Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.
Author(s): Charrez, Berenice Lucie Mechthild | Advisor(s): Healy, Kevin E | Abstract: With highly regulated safety requirements, the average cost of drug development is $2.5B, which has increased by more than 9-fold since 1979. Preclinical studies include both in vitro and in vivo studies in non-human models to determine the efficacy and toxicity of the compound before any administration to people. However, no commonly used platforms accurately predict drug effects on patients, since they do not mimic human physiology. The fourth reason for drug withdrawal from the market in the last 70 years were unexpected side effects on the cardiovascular system, such as toxicity or dysfunction of the heart, associated with life-threatening conditions. Accordingly, the FDA has mandated that all drugs must be screened for the potential to alter ventricular repolarization prior to human studies. There is an urgent need to develop tools to diminish time and costs of the drug development pipeline while getting safer and more efficient cardiotoxicity screening. A big advance for 2D in vitro assays for cardiotoxicity screens was the discovery of human induced pluripotent stem cells (hiPSC) from which cardiomyocytes with patients genetics could be derived. However, one challenge remains : hiPSC-derived cardiomyocytes (CM) show immature metabolic and mechanical properties, questioning their prognostic capabilities. In this thesis, we demonstrated that the combination of 3D culture of hiPSC-CM in microphysiological systems (heart-on-a-chip) with fatty-acid based media, synergized to promote maturation of hiPSC-CM metabolism and electrophysiology. To further improve the maturation of the tissues we screened through different confinement chamber designs and selected for parameters that enhance mechanical, morphological and contractility outcomes of tissues. As the COVID-19 pandemic hit, we saw the opportunity for our matured cardiac system to rapidly predict cardiotoxicity associated with potential therapeutics (hydroxychloroquine combination with azithromycin) in a mock clinical trial experimental design as well as in an acute dose escalation study. The high content system can help clinicians rapidly evaluate safety properties of potential therapeutics in crisis times to hopefully accelerate access of patients to treatment.
Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/μL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.
Abstract Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are a promising in vitro tool for drug development and disease modeling, but their immature electrophysiology limits diagnostic utility. Tissue engineering approaches involving aligned 3D cultures enhance hiPSC-CM structural maturation but are insufficient to induce mature electrophysiology. We hypothesized that mimicking post-natal switching of the heart’s primary ATP source from glycolysis to fatty acid oxidation could enhance electrophysiological maturation of hiPSC-CM. We combined hiPSC-CM with microfabricated culture chambers to form 3D cardiac microphysiological systems (MPS) that enhanced immediate microtissue alignment and tissue specific extracellular matrix (ECM) production. Using Robust Experimental design, we identified a maturation media that improved calcium handling in MPS derived from two genetically distinct hiPSC sources. Although calcium handling and metabolic maturation were improved in both genotypes, there was a divergent effect on action potential duration (APD): MPS that started with abnormally prolonged APD exhibited shorter APD in response to maturation media, whereas the same media prolonged the APD in MPS that started with aberrantly short APD. Importantly, the APD of both genotypes was brought near the range of 270-300ms observed in human left ventricular cardiomyocytes. Mathematical modeling explained these divergent phenotypes, and further predicted the response of matured MPS to drugs with known pro-arrhythmic effects. These results suggest that systematic combination of biophysical stimuli and metabolic cues can enhance the electrophysiological maturation of hiPSC-derived cardiomyocytes. However, they also reveal that maturation-inducing cues can have differential effects on electrophysiology depending on the baseline phenotype of hiPSC-CM. In silico models provide a valuable tool for predicting how changes in cellular maturation will manifest in drug responsiveness.
Abstract Low-temperature ex vivo preservation and tissue engineering based on human induced pluripotent stem cells (hiPSC) represent two of the most promising routes towards on-demand access to organs for transplantation. While these fields are often considered divergent from one another, advances in both fields present critical new opportunities for crossover. Herein we demonstrate the first-ever sub-zero centigrade preservation and revival of autonomously beating three-dimensional hiPSC-derived cardiac microtissues 1 via isochoric supercooling 2 , without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study organ preservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.
Hepatocellular carcinoma (HCC) is now the fifth cancer of greatest frequency and the second leading cause of cancer related deaths worldwide.Chief amongst the risks of HCC are hepatitis B and C infection, aflatoxin B1 ingestion, alcoholism and obesity.The latter can promote non-alcoholic fatty liver disease (NAFLD), that can lead to the inflammatory form non-alcoholic steatohepatitis (NASH), and can in turn promote HCC.The mechanisms by which NASH promotes HCC are only beginning to be characterized.Here in this review, we give a summary of the recent findings that describe and associate NAFLD and NASH with the subsequent HCC progression.We will focus our discussion on clinical and genomic associations that describe new risks for NAFLD and NASH promoted HCC.In addition, we will consider novel murine models that clarify some of the mechanisms that drive NASH HCC formation.
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