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News Article | January 26, 2016
Site: www.rdmag.com

A team of scientists at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School of Engineering and Applied Sciences has evolved their microscale 3D printing technology to the fourth dimension, time. Inspired by natural structures like plants, which respond and change their form over time according to environmental stimuli, the team has unveiled 4D-printed hydrogel composite structures that change shape upon immersion in water. "This work represents an elegant advance in programmable materials assembly, made possible by a multidisciplinary approach," said Jennifer Lewis, Sc.D., senior author on the new study. "We have now gone beyond integrating form and function to create transformable architectures." Lewis is a Core Faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Hansjörg Wyss Professor of Biologically Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Science (SEAS). L. Mahadevan, Ph.D., a Wyss Core Faculty member as well as the Lola England de Valpine Professor of Applied Mathematics, Professor of Organismic and Evolutionary Biology, and Professor of Physics at Harvard University and Harvard SEAS, is a co-author on the study. Their team also includes co-author, Ralph Nuzzo, Ph.D., the G.L. Clark Professor of Chemistry at the University of Illinois at Urbana-Champaign.   In nature, flowers and plants have tissue compositions and microstructures that result in dynamic morphologies that change according to their environments. Mimicking the variety of shape changes undergone by plant organs such as tendrils, leaves, and flowers in response to environmental stimuli like humidity and/or temperature, the 4D-printed hydrogel composites developed by Lewis and her team are programmed to contain precise, localized swelling behaviors. Importantly, the hydrogel composites contain cellulose fibrils that are derived from wood and are similar to the microstructures that enable shape changes in plants.   Reported on January 25 in a new study in Nature Materials, the 4D printing advance combined materials science and mathematics through the involvement of the study's co-lead authors A. Sydney Gladman, who is a graduate research assistant advised by Lewis and specializing in the printing of polymers and composites at the Wyss Institute and SEAS, and Elisabetta Matsumoto, Ph.D., who is a postdoctoral fellow at the Wyss and SEAS advised by Mahadevan and specializing in condensed matter and material physics.   By aligning cellulose fibrils during printing, the hydrogel composite ink is encoded with anisotropic swelling and stiffness, which can be patterned to produce intricate shape changes. The anisotropic nature of the cellulose fibrils gives rise to varied directional properties that can be predicted and controlled. This is the reason that wood can be split easier along the grain rather than across it. Likewise, when immersed in water, the hydrogel-cellulose fibril ink undergoes differential swelling behavior along and orthogonal to the printing path. Combined with a proprietary mathematical model developed by the team that predicts how a 4D object must be printed to achieve prescribed transformable shapes, the new method opens up many new and exciting potential applications for 4D printing technology including smart textiles, soft electronics, biomedical devices, and tissue engineering. "Using one composite ink printed in a single step, we can achieve shape-changing hydrogel geometries containing more complexity than any other technique, and we can do so simply by modifying the print path," said Gladman. "What's more, we can interchange different materials to tune for properties such as conductivity or biocompatibility."   The composite ink that the team uses flows like liquid through the printhead, yet rapidly solidifies once printed. A variety of hydrogel materials can be used interchangeably resulting in different stimuli-responsive behaviors, while the cellulose fibrils can be replaced with other anisotropic fillers of choice, including conductive fillers.   "Our mathematical model prescribes the printing pathways required to achieve the desired shape-transforming response," said Matsumoto. "We can control the curvature both discretely and continuously using our entirely tunable and programmable method."   Specifically, the mathematical modeling solves the "inverse problem", which is the challenge of being able to predict what the printing toolpath must be in order to encode swelling behaviors toward achieving a specific desired target shape. "It is wonderful to be able to design and realize, in an engineered structure, some of nature's solutions," said Mahadevan, who has studied phenomena such as how botanical tendrils coil, how flowers bloom, and how pine cones open and close. "By solving the inverse problem, we are now able to reverse-engineer the problem and determine how to vary local inhomogeneity, i.e. the spacing between the printed ink filaments, and the anisotropy, i.e. the direction of these filaments, to control the spatiotemporal response of these shapeshifting sheets."   "What's remarkable about this 4D printing advance made by Jennifer and her team is that it enables the design of almost any arbitrary, transformable shape from a wide range of available materials with different properties and potential applications, truly establishing a new platform for printing self-assembling, dynamic microscale structures that could be applied to a broad range of industrial and medical applications," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital and Professor of Bioengineering at Harvard SEAS.   This work was supported by funding from the Army Research Office (ARO) and the National Science Foundation's Materials Research Science and Engineering Center (MRSEC).


