Date of Award
Doctor of Philosophy (PhD)
3D scaffold-free spherical micro-tissue (spheroids) holds great potential in tissue engineering as building blocks to fabricate the functional tissues or organs in vitro. To date, agarose based hydrogel molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we developed an alginate-based, direct 3D mold-printing technology: 3D printing micro-droplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit micro-droplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D agarose hydrogel molds. Tissue spheroids composed of 50% human endothelial cells and 50% human smooth muscle cells were robotically dispensed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation.
The current inability to derive mature cardiomyocytes (CMs) from human pluripotent stem cells (hiPSC) has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of CMs derived from hiPSC. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids prepared by seeding CMs into agarose microwells were shown to improve cardiac functions. However, CMs within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here we show, for the first time, that incorporation of a trace amount (i.e., ~0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.
Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (~0.5k, ~1k, ~3k, ~7k cells/spheroid). Spheroids with ~3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases.
We reasoned that the presence of e-SiNWs in the injectable spheroids improves their ability to receive exogenous electromechanical pacing from the host myocardium to enhance their integration with host tissue post-transplantation. In this study, we examined the cardiac biocompatibility of the e-SiNWs and cell retention, engraftment and integration after injection of the nanowired hiPSC cardiac spheroids into adult rat hearts. Our results showed that the e-SiNWs caused minimal toxicity to rat adult hearts after intramyocardial injection. Further, the nanowired spheroids were shown to significantly improve cell retention and engraftment, when compared to dissociated hiPSC-CMs and unwired spheroids. The 7-days-old nanowired spheroid grafts showed alignment with the host myocardium and development of sarcomere structures. The 28-days-old nanowired spheroid grafts showed gap junctions, mechanical junctions and vascular integration with host myocardium. Together, our results clearly demonstrate the remarkable potential of the nanowired spheroids as cell delivery vehicles to treat cardiovascular diseases.
Tan, Yu, "Nanowired Human Cardiac Spheroids for Cardiac Regenerative Medicine" (2017). All Dissertations. 2545.