The publication aspect of my career development is under construction. Please check back in regularly as I work to make this list grow. In the meantime, toggle the buttons on the left to read the abstract and access a quick link to my first exciting publications and grants.
Empowered by emerging concepts from physics, chemistry, and bioengineering, learning-by-building approaches have found increasing application in the life sciences. Particularly, they are directed to tackle the overarching goal of engineering cellular life from scratch. The SynCell2020/21 conference brought together a diverse group of researchers to share progress and chart the course of this field. Participants identified key steps to design, manipulate, and create cell-like entities, especially those with hierarchical organization and function. This article highlights achievements in the field, including areas where synthetic cells are having socioeconomic and technological impact. Guided by input from early-career researchers, we identify challenges and opportunities for basic science and technological applications of synthetic cells. A key conclusion is the need to build an integrated research community through enhanced communication, resource-sharing, and educational initiatives. Development of an international and interdisciplinary community will enable transformative outcomes and attract the brightest minds to contribute to the field.
Work in Progress: Organ architecture and performance are some of life’s most challenging self-assembling features to reduce into the minimal essential building blocks required for form and function. Organoid biology has gained scientific attention as an approach to study tissue formation and function using miniaturized versions of vital organs grown from stem cells in the laboratory. However, organoid methodology is in need of innovation to gain fundamental understanding and control over adhesion mediated interactions (e.g. cell/cell and cell/ECM) that influence stem cell programming. This action will combine organoid biology with bottom-up synthetic biology to design minimalist light-controlled and modular interactions between stem cells, synthetic cells, and ECM thereby leading to the innovation of programmable synthetic stem cell niches that will produce predictable and uniform organoid populations. I will develop this programmable hybrid organoid approach using human inducible pluripotent stem cells (hiPSCs), syncells with light-controllable cell/cell adhesion modules (optoSynCells), and droplet-based microenvironments comprising light-sensitive extracellular matrix (optoECM). I will use these new hybrid organoids to understand the impact of juxtacrine signalling on programming the differentiation and spatial organization of hiPSCs into hybrid organoids. The action is designed to produce bifunctional pancreatic organoids with the state-of-the-art implementation demonstrating exocrine and endocrine phenotypes, although the knowledge gained by the work packages will be far reaching and applicable to other organoid types. The new understanding of adhesion interactions that will be uncovered by my experiments will lay the foundation for applications in high-content drug screening, reduce the use of animal models in biomedical research, and generate of patient-derived transplant materials.
Formation of condensed phase nucleoprotein assemblies, such as membraneless organelles (MLOs), that contribute to gene regulation and signaling within the cell is garnering widespread attention. A critical technical challenge is understanding how interactions between intrinsically disordered protein (IDP) and nucleic acid molecular components affect liquid-liquid phase separation (LLPS) into nucleoprotein condensates. To better understand the physics of LLPS that drive the formation of biomolecular condensates (known as coacervates), we investigate a model IDP system using a cationic elastin-like polypeptide (ELP), “E3”, that is engineered to phase separate and bind DNA upon coacervate formation. Using mean field Flory-Huggins (FH) theory, we create ternary phase diagrams to quantify DNA component partitioning within discrete protein and solvent rich phases across a range of salt and E3 compositions. We suggest a modified FH theory that combines canonical FH interaction parameters with an approximation of the Debye-Hückel theory to predict the strength of E3-DNA interactions and partitioning with variable salt concentration. Finally, we establish a simple two-step DNA solution separations/purification assay to highlight the potential utility of our system. This model LLPS biopolymer platform represents an important chemical engineering-based contribution to synthetic biology and DNA technologies, with possible implications toward origin of life discussions.
The interplay between academics and society within the environment of the COVID-19 pandemic has impacted on scientists across the world, prompting reevaluation of how virtual toolboxes can be used to support responsible collaborative research practices. We provide awareness of virtual resources and activities that enable scientific discovery using safe and efficient practices.
Quickly and easily producing uniform populations of microsphere-based 3D cell cultures using droplet-based templating methods has the potential to enable widespread use of such platforms in drug discovery or cancer research. Here, we advance the design of centrifuge-based droplet generation devices, describe the use of this platform for droplet generation with controlled cell occupancy, and demonstrate weeklong culture duration. Using simple-to-construct devices and easily implemented protocols, the initial concentration of encapsulated cells is adjustable up to hundreds of cells per microsphere. This work demonstrates the first instance of using centrifugal droplet-generating devices to produce large numbers of cell-encapsulating microspheres. Applications of this versatile methodology include the rapid formation of templated 3D cell culture populations suitable for suspension culture or large batch bioreactor studies that require uniform populations.
