Optimized delivery of skin keratinocytes by aerosolization and suspension in fibrin tissue adhesive

Aerosolized suspensions of keratinocytes provide a potential therapy for wounds, but the effects of aerosolization on cell viability remain unclear. Likewise, little is known of the resulting cell distribution pattern and how this compares to the density required for epithelialization. The potential benefits of cospraying cells in the presence of fibrin adhesive are equally uncertain. Thus, in the present study we have optimized conditions for the aerosolization of cultured keratinocytes using a device (Tissomat (TM)) that supports the option for coapplication with fibrin (Tisseel (TM)). Cell viability was unaffected when sprayed at 10 psi, but a significant reduction in metabolic activity, as determined by the methylthiazoyldiphenol-tetrazolium assay, was observed at higher pressure. Bursts of 0.2 mL cell suspension (1.5 x 10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm(2) within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm(2) at 24 hours. Optimal cell growth in Tisseel (TM) was achieved through dilution of fibrinogen (1-3 mg/mL) and thrombin (2-5 IU/mL). This optimized formulation eliminated fluid run-off postspraying and stimulated a twofold increase in cellular response. Therefore, our in vitro data supports the theory that aerosolized suspensions of keratinocytes in fibrin will benefit healing.