Jennifer A. Lewis - US grants

NON-TECHNICAL DESCRIPTION: Three-dimensional (3D) printing offers the potential to digitally specify the form and function of materials. This project focuses on patterning 3D ceramic architectures by designing and implementing microfluidic printheads for use with model colloidal suspensions - i.e. concentrated fluids, gels, and biphasic mixtures - whose flow behavior can be directly imaged within microfluidic core-shell and multinozzle printheads. Advances from this research effort are expediting the transformation of 3D printing from rapid prototyping to a true manufacturing platform. The fundamental knowledge gained from this program of research will enable low-cost, high-throughput printing of designer ceramics that may find application as 3D ceramic composites, membranes, and battery electrodes. In addition, new scientific understanding of the flow and mixing of concentrated colloidal suspensions within confined microfluidic geometries is emerging from this effort.

TECHNICAL DETAILS: This project focuses on 3D printing of concentrated colloidal inks in interspersed and interpenetrating motifs using microfluidic core-shell and multinozzle printheads. Fundamental relationships between ink rheology, printhead designs, and printing behavior are being systematically investigated. The resulting 3D ceramic architectures may offer significant performance advantages in a broad range of applications, including lightweight composites, membranes, and batteries. The project integrates multiple education and outreach activities aimed at expanding the number of underrepresented groups in science, technology, engineering and mathematics (STEM) through public lectures and hands-on activities as well as by creating scientific videos of our research targeted for the DIY (do-it-yourself) and maker communities.

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The Harvard MRSEC focuses on highly collaborative multidisciplinary research with participants drawn from five departments across the university. The overarching goal of the Center is to perform transformative research that significantly advances the state of knowledge in several areas of soft matter science, and to educate the next generation of leaders in materials science and engineering. The MRSEC has three interdisciplinary groups focused on very large deformations in soft materials, creating new materials for 3D printing and creating novel forms of soft machines. An essential contribution of the Center is to increase diversity of participants in its science at all levels, from undergraduate through graduate and postdoctoral to faculty. The Harvard MRSEC also sponsors a robust suite of activities to share the excitement of its science with the broader scientific community, and with the public at large. By combining soft matter science with cooking the Center elicits exhilaration through a public lecture series that combines science with talks by famous chefs; these are also posted online where there is an avid following, with the videos already attracting over 1.5M hits. The MRSEC is also developing special initiatives to bring returning veterans into STEM fields. By pooling resources for shared experimental facilities, the MRSEC supports a more extensive research effort. The resultant teamwork enables the Center to address problems that are larger in scale, and to produce results that are truly transformative. The MRSEC sets a standard of excellence for highly collaborative, multidisciplinary research.

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The Harvard MRSEC supports highly collaborative, interdisciplinary research in several areas of soft matter science. These include investigations of the properties of soft materials subjected to very large deformations; development of fundamental knowledge essential to create and rapidly transform diverse classes of soft materials into 3D functional architectures; and development of the science of soft, non-linear unstable elastomeric materials and the use of these material instabilities to develop devices with high-value performance at lower cost. The Center also supports seed projects to enable rapid response to new developments; currently it supports an effort in developing tough fabrics to elicit interest among returning veterans, a group targeted by the MRSEC to help integrate into STEM careers. The MRSEC establishes a culture of innovation and entrepreneurship, enhancing the competitiveness of the national economy through industrial partnerships and formation of numerous start-up companies, creating more than 350 new high-tech jobs based directly on NSF support. Graduates of the Harvard MRSEC continue to be leaders in materials science and engineering, both nationally and internationally.

The ability to fabricate vascularized living tissues would enable critical advances in drug screening, tissue repair and regeneration. Tissue engineering has traditionally relied on the use of acellular scaffolds. However, this approach vastly limits the size and complexity of the tissues that can be created due to the lack of stable, perfusable vasculature and the inability to replicate intricate multicellular configurations. Vasculature networks are central to living tissues, since all cells must reside within several hundred micrometers of a nutrient supply to survive. This award supports scientific investigations on a new additive manufacturing process for fabricating three-dimensional, vascularized living tissues composed of cells, extracellular matrix, and embedded blood vessels. Results from this research will enable broader use of 3D living tissues in the pharmaceutical industry for drug safety and toxicity screening and, ultimately, in the medicine for tissue repair and regeneration.

