Why did we fund this project?
This award aims to reduce the number of rats and rabbits in cardiac injury research by establishing protocols to generate a cardiac injury in vitro using living myocardial slices (LMSs).
Cardiac injury research has a heavy reliance on animal models, particularly rats and rabbits, due to a lack of in vitro models that accurately replicate the complexity of the heart. In vivo studies of ischemia temporarily block an artery under general anaesthesia, to replicate the temporary loss of blood supply, and the effects are then observed after blood flow is restored. These studies are classified as severe under the UK’s Animals (Scientific Procedures) Act 1986 because of the level of suffering caused. Professor Cesare Terracciano and colleagues have previously developed LMSs, where thin heart slices from mammals are kept in culture and are viable for up to five days. Using slice culture preserves the 3D tissue structure and cellular heterogeneity of the heart, providing a better in vitro representation of the organ compared to other in vitro models, which are typically 2D. LMSs can be produced from a number of mammals, including rats, rabbits and humans, and each heart can be used to prepare up to 8, 15 and 50 slices respectively.
Studying cardiac injuries in the LMS currently requires the injury to be induced in the animal before the tissue slices are prepared. Cesare will optimise techniques to generate the injury in vitro, using cryoinjury to replicate myocardial necrosis and an ischemic solution to induce global ischemia/reperfusion. The processes leading to heart failure will also be examined by mechanically overloading the tissue. Workshops will be conducted at Cesare’s laboratory to disseminate and build confidence in the model and injury techniques.
The use of living animals in cardiovascular research is currently necessary due to the lack of appropriate and relevant in vitro models. Current in vitro models suffer from being reductionists (cell lines and stem cell-derived cardiac cells, often immature, no 3D structure) and/or are subjected to rapid changes induced by culture (can only be used for acute studies). The development of the living myocardial slice (LMS) model with its preserved tissue structure and ability to remain stable in culture for several days/months, therefore has the potential to have a significant impact upon the 3Rs.
Several slices can be prepared from the same heart so that, for instance, one rat heart used in LMSs experiments can provide the same number of experimental repeats of 6-8 rat hearts used for experiments with other preparations. In addition, LMSs are ideal to study both structure and function and, with one LMS, one can perform several experiments compared with other models which need several hearts. Because slices are prepared by pure mechanical dissociation, the same slices analysed for contractility experiments can then be dissected in smaller slices and used for biochemistry, imaging and other studies. This ability to optimise the use of tissue can bring about a minimum of 50% reduction of the number of animals needed.
In our project we will test the feasibility to induce injury on slices in vitro and not in living animals.
This aspect is likely to have a profound impact on cardiovascular research in many academic labs and industry. The ability to induce disease "in a dish" means that laboratories can avoid performing severe regulated procedures aimed at inducing cardiovascular disease in living animals. Furthermore, by using the LMS model it is now possible to optimise the use of available human heart tissue. While this is limited, transplant centres perform approximately 20-30 heart transplants per year. Another expanding source of human heart tissue is the implantation of left ventricular assist devices (LVADs), now performed routinely in many cardiac surgery centres (20-30 patients per year/per centre).
The implantation of LVADs is accompanied by the removal of a core of ventricular tissue that is amenable to the preparation of the LMS model. In addition, unused donor human hearts are available through dedicated research programmes, making the use of human LMSs not unrealistic and particularly relevant. We will test several kinds of disease in vitro using LMSs, and we will validate the use of this technique to study the effects of drugs that are toxic for the heart (some treatments for cancer bring about cardiac disease) and drugs that can be used to treat cardiac disease. Overall, we expect that the method developed by this study will became a major tool in cardiovascular research with important implications for the use of living animals in research.