Introduction: Cardiovascular disease is the leading cause of death in the US. Patients with CVD face chronic symptoms requiring lifelong pharmaceutical usage [1]. On the other hand, cardiovascular toxicity is a leading cause of drug candidate failure. New drug development is extremely slow, with success rates in the drug discovery pipeline abysmally low. One factor affecting the low success rate is the translation of pharmaceutical effects in animal models to clinical testing. To circumvent the physiological differences between humans and animals, researchers have been developing multiple models to predict pharmaceutical efficacy and safety [2]. Human organs-on-a-Chip (OOC) are comprised of microfluidic platforms that facilitate a 3D culturing environment, the inclusion of heterogenous cell populations, and biophysical stimuli. Pairing the advantages with a 3D culturing environment and including human stem cells creates a pharmaceutical screening tool capable of in situ imaging, high throughput screening, and improved physiological relevancy. Traditional OOC platforms are constructed via soft lithography with PDMS. However, the use of PDMS allows for lipophilic materials to diffuse into the system, adding confounding variables to dose-dependent pharmaceutical trials. New techniques developed in the lab utilize the “laser cut and assembled method to fabricate our microfluidic platforms. Previous models are primarily limited to cardiomyocytes and fibroblasts. However, few include other cell types found in the myocardium. The presented work characterizes an OOC model composed of human-derived cardiomyocytes and neurons. [1] Martin, S.S., et al. (2024) [2] Fine, B.;G.A.-O. Vunjak-Novakovic (2017) [3] Bhatia, S.N.; Ingber, D.E (2014) [4] Hayes, J.A. et al. (2023)
Materials and
Methods: Chip Fabrication: The heart OOC platform consists of the following layers: a top 3/16” PMMA cover with access ports to the media channel, a 1/16” PMMA layer providing the depth of the media channel, a 10 μm PET membrane (1 μM pore size) to serve as the interface between the media channel and the cell culture space, a 60 μm layer of 3M tape, a 76 μm PET GelPin to create distinct but continuous chambers in the chip, a 120 μm layer of 3M tape, and a glass coverslip to enclose and allow imaging of the cell culture region Cardiomyocyte Differentiation: The cardiomyocyte differentiation will follow Lian et al (2013). To summarize the protocol, hiPSCs are cultured on a Matrigel plate until the culture reaches approximately 90% confluence. The temporal addition of inhibitors directs the differentiation, with successful differentiation occurring after ~15 days since initiation. Chip seeding and culturing: The cardiomyocyte chamber will be seeded with approximately 1×10^6 cells suspended in 20 μl of gelatin methacryloyl (GelMA) hydrogel precursor. This hydrogel precursor comprises o.5% w/v of GelMA, 0.5% w/v LAP, and the desired volume of cell culture media. The precursor is crosslinked using 405 nm light for 60 seconds. Following the polymerization in the cardiomyocyte chamber, the neurons are seeded at 1×10^5 cells in 10 μl of GELMA precursor per neuron chamber which is also polymerized for 60 seconds.
Results, Conclusions, and Discussions: Following the described methods, hiPSCs were differentiated into cardiomyocytes that spontaneously contracted within well plates. These cells were successfully seeded into our custom built organ chip system and cultured, retaining their spontaneous contraction phenotype. The entire chip was fixed and stained after 3 weeks of culture to investigate how the cardiomyocytes populate the system (Figure 1). Immunofluorescent staining demonstrates that hiPSC-derived cardiomyocytes can readily grow in our chip system and network with one another in a 21μm film. Further development of the human heart chip included the introduction of posterior CNS neurons sourced from the Iyer lab at Tufts University. The neurons successfully extended neurites into the cardiac chamber, potentially creating neurocardiac junctions for complex electrophysiological communication (Figure 2). The success of culturing cardiomyocytes was followed with preliminary validation of cardiomyocyte behavior in a heart chip consisting of only cardiomyocytes. To validate mature cardiomyocytes and the expected biophysical response to exogenous compounds, epinephrine (5 μM) was dosed onto the chip. The heart chip responded to the dosed epinephrine through a significant increase in beat rate (Figure 3). These experiments show that the platform has a promising future for further pharmaceutical drug testing and investigations into cardiomyocyte behavior changes in the presence of autonomic neurons.
