Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart-lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart-lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ-specific composition, as evidenced by differences in organ-specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics.

In vitro stability of therapeutically relevant, internally truncated dystrophins.

BACKGROUND: The X-linked recessive disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin. Despite its large size, dystrophin is a highly stable protein, demonstrating cooperative unfolding during thermal denaturation as monitored by circular dichroism spectroscopy. In contrast, internal sequence deletions have been associated with a loss of the cooperative unfolding and cause in vitro protein aggregation. Several emerging therapy options for DMD utilize internally deleted micro-dystrophins and multi-exon-skipped dystrophins that produce partially functional proteins, but the stability of such internally truncated proteins has not been investigated. METHODS: In this study, we analyzed the in vitro stability of human dystrophin constructs skipped around exon 45 or exon 51, several dystrophin gene therapy constructs, as well as human full-length and micro-utrophin. Constructs were expressed in insect cells using the baculovirus system, purified by affinity chromatography, and analyzed by high-speed sedimentation, circular dichroism spectroscopy, and differential scanning fluorimetry. RESULTS: Our results reveal that not all gene therapy constructs display stabilities consistent with full-length human dystrophin. However, all dystrophins skipped in-frame around exon 45 or exon 51 show stability profiles congruent with intact human dystrophin. Similar to previous studies of mouse proteins, full-length human utrophin also displays stability similar to human dystrophin and does not appear to be affected by a large internal deletion. CONCLUSIONS: Our results suggest that the in vitro stability of human dystrophin is less sensitive to smaller deletions at natural exon boundaries than larger, more complex deletions present in some gene therapy constructs.