Autologous Cellular Graft in Surgical Treatment of Vitiligo

Prior to surgery, two comparable lesions will be selected.

Lesions will be assessed for:

1. Surface Area using point counting technique.
2. Any attempts of pigmentation using VESTA score. Lesions will be derma braded under local anesthesia using fractional laser CO2 resurfacing, Using dot mode off resurfacing will be performed at a power of 18-20 W, dwell time 1,000 milliseconds (DEKA, Florence, Italy). One to three passes will be performed until the epidermis is removed uniformly.

Autologous Cellular Micrografts (ACM) suspension generated by regenera technique 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 melanocyte pellet generated by NCES technique and will be spread on one of the derma braded vitiliginous patches.

Non cultured Epidermal Suspension (NCES):

Melanocyte keratinocyte pellet will be prepared. Donor skin will be harvested from the gluteal region after anaesthetizing the area with intra-lesional Mepicaine L; using a sterile shaving blade mounted on a Kocher's forceps. An expansion ratio of 1:2 was used for each lesion separately to ensure equivalent melanocyte concentration in all transplanted lesions. Cell Suspension will be prepared by placing the donor skin in 0.25% trypsin-EDTA (GIBCO) solution for 40 minutes at 37 ᴼC. Each 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 two labelled 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. The resultant pellet will be suspended once in ACM generated by regenera technique, once in hair follicle suspension and once in 1 ml of ringer's lactate.

Follicular Cellular Suspension:

• 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 pigmented follicles will be extracted 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 60 min divided in to 3 intervals to prepare the single cell suspension. The first interval lasted 30 minutes after which the supernatant fluid containing separated cells was poured into a new falcon tube and neutralized by 1% fetal bovine serum. Fresh trypsin was then added to hair follicles and reincubated for 15 minutes twice.
• 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 melanocyte pellet generated by NCES technique and will be spread on a comparable derma braded lesion.

The resultant suspension will be checked for melanocyte count and viability using trypan blue dye exclusion method to ensure above 250 viable cells/mm2 of the recipient area prior to its spreading on the derma braded vitiligo patch.

Both sites will be covered with a dry collagen sheet followed by sterile surgical pad and tegaderm.

The area will be immobilized after bandaging and the patient will be advised to restrict movement at the operated site.

Patients will start a 1-week course of antibiotics. Dressings will be removed after 1 week, and patients will receive excimer laser sessions, 3 sessions per week for 3 months.

Patients will be followed up monthly for 3 months following the first visit at 1 week.

At 3 months, both qualitative and quantitative assessment will be done. Qualitatively, blinded physician assessment of repigmentation as well as color match using serial photographs will be done. Quantitatively, point counting method as well as VESTA score will be used as well to assess degree of repigmentation.