embryonic stem cells (ESCs)—stem cells derived from the inner blastocyst of in vitro fertilized embryos—they have been shown to retain epigenetic memory of their tissue origin and have been shown to form teratomas. iPS cells are typically derived from dermal fibroblasts, and thus, differentiating them into the various lung cell types has been challenging. However, despite this limitation, recent work has demonstrated that human iPS cells can be differentiated into cells expressing a distal pulmonary epithelial cell phenotype and seeded into acellular human lung scaffolds (Ghaedi et al. 2013; Huang et al. 2014). These results further encourage the use of this approach in moving towards the clinic.

Other potential approaches include the use of fetal homogenates or ESCs. As previously mentioned, ethical concerns remain for either of these approaches, as well as the potential for teratoma formation with ESCs. While initial studies have shown that ESCs can engraft in acellular murine lungs (Longmire et al. 2012; Cortiella et al. 2010), seeding into acellular lungs was not sufficient to induce differentiation and thus optimized in vitro differentiation protocols must be used in conjunction with seeding and repopulation strategies. ESC-derived murine Nkx2-1GFP+ progenitor cells were able to recellularize acellular murine lungs and form alveolar structures, while in contrast, seeding with undifferentiated ESCs resulted in nonspecific cell masses in distal regions of acellular lungs. Fetal homogenates have the distinct advantage of containing all necessary cell populations which have been shown to have some capacity for self-assembly. Further, these cells have been successfully used in the current rodent models of ex vivo regeneration and transplantation. However, in both instances, ethical concerns remain in obtaining these cells and the use of immunosuppressive drugs post-transplantation remains unknown. Tables 8.5 and 8.6 are a summary of recellularization approaches in animal and human models and the phenotypic adoption of seeded cells.