THE USE OF MELANOCYTE/KERATINOCYTE CO-SUSPENSION FOR THE RE-PIGMENTATION OF AMELANOTIC PATCHES IN VITILIGO

Keratinocyte/melanocyte co-suspension preparation ‘CellStat’ is a one-step keratinocyte/melanocyte isolation process, which is performed under aseptic conditions in the operating theatre. The skin biopsy is taken from the patient, processed and the keratinocyte/melanocyte co-suspension generated is sprayed back onto the site requiring coverage. This process is entirely autologous and uses medium supplemented with patient’s serum prepared on the day. SPECIMEN REQUIREMENTS The skin biopsy is to be taken from an area of normo-pigmented skin adjacent to, but not within 2cm of, the leukodermic patch to enable the closest colour-matched start point (See D, Figure 1). The donor area will be subcutaneously infiltrated with 0.25% bupivacaine with 1:400,000 adrenaline and the biopsy taken using the Zimmer electric dermatome with 1 inch plate and set at 7/1000ths of an inch thick. The donor area will be dressed with a thin hydrocolloid dressing (DuodermTM Thin, 3M Healthcare, St Paul, Minnesota, USA). Four tubes of blood will be taken from the patient before the procedure begins from which serum will be prepared. The blood samples will be centrifuged at 3000rpm for 15 minutes to separate the serum from the cellular components. The autologous serum (AS) is pooled into a sterile 50ml tube using a transfer pipette. Ten millilitres of this serum is added to a bottle of sterile Dulbecco’s Modified Eagle’s Medium (DMEM) to make the DMEM-2%AS required for re-suspension of cells. The skin biopsy will be rinsed thoroughly in phosphate buffered saline (PBS) before being cut into small, narrow strips (~0.5cm x 2cm). These skin pieces will then be transferred into a petri dish containing warm Dispase II solution (Gibco Invitrogen; 4mg/ml in PBS) and incubated for 15-20 minutes on a heating block set at 37oC. Dispase cleaves the adhesion molecules at the dermo-epidermal junction allowing separation of the epidermal sheets from the dermal tissue. At the end of the Dispase II treatment, the epidermal sheets can be gently peeled from the dermal tissue and kept transiently in a PBS containing dish until all the epidermal sheets have been obtained. The sheets are then transferred into a 70ml container containing a magnetic stirrer and 20mls of 0.05% trypsin-0.53mM EDTA (Gibco Invitrogen) and trypsinised for 5 minutes on the magnetic plate set at 700rpm. This level of agitation and length of trypsinisation allows for the isolation of the majority of the keratinocytes and melanocytes from the basal epidermal layer without isolating a large number of the differentiated suprabasal cells. The trypsin is quenched with 20mls of soybean trypsin inhibitor (Sigma; 0.1mg/ml in DMEM). The resultant cell suspension is passed through a 70mm cell to remove the undigested cornified sheets and large cell clumps and centrifuged at 1200rpm. The cells are re-suspended in the required volume of media for administration. SURGICAL PROCEDURE The site to receive the cell suspension (A or B) is randomised by sealed envelopes produced before the commencement of the study. The randomisation will be double-blinded to surgeon and patient, known only to the technicians preparing the treatment samples. Three areas within the study patch will be delineated by ink lines. Each area will be 1cm x 1cm in area and separated from each other by at least 2 cm. Two of the areas will be wholly within the study patch and not encroaching at any point on the normo-pigmented skin at the patch edge. The third area will cross the edge of the patch onto normo-pigmented skin at one of its faces. The treatment will be organised as follows: The three treatment areas will be subcutaneously infiltrated with 0.25% bupivacaine with 1:400,000 adrenaline; for local anaesthesia, patient comfort post-operatively and to ensure a bloodless bed for study dressing application. Patches A and B – these will be sited entirely within the leukodermic area. Both will be dermabraded using a diamond-tipped burr. Both patches will be ‘treated’; one with medium PLUS keratinocyte/melanocyte cell suspension, the other with medium alone. Which patch receives the suspension will be randomised and double-blinded. Post-dermabrasion, the test areas will be covered with individual adhesive, occlusive, transparent film dressings (TegadermTM, 3M Healthcare, St Paul, Minnesota, USA). A sterile 25G (orange-hubbed) needle will be used to puncture the film over the dermabraded area and the treatment fluid will be injected until the dermabraded area is completely covered. Area C will be located so that the dermabraded area overlaps the edge of the patch onto normo-pigmented skin and will be treated by dermabrasion alone and similarly covered with TegadermTM. A gentle compression dressing will be applied over the whole treatment area to prevent reactive haemorrhage at the treatment sites and consequent haematoma formation under the occlusive dressings.