We present an easy-to-assemble microfluidic system for synthesizing cell-loaded dextran/alginate (DEX/ALG) hydrogel spheres using an aqueous two-phase system (ATPS) for templated fabrication of multicellular tumor spheroids (MTSs). An audio speaker driven by an amplified output of a waveform generator or smartphone provides acoustic modulation to drive the breakup of an ATPS into MTS template droplets within microcapillary fluidic devices. We apply extensions of Plateau–Rayleigh theory to help define the flow and frequency parameter space necessary for acoustofluidic ATPS droplet formation in these devices. This method provides a simple droplet microfluidic approach using off-the-shelf acoustic components for quickly initiating MTSs and subsequent 3D cell culture.
Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation techniques have the ability to encapsulate cancer cells in droplets for standardized and more realistic 3D cell culture and cancer biology applications. Three individual droplet generating platforms have been designed and optimized for droplet-based cell encapsulation. Each has its own advancements and challenges. Together, however, these technologies accomplish medium to high-throughput generation (10 droplets/second to 25,000 droplets/second) of biomaterial droplets for encapsulation of a range of cell occupancies (5 cells/droplet to 400 cells/droplet). The data presented also demonstrates the controlled generation of cell-sized small droplets for biomolecule compartmentalization, droplets with diameters ranging between 100-400 μm depending on device parameters, and the generation of instant spheroids. Standardized assays for analyzing cells grown within these new 3D environments include proliferation assays of cells grown in mono- and co-cultures, the generation of large and uniform populations of scaffold supported multicellular spheroids, and a new system for culturing encapsulated cells in altered environmental conditions.
Adaptation of cancer cells to changes in the microenvironment in an expanding tumor mass is a crucial aspect of malignant progression. To proliferate, cells depend on nutrient and waste transport from surrounding vasculature that becomes less orderly as tumor volume increases, creating chemical gradients in extracellular pH (pHe) and oxygen (O2). Hypoxia inducible factor 1α (HIF-1α), a monomer of a heterodimeric transcription factor regulated by a complex network, mediates cellular responses to physiologic and pathologic processes that allow changes in angiogenesis, cell proliferation and survival, chemosensitivity, and metabolism. The activation of the glycolytic pathway even in the presence of oxygen is a characteristic of many cancer cells, leading to an increase in metabolic consumption rates that can be directly correlated to pHe and O2. HIF-1α regulates energy metabolism by activating the gene expression of glycolytic enzymes, regulatory enzymes, and glucose transporters. HIF-1α is itself post-transcriptionally regulated through blockage of proteosomal degradation as it depends on oxygen concentration. We hypothesize that pHe also plays a role in the regulation of HIF-1α by influencing the half-life of the transcription factor potentially explaining clinical observations of increased chemosensitivity even under hypoxic conditions traditionally thought to cause chemoresistance. Currently, there are no experimental platforms that provide the tools to test such a hypothesis. The fellowship-training plan, through three specific aims, will develop a new in vitro 3D cell culture perfusion system with integrated fluorescence nanosensing capabilities for in situ detection of pHe and O2 gradients. The instrument is designed to enable spatially correlated investigation of cellular responses to the measured pHe and O2 gradients by determination of metabolic consumption rates using a simple transport model. Recovering cells and supernatants from defined regions within the device allows for biochemical and molecular analysis of HIF-1α, proteins regulated by HIF-1α, cell proliferation and survival, and VEGF as adapted by cells in different microenvironments. The three aims will ultimately yield a new integrated model system that will produce advances in: 1) quantitative, manipulable in vitro 3D models of the tumor microenvironment; 2) direct spatial correlation of pHe and O2 gradients with cellular adaptations; and 3) understanding tumor cell response to gradients of pHe and O2 as a function HIF-1α.
Rapid expansion of cancer cells in solid tumors and the impact on interactions in the tumor microenvironment is a dynamic and multiscale process. The integration of improved tools and hypothesis driven experimentation will lead to a better understanding of these interactions and allow progress in the tumor microenvironment field. The goal of this fellowship-training proposal is to utilize this integrated approach to address the interacting dynamics of HIF-1α mediated responses to pH and oxygen microenvironments.
Grant Report: https://reporter.nih.gov/project-details/9071293
Poly(N-isopropyl acrylamide) (pNIPAM) is a “smart” polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinningparameters were varied (needle gauge, collection time, and molecular weight of the polymer)to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.