3D-Printing in the Life Sciences Agenda

Conference Chairperson
Nancy Allbritton, Kenan Professor of Chemistry and Biomedical Engineering and Chair of the Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, United States of America

Conference Chairperson
Y. Shrike Zhang, Assistant Professor of Medicine, Harvard Medical School, Associate Bioengineer, Division of Engineering in Medicine, Brigham and Women’s Hospital, United States of America

Pumps, Valves, and Fluidic Connectors for Automating and Integrating Microphysiological Systems
John Wikswo, Gordon A. Cain University Professor, A.B. Learned Professor of Living State Physics; Founding Director, Vanderbilt Institute for Integrative Biosystems, Vanderbilt University, United States of America

Much of the organ-on-chip and tissue-chip research to date has relied on very simple perfusion schemes that include surface tension, hydrostatic pressure, rocking plates, syringe pumps, pressurized reservoirs, and on-chip pneumatic or electromagnetic peristaltic pumps. A number of commercial microphysiological systems are moving towards high-throughput well-plate formats. Each of these fluid handling technologies presents different advantages in cost, convenience, and simplicity, and disadvantages regarding reservoir filling, media change, recirculation and volume minimization, organ-organ integration, bubble- and microbe-free addition and removal of chips from multi-organ microphysiological systems, temporal control of drug concentrations, adjusting/balancing flow rates, and controlling sensors for long-term use. While a common trend is towards high throughput and low cost, there are critical applications where high content is more important than high throughput, particularly those requiring multi-omic characterization of physiological responses over time to drugs and toxins for both single and multiple organs. We review how our computer controlled, modular, rotary planar peristaltic micropumps and twenty-five port rotary planar valves can be used to create autocalibrating electrochemical MicroClinical Analyzers, multi-well MicroFormulators that can create in vivo the pharmacokinetics observed in animals and humans, and perfusion controllers for multiple NeuroVascular Unit, Gut-in-a Puck, and Thick Tissue Mammary Bioreactors. Pumps and valves are useful for compact perfusion controllers that operate in microgravity environments. Miniature rotary valves will enable bubble-free, sterile connection and disconnection of individual organ chips to other organs and analyzers. Ongoing and future applications include 100-port valves and systems for optimizing the differentiation of induced pluripotent stem cells, a 10,000 channel yeast MicroChemostat, and a MicroDialysis imager, all with direct injection into a mass spectrometer. The cost and complexity of these systems over present technologies is balanced by sophisticated computer control and sensor integration, reduced technician effort, high content results, and parallelization.

Intestinal Epithelium for Basic Physiology and Drug Assays
Nancy Allbritton, Kenan Professor of Chemistry and Biomedical Engineering and Chair of the Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, United States of America

Organ-on-chips are miniaturized devices that arrange living cells to simulate functional subunits of tissues and organs. These microdevices provide exquisite control of tissue microenvironment for the investigation of organ-level physiology and disease. Human organ-on-chips are expected to transform biomedical research providing platforms that accurately replicate human tissues, enable a better understanding of human-to-human physiologic variations, and even permit patient-specific organ mimics. These human organ facsimiles will fundamentally alter drug discovery and development by providing human constructs for screening assays, toxicity measurements and investigation of molecular-level drug actions. Breakthroughs in stem-cell biology now enable single stem cells or intestinal crypts isolated from primary mouse or human intestine or differentiated from induced pluripotent stem cells (iPSCs) to grow and persist indefinitely in defined 3D culture conditions to form organotypic structures generically termed enteroids. We have developed a long-lived, self-renewing monolayer culture format from primary intestinal cells. A surface matrix and chemical factors sustain the epithelial cell monolayers so that they possess all cell repertoires found in vivo. This technical advance has made it possible to create primary tissues on platforms that are compatible with high-content screening strategies. For example, the screening of 77 dietary compounds revealed that these compounds altered proliferation to increase stem cell numbers or increased cell differentiation with formation of increased numbers of goblet cells or enterocytes. Measurement of drug transport and metabolism has also been demonstrated in these human intestinal monolayer systems as well as formation of physiologic mucus layers many hundreds of microns thick. Culture of these monolayers on a shaped scaffold under chemical gradients replicates much of the cell compartmentalization and physiology observed in vivo. This bioanalytical platform is envisioned as a next-generation system for assay of microbiome-, drug- and toxin-interactions with the intestinal epithelia. Finally, intestinal biopsy samples can be used to populate these constructs with cells producing patient-specific tissues for personalized medicine that can be applied to emerging areas of disease modeling and microbiome studies.

