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Johns Hopkins Bloomberg School of Public HealthCAAT

CAAT Grants Program

Research Grants 2019-2020: Summaries

  • Susan Tilton,Oregon State University (new)
    Understanding Combined Effects of Chemical and Non-Chemical Stressors in an In Vitro Respiratory Model
  • Matthias Gossmann, innoVitro GmbH (new)
    IN FACT: An In vitro Functional Assay for Cardio Toxicity
  • Andrew John Smith, University of Leeds (new)
    Utilizing novel 3-D bioprinting technology to translate in vitro toxicology findings into complex structures


Susan Tilton, Oregon State University (new)
Understanding Combined Effects of Chemical and Non-Chemical Stressors in an In Vitro Respiratory Model

There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interaction and potential mechanisms of action. We propose to utilize primary human bronchial epithelial cells cultured in 3D at the air-liquid intercase as a physiologically relevant model to evaluate the effects of inflammation on toxicity of PAH chemicals in air pollution. In particular, we will investigate the role of exosomal miRNAs as potential primary 3D human bronchial epithelial cells (EBECs) collected from m=normal and asthmatic donors. We will also treat HBECs from normal donors with IL-13 to generate a profibrotic phenotypic similar to asthma. Normal, asthmatic and normal/IL-13 cells will be treated with a dose-response of benzo[a]pyrene (BAP) and a simulated air mixture (AM) containing a representative mixture of PAHs identified from air filters in Beijing China for evaluation of cytotoxic, oxidative stress, cytokine production, barrier integrate and DNA damage 48 hours after exposure. We will further measure cellular mRNA and exosomal miRNAs from apical mucous by RNAseq. Non-invasive biomarkers, such as miRNAs, are particularly important because they allow for quantitative experimental evaluation and validation in vitro, but are easily translated to humans for bio monitoring. We will utilize a systems biology approach to develop integrated miRNA networks for each condition to determine the functional consequences for miRNAs in HBEC. These data will be the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease states and chemical exposure on pulmonary toxicity in a physiologically relevant human in vitro model. 

Matthias Gossmann, innoVitro GmbH (new)
IN FACT: An In vitro Functional Assay for Cardio Toxicity

Despite rapid progress in the production and application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), their complete utilization for the quantification of toxic effects on cardiac contractility remains a challenge. The main reason is that common in vitro systems for contractility measurement tend to be complex, poorly controllable and labor-intense. Consequently drug discovery and development still rely on ethically and physiologically questionable, cost-intensive whole-animal experiments and ex vivo studies. 

We developed the CellDrum technology that provides a scalable, human-based heart muscle model with hiPSC-derived cardiomyocytes. The cells are cultured on a freely swinging, ultra-thin and hyper-elastic silicone membrane to form a bio hybrid tissue construct. The mechanical stiffness of the tissue can be dynamically adjusted to optimally reflect the microenvironment of either native or diseased human cardiac and pressure changes during spontaneous contractions of the bio hybrid tissues, the assay system enables true contractility measurements under defined and physiologically relevant conditions. We have scaled our technology to the 96-well-format in cooperation with Nanion Technologies, one of the leading providers of cell-based assay systems. With the newly developed cell-device-architecture, pharmacological analyses with commercially available hiPSC-derived cardiomyocytes can be performed with very low expenses. 

In the proposed study, we want to examine cardiotoxic effects of 20 kinase Inhibitors on hiPSC-derived cardiomyocytes as a benchmark for the applicability of the CellDrum technology in safety pharmacological assessment/ Kinase inhibitors show a high potential for cardiotoxic side effects and many of these effects could not be anticipated during animal-based preclinical safety evaluation. Thus, high drug attrition rates and their corresponding costs drive the pharmaceutical industry to search for alternatives to animal tests. 

With the data obtained in the proposed study, we want to directly address the respective individuals in the safety pharmacological departments of pharmaceutical companies. Our aim is to convince decision-makers and opinion-leaders that the productivity of our model is comparable or even superior to animal experiments due to the use of human tissue in a physiologically relevant environment. 

Andrew Smith, University of Leeds (new)
Utilizing novel 3-D bioprinting technology to translate in vitro findings into complex structures. 

This project builds upon our work examining receptor tyrosine kinase inhibitor (RTKI) cardiotoxicity, expanding this into a three-dimensional multicellular in vitro model. We will use the in-lab bioprinting technology available from Advanced Solutions Inc. (BioBotTM), which is capable of generating operator-designed three-dimensional structures, using up to five biomaterials (including cell suspensions) in a multi-well format. This provides a novel model to advance in vitro toxicology analyses: we propose to examine RTKI impacts on human endothelial cells and cardiomyocytes derived from induced pluripotent stem cells (iPS-CMs). 
Objective 1. Generate three-dimensional structures with human endothelial cells in hydrogel, dispensed as tubular structures to form a ‘vascular network’, with cell-free hydrogel in spaces between the ‘vessels’: this will form the structure’s lower layer. The upper structure layer will be then be overlaid, by dispensing narrow grooves of hydrogel and then placing iPS-CMs in hydrogel suspension between and above these. This will form a contractile ‘syncytium’ of cardiomyocytes in close proximity to the vascular network. Once these structures are generated, they will be assessed for maturity and function: 1) fixation, sectioning and immunocytochemistry to assess vessel network and iPS-CM syncytium structural maturity and 2) functional assessment by microdissection, then analyse calcium and electrophysiological responses to stimulation. Objective 2. Application of RTKIs by infusion through vessel lumen, identifying impacts on endothelial cell structure and function (if no discernible impact is seen, RTKIs will be applied in solution to the structure surface). To identify impact upon vessel formation, RTKIs will be mixed into the cell suspension prior to bioprinting the vascular network. Impacts on cell/syncytium phenotype will be assessed by direct comparison with controls, using the techniques described for Objective 1. 
We do not propose this model as final: the study will establish proof-of-concept for drug application in a three-dimensional multicellular model representing some salient features of tissue. Striking findings in iPS-CMs indicate their methods could allow generation of more mature iPS-CMs for our structure’s upper layer. However, to obtain funding for toxicology-focused studies, proof-of-concept is required, hence a simpler iPS-CM layer. This study will proceed with the expert advice of Advanced Solutions Inc. as part of their normal support, offering an opportunity to foster a trans-Atlantic collaboration between academia and commercial science. This expands the programme we are building to utilise this versatile technology in stem cell and translational research.