This research will establish the fundamental scientific understanding required for bioprinting of vascularized living tissue at organ scale. The complex interplay between cells, extracellular matrix, and vasculature in printed tissues will be determined. These relationships will be established by quantifying cell viability via live/dead staining as a function of varying cell type, concentration, and extracellular matrix composition. The effects of vascular network architecture, including blood vessel size, spacing, and degree of branching, on cell viability and function will also be quantified by live/dead and, concurrently, barrier function of the vascular channels will be measured by a standard leak test as a function of different architectural motifs. Finally, the relationship between nozzle size, design, and printing speed will be determined for cell-laden inks of varying cell type, concentration, and extracellular matrix composition to identify the requisite conditions that promote maximum cell viability, as determined by live/dead staining, during bioprinting and perfusion over long time periods.

NON-TECHNICAL SUMMARY
Materials that satisfy society's increasing demand for technological innovation and that provide solutions to major global challenges of the 21st century in the fields of energy efficiency, resource management, technology development, human health, and world security are frequently required to simultaneously exhibit multiple functions with superior performance. Through the course of natural evolution, a plethora of organisms have conceived material solutions that show exemplary performance characteristics across multiple property classes, including mechanics, optics, actuation and chemistry. These organisms thus provide an advantageous starting point for studying the role of morphology, morphogenesis, and material composition on emerging material properties. The project will explore the causalities between hierarchical material architectures, composition and morphogenesis, and the emerging functionalities in a set of exemplary biological systems. This will enable the identification of a generalized set of rules for guiding the design and fabrication of multifunctional 21st century materials.

TECHNICAL SUMMARY
This research is inspired by the vision that an understanding of the material solutions and design criteria used by Nature's finest multitasking artists in combination with novel analytical and computational materials evolution tools can provide insight into functional synergies and trade-offs in multifunctional materials and result in revolutionary biomimetic material platforms. The research team proposes to study the causalities between hierarchical material architectures, composition, and morphogenesis and the emerging properties in a set of exemplary biological systems by analytical and computational analysis of the multi-faceted material parameter interactions underlying true multifunctionality. Building on knowledge about design paradigms prevalent in biological multifunctional materials, analytical algorithms, computational routines, and virtual material design environments will be conceived that will allow the characterization of the phase space of possible material solutions as a function of user-prescribed performance criteria. This will permit the team to identify a generalized set of rules for guiding the design and fabrication of multifunctional new materials. The particular emphasis is on identifying synergies and trade-offs between mechanical functionalities, optical properties, actuation behavior, fluidics, and surface-chemistry induced effects. Based on this set of design rules, the PIs will fabricate material prototypes using state-of-the-art additive manufacturing, self-assembly, and microfabrication strategies. A detailed characterization of the performance of these prototypes and comparison to the parent biological system(s) will enable evaluation of the validity and prediction capabilities of the design rules and allow for their refinement in an iterative process. In summary, the PIs propose to tackle the challenges of multifunctional material design using a feedback oriented "evolutionary research algorithm" with focus on the realization of dynamic multifunctional materials capable of fast autonomous or controlled functional morphing stimulated by external influences or user input.

