Figure 4 Surgical technique: laser ablation, lipotransplantation and epidermal cell suspension graft. Intraoperative views: A) the skin scarred area was prepared by a soft laser superficial ablation then fat injections have been performed using a spoon-tip blunt micro-cannula (1 mm). B) deeper Co2 laser ablation at the end of lipofilling prepared a bleeding dermal graft recipient site. C) epidermal non cultured cells were slowly dropped on the dermal bed (total volume of suspension dropped 1.3 ml). Laboratory phase 1. Plasma preparation: patient plasma

was obtained by collecting 7 ml of whole blood into heparin-treated tubes after centrifugation.   2. Preparation of single cell suspension: under sterile conditions, skin samples were broken into small pieces and incubated with 0.25% trypsin-0.05% ethylenediamine tetraacetic acid (EDTA) (Gibco BRL, Milan Italy) at 37°C for GDC-0449 in vivo 30 min whilst the recipient site was prepared. In order to prevent digestion of separated cells, the reaction of trypsin-EDTA Regorafenib chemical structure was stopped by adding one volume of patient plasma and cell suspension was then filtered through a 70-μm cell strainer (BD Bioscences, Milan Italy). Finally,

the cell suspension was centrifuged for 5 min at 800 rpm to obtain a cell pellet, which was suspended in 0.4 ml of patient plasma. It was then transported to the operation theatre where the cell suspension was aspirated and drawn up into a clean pentoxifylline syringe, ready for application. To monitor cell viability about 10% of cell suspension preparation was seeded into cell culture plates. Fibroblasts, keratinocytes and melanocytes were cultured separately for a week [8, 9], morphological observations documented the presence of active replicating cells (Figure 5 A,B,C).   Figure 5 Microscopic assay of epidermal cell suspension viability. Microscopic observation of cell cultures. Melanocytes (A), Keratinocytes (B) and Fibroblasts (C)

were maintained in specific commercial culture medium and routinely https://www.selleckchem.com/products/su5402.html observed under contrast microscope. Specific morphologic analysis confirmed the presence of epidermal cells and dermal fibroblast. The capacity to seed and to proliferate demonstrated that cell suspension contained mostly viable cells. Original magnification 20×. Results Five days after surgical treatment, all the medications were gently removed by 0.9% NaCl solution moistening. At the time of the first medication the cell graft demonstrated to be well integrated, in all patients. Veloderm™ membranes have been applied once more at medication time, on all the grafts for seven days more. At the second medication, twelve days after the surgical treatment, the grafts were fully integrated and the treated areas were unnoticeable if compared to the surrounding untreated skin. Only in one patient a small area (about 2 mm) in the peripheral region lightly bleeding, was successfully treated with a zinc oxide moisturizer.