In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment

Abstract

Event Abstract Back to Event In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment Michiya Matsusaki1, Yuto Amano1, Manabu Seo2*, Takashi Yamaguchi2, Shigeru Miyagawa3, Yoshiki Sawa3* and Mitsuru Akashi1 1 Osaka University, Department of Applied Chemistry, Graduate School of Engineering, Japan 2 Ricoh Company, Ltd., Advanced Technology Research & Development Center, Ricoh Institute of Technology, Japan 3 Osaka University Graduate School of Medicine, Department of Cardiovascular Surgery, Japan Introduction: Recent drug discovery systems are time-consuming and costly because the pharmaceutical assays using cell monolayer and animal models have many issues due to different drug responses as compared to human body. Therefore, the use of induced pluripotent stem cell (iPSC), which can provide many types of normal and diseased human cell sources, has enormous potential for pharmaceutical assays. However, since nearly all tissues are integrated three-dimensional (3D) structures of multiple types of cells and extracellular matrices (ECMs), and since intercellular signaling is important for tissue functions, it is difficult to evaluate actual tissue functions by 2D-culture method. In this study, we aim to develop 3D-cardiac myoblast tissues (3D-iPSC-CM) composed of iPS-CMs, cardiac fibroblast, and cardiac endothelial cells by the cell-accumulation technique (Figure 1). We reported a bottom-up approach, termed “accumulation technique”[1] which was improved method of our previous technique, hierarchical cell manipulation[2], to develop multilayered thick tissues (>100 µm) by cell surface coating with nanometer-sized ECM-films[3]. Less than 10 nm sized ECM-films, such as fibronectin (FN)-gelatin (G), induced cell-cell interaction in three dimensions. By this method, iPSC-derived 3D-cardiac muscle tissues were successfully fabricated. The obtained tissues showed the synchronized contractions, and the tolerance to the cytotoxicity of anticancer drugs[4]. The iPSC-derived 3D-cardiac muscle tissues have a great potential for pharmaceutical applications. Figure 1. Schematic illustration of the vascularized 3D-iPSC-CM for drug assessments. Methods: iPSC-CM with 50~60% differentiation was used in this study. After repeating the nine steps of layer-by-layer (LbL) deposition, the (FN/G)4 FN films with about 10 nm thickness were prepared on the cell surface by cell accumulation technique. Normal human cardiac fibroblasts (NHCF) and normal human cardiac endothelial cells (NHCMEC) were also coated as the same way. The coated cells were seeded into a 24 micro well cell culture insert (1 x 105 cells/layers) and cultured to construct multilayered tissues. The ratio of NHCF was varied to optimize for the highest beating number, synchronization, and vascularization. 10% NHCMEC was added for blood capillary formation. Results: The ratio of NHCF was varied to 0, 25, 50, 75, and 100% to optimize beating number and synchronization of the tissues. The 3D-iPSC-CM tissues with 25~50% of NHCF clearly showed the highest beating number and synchronization. In the case of blood capillary formation, the network clearly found at over 25% NHCF condition and the network area increased with increasing NHCF ratio. Accordingly, 50% NHCF was used for the drug assessment. Interestingly, the vascularized 3D-tissues showed higher tolerance to the drug as compared to the monolayer culture (2D). We also found that the capillary was damaged by the treatment of doxorubicin in dose dependent manner. These results indicated that the vascularized 3D-iPSC-CM tissues might be useful for drug assessment. Figure 2. (a) Beating number of 3D-iPSC-CM tissues (NHCF50) and 2D monolayr of iPSC-CMs (NHCF50) after 3 days of incubation with 1, 50, or 1000 nM doxorubicin. *indicates 0. (b) CLSM images of the 3D-tissues and 2D-monolayer after incubation with doxorubicin by immunostaining with troponin T antibody. (c) CLSM images of blood capillary networks in 3D-iPSC-CM tissues after treatment with doxorubicin. The networks were immunostained with anti CD31 antibody. (d) Quantified capillary area in images of (c). ††p < 0.01 statistically significant difference as compared to 2D. Conclusions: We successfully constructed vascularized iPSC-CM tissues by coating of FN-G nanofilms. Drug responses and tolerance mechanism of the 3D-tissues will report in the conference. NEXT Program (LR026); Grant-in-Aid for Scientific Research (S) (A232250040); Grant-in-Aid for Scientific Research (B) (26282138); Grant-in-Aid for Scientific Research on Innovative Areas (26106717); SENTAN-JST Program (13A1204); Health Labour Sciences Research Grant from the Japanese Ministry of Health, Labour and Welfare, and Grand-in-Aid for JSPS Fellows (24・622)