Investigating Non-thermal Plasma Induced Calcium ion Flux in an in vitro Human Keratinocyte Wound Closure Model for the Development of a Closed Loop Control System to Regulate Plasma Delivery

Non-thermal plasma (NTP) generated through dielectric barrier discharge is an innovative treatment for wounds. An amalgam of short- and long-lived reactive oxygen and nitrogen species generated by NTP alter cellular redox processes that enhance wound closure. However, the establishment and application of appropriate NTP dosage for clinical use is challenging due to variation in devices used and biological responses. One such response is the flux of cytoplasmic calcium released from intracellular stores within minutes of treatment. As calcium acts as a second messenger during wound healing, it is expected that an NTP-induced calcium release will influence wound closure. Therefore, we anticipate that detection of calcium in real-time will provide feedback on cellular responses that enhance wound closure. Our research utilizes an in vitro wound healing model that incorporates the HaCaT human keratinocyte cell line. A linear gap (~0.75 mm wide) is created in a confluent cell layer using the Aglient BioTek Autoscratcher. Acute changes in calcium flux and subsequent wound closure are evaluated using the Aglient BioTek Cytation-5 cell imaging and multimode reader, a high content automated microscope that images cells (in visible and fluorescent wavelengths). Calcium flux is measured in conjunction with Fluo-4 NW, a calcium sensitive fluorescent dye, through imaging and fluorescence intensity. Wound closure is measured through kinetic imaging and automatic detection of the leading edge over a 24-hour period using the Aglient BioTek Gen5 software. This enables us to quantify changes in area and closure of the generated gap in real time. With our collaborators at Rutgers University and North Carolina State University, we will optimize this methodology in a wound healing Balb/c mouse model to develop a sensor-based, closed loop control system capable of regulating NTP dosage using real-time feedback from biological signals such as calcium flux.