Ex vivo model for the study of epicardium-targeted therapies
Lead Research Organisation:
University of Surrey
Department Name: Biochemistry & Physiology
Abstract
The recovery capacity of the adult heart after injury is severely limited by the low number of regenerating cells within this tissue. The epicardium, the most external layer of the heart, contains cells able to home to the injured heart muscle and promote its recovery. The models available to the scientists to study this phenomenon and develop strategies aimed at facilitating and improving the reparative process are limited and mainly based on regulated procedures (surgeries) performed on animals. In this project we will develop a model based on leftover pig heart tissues to mimic in a dish the behaviour of the epicardium after stimulation.
Pig hearts will be reduced to thin slices including both the epicardium and underlying heart muscle; the slices will be subjected to treatments to simulate the aftermath of heart disease and stimulated with known therapeutic substances to assess the capacity of our system to reproduce what has been observed in the animal models.
Once our model is tested, it will be used to develop new therapies harnessing the reparative potential of the epicardium.
Pig hearts will be reduced to thin slices including both the epicardium and underlying heart muscle; the slices will be subjected to treatments to simulate the aftermath of heart disease and stimulated with known therapeutic substances to assess the capacity of our system to reproduce what has been observed in the animal models.
Once our model is tested, it will be used to develop new therapies harnessing the reparative potential of the epicardium.
Technical Summary
The emerging role of the epicardium in heart regeneration points at a largely unexplored reparative potential, boosting the number of studies focusing on this tissue. This project aims at developing an ex vivo model to study the epicardial cell behaviour in order to understand the observed in vivo reparative effect and provide a platform to enable future screening of arrays of gene/drug therapy candidates ex vivo, replacing animals in this type of studies.
We will base our model on the ex vivo culture of porcine superficial cardiac slices, comprising the epicardial layer and the underlying myocardial tissue (Epicardial/Cardiac-Tissue Slices, EpCardio-TS). Cardiac slices mirror the complexity of the heart tissue while allowing the high throughput approach needed for gene/drug discovery. Selective gene transfer to the epicardial cells is obtained by localised delivery of plasmid DNA mediated by a nanomaterial tool, the nanoneedles. The process of nanoneedles-mediated gene transfer (nanoinjection) efficiently delivers DNA to the cytoplasm of epicardial cells. Nanoinjection of reporter genes will allow the visualisation the cells within the tissue upon culture and will establish a proof of concept for future gene therapy applications. Post-fixation treatments leading to high tissue transparency will enable enhanced imaging of the epicardial cells throughout the cardiac slices. All these methods have been developed and optimised by the applicants and the named collaborators and will constitute the basis for the current project. This enabling model will contribute to further our understanding of the contribution of the epicardium to cardiac repair and improve the outcomes of therapies targeted to this tissue, reducing the burden to animals. The use of porcine heart slices will determine reduction of the number of small animals involved these in preclinical studies by substantially replacing the in vivo whole heart approach.
We will base our model on the ex vivo culture of porcine superficial cardiac slices, comprising the epicardial layer and the underlying myocardial tissue (Epicardial/Cardiac-Tissue Slices, EpCardio-TS). Cardiac slices mirror the complexity of the heart tissue while allowing the high throughput approach needed for gene/drug discovery. Selective gene transfer to the epicardial cells is obtained by localised delivery of plasmid DNA mediated by a nanomaterial tool, the nanoneedles. The process of nanoneedles-mediated gene transfer (nanoinjection) efficiently delivers DNA to the cytoplasm of epicardial cells. Nanoinjection of reporter genes will allow the visualisation the cells within the tissue upon culture and will establish a proof of concept for future gene therapy applications. Post-fixation treatments leading to high tissue transparency will enable enhanced imaging of the epicardial cells throughout the cardiac slices. All these methods have been developed and optimised by the applicants and the named collaborators and will constitute the basis for the current project. This enabling model will contribute to further our understanding of the contribution of the epicardium to cardiac repair and improve the outcomes of therapies targeted to this tissue, reducing the burden to animals. The use of porcine heart slices will determine reduction of the number of small animals involved these in preclinical studies by substantially replacing the in vivo whole heart approach.
Planned Impact
Epicardial/Cardiac-Tissue Slice (EpCardio-TS) is an ex vivo model for the study of the epicardial cell reparative potential and the development of targeted therapeutic approaches. Current models are based on simplified in vitro systems (isolated epicardial cells) or on small animal models of cardiovascular diseases.
EpCardio-TS is based on the nanomaterial-based localised delivery of reporter genes to epicardial cells within hybrid porcine cardiac slices, incorporating both epicardium and myocardium. EpCardio-TS provides the complexity of a multicellular preparation with physiological and electrical properties similar to the whole heart, combined with the simplicity of an in vitro system.