Digital Manufacturing of Microfluidic Tissue Chips
Albert Folch, Professor of Bioengineering, University of Washington, United States of America

The microfluidics field is at a critical crossroads. The vast majority of microfluidic devices are presently manufactured using micromolding processes that work very well for a reduced set of biocompatible materials, but the time, cost, and design constraints of micromolding hinder the commercialization of many devices. PDMS, in particular, is extremely popular in academic labs, yet the fabrication procedures are based on cumbersome manual methods and the material itself strongly absorbs lipophilic drugs. As a result, the dissemination of many cell-based microfluidic chips – and their impact on society – is in jeopardy. Digital Manufacturing (DM) is a family of computer-centered processes that integrate digital 3D designs, automated (additive or subtractive) fabrication, and device testing in order to increase fabrication efficiency. Importantly, DM enables the inexpensive realization of 3D designs that are impossible or very difficult to mold. The adoption of DM by microfluidic engineers has been slow, likely due to concerns over the resolution of the printers and the biocompatibility of the resins. We have developed microfluidic devices by SL in PEG-DA-based resins with automation and biocompatibility ratings similar to those made with PDMS. I will also present our work on our microfluidics platform (digitally-manufactured in thermoplastics) for cancer diagnostics using live tumor biopsies and I will review the bright future ahead for the promising, fertile field of DM.

Models of Neurological Disease
Roger Kamm, Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering, Massachusetts Institute of Technology (MIT), United States of America

Many of the most debilitating and life-threatening diseases are associated with the central nervous system. Some, such as neurodegenerative diseases, predominately afflict our aging population. Yet others such as brain cancers and ALS are prevalent among young and old alike. In this presentation, models will be presented that attempt to recapitulate certain aspects of the brain and these diseases, both to probe the disease process, and as a platform to screen for new therapeutics. Three examples will be presented. First, a model for the healthy blood-brain barrier and neurovascular unit have been developed in order to capture the essential aspects associated with these diseases. Second, the blood-brain barrier model is used to study metastasis of cancers to the brain, as well as primary glioblastoma. In the third part of the talk, a model for the healthy and diseased neuromuscular junction will be presented as a step toward developing therapeutic strategies for treating ALS.

“Bone Marrow-on-a-Chip”: A Strategy to Replicate Hematopoiesis and Cancer Cell Dormancy
Steven C. George, Edward Teller Distinguished Professor and Chair, Department of Biomedical Engineering, University of California-Davis, United States of America

Tissue engineering holds enormous potential to not only replace or restore function to a wide range of tissues, but also to capture and control 3D physiology in vitro (e.g., microphysiological systems or “organ-on-a-chip” technology).  The latter has important applications in the fields of drug development, toxicity screening, modeling tumor metastasis, and repairing damaged cardiac (heart) muscle. In order to replicate the complex 3D arrangement of cells and extracellular matrix (ECM), new human microphysiological systems must be developed.  The past decade has brought tremendous advances in our understanding of stem cell technology and microfabrication producing a rich environment to create an array of “organ-on-a-chip” designs.  Over the past six years we have developed novel microfluidic-based systems of 3D human microtissues (~ 1 mm³) that contain features such as perfused human microvessels, primary human cancer, mouse tumor organoids, stem cells, and spatiotemporal control of oxygen.  The technologies are part of two early start-up companies, Kino Biosciences and Immunovalent Therapeutics, based in Irvine, CA and St. Louis, MO.  This seminar will describe our approach and early results on a strategy to create a 3D in vitro model of human bone marrow with applications in hematopoiesis and cancer cell dormancy.

Human Tissue Chips for Drug Development, Disease Modeling, and More…
Kevin Healy, Jan Fandrianto and Selfia Halim Distinguished Professorship in Engineering, University of California, Berkeley, United States of America

Our work has emphasized creating both healthy and diseased model organ systems, we call microphysiological systems or ‘tissue chips’, to address the costly and complex drug discovery process. The average time to develop and launch a new drug in the United States is 10-15 years, and costs ~ $2-5b. The poor efficiency and high failure rates are attributed to the heavy reliance on non-human animal models employed during safety and efficacy testing that poorly reflect human disease states. With the discovery of human induced pluripotent stem cells, we can now develop tissue chips to be used for high content drug screening, disease modelling, and numerous other applications. While tissue chips are poised to disrupt the drug development process and significantly reduce the cost of bringing a new drug candidate to market, tissue chip technology is much more robust and creates a whole new paradigm in how to conduct biological science, and advances medicine in revolutionary ways. Ultimately, the vision is to reduce or eliminate the use of animals in drug discovery, and conduct ‘clinical trials’ in patient-specific tissue chips that can accommodate variations in genetics, environment, and lifestyle.