News Article | March 28, 2016
Site: phys.org

With super resolution microscopy and qPAINT analysis, researchers will be able to quantify individual molecules at specific locations in the cell. These images show varying copy numbers (shown are three, four, five, and six in the bottom images) of a protein residing in small so-called nuclear pore complexes that permit shuttling of various molecules in and out of the cell's nucleus. The arrows indicate pores with only a single protein that serve to calibrate the counting method. Credit: Wyss Institute at Harvard University. Many biological and pathological processes are not strictly controlled by the presence, absence or function of biomolecules such as proteins or nucleic acids but rather by subtle changes in their numbers at specific locations within cells. However, despite the recent revolution of optical imaging technologies that has enabled the distinction of molecular targets residing less than 200 nm apart from each other, modern super-resolution techniques still face the challenge to accurately and precisely count the number of biomolecules at cellular locations. A new analytical tool developed by a team at the Wyss Institute for Biologically Inspired Engineering solves this problem. The team led by Peng Yin, Ph.D., a Core Faculty member at the Wyss Institute and Professor of Systems Biology at Harvard Medical School, has forged ahead with its previously developed DNA-PAINT and Exchange-PAINT super-resolution microscopy platform to now count different molecular species in biological samples with high accuracy and precision. DNA-PAINT affords higher resolution than costly super-resolution microscopes and Exchange-PAINT can survey multiple different molecules in the same biological sample. The method is reported in the March 28 issue of Nature Methods. "We now have enhanced our DNA-powered super-resolution microscopy methods with a highly quantitative analytical tool kit. qPAINT, as we named it, can accurately count the actual numbers of specific molecules at specific locations inside the cell," said Yin. "Introducing this quantitative power has crucially extended the spectrum of imaging capabilities of this comprehensive and inexpensive technology so that it can be applied in many areas of biological and clinical research." Key to the DNA-driven imaging technology is the transient interaction of two short strands of DNA, one called the "docking strand" that is attached to the molecular target to be visualized and the other, called the "imager strand", which carries a light-emitting dye. "We can precisely program the time interval for which the two complementary DNA strands transiently interact with each other so that when the pair of strands goes through binding and dissociation, the dye will blink at a specific frequency. From an increase of this frequency, we can then deduce with qPAINT analysis how many targets exactly are located at a specific cellular location without spatially resolving each target," said Ralf Jungmann, Ph.D., one of the two co-first authors of the study, a former Postdoctoral Fellow in Yin's lab and now a Group Leader at the Max Planck Institute of Biochemistry at the Ludwig Maximilian University in Munich, Germany. Applying this kind of binding analysis to DNA-PAINT and Exchange-PAINT allows the Wyss team to disregard common problems that fluorescent dyes pose for achieving truly quantitative potential in super-resolution microscopy, like their hard-to-model photophysical properties and tendency to wane under the influence of light, a phenomenon known as photobleaching. In earlier proof-of-principle studies, the team integrated DNA-powered super resolution microscopy with highly specific and broadly available detection reagents, for example, by attaching docking strands to antibodies that specifically bind molecules in various cellular structures and complexes or to DNA probes that bind to specific messenger RNA molecules shuttling genetic information inside cells. "With qPAINT, we counted the numbers of proteins targeted by antibodies at sites such as a cell surface, or the membrane that surrounds the cell nucleus, and even at the nerve endings that stimulate muscles to twitch. The technology can be integrated with a large array of detection reagents to eventually count diverse molecules of interest at the cellular sites where they perform their tasks," said Maier Avendaño, Ph.D., the work's other co-first author and Postdoctoral Fellow in Yin's team. "qPAINT adds a powerful new tool to this simple super-resolution microscopy platform, which now gives researchers the extraordinary capability to quantify how changes in molecule numbers at specific locations influence cell signaling and function. And what is most amazing is that they do this without requiring a highly expensive microscope, and so it should be useable by virtually any biological or clinical laboratory," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences. More information: Quantitative super-resolution imaging with qPAINT using transient binding analysis, DOI: 10.1038/nmeth.3804


