Femke Mathot

Chapter 3 48 Seeding efficiency, cell adhesion, distribution and migration A dynamic seeding strategy that was previously described for undifferentiated MSCs was used in this study. 29 In total, 36 processed nerve graft segments of 10mm in length were soaked in growth medium for two hours to remove any toxic decellularization agents and divided among conical tubes containing 10mL growth medium with one million undifferentiated MSCs or differentiated MSCs per nerve. These tubes were placed on a rotating system which was rotated for six, 12 or 24 hours at 37°C (n = 6 per group per seeding duration). Seeding efficiency on each time point was determined by cell counts in the cell supernatant of all the different samples; this provided the average number of free floating cells in the tubes and the average number of cells that were adherent to the nerve. Two supernatant samples (10 m L) were taken out of each of the conical tubes after the seeding duration had passed. Subsequently, one investigator performed three cell counts on each of the supernatant samples. So for each nerve sample (n=6 per group per time point), 6 cell counts were averaged for final analysis in order to minimize potential error. The viability and distribution of the cells seeded on the nerve grafts was studied by live/ dead Cell Viability Assays (Invitrogen, Life Technologies Corporation, NY, USA) and Hoechst stain (Hoechst stain solution; Sigma-Aldrich Corp., MO, USA) and visualized using a confocal microscope (Zeiss LSM 780 confocal microscope). The live/dead and Hoechst stain assays were prepared by one investigator. Both the Live/dead stain and the Hoechst stain were obtained according to standardized protocols; incubation of the samples with live/dead stain (Invitrogen, Life Technologies Corporation, NY, USA) or Hoechst stain mixture (Hoechst stain solution; Sigma-Aldrich Corp., MO, USA) for 20 minutes after which the samples were washed with PBS. Both stains were performed on three samples per group per seeding duration. Cell shape and distribution was evaluated by Scanning Electron Microscopy (SEM) of the three remaining seeded samples per group per time point. To obtain SEM images, the samples were transferred to 2% Trump’s fixative solution (37% formaldehyde and 25% glutaraldehyde) overnight, washed in phosphate buffer, and rinsed in water and dehydrated through a graded series of ethanol. Subsequently, the samples were critical point dried using carbon dioxide, mounted on an aluminum stub and sputter-coated for 60 seconds using gold-palladium. The samples were imaged in a Hitachi S-4700 cold field emission scanning electron microscope (Hitachi High Technologies America, Inc., IL, USA) at 5kV accelerating voltage. The preparation and the imaging of the SEM samples were performed by the Microscopy and Cell analysis core lab of the Mayo Clinic. The distribution of cells on the outer surface was only assessed and described subjectively and were therefore not blinded. An overview of the experimental design is depicted in table 2 . Two extra processed nerves per group were seeded with the previously estimated optimal seeding duration and transferred to 10% formalin and processed and embedded in paraffin to evaluate the migration of cells into the nerve grafts. Three 5µM sections of the proximal- and mid-nerve segment were sectioned and Hoechst stained. The Hoechst-stained cross- sectional segments of the nerves were blinded for the objective assessment of present cells on the inner surface of the samples (present versus absent). The cells were visualized with a confocal microscope (Zeiss LSM 780 confocal microscope; Zeiss, Germany).