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Biopreservation of Living Tissue Engineered Nerve Grafts Built from Clinically Relevant Biomass
Robert B. Shultz, PhD1; Franco A. Laimo, BS2; Zarina S. Ali, MD, MS2; D. Kacy Cullen, PhD2; Kritika Katiyar, PhD?
1Axonova Medical, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA

Introduction: Tissue engineered nerve grafts (TENGs) built from living neurons and axons have been previously shown to repair critical gap-length (1cm-5cm) peripheral nerve defects in rodents and swine, respectively, at levels comparable to gold-standard sensory nerve autografts. To translate this promising technology, grafts have been built from GalSafeŽ porcine neurons (engineered to elicit minimal immune responses in humans). In order to facilitate graft shipping, handling and storage, however, biopreservation protocols capable of extending graft viability and sustaining graft potency are essential. We have previously shown that TENGs built from embryonic rat tissue can survive and remain efficacious after up to 28 days of storage at 4oC in commercially available preservation media; here we assess the impacts of this protocol on TENGs built from GalSafeŽ porcine tissue.
Materials & Methods: Embryonic day 40 GalSafeŽ dorsal root ganglia were harvested, plated in custom mechanobioreactors, and subjected to stretch-growth, resulting in grafts of 4cm length according to previously established protocols. Cultures were then encapsulated in a matrix of porcine type I collagen and transferred into commercially available nerve guidance tubes to form TENGs. These TENGs were then stored in commercially available biopreservation media at 4oC. Media was sampled at regular intervals and tested for the presence of lactate dehydrogenase, an intracellular enzyme normally sequestered in cells with intact plasma membranes, as well as neurofilament light fragment, an indicator of cytoskeletal damage. Finally, at the terminal timepoint, TENGs will be fixed and subjected to histological analysis.
Results: GalSafe TENGs spanning up to 4 cm in length and containing robust, fasciculated, and aligned axon tracts could be repeatedly fabricated in just a few weeks. By growth, morphometric, and other metrics, GalSafeŽ TENGs were found to be indistinguishable from TENGs built from wild-type porcine DRG. Stretch-grown GalSafeŽ neurons and axon tracts were found to remain viable after up to 55 days of cold storage. Biochemical characterization of biopreserved GalSafeŽ TENGs remains ongoing.
Conclusions: Biopreservation of GalSafeŽ TENGs can be routinely achieved using commercially available reagents, advancing TENGs as an off-the-shelf repair strategy for major PNI. Notably, because 4oC can be reached in standard refrigerators, our protocol does not necessitate the use of specialized equipment.


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