Electrical Cell Impedance Sensing (ECIS): Feasibility of a Novel In Vitro Approach to Studying Venom Toxicity and Potential Therapeutics

Abstract

Snakebite envenoming is often discussed in terms of lethality and limb loss, but local tissue injury and coagulotoxic effects of venom are significantly more common acute manifestations of snakebite envenoming (SBE). Local tissue injury and the hemorrhagic and coagulotoxic effects of venom are challenging to study in live animals and can be ethically fraught due to animal welfare concerns such that attention to the 3Rs of animal welfare motivates the development of in vitro techniques in this arena. Herein, we tested the use of a wound-healing study technique known as Electric Cell-Substrate Impedance Sensing (ECIS) to assess populations of cultured cells exposed to venom with or without sPLA2 and/or metalloprotease inhibitors (varespladib and marimastat, respectively). For comparison, the StarMax coagulation analyzer for coagulotoxicity was further used to evaluate the venoms and the neutralizing capabilities of the abovementioned direct toxin inhibitors (DTIs) against the same venoms examined using ECIS. Three viper and three elapid venoms that were examined for their effects on H1975 cells were Agkistrodon contortrix (Eastern Copperhead), Crotalus helleri (Southern Pacific Rattlesnake), and Vipera ammodytes (Horned Viper) and Naja atra (Chinese Cobra), Naja mossambica (Mozambique Spitting Cobra), and Naja nigricollis (Black-necked Spitting Cobra), respectively. The combination of cellular and coagulation techniques appears to usefully discriminate the in vitro capabilities and limitations of specific inhibitors to inhibit specific venom effects. This study suggests that ECIS with or without concomitant coagulation testing is a feasible method to generate reproducible, meaningful preclinical data and could be used with any type of cell line. Importantly, this approach is both quantitative and has the potential of reducing animal use and suffering during the evaluation of potential therapeutics. To further evaluate the potential of this method, rescue studies should be performed.

Keywords

• snakebite envenoming
• direct toxin inhibitors (DTIs)
• cytotoxicity
• detachment
• basement membrane
• coagulotoxicity