News Article | November 29, 2016
Site: www.eurekalert.org

A Wyss Institute team has engineered a method for directly bonding biocompatible chitosan materials to living tissues, which could be used to quickly patch life-threatening wounds (BOSTON) - Chitosan, a biomaterial derived from the chitin shells of crustaceans and insects, has already been developed by scientists at Harvard's Wyss Institute for Biologically Inspired Engineering into an environmentally-friendly and fully biodegradable substitute for plastic. It is only natural that the team, led by Wyss Institute Founding Director Donald Ingber, has also become interested in extending chitosan's usefulness into the clinical realm. "What's good for the environment is also good for us," said Javier Fernandez, who first developed a chitosan bioplastic called 'Shrilk' with Ingber back in 2014. Now Ingber and Fernandez have unveiled a new study in the journal Tissue Engineering that demonstrates biodegradable chitosan bioplastics can be used to bond bodily tissues to repair wounds or even to hold implanted medical devices in place. As chitosan is already approved for clinical use and it has antimicrobial properties, the approach could one day be utilized to immediately seal tissue tears or other serious injuries, preventing infection from setting in before a patient can be moved to a hospital for more in-depth care. "This work really spans the entire mission of the Wyss, as we have developed a biomaterial that could be used in sustainable consumer products and packaging or, as we now show, be adapted for clinical uses," said Fernandez, Ph.D., the first author on the new study, who is a former Wyss Institute Postdoctoral Fellow and is currently an Assistant Professor at Singapore University of Technology and Design. "The material is non-toxic and biodegradable, leaving behind no trace once it has served its purpose." To adapt chitosan to seal wounds and surgical incisions, Ingber and Fernandez searched for a way to quickly and tightly bond chitosan materials to living tissues. They zeroed in on transglutaminase (TG), a naturally occurring enzyme found in the body - where it keeps skin strong and strengthens blood clots - that has also been adopted to bond proteins together during commercial food processing. "As we starting thinking about going in vivo, we faced the challenge of how to adhere chitosan to living tissues," said Ingber, M.D., Ph.D., who is senior author on the new study and in addition to directing the Wyss Institute is Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital as well as Professor of Bioengineering at Harvard's John A. Paulson School of Engineering an Applied Sciences. "We explored using different formulations of transglutaminase to bond various forms of chitosan materials, including sheets, foams and sprays, to many different types of tissues." A sheet of chitosan may be applied with a transglutaminase powder to patch wounds, as the team demonstrated using an ex vivo porcine intestine with a large hole in it. A pressure test revealed that the chitosan patch was even stronger than the native intestinal tissue. For the spray, a stream of liquid chitosan and liquid transglutaminase combine during application to quickly bond chitosan to tissue and close wounds. The team used this approach to seal a porcine lung that had sustained a puncture wound while it was cyclically insufflated with air to mimic inspiration and expiration. The spray application could also be useful for covering large areas of vulnerable tissue, like might be found on someone whose skin had sustained serious burns. To treat even larger and more traumatic wounds like those that might occur on the battlefield or during a motor vehicle accident, Ingber and Javier formulated a chitosan foam that could potentially be used to fill and seal larger wound cavities until a patient can be transported to a hospital for surgical intervention. The team's findings also suggest that their approach could be tailored to bond inorganic surfaces - which make up crucial components of many different kinds of biomedical implants and microfluidic devices - to tissue or chitosan. "Right now our approach is very general, but we could theoretically take this concept and mold it into almost any form imaginable for a broad number of possible uses," said Fernandez. Looking ahead, the team hopes to develop an array of specific applications through collaboration with clinical partners. Additional co-authors on the study include Wyss Institute researchers Suneil Seetharam, Christopher Ding, and Edward Doherty. The Wyss Institute for Biologically Inspired Engineering at Harvard University uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new startups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard's Schools of Medicine, Engineering, Arts & Sciences and Design, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana-Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charité - Universitätsmedizin Berlin, University of Zurich and Massachusetts Institute of Technology.


News Article | October 31, 2016
Site: www.eurekalert.org

(CAMBRIDGE, Massachusetts) - Alginate hydrogels - which are derived from the polysaccharide found in brown seaweed - have emerged as an effective material for manipulating cells and tissues due to their biocompatibility and the ability to tune their mechanical and biochemical properties to match physiological conditions found inside the body. Already they have been demonstrated to influence the differentiation of stem cells, incite immune attacks on cancer cells, and weaken tumors' resistance to chemotherapy, but as of yet, hydrogels have mostly been useful for controlling groups of cells at large rather than individual cells. For example, alginate capsules filled with hundreds of pancreatic islet cells can be implanted in diabetic patients. However, these capsules are millimeters in size and eventually become surrounded by thick scar tissue that blocks the biological signals of islet cells and renders the implant ineffective. Now, thanks to the joint efforts of a team from the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), a new and highly effective microfluidic method for encapsulating single cells in microscale hydrogels sets the stage for a dramatic increase in the specificity of control that can be exerted upon cells and their ability to survive implantation. The research was reported October 31 in the scientific journal Nature Materials. "There's been a tremendous amount of work to try and understand how biomaterials can determine cell function and fate, but the majority of that work has been done in populations of cells," said David Mooney, Ph.D., a Wyss Core Faculty member and the Robert P. Pinkas Family Professor of Bioengineering at SEAS, who is the corresponding author on the new study. "With this work, we take everything we have learned and take it down to the single cell level, enabling us to influence cell behavior on a whole different scale." Mooney teamed with fellow Wyss Core Faculty member David Weitz, Ph.D., who is the Mallingkrodt Professor of Physics and Applied Sciences at Harvard University and SEAS and who is co-author on the study, to achieve the novel microfluidic-based method for encapsulating single cells within microgel capsules. The co-first authors on the study are Angelo Mao, a graduate researcher at Wyss and SEAS, and Jae-Won Shin, Ph.D., who was formerly a Wyss Institute Postdoctoral Fellow and is currently Assistant Professor of Pharmacology and Bioengineering at University of Illinois at Chicago. "This is an exciting and important extension of cell-based biomaterials to the level of single cells, which can then serve both as a precise building block for larger cell structures and as a means of investigating the behavior at the level of single cells, providing unprecedented insight into cell function and properties," said Weitz. Pre-existing single cell encapsulation methods result in relatively large gel capsules - consisting of a very thick hydrogel layer around encapsulated cells - in proportion to the size of the cell captured inside. On average, encapsulated cells take up a mere four percent of the volume of these larger capsules, meaning there is an extremely excessive hydrogel layer. And these pre-existing methods often fail to capture cells at all, resulting in many, many empty capsules and therefore an inefficient process. In contrast, the microfluidic-based method described by the Mooney and Weitz team achieves a much thinner hydrogel layer around encapsulated cells. These aptly-called "microgels" have a volume, on average, that consists 40 percent of a single cell and 60 percent hydrogel layer, resulting in a much smaller capsule size. What's more, the method results in formation of far fewer, if any at all, empty capsules. At that small size, microgel-encapsulated cells can be delivered intravenously, opening new pathways for therapeutic interventions to treat cancers, tissue injuries, and a wide variety of immune disorders. With a thinner hydrogel layer between encapsulated cells and the body's environment, cell therapies can exert influence on the body faster, kicking their disease-fighting effects into action sooner. Microgel-encapsulated cells also stand a better chance of thriving inside the body after injection; currently stem cell therapeutics are challenged by how quickly the body clears cells that are injected 'naked'. Yet microgels infused with growth and anti-inflammatory factors could act as life-sustaining rafts for injected cells, ensuring their survival and ability to carry out their therapeutic purpose. As done in pre-existing techniques, the team first coated cells in calcium carbonate nanoparticles, a step that facilitates cell encapsulation when mixed with an alginate polymer solution. But for the first time, before mixing with a polymer solution, the team washed away the nanoparticles that had not adhered to cells using a water and oil emulsion inside a microfluidic device. What remained were predominantly microgel-encapsulated single cells. "Even though each cell is encapsulated in its own individual thin hydrogel layer, the process is extremely fast and can encapsulate one thousand cells per second inside one microfluidic channel," said Mao. The researchers envision that their method can improve cell-based therapies, help explore heterogeneity between cell populations that underlie tumors and other abnormalities, and even enable a paradigm shift in precision tissue engineering. "Mini tissues could plausibly be formed from meticulous cell-by-cell construction, giving us scrupulous control over the composition of engineered tissues that has not been yet been possible," said Shin. The promising development would not have happened without collaboration between Mooney, who is an expert in tissue engineering and biocompatible hydrogels, and Weitz, who is an expert in using 'designer' emulsions inside microfluidic devices to encapsulate active materials drop by drop. "It's really a great example of what can happen at the Wyss when people can ally with colleagues of different expertise and really rally around a shared goal," Mooney said. "Enabling microgel encapsulation of single cells should allow much better integration and vascularization of implanted cellular therapies, for example in treatment of diabetes or Parkinson's disease, and provide new ways to study and control behavior of individual cells both inside and outside our bodies," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard SEAS. The Wyss Institute for Biologically Inspired Engineering at Harvard University uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new startups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard's Schools of Medicine, Engineering, Arts & Sciences and Design, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana-Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charité - Universitätsmedizin Berlin, University of Zurich and Massachusetts Institute of Technology. The Harvard John A. Paulson School of Engineering and Applied Sciences serves as the connector and integrator of Harvard's teaching and research efforts in engineering, applied sciences, and technology. Through collaboration with researchers from all parts of Harvard, other universities, and corporate and foundational partners, we bring discovery and innovation directly to bear on improving human life and society.


Already they have been demonstrated to influence the differentiation of stem cells, incite immune attacks on cancer cells, and weaken tumors' resistance to chemotherapy, but as of yet, hydrogels have mostly been useful for controlling groups of cells at large rather than individual cells. For example, alginate capsules filled with hundreds of pancreatic islet cells can be implanted in diabetic patients. However, these capsules are millimeters in size and eventually become surrounded by thick scar tissue that blocks the biological signals of islet cells and renders the implant ineffective. Now, thanks to the joint efforts of a team from the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), a new and highly effective microfluidic method for encapsulating single cells in microscale hydrogels sets the stage for a dramatic increase in the specificity of control that can be exerted upon cells and their ability to survive implantation. The research was reported October 31 in the scientific journal Nature Materials. "There's been a tremendous amount of work to try and understand how biomaterials can determine cell function and fate, but the majority of that work has been done in populations of cells," said David Mooney, Ph.D., a Wyss Core Faculty member and the Robert P. Pinkas Family Professor of Bioengineering at SEAS, who is the corresponding author on the new study. "With this work, we take everything we have learned and take it down to the single cell level, enabling us to influence cell behavior on a whole different scale." Mooney teamed with fellow Wyss Core Faculty member David Weitz, Ph.D., who is the Mallingkrodt Professor of Physics and Applied Sciences at Harvard University and SEAS and who is co-author on the study, to achieve the novel microfluidic-based method for encapsulating single cells within microgel capsules. The co-first authors on the study are Angelo Mao, a graduate researcher at Wyss and SEAS, and Jae-Won Shin, Ph.D., who was formerly a Wyss Institute Postdoctoral Fellow and is currently Assistant Professor of Pharmacology and Bioengineering at University of Illinois at Chicago. "This is an exciting and important extension of cell-based biomaterials to the level of single cells, which can then serve both as a precise building block for larger cell structures and as a means of investigating the behavior at the level of single cells, providing unprecedented insight into cell function and properties," said Weitz. Pre-existing single cell encapsulation methods result in relatively large gel capsules - consisting of a very thick hydrogel layer around encapsulated cells - in proportion to the size of the cell captured inside. On average, encapsulated cells take up a mere four percent of the volume of these larger capsules, meaning there is an extremely excessive hydrogel layer. And these pre-existing methods often fail to capture cells at all, resulting in many, many empty capsules and therefore an inefficient process. In contrast, the microfluidic-based method described by the Mooney and Weitz team achieves a much thinner hydrogel layer around encapsulated cells. These aptly-called "microgels" have a volume, on average, that consists 40 percent of a single cell and 60 percent hydrogel layer, resulting in a much smaller capsule size. What's more, the method results in formation of far fewer, if any at all, empty capsules. At that small size, microgel-encapsulated cells can be delivered intravenously, opening new pathways for therapeutic interventions to treat cancers, tissue injuries, and a wide variety of immune disorders. With a thinner hydrogel layer between encapsulated cells and the body's environment, cell therapies can exert influence on the body faster, kicking their disease-fighting effects into action sooner. Microgel-encapsulated cells also stand a better chance of thriving inside the body after injection; currently stem cell therapeutics are challenged by how quickly the body clears cells that are injected 'naked'. Yet microgels infused with growth and anti-inflammatory factors could act as life-sustaining rafts for injected cells, ensuring their survival and ability to carry out their therapeutic purpose. As done in pre-existing techniques, the team first coated cells in calcium carbonate nanoparticles, a step that facilitates cell encapsulation when mixed with an alginate polymer solution. But for the first time, before mixing with a polymer solution, the team washed away the nanoparticles that had not adhered to cells using a water and oil emulsion inside a microfluidic device. What remained were predominantly microgel-encapsulated single cells. "Even though each cell is encapsulated in its own individual thin hydrogel layer, the process is extremely fast and can encapsulate one thousand cells per second inside one microfluidic channel," said Mao. The researchers envision that their method can improve cell-based therapies, help explore heterogeneity between cell populations that underlie tumors and other abnormalities, and even enable a paradigm shift in precision tissue engineering. "Mini tissues could plausibly be formed from meticulous cell-by-cell construction, giving us scrupulous control over the composition of engineered tissues that has not been yet been possible," said Shin. The promising development would not have happened without collaboration between Mooney, who is an expert in tissue engineering and biocompatible hydrogels, and Weitz, who is an expert in using 'designer' emulsions inside microfluidic devices to encapsulate active materials drop by drop. "It's really a great example of what can happen at the Wyss when people can ally with colleagues of different expertise and really rally around a shared goal," Mooney said. "Enabling microgel encapsulation of single cells should allow much better integration and vascularization of implanted cellular therapies, for example in treatment of diabetes or Parkinson's disease, and provide new ways to study and control behavior of individual cells both inside and outside our bodies," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard SEAS. Explore further: Researchers develop a way to predict how a tumor tissue's physical properties affect its response to chemotherapy drugs More information: Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery, Nature Materials (2016) DOI: 10.1038/nmat4781


News Article | November 1, 2016
Site: www.sciencedaily.com

Alginate hydrogels -- which are derived from the polysaccharide found in brown seaweed -- have emerged as an effective material for manipulating cells and tissues due to their biocompatibility and the ability to tune their mechanical and biochemical properties to match physiological conditions found inside the body. Already they have been demonstrated to influence the differentiation of stem cells, incite immune attacks on cancer cells, and weaken tumors' resistance to chemotherapy, but as of yet, hydrogels have mostly been useful for controlling groups of cells at large rather than individual cells. For example, alginate capsules filled with hundreds of pancreatic islet cells can be implanted in diabetic patients. However, these capsules are millimeters in size and eventually become surrounded by thick scar tissue that blocks the biological signals of islet cells and renders the implant ineffective. Now, thanks to the joint efforts of a team from the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), a new and highly effective microfluidic method for encapsulating single cells in microscale hydrogels sets the stage for a dramatic increase in the specificity of control that can be exerted upon cells and their ability to survive implantation. The research was reported October 31 in the scientific journal Nature Materials. "There's been a tremendous amount of work to try and understand how biomaterials can determine cell function and fate, but the majority of that work has been done in populations of cells," said David Mooney, Ph.D., a Wyss Core Faculty member and the Robert P. Pinkas Family Professor of Bioengineering at SEAS, who is the corresponding author on the new study. "With this work, we take everything we have learned and take it down to the single cell level, enabling us to influence cell behavior on a whole different scale." Mooney teamed with fellow Wyss Core Faculty member David Weitz, Ph.D., who is the Mallingkrodt Professor of Physics and Applied Sciences at Harvard University and SEAS and who is co-author on the study, to achieve the novel microfluidic-based method for encapsulating single cells within microgel capsules. The co-first authors on the study are Angelo Mao, a graduate researcher at Wyss and SEAS, and Jae-Won Shin, Ph.D., who was formerly a Wyss Institute Postdoctoral Fellow and is currently Assistant Professor of Pharmacology and Bioengineering at University of Illinois at Chicago. "This is an exciting and important extension of cell-based biomaterials to the level of single cells, which can then serve both as a precise building block for larger cell structures and as a means of investigating the behavior at the level of single cells, providing unprecedented insight into cell function and properties," said Weitz. Pre-existing single cell encapsulation methods result in relatively large gel capsules -- consisting of a very thick hydrogel layer around encapsulated cells -- in proportion to the size of the cell captured inside. On average, encapsulated cells take up a mere four percent of the volume of these larger capsules, meaning there is an extremely excessive hydrogel layer. And these pre-existing methods often fail to capture cells at all, resulting in many, many empty capsules and therefore an inefficient process. In contrast, the microfluidic-based method described by the Mooney and Weitz team achieves a much thinner hydrogel layer around encapsulated cells. These aptly-called "microgels" have a volume, on average, that consists 40 percent of a single cell and 60 percent hydrogel layer, resulting in a much smaller capsule size. What's more, the method results in formation of far fewer, if any at all, empty capsules. At that small size, microgel-encapsulated cells can be delivered intravenously, opening new pathways for therapeutic interventions to treat cancers, tissue injuries, and a wide variety of immune disorders. With a thinner hydrogel layer between encapsulated cells and the body's environment, cell therapies can exert influence on the body faster, kicking their disease-fighting effects into action sooner. Microgel-encapsulated cells also stand a better chance of thriving inside the body after injection; currently stem cell therapeutics are challenged by how quickly the body clears cells that are injected 'naked'. Yet microgels infused with growth and anti-inflammatory factors could act as life-sustaining rafts for injected cells, ensuring their survival and ability to carry out their therapeutic purpose. As done in pre-existing techniques, the team first coated cells in calcium carbonate nanoparticles, a step that facilitates cell encapsulation when mixed with an alginate polymer solution. But for the first time, before mixing with a polymer solution, the team washed away the nanoparticles that had not adhered to cells using a water and oil emulsion inside a microfluidic device. What remained were predominantly microgel-encapsulated single cells. "Even though each cell is encapsulated in its own individual thin hydrogel layer, the process is extremely fast and can encapsulate one thousand cells per second inside one microfluidic channel," said Mao. The researchers envision that their method can improve cell-based therapies, help explore heterogeneity between cell populations that underlie tumors and other abnormalities, and even enable a paradigm shift in precision tissue engineering. "Mini tissues could plausibly be formed from meticulous cell-by-cell construction, giving us scrupulous control over the composition of engineered tissues that has not been yet been possible," said Shin. The promising development would not have happened without collaboration between Mooney, who is an expert in tissue engineering and biocompatible hydrogels, and Weitz, who is an expert in using 'designer' emulsions inside microfluidic devices to encapsulate active materials drop by drop. "It's really a great example of what can happen at the Wyss when people can ally with colleagues of different expertise and really rally around a shared goal," Mooney said. "Enabling microgel encapsulation of single cells should allow much better integration and vascularization of implanted cellular therapies, