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

Abstract

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) that supports the option for coapplication with fibrin (Tisseel). 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 (1.5x10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm2 within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm2 at 24 hours. Optimal cell growth in Tisseel 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.

Transplantation of cultured human keratinocytes in single cell suspension: A comparative in vitro study of different application techniques

“…adhere, but the proliferation capacity seemed poor and the cultures never recovered to reach the cell yield of the other methods even after 14 days of culture. This could be the result of different stresses, presented by Duncan et al [12], as it is likely that the cells sustained at least elongation stress by being smeared across the surface and that this in turn would lead to reduced cellular metabolism and proliferative capacity, which was also described by Harkin et al [11]. The Harvest ® technique gave a cell survival of about 70%…”

“…This has long been thought to damage the cells, both by the passing of the spray nozzle and by the high velocity impact on to the wound bed. Harkin et al [11] recently examined the viability of keratinocytes delivered by aerosol, using the Tissomat applicator and found that the viability after transplantation (93.7% at 70 kPa and 90% at 138 kPa) was similar to the viability of the cells just recently detached from the culture dish (94%). When they adjusted the Tissomat applicator to deliver 207 kPa, they showed that fewer cells survived (73.3%), but not significantly so.…”

Wound Stimulation by Growth-Arrested Human Keratinocytes and Fibroblasts: HP802-247, a New-Generation Allogeneic Tissue Engineering Product

“…observations, data on file]. The sequential fibrinogen and thrombin spray application, which in line with the literature [25] had a limited impact on cell viability in vitro, offers an elegant nontouch approach to evenly deliver the cells to the wounds. Together, the in vitro data and the reported explorative phase II study allow for a rational final product design of HP802-247 to be tested in further clinical studies for its efficacy in wound healing.…”

Successful application of keratinocyte suspension using autologous fibrin spray

“…There is no wastage of the sprayed cells, unlike some of the digitalpressured aerosolised spray which are known to clog up around the nozzle when the cell suspension concentration is too thick. The cell viability was demonstrated to be unaffected with air pressure up to 10 psi [15], with numerous studies demonstrating the viability of the epidermal cells afteraerosolisation [11,15,16].…”