From our recent experience, we calculate that an average lab might use 5-600 animals per year (5/6 experiments; 100 animals/experiment). Labs focusing almost entirely on this subject use up to 3,000 animals/year, as reported by Dr. Smart. Considering the 4 groups working on the epicardium in the UK with whom we have direct contact, we can estimate that over 5,000 animals might be used per year. Extrapolating from our experimental design and the data reported by Dr. Smart, we assume that 20% of these mice and rats could be replaced by EpCardio-TS, totalling to 1,000 animals in the UK. Literature search retrieves an average of 20 publications per year on this topic, using 50-150 animals per study. If we assume 20 labs working on the epicardium, performing an average of 5 experiments/year and using 100 animals/experiment, we can calculate the replacement of an additional 2,000 animals worldwide (20 groups x 5 experiments x 100 animals = 10,000; 20%x10,000=2,000).
EpCardio-TS employs multicellular cardiac slices derived from pigs sacrificed for other projects, maximising the use of the sacrificed animal. Furthermore, each pig heart will originate 15-20 slices reducing the number of animals required per experiment. The potential to apply the protocols optimised during this project to human cardiac slices from donor hearts, suggests the possibility to eliminate completely the use of animals in this context.
The strategy we propose to achieve our replacement target includes the following steps: (1) immediate implementation of EpCardio-TS in our labs; (2) practical workshop to facilitate adoption by our collaborators' labs; (3) wider promotion of the model by production of papers, website contents and posters/presentations; (4) highly specialised training of one early career researcher during the project; (5) engagement of the public by uploading special contents on the website and organising outreach activities.
We envision EpCardio-TS having an economic impact by reducing the cost of the projects by limiting the animal expenditure, therefore benefitting the research groups and the founders. We also expect the translatability of the results obtained using EpCardio-TS to human subjects to be superior, as compared to small animal studies. This is an extremely relevant issue in the pharmaceutical industry, as explained in Dr. Minger's letter. Therefore, the interest for EpCardio-TS might extend to the industry.
Our system is based on commonly available cell culture and imaging facilities; the only limit to diffusion might be represented by the availability of a vibratome. However, this is a relatively cheap piece of equipment and is available in most major universities in the UK. The technical challenges normally encountered in the cutting and handling of the slices will be eliminated thanks to the planned workshops and videos.
The nanoneedles will be provided freely through collaborations with King's College London, subject to Material Transfer Agreement.
EpCardio-TS is based on the nanomaterial-based localised delivery of reporter genes to epicardial cells within hybrid porcine cardiac slices, incorporating both epicardium and myocardium. EpCardio-TS provides the complexity of a multicellular preparation with physiological and electrical properties similar to the whole heart, combined with the simplicity of an in vitro system.
From our recent experience, we calculate that an average lab might use 5-600 animals per year (5/6 experiments; 100 animals/experiment). Labs focusing almost entirely on this subject use up to 3,000 animals/year, as reported by Dr. Smart. Considering the 4 groups working on the epicardium in the UK with whom we have direct contact, we can estimate that over 5,000 animals might be used per year. Extrapolating from our experimental design and the data reported by Dr. Smart, we assume that 20% of these mice and rats could be replaced by EpCardio-TS, totalling to 1,000 animals in the UK. Literature search retrieves an average of 20 publications per year on this topic, using 50-150 animals per study. If we assume 20 labs working on the epicardium, performing an average of 5 experiments/year and using 100 animals/experiment, we can calculate the replacement of an additional 2,000 animals worldwide (20 groups x 5 experiments x 100 animals = 10,000; 20%x10,000=2,000).
EpCardio-TS employs multicellular cardiac slices derived from pigs sacrificed for other projects, maximising the use of the sacrificed animal. Furthermore, each pig heart will originate 15-20 slices reducing the number of animals required per experiment. The potential to apply the protocols optimised during this project to human cardiac slices from donor hearts, suggests the possibility to eliminate completely the use of animals in this context.
The strategy we propose to achieve our replacement target includes the following steps: (1) immediate implementation of EpCardio-TS in our labs; (2) practical workshop to facilitate adoption by our collaborators' labs; (3) wider promotion of the model by production of papers, website contents and posters/presentations; (4) highly specialised training of one early career researcher during the project; (5) engagement of the public by uploading special contents on the website and organising outreach activities.
We envision EpCardio-TS having an economic impact by reducing the cost of the projects by limiting the animal expenditure, therefore benefitting the research groups and the founders. We also expect the translatability of the results obtained using EpCardio-TS to human subjects to be superior, as compared to small animal studies. This is an extremely relevant issue in the pharmaceutical industry, as explained in Dr. Minger's letter. Therefore, the interest for EpCardio-TS might extend to the industry.
Our system is based on commonly available cell culture and imaging facilities; the only limit to diffusion might be represented by the availability of a vibratome. However, this is a relatively cheap piece of equipment and is available in most major universities in the UK. The technical challenges normally encountered in the cutting and handling of the slices will be eliminated thanks to the planned workshops and videos.
The nanoneedles will be provided freely through collaborations with King's College London, subject to Material Transfer Agreement.