Manufacturing of fiber scaffolds and cell seeding

Polycaprolactone (PCL) fiber materials are fabricated using an electrospinning method. Molecular weight of PCL is 45 000 Da (Sigma-Aldrich). The electrospinning process is performed using 18wt% PCL solution dissolved in chloroform:ethanol at a ratio of 9:1. Two high-voltage sources are used to generate positive and negative potentials. The positive source is connected to a syringe needle, whereas the negative source is connected to a collector. Voltage applied to the syringe needle is 20 kV, as well as voltage of the collector. Distance between syringe needle and collector was approximately 20 cm. Obtained 3D PCL fiber materials can be stored until use. From the bulk material of electrospun nanofiber mats, small discs with areas of approximately 15 cm2 are cut out and placed in 24-well plastic cell culture plates. Scaffolds are sterilized by gamma sterilization in 70% filtrated ethanol for 30 minutes and further left under UV-light for 30 minutes. All scaffolds are incubated in cell culture medium at 37°C in a humidified 5% CO2 incubator for 1 hour prior to cell seeding to facilitate cell attachment onto the nanofiber. Human skin derived precursors (hSKP) are seeded and cultured on the on fiber scaffolds for 7 days.