Figure 1 Porcine liver decellularization. Whole native liver (A) before perfusion with Triton X-100 (B) and SDS (C). Indicated by panels on the SDS-treated liver, the preserved major (D) and minor (E) vessels are visible at the end of the protocol. Native liver sections stained with H&E (F) and Masson’s trichrome (G) help illustrate the complete cellular removal seen in their respective decellularized counterparts (H and I). Arrows indicate preserved vessels in decellularized matrices compared with native structures. Scale bar Z 100 mm (FeI).

detergents and, in some cases, enzymatic means, but the resultant matrices must demonstrate noncytotoxocity and, moreover, scaffolds must support cell growth to be considered for organ bioengineering purposes (Figure 3). HepG2 cells were statically seeded onto decellularized liver scaffolds and maintained for

Figure 2 Preservation of discreet ultrastructural components. Positive immunostaining for vascular elements elastin (ELN; A) and laminin (LN; B). C: Scanning electron micrographs of the decellularized porcine liver showed distinct areas of the capsule, vessels, and parenchyma, shown under higher magnification in DeF, respectively. Original magnification: 50 (C); 200 (DeF). Scale bar Z 200 mm (A and B).

Figure 3 In vitro noncytotoxicity of porcine liver matrix scaffolds. HepG2 cells were statically seeded onto scaffolds and cultured out to 21 days to determine whether any cytotoxic compounds would be released from the decellularized matrices. TUNEL staining of the scaffolds at 7 (A) and 21 (B) days indicated minimal apoptotic response of HepG2 cells to liver scaffolds, in both close contact with scaffolds and within cell masses (arrows), compared with DNase-treated positive control samples (C). LM, liver matrix. Scale bar Z 50 mm (AeC).

up to 21 days, when dense cell layers were observed. Cells at day 7 (Figure 3A) and day 21 (Figure 3B) did not exhibit apoptotic markers, as displayed in control samples with degraded DNA (Figure 3C) and confirmed by TUNEL staining. Cells were observed attached to the surfaces of matrices, with minimal penetration into the liver matrix scaffold.

Immunogenicity of Liver Matrix Scaffolds

To investigate the immunogenicity of our decellularized scaffolds (rodent and porcine), we implanted naked scaffolds (Figure 4, C and D) into the s.c. dorsal adipose tissue of rats (Figure 4A). Scaffolds were recovered (Figure 4B) with surrounding tissue intact to measure cellular infiltration and host response, with no visible fibrous encapsulation of scaffolds or exudates observed at implantation sites. After 7 days (Figure 4, E and F), cells had migrated into both allogeneic (rat) and xenogeneic (porcine) scaffolding, with no signs of an inflammatory response. Over 28 days (Figure 4, G and H), host cells continued to populate each recovered scaffold, with no noticeable adverse host response surrounding the matrices. Systemic white blood cell counts were collected from animals during the experiment and compared with rodents who underwent implantation surgeries, but received no matrix implant (sham). Total white blood cell counts (Figure 4I), lymphocyte counts (Figure 4J), and monocyte counts (Figure 4K) were not significantly different between groups (P > 0.05) at 2, 7, 14, and 28 days, indicating no major systemic host response compared with normal surgical recovery. Lymphocyte count results were supported by little to no CD3þ T-cell activation at the implantation site, as seen over the 28 days in either group (Figure 5, A and B). Many cells that infiltrated the implanted scaffolds were positive for the panmacrophage phenotypic marker, CD68 (Figure 5, C and D). However, as seen previously, neither the monocyte counts (as macrophage precursors) (Figure 4K) nor the cells expressing markers indicating adoption of the M1 (CD86þ) or M2 (CD206þ) phenotypes (Figure 5, EeH) showed an increase.

