This project focuses on developing a screen printing process to build models of both neural networks and the innervated colon (i.e., a colon with imbedded nerves to mimic the natural structure). The screen printing process has been around for thousands of years and is used to make art, to mass produce t-shirts, and to make microelectronics. It is simple, low cost, reproducible, and scalable, making it ideal for printing highly accessible tissue models. While there have been many exciting approaches to print tissue models, most involve expensive equipment and subject the cells to either ultraviolet rays or shearing through fine needles, both of which can impact cell survival and behavior. The objective of this project is to determine the resolution and reproducibility of the screen printing approach and then use the process developed to make the two new models. The neural network model (based on human derived stem cells) will lay the groundwork to study diseases and investigate therapies relevant to nerve tissue in a more physiology-based/meaningful way that is still suitable for high throughput systems. The colon model (consisting of epithelial cells, goblet cells producing mucous, immune cells, and neurons) will provide one of the first innervated gut models that is suitable for high throughput techniques. High throughput screening opens the possibilities of investigating the interplay between these cells in the development of food allergies and conditions such as colitis. The research will provide new tools for researchers interested in understanding the complex cross-talk between cells in these tissues and developing new therapies for insults to these systems. The screen printing technique will also be used to engage young researchers. Screen printing demonstrations will be built into outreach programs in schools and utilized to educate researchers in classrooms and labs. Screen printing is exceptionally familiar and "user-friendly," thus a great platform for engaging and keeping people engaged in science. For example, Jello will be screen printed on edible paper to explain methods to make tissues as part of outreach program demonstrations in the Baltimore and Boston public schools. Educational and outreach efforts will be complemented by a website that highlights scientists from diverse backgrounds and the accessible, low tech parts of their work, like screen printing.
The objective of this project is to determine the resolution and reproducibility of a screen printing approach to making highly scalable hydrogel-cell tissue models and to then use the system developed to make new models of neural networks and the innervated colon. The investigators hypothesize that screen-printing will provide a cost-effective approach to making tissue models that surpass those made via bioprinting approaches in terms of ease, cost and cell survival and thus enable more relevant models of tissues from cells that are highly sensitive to shear. The Research Plan is organized under four aims. THE FIRST AIM is to characterize the screen-printed materials (PLL(poly(L-lysine))/protein and hydroxy appetite-based gels functionalized with PEG-thiol groups printed on glass slides) with regards to the resolution, repeatability and lamination of layers. Expected aim outcomes include: determining the gelation time and mechanics of the different materials, confirming the concentration and distribution of proteins on the gels, determining the strength of lamination between similar and different layers, and knowing the limits on how thin gels can be reproducibly screen printed. THE SECOND AIM is to characterize the multilamellar, patterned gels with regards to architecture including resolution and reproducibility of features in the printing plane. Expected aim outcomes include: determining the finest resolution that can be reproducibly achieved for a range of gels, confirming the concentration and distribution of proteins on and in the gels and determining if there are any types of features that are more or less easily printed at high resolution. THE THIRD AIM is to determine the relationship between printing resolution, cell survival, and cellular behavior using human induced pluripotent stem (iPS) cells and human iPS-derived neurons to build neural circuits including excitatory and inhibitory neurons. Expected aim outcomes include: knowing which hydrogel augments iPS cell survival the most, knowing the impact of the matrix on the genetic and phenotypic cellular behavior of iPS and their progeny, knowing the impact screen printing has on the genetic and phenotypic cellular behavior of iPS cells and their progeny and developing a reproducible, scalable system for high throughput screening of 3D neural interactions that lead to neural circuits. THE FOURTH AIM is to build a model of the innervated colon to investigate the application of screen printing in a multicellular, physiologically relevant, high throughput model. Cell lines include enterocytes and goblet cells derived from Caco-2 cells, primary human intestinal epithelial cells, immune cells (dendritic cells) and neural cells (from Aim 3). Matrix materials include PLL-PEG gel systems printed with laminin and collagen I. The matrix optimized in Aim 3 will be used for the neural layer. Expected aim outcomes include: the development of a novel gut/colon model 1) that lays the foundation for study and enhanced understanding of the role of the enteric nervous system, 2) that, even without the neural component, will be one of the first colon models that can be prepared in a high throughput manner and interface with real time electrical recording and imaging modalities without a substantial investment in materials or equipment and 3) that lays for foundation for adding the next critical component--the microbiome.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
