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Schwann Cell Transfer: Does Source and Passage Matter?
Bin Chu, MD1; Robert J. Spinner, MD1; Victor H. Molina, BS2; Shaonan Hu, MD, PhD3; Michael J. Yaszemski, MD, PhD4; Anthony J. Windebank, MD5; Huan Wang, MD, PhD1
1Department of Neurologic Surgery, Mayo Clinic, Rochester, MN; 2University of Puerto Rico School of Medicine, San Juan; 3Department of Hand Surgery, Huashan Hospital, Fu Dan University, Shanghai, China; 4Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN; 5Department of Neurology, Mayo Clinic, Rochester, MN
Introduction: Schwann cells (SCs) have been shown to facilitate regeneration when applied locally at the nerve repair site or inside artificial conduits. Due to its antigenicity autologous cells should be used. In current literature only SCs derived from sciatic nerves have been used in both in vitro and in vivo studies. This poses an obstacle for clinical translation. We developed a protocol to isolate and expand SCs from sural nerve, the most common donor nerve, and compared the yield, purity and bioactivity of these cells to sciatic nerve derived SCs.
Methods: Sciatic nerve and sural nerve were harvested from adult female Lewis rats. A course of predegeneration of the nerves was employed before tissue digestion. Dissociated cells were plated and expanded. Serum tapering was used to eliminate fibroblast contamination. Sural nerve derived SCs (SuSCs) and sciatic nerve derived SCs (SciSCs) were harvested when culture reached subconfluency. Cells were counted and SC purity quantified. Expressions of P75, S100 and GFAP were measured via immunofluorescence staining. Cell bioactivity was determined by the extent of PC12 cell differentiation when co-cultured with SCs. Percentage of neurite-bearing PC12 cells, number of neurites per cell, and neurite length were used as indicators. These parameters were compared between SuSCs and SciSCs. Furthermore, SuSCs culture was passaged till 3rd passage. SC purity, biomarker expression and bioactivity of different passages were compared.
Results: The amount of cells harvested from per mg of sciatic nerve was much more than that from sural nerve (1.52E+05 vs. 3.34E+04). Purity of SciSCs and SuSCs were both above 94%. Expressions of P75, S100 and GFAP were 94.22%, 95.07%and 94.34% for SciSCs and 97.19%, 97.37%, and 98.06% for SuSCs. PC12 cell differentiation in SuSCs co-culture and in SciSCs co-culture was similar. Percentage of neurite-bearing PC12 cells, number of neurites per cell, and neurite length were 40%, 4.74/cell and 61µm for SuSCs and 42%, 3.78/cell and 68µm for SciSCs. For multiple passage cultures, cell purity ranged from 97.2% to 88.6% from P0 to P3. Cell marker expression showed similar trend. Quantification and comparison of bioactivity of cells from different passages is ongoing.
Conclusions: Culturing SCs from sural nerve can achieve comparable purity, cell marker expression and cell bioactivity. Cell yield from sural nerve is lower than that from sciatic nerve. Passaging of cells can increase cell number from sural nerve culture without significantly compromising SC purity, phenotype and bioactivity.
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