Autologous Cellular Micro-graft in Surgical Treatment of Stable Resistant Vitiligo: A Randomized Self-controlled Trial

Under local anesthesia, a 2.5 mm punch biopsy will be used to extract 3 scalp tissue specimens from the patient's occiput behind the ear, using Rigeneracons medical device (CE certified class I; Human Brain Wave, Turin, Italy). The collected specimens will be placed in Rigeneracons by adding 1.5 mL of sterile physiologic solution to the device. The device then generates a cellular suspension by rotation of Rigeneracons at 80 RPM for 2 minutes. Subsequently, the obtained suspension is diluted with an additional 3 mL sterile physiologic solution. The resulting suspension will be added to NCES and placed on one of the derma braded vitiligo patches

• Under local anesthesia, 1 mm punch will be used to extract hair-follicles from occipital scalp behind the ear. Depending on the area to be transplanted, one hair follicle will be etracted for each 1 cm2. The extracted hair-follicles are washed with phosphate buffered saline (PBS) for about 3 times. The hair-follicles are then incubated with 0.25% trypsin - 0.05% ethylene diamine tetra acetic acid (EDTA) at 37°C for 90 min to prepare the single cell suspension. Within 15-20 min of incubation, the cells start loosening from each other. The hair-follicles are subsequently placed in another tube of trypsin and EDTA. At the end of three such cycles, a thin keratinous shaft is left behind. The cell suspensions of all the tubes are added in a single tube. The final cell pellet is obtained by centrifuging the combined cell suspension at 1000 rpm for 5 min. The resulting suspension will be added to NCES and placed on a comparable derma braded lesion

Melanocyte keratinocyte pellet will be prepared. Donor skin will be harvested from the gluteal region ; using a sterile shaving blade mounted on a Kocher's forceps. Cell Suspension will be prepared by placing the donor skin in 0.25% trypsin-EDTA (GIBCO) solution for 40 minutes at 37 ᴼC. sample and trypsin will be poured into a labelled petri dishes then neutralized using ringer's lactate. All the steps will be performed in a laminar air flow bench under strict aseptic conditions. The epidermis will be detached from the dermis which will be discarded. Then the epidermis will be cut into tiny pieces and scrapped so that no pigment is left on the surface. Afterwards, they will be transferred to sterile falcon tubes and centrifuged for 10 minutes at 1,000 rpm. The floating epidermal fragments and supernatant fluid will be removed, leaving the cell pellet containing epidermal cells rich in melanocytes and Keratinocytes at the bottom.