Objective

Introduction:

Ischaemia-reperfusion injury (IRI) is a major mediator of cardiac damage during heart attack. IRI occurs when cells deprived of oxygen and metabolic factors following ischaemia are reperfused with oxygen-rich blood. The reperfusion causes reactive oxygen species (ROS) generation and mitochondrial permeability transition pore opening, leading to apoptotic cell death and loss of heart tissue. Activation of the RISK (reperfusion injury salvage kinase) signalling pathway, such as by insulin, has been demonstrated to be protective against IRI in animal models. Current models of IRI rely on hypoxia chambers, endpoint measures or extensive use of animal models due to the difficulty of initiating, maintaining and reversing an acute hypoxic insult whilst evaluating cell viability in vitro. The large contribution of IRI to heart attack means better in vitro models are needed to reduce animal usage and better inform hit-to-lead drug discovery.

Methods:

Here we describe an in vitro model of IRI in 96-well format suitable for medium throughput small molecule drug screening. This model arises from a collaboration between Metacell partners (BMG Labtech and Agilent Technologies (previously Luxcel)) and UCL. This model employed a BMG Clariostar reader with atmospheric control unit which enabled the rapid elimination of chamber O₂ to initiate and maintain a hypoxia-reperfusion insult. Atmospheric control in the plate reader allowed for real-time measurement of Chamber O_2, Internal cell O₂ (Carey et al. 2017) and cell death throughout the simulated IRI. Following the IRI protocol, the cell plate was suitable for end-point assay measurements.

Results:

Chamber O₂ levels drop to 0.1% in 20 minutes and intracellular O₂ to <0.1% within 25 minutes. Limited cell death occurs during 1 hour of ischaemia, but increases 2 hours after reperfusion and plateaus after 4 hours. Insulin treatment results in a delayed and diminished extent of cell death.  We have developed a sensitive model of IRI that enables multiplexed kinetic monitoring of cell death and internal O₂, is available for endpoint assaying of cell viability or ROS, and is suitable for drug screening in 96-well plate format. 

References: Carey et al., High-throughput in vitro ischemia-reperfusion model with real-time monitoring of cellular oxygenation and reactive oxygen species generation, BSCR Annual Meeting 2017.