Femke Mathot

Chapter 8 130 supported by MSCs. In order to determine the clinical potential of dynamic seeding of MSCs, this study was designed to examine the interaction between MSCs and two commercially available nerve graft substitutes; the Avance® Nerve Graft and the NeuraGen® Nerve Guide. The purpose of this study was to determine (I) if the interaction of human adipose derived MSCs with the NeuraGen® Nerve Guide and the Avance® Nerve Graft influences cell viability, (II) if human adipose derived MSCs can be dynamically seeded and distribute uniformly onto these nerve substitutes, and (III) if dynamic seeding and optimized timing improves the efficiency of MSC- seeding. METHODS General design Two experiments were designed to ascertain the interaction of the nerve graft substitutes with human adipose derived MSCs and to determine the optimal seeding times, survivability and distribution of MSCs. Adipose Derived Mesenchymal Stem Cell Collection and Preparation Passage five human MSCs, isolated from abdominal lipo-aspirates of a male donor were used in this experiment. Cells were provided by the Mayo Clinical Human Cellular Therapy Laboratory (Rochester, Minnesota, USA). These MSCs have been tested extensively for multi-lineage potential, cell surface markers (CD73, CD90, CD105, CD44, CD14, CD45) and RNA-sequence transcriptome profiles previously.(23-25) For MSC culture, growth media consisting of a-MEM (Advanced MEM (1x); Gibco by Life TechnologiesTM, Cat #12492013), 5% platelet lysate (PLTMax®; Mill Creek Life Sciences), 1% penicillin/streptomycin (Penicillin- Streptomycin (10.000 U/mL); Gibco by Life TechnologiesTM Cat #15140148), 1% GlutaMAX (GlutaMAXTM Supplement 100X; Gibco by Life Technologies, Cat #35050061) and 0.2% heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; NOVAPLUS®) was used and media was changed every 72 hours. 29-31 Experimental Design and Measurement of Mesenchymal Stem Cell Viability To test whether the chemical products used during processing of the Avance® Nerve Graft and the NeuraGen® Nerve Guide are harmful to MSCs, cell metabolic activity was measured using (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) (MTS) assays that were performed according to the manufacturer’s protocol (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega®). Twenty-four 2mm-segments of the Avance® Nerve Graft and the NeuraGen® Nerve Guide were soaked in a-MEM for two hours prior to the MTS assay. The soaked nerve substitute segments and 5,000 MSCs dissolved in 100μL growth medium were placed into wells which were coated with pHEMA (Poly 2-hydroxyethyl methacrylate; Sigma Cat # P3932) to prevent migration of the MSCs to the plastic well. Any influence of the pHEMA coating on cell viability, was