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Dose-response Analysis after PEP Administration on Nerve Regeneration in Vitro
Daan J Rademakers, BSc1; Tara Sara Saffari, MD, MSc1; Nicholas Pulos, MD1; Shin Y Alexander, MD2
1Mayo Clinic, Rochester, MN; 2Orthopedics, Mayo Clinic, Rochester, MN

Background Application of Purified Exosome Product (PEP), an off the shelve product, to enhance nerve regeneration has not been investigated before. Exosomes are extracellular vesicles, which play an important role in intercellular communication due to their specific bioactive cargo and are postulated to overcome limitations associated with current cell-based therapy. The aim of this study was to determine the optimal concentration of PEP on nerve regeneration in vitro using a dose-response analysis.
Materials and methods To determine the optimal concentration of PEP on nerve regeneration, Neonatal dorsal root ganglia (DRG) were incubated with (i) 5%, (ii) 10%, (iii) 15% and (iv) 20% PEP (N=6/group). After 72 hours of incubation, the average neurite length of PEP treatment concentrations was determined to quantify nerve regeneration, compared to negative controls. Samples were stained against neurofilament-160. and the average neurite outgrowth was measured three times per sample. Live cell imaging was used to visualize neurite outgrowth every three hours until the end of incubation.
Results The macroscopic appearance of neurites, stained against neurofilament-160, was most evident in the 5% PEP group (figure 1). Compared to untreated DRGs, neurite outgrowth was found superior in the 5% PEP group (P<0.005), and DRGs treated with 10%, 15% and 20% PEP concentration did not show any significant differences (respectively P=0.59, P=0.74, P=0.99). Moreover, no decreasing or increasing trend in neurite length was found after administration of higher concentrations of PEP
Conclusion This study highlights the potential of PEP, an off the shelf Purified Exosome Product, on nerve regeneration. The optimal dosis of 5% PEP was found to be optimal on nerve regeneration an in vitro dose-response analysis. This optimal dosis could be used for further in vivo peripheral nerve basic science research using PEP.
Figure 1: Macroscopic appearance of neurites stained against neurofilament-160 from dorsal root ganglia (DRG). >Neurite outgrowth of DRGs is visualized after incubation with no treatment (A), 5% PEP (B), 10% PEP (C), 15% PEP (D) and 20% PEP (E). The scale bar is set at 200 ?m.


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