Cancer immunotherapy is currently providing exciting new treatment options for patients. However, the majority of patients still do not respond to current immunotherapies, and this failure likely results, at least in part, from an inability to generate potent cytotoxic T lymphocyte (CTL) responses against cancer antigens, and the tolerizing effects of the tumors. Therapeutic vaccines may be needed to generate robust CTL responses, and we have recently developed a new biomaterials strategy for vaccination that led to unprecedented ability to eradicate established tumors in preclinical models. However, the development of next generation vaccines based on this concept, and therapeutic cancer vaccines more generally, is significantly impaired by the limitations of current model systems available to explore and test these types of therapies. Preclinical studies typically utilize mouse models, but even humanized mouse models do not capture key aspects of human biology relevant to immunotherapies. Cell culture studies can be used to explore human immune cell biology, but standard human cell culture models do not recreate the 3D, multicellular interactions that direct the immune response against cancer nor the tumor cell-immune cell interactions that dictate vaccination success. This application proposes to create 3D models of human biology that enable one to study key aspects of vaccination. These models will replicate, in vitro, the vaccine site itself, where the immune response to cancer antigens is initiated, and the tumor, where immune cells encounter cancerous cells, and the function of the immune cells is typically down-regulated by the cells within the tumor. In order to thoroughly characterize and validate our approach, we will first create 3D mouse models of the vaccine site and the tumor, as this will allow direct comparison between the 3D in vitro model and the in vivo tissue of the same type. These studies will be key to validate the models. We will then create the human models, using tumor, vascular and immune cells all derived from the same patient. These human models will be used to begin exploring several key issues in therapeutic cancer vaccination, including the role of checkpoint blockade and angiogenic factors on the tumors, and the impact of vaccination intratumorally on the immune cell response. At the completion of this project we will have developed and thoroughly characterized novel, 3D models of both mouse and human biology that will replicate the vaccination site and vascularized tumors. These models will allow us to explore key questions relevant to human cancer immunotherapy, and provide a means to screen the impact of immunomodulatory agents (e.g., various adjuvants) in the future as we and others develop new cancer immunotherapies.

PROJECT SUMMARY In the U.S. alone, up to 26 million people have chronic kidney disease, over 460,000 people are on dialysis, and 100,000 people await kidney transplants with 3,000 new patients added monthly. Given the growing lack of transplantable organs, patients typically require renal replacement therapies that themselves lead to substantial morbidity and mortality. We posit that biomanufactured kidney tissues, and ultimately, organs may offer an important solution to this growing problem. Indeed, recent protocols in developmental biology are unlocking the potential for stem cells to undergo differentiation and self-assembly to form ?mini-organs?, known as organoids. Kidney organoids exhibit remarkable tissue microarchitectures with high cellular density and heterogeneity akin to their in vivo counterparts. To bridge the gap from renal tissue building blocks to therapeutic organs, integrative approaches that combine bottom-up organoid assembly with top-down bioprinting are needed. It is difficult, if not impossible, to imagine how either organoids or bioprinting alone would fully replicate the complex multiscale features required for kidney function. Yet their combination could provide an enabling foundation for de novo organ manufacturing. To biomanufacture 3D vascularized kidney tissues for potential therapeutic applications, in Specific Aim 1, we will create microvascularized kidney organoids perfused by luminal connection with bioprinted macrovessels under controlled flow in vitro. We will produce iPSC-derived kidney organoids and subject them to fluid flow during their differentiation and maturation on an adherent extracellular matrix (ECM). We will then investigate the integration of their endogenous microvasculature with printed macrochannels by establishing a controlled VEGF gradient to guide anastomosis on a customized perfusion chip. This proof-of- concept experiment is a necessary first step towards a scalable tissue biomanufacturing process. In Specific Aim 2, we will evaluate the effects of macro-microvascular integration and perfusion on kidney organoid structure, glomerular filtration and tubular maturation. Both qualitative and quantitative analysis of vasculature and nephron development will be carried out to assess their morphology and function. We will then assess perfusion and transport through the vascular and tubular network(s) using a combination of bead flow, microCT, microperfusion, and micropuncture experiments. We will analyze the composition of primitive urine collected via a printed drainage channel co-localized near the organoid bed. To create 3D vascularized kidney tissues in a scalable manner, in Specific Aim 3, we will form tissue matrices composed of kidney organoids (1 mL or higher) within which a perfusable macrovasculature network will be patterned by embedded bioprinting. Kidney tissue viability, structure, maturation and function will be qualitatively and quantitatively analyzed following the protocols used in Specific Aim 2. If successful, our proposed discovery-based project will provide a foundational advance in kidney organ engineering for potential renal therapeutic applications.

