Femke Mathot

Chapter 9 146 and immune responses in human MSCs as a function of time after dynamic seeding onto Avance® Nerve Grafts and NeuraGen® Nerve Guides. MATERIALS AND METHODS General design Human adipose derived mesenchymal stem cells were cultured and seeded onto 30 Avance® Nerve Grafts (AxoGen, Inc., Alachua, Florida, USA) and 30 NeuraGen® Nerve Guides (Integra LifeSciences Corporation, Plainsboro, New Jersey, USA). At 6 different time points after seeding, quantitative polymerase-chain-reaction (qPCR) analyses were performed (5 samples per group). Five additional unseeded Avance® Nerve Grafts and 5 unseeded NeuraGen® Nerve Guides provided the baseline gene expression of the nerve substitutes; 5 samples of unseeded MSCs provided the reference gene expression of MSCs. Human mesenchymal stem cells The Mayo Clinical Human Cellular Therapy laboratory (Rochester, Minnesota, USA) provided the human MSCs used in this experiment. They complied with the criteria defined by the International Society for Cellular Therapy. 20 Multi-lineage potential, presence of cell surface markers (CD73, CD90, CD105, CD44, CD14 and CD45) and RNA-sequence transcriptome profiles have all been tested previously.(23-25) MSCs were cultured in growth media composed of Advanced MEM (a-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®). The MSCs were cultured in an incubator at 37 ° C (5% CO2), growth medium was changed every 72 hours and cells were split at 80% confluence. Passage 5 MSCs were used in this study. 21-23 Nerve allografts and guides Avance® Nerve Grafts are human nerve allografts that have been decellularized and irradiated to obtain non-immunogenic, sterile human nerves with remaining ultrastructure. The NeuraGen® Nerve Guides are composed of purified bovine type I collagen and are empty conduits that do not contain any ultrastructure in the central portion of the guide. 24 Both the Avance® Nerve Graft and the NeuraGen® Nerve Guide have been approved by the Food and Drug Administration for human clinical use since 2007 and 2001, respectively. A total of 35 Avance® Nerve Grafts and 35 NeuraGen® Nerve Guides of 15mm in length were voluntarily provided by Axogen® (Axogen Corporation, Alachua, Florida, USA) and Integra (Integra LifeSciences Corporation, Princeton, New Jersey, USA) respectively. Five samples of each group were used to determine the baseline gene expression and 30 of