Discussion

In this study, we developed a successful protocol for the generation of clinically relevant sized decellularized porcine

livers, as derived from our previous rodent liver decellularization method.28 These studies demonstrated, after detergent perfusion and rinsing, cellular clearance and preservation of the vasculature tree and ECM proteins, as shown by our

previous scaffolds24,28 or scaffolds generated by other groups.26,30,42 Maintenance of patent blood vessel structures

provides an essential nutrient distribution network for whole organ regenerative studies. Without blood vessel structures, static transport limitations within the scaffolding material will generate necrotic pockets because cell proliferation would leave the cells most distant from nutrient sources starved.38 With blood vessel structures, we avoid such a

detrimental situation and instead replicate the body’s nutrient delivery and toxin removal by perfusing the vasculature at model pressure and flow conditions.

The vasculature also provides a lattice structure for endothelial cell attachment and proliferation that will be crucial for prevention of thrombosis on vascular anastomosis within the recipient. Specifically for the liver, an implantable unit will need to be directly connected to the host vasculature. A previous study demonstrated the successful decellularization and implantation25 of porcine kidney scaffolds, which con- firmed that an acellular matrix could withstand the strain of surgical implantation and exposure to arterial blood flow. Our

liver scaffolds have also maintained the integrity of the organ’s capsule, critical for preventing increases in permeability and circulation leaks that would be detrimental in maintaining the closed-loop vasculature.

The preservation of basement membrane proteins, such as collagens, fibronectin (data not shown), elastins, and laminins, has been shown to maintain hepatic cell function for longer time periods than classic substrates.43e45 As well as primary cells, embryonic and mesenchymal stem cells have been infused into decellularized scaffolds and shown to develop organ-specific phenotypes and functions simply through scaffold interaction.7,42 Our present studies focused on minimizing the cytotoxicity of our scaffold material and demonstrated no contact or soluble toxicity, with maintenance of HepG2 hepatoblastoma cells over 21 days. Our follow-up research is focused on the co-culture of primary hepatic and endothelial cells on the matrix material for the development of a functional and implantable liver device.

Related to cellular engraftment, viability, and proliferation, there are ongoing investigations into the effects of g sterilization and peracetic acid chemical sterilization on scaffold elasticity.46 It has been reported that the elastic modulus of the underlying substrate can directly affect cell attachment, behavior, and viability.47,48 Anecdotal observations suggested significant alteration of the physical scaffold

stiffness pre-g and post-g sterilization, supporting previous studies.49,50 However, specifics into the impact these changes

have on tissue regeneration are limited.8 We believe each step in our decellularization protocol should focus only on the removal of the donor cellular material, while minimizing the impact on inherent mechanical properties of the substratum. Ongoing studies are examining alternatives to irradiation to achieve whole organ sterilization before reseeding.

Before progressing to the transplantation of regenerated liver tissue, a basic understanding of the host immune response toward the scaffolding material is essential to determine whether a decellularized biomaterial is able to affect proin- flammatory macrophage activation.38,51 Our studies assessed the response to rodent and porcine decellularized liver biopsy specimens within a rodent model (allograft versus xenograft proteins). In doing so, we reaffirmed the similarity of the scaffold components between species and reduced future concerns over matrix properties when evaluating host immune response to a recellularized scaffold. The influx of macrophages without activation, as seen with our scaffold materials, is not uncommon and may, instead, be an indication of the influence of potential factors stored in the matrix material itself.32,35

In summary, our protocol adaptation was able to generate large-scale organ scaffolds of a clinically relevant size for further investigations of organ regeneration. Our matrices preserved essential ECM proteins for cell engraftment and function, as well as the vasculature required for nutrient distribution for whole organ reseeding. Neither our previously developed rodent scaffolds nor newly prepared porcine scaffolds elicited a host immune response, while also readily being colonized by host cells. These results indicate that a naturally derived ECM scaffold is viable for the next stages of regeneration of a bioengineered hepatic tissue.

Acknowledgments

We thank Dr. James Jordan and Magan Lane (Wake Forest University, Winston-Salem, NC) for the provision of pig livers and Daniel Deegan, Erica Wieser, and Kayla Trivette for technical support.

Supplemental Data

Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.05.002.

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