(L-468) Multiwell Platform to Elucidate Reciprocal Interactions Between Human Cardiomyocytes and Hepatocytes

Introduction:: The complex interplay between the heart and liver in physiology, drug toxicity, and disease is still not fully understood, but has important implications for drug discovery and personalized medicine. Specifically, congestive heart failure can lead to hepatic dysfunction, while nonalcoholic fatty liver disease can impair cardiac electrical conduction, metabolism, and contractility, thus resulting in adverse patient outcomes. Furthermore, the majority ( >70%) of drugs on the market are metabolized by the liver prior to exerting their effects on other organs, including the heart. Animal liver and heart functions are not fully representative of human outcomes, which necessitates the development of human-relevant models to study the above phenomena. However, current platforms either rely on immortalized/cancerous cell lines or are too low throughput to be utilized for routine drug discovery. Therefore, here we sought to engineer a heart-liver model in a multiwell format utilizing long-term cultures of primary human hepatocytes (PHH) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM).

Materials and Methods:: Tissue culture polystyrene (TCPS) or oxygen-permeable polydimethylsiloxane (PDMS)-bottom plates were subjected to PDMS-based soft lithography to pattern collagen I into 2D circular domains (500 µm diameter, 1200 µm center-to-center spacing, previously shown to be optimal to maintain hepatocyte polarity). PHHs selectively attached to the collagen-coated domains and were subsequently surrounded by 3T3-J2 fibroblasts that were previously shown to induce long-term functions in hepatocytes. After 1 week of culture, Transwell^TM inserts containing co-cultures of hiPSC-CM and cardiac fibroblasts (CF) were combined with MPCCs and co-cultured for 2 weeks (Fig. 1A); control liver- or heart-only models were also created. Urea secretion, a marker of liver nitrogen metabolism, and cytochrome P450 3A4 (CYP3A4) enzyme activity, a marker of liver drug metabolism capacity, were assessed using published assays, while expression of cardiomyocyte genes was measured via qPCR.

Results, Conclusions, and Discussions:: MPCCs were successfully fabricated on both TCPS and PDMS-bottom plates (Fig. 1B); however, CYP3A4 protein expression was better retained on the PDMS bottom plates (Fig. 1C), likely due to better delivery of oxygen to MPCCs. PDMS bottom plates also allowed stable CYP3A4 enzyme activity (Fig. 1D) and urea secretions (Fig. 1E) in MPCCs. While the co-culture of liver MPCCs with iPSC-CM/CF co-cultures caused some downregulation of liver functions as compared to the control MPCCs, likely due to the presence of the Transwell and mixing of liver and heart media types, MPCC functions were nonetheless stable throughout (Fig. 1D-E). Interestingly, urea synthesis in MPCCs was enhanced, after 3 weeks in the presence of the iPSC-CM/CF co-cultures relative to the appropriate empty Transwell controls. Similarly, cardiac troponin T (cTnT) in iPSC-CMs was enhanced with MPCCs relative to the control.
We engineered a multiwell co-culture platform containing PHH/fibroblast MPCCs and iPSC-CM/CF; such co-cultures led to reciprocal modulation of tissue specific functions and gene expression over several weeks in vitro. Future work will focus on further characterization of phenotype of each tissue compartment, testing drug metabolism and toxicity on the co-cultures, and elucidating the effects of liver disease on cardiac function.


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