PROJECT SUMMARY In the U.S. alone, up to 26 million people have chronic kidney disease, over 460,000 people are on dialysis, and 100,000 people await kidney transplants with 3,000 new patients added monthly. Given the growing lack of transplantable organs, patients typically require renal replacement therapies that themselves lead to substantial morbidity and mortality. We posit that biomanufactured kidney tissues, and ultimately, organs may offer an important solution to this growing problem. Indeed, recent protocols in developmental biology are unlocking the potential for stem cells to undergo differentiation and self-assembly to form ?mini-organs?, known as organoids. Kidney organoids exhibit remarkable tissue microarchitectures with high cellular density and heterogeneity akin to their in vivo counterparts. To bridge the gap from these kidney organoid building blocks (OBBs) to therapeutic organs, integrative approaches that combine bottom-up organoid assembly with top-down bioprinting are needed. While it is difficult, if not impossible, to imagine how either organoids or bioprinting alone would fully replicate the complex multiscale features required for kidney function ? their combination could provide an enabling foundation for de novo organ manufacturing. To generate 3D functional kidney tissues ex vivo for potential transplantation, our highly collaborative research team will undertake two primary aims. In Specific Aim 1, we will create kidney organoids enhanced by multilineage induction that display functional differentiation of nephrons. We will produce iPSC-derived kidney organoids and subject them to fluid flow during their differentiation and maturation on an adherent extracellular matrix (ECM). Through multilineage induction, we will also induce collecting duct cells that self-assemble and structurally bridge other tubular nephron segments. We will evaluate the effects of mimicking kidney organogenesis on kidney organoid structure and function using microperfusion and micropuncture methods. In Specific Aim 2, we will create 3D functional kidney tissues composed of these optimized kidney OBBs with embedded macrochannels produced by bioprinting that serve as both vascular and urinary output conduits. We will first produce a densely cellular, tissue matrix composed of kidney OBBs that facilitates bioprinting of embedded macrochannels. We will then establish connections between the printed macrochannels embedded in this OBB-laden matrix and the self-assembled microvascular and collecting duct networks within individual OBBs. Finally, we will assess the glomerular filtration, tubular maturation, and primitive urinary production of these 3D kidney tissues. If successful, our proposed project will provide a foundational advance in kidney organ engineering for potential renal therapeutic applications.

Experimental models of the human developing brain are needed to investigate human-specific aspects of brain development, evolution, and neurological disease. Progress in the field has been hampered by the lack of models, considering that the endogenous developing human brain cannot be directly investigated; animal models often fail to recapitulate human disorders and cannot feasibly be used to study complex polygenic states spanning many genes. While reductionist in nature, stem-cell derived 3D human brain organoids offer a first-of- its-kind opportunity to study processes of human brain formation and wiring that are otherwise not accessible. However, there is an unmet need for organoid models that are cellularly complete and reproducible and for methodology to decode the establishment, connectivity and dynamics of neural circuits in organoids, at scale and with high fidelity. If we could map the activity and connectivity of organoids at scale, both to understand circuit function/dysfunction and to guide further development of organoids, we could close the loop on organoid design and application. Towards this goal, we have developed many molecular and imaging tools for high-throughput analysis of neural activity and connectivity, which we propose to apply to new, next-generation organoid models. Here, we propose a collaborative approach among four groups (Arlotta - brain organoids and human brain development; Boyden - circuit physiology and neural imaging technology; Lewis - material science and bioengineering and Insoo Hyun- bioethics) to pioneer a robust organoid system that combines the development of vascularized brain organoids incorporating more complete cell diversity and maturation with advanced high-throughput functional molecular and imaging tools to enable interrogation of circuit activity, connectivity, and molecular changes in cells participating in physiologically relevant circuits. We will build on a highly reproducible brain organoid model that we recently developed to promote the generation of cell types that are currently absent in organoids but needed for circuit maturation, refinement, and functionality. This work is intended to generate more advanced organoid models designed to promote maturation and robust network activity. In parallel, we will develop a pipeline to record neural activity from intact organoids using all- optical-electrophysiology techniques at scale, and optimize epitope-based barcoding and expansion microscopy to enable molecularly-annotated connectomics of brain organoids. The work proposed here will enable the use of human organoid models to study human circuit formation, plasticity, and function, analyses that are currently hampered by the lack of technologies and assays for high-throughput measurements of circuit physiology and connectivity in organoids. Beyond the work proposed here, these methods will directly enable investigation into how disease states alter information processing in the brain; for example, linking mutations in disease-associated genes with specific abnormalities in human neurons and circuits to inform the identification of molecular targets for therapeutic intervention.