Computed Axial Lithography For Volumetric Printing of Soft Structures
Hayden Taylor, Assistant Professor, Mechanical Engineering, University of California-Berkeley, United States of America

Volumetric additive manufacturing is defined as producing the entire volume of a component or structure simultaneously — rather than by layering — and has been envisioned as a possible way to increase the speed of additive manufacturing. Until recently, however, no practicable technique existed for creating arbitrary 3D geometries volumetrically. Recently, we have demonstrated Computed Axial Lithography (CAL) to meet this need. CAL essentially reverses the principles of computed tomography (widely used in imaging, but not previously in fabrication) to synthesize a three-dimensionally controlled illumination dose within a volume of photocurable resin. The photosensitive volume rotates steadily while a video projector illuminates the material from a direction perpendicular to the axis of rotation. The illumination pattern typically changes >1000 times per revolution, so that light from many different projection angles contributes to the cumulative dose. Where the dose exceeds a threshold, the resin solidifies and the part is formed. In this talk I will discuss the CAL process in the context of its possible bioprinting applications. The CAL printing technique has several potential advantages. Because there is no relative motion between the component being printed and the resin during printing, the printing speed is not limited by resin flow effects, as it can be in layer-by-layer photopolymerization-based printing. The absence of relative motion also allows highly viscous resins or soft, highly compliant materials such as hydrogels to be printed. Because layers are not used in CAL, the surfaces of printed components are very smooth (c. 1–4 µm roughness in preliminary experiments), which may open up new applications. Additionally, it is possible to print objects around pre-existing solid objects that could have been made using a different material or process. This ‘overprinting’ capability suggests applications in mass-customization for end users and also potentially in fabricating biological structures with highly heterogeneous mechanical properties.

Handheld Skin Printer: In-Situ Formation of Biomaterials and Skin Tissues
Axel Guenther, Professor and Co-Lead, Collaborative Centre for Research and Applications in Fluidic Technologies (CRAFT), University of Toronto, Canada

I discuss the in-situ delivery of biomaterials and cells for skin tissue repair. We developed a handheld skin bioprinter, specifically for intraoperative use. I will discuss unique features of the handheld bioprinter instrument, physical and in vitro characterization of in-situ deposited biomaterials and tissues, as well as in vivo results that were obtained in porcine wound in collaboration with Dr. Marc Jeschke’s team at Sunnybrook Health Sciences Center in Toronto.

Innovations in Bioprinting
Ali Khademhosseini, Professor, Department of Bioengineering, Department of Radiology, Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, United States of America

Putting 3D Bioprinting to the Use of Tissue Model Fabrication
Y. Shrike Zhang, Assistant Professor of Medicine, Harvard Medical School, Associate Bioengineer, Division of Engineering in Medicine, Brigham and Women’s Hospital, United States of America

The talk will discuss our recent efforts on developing a series of bioprinting strategies including sacrificial bioprinting, microfluidic bioprinting, and multi-material bioprinting, along with various cytocompatible bioink formulations, for the fabrication of biomimetic 3D tissue models. These platform technologies, when combined with microfluidic bioreactors and bioanalysis, will likely provide new opportunities in constructing functional microtissues with a potential of achieving precision therapy by overcoming certain limitations associated with conventional models based on planar cell cultures and animals.

Modulating Physical, Chemical, and Biological Properties of Tissue Constructs via Rapid 3D Bioprinting
Shaochen Chen, Professor and Chair, NanoEngineering Department, University of California-San Diego, United States of America

In this talk, I will present our laboratory’s recent research efforts in rapid 3D bioprinting to create 3D tissue constructs using a variety of biomaterials and cells. These 3D biomaterials are functionalized with precise control of micro-architecture, mechanical (e.g. stiffness), chemical, and biological properties. Such functional biomaterials allow us to investigate cell-microenvironment interactions at nano- and micro-scales in response to integrated physical and chemical stimuli. From these fundamental studies we have been creating both in vitro and in vivo precision tissues for tissue regeneration, disease modeling, and drug discovery. To vascularize these engineered tissues, we have also developed a pre-vascularization technique by using the rapid 3D bioprinting method. Multiple cell types mimicking the native vascular cell composition were encapsulated directly into hydrogels with precisely controlled distribution.