Fabrication of perfusion microbioreactor for culturing engineered heart tissue with electrical stimulation

This thesis focuses on fabricating a perfused microbioreactor platform to generate functional human engineered heart tissue to be used for drug testing. To evaluate the feasibility of this system and make the conditions optimized for culturing cardiomyocytes in 3D constructs, rats’ heart tissue models were firstly set up to test the efficacy for long-term culture. Myocytes from embryonic rats were cultured in 200mg/ml fibrinogen gel with 5 million/ml cell densities. The tissues were formed in 24-well culture dish and then transferred into the micro-bioreactors with carbon electrodes to provide electrical stimulations. Culture medium was perfused to maintain chemostat environment. To measure contractile force, the key function of cardiomyocytes in vivo, rod-shaped tissue constructs were generated and attached onto two flexible PDMS pillars (1mm diameter and 12mm length). They worked as a force transducer and their deformation was measured to estimate cellular contractile force with accurate mechanical model. The dynamic perfusion system was stimulated to determine optimal design and operation of microbioreactor. By comparing the functions of the cells cultured in static and perfused environment, it was concluded that the perfused system would be necessary to generate more representative 3D in vitro models Electrical stimulation is an important tool to induce functions of cardiomyocytes cultured in vitro. To investigate the electrophysiological activities of engineered heart tissue, different levels of electrical stimulations were applied onto the engineered heart tissue to mimic those in native heart. All the tissues were cultured 5 days before electrical stimulations were initialled to ensure tissues are sufficiently stable. This can avoid the influence onto protein assembly due to early electrical stimulation. In the experiments, monophasic electrical signals were applied onto the samples with amplitudes ranging from 1V/cm to 5V/cm and frequencies varying between 1Hz and 5Hz. While comparing the stimulated and non-stimulated samples, the optimal electrical stimulation condition is 3V/cm, 2Hz on the basis of performance of rEHT in terms of contractile force, beating rate and microstructures. It is then used to induce higher maturation of iPSC-CMs for human EHT generation. After 20 days of long-term culture, the human engineered heart tissues are mature and functional and can be used for testing the effect of drugs. Five drugs, including Ouabain, Bay k8644, Milrinone, Amlodipine and Ryanodine, all clinically approved to treat various cardiovascular diseases, were used for the evaluation tests. The tested drugs at different dosages were added into the culture medium and the latter was perfused into the microbioreactor hosting the human engineered heart tissues. The dynamic responses of the hEHTs were recorded over 24 hours experiments. The responses were shown to be clinically relevant. Although further tests are required to validate the efficiency and efficacy of the developed microbioreactor platform, it showed potentials for high throughput testing and refining, reducing and replacing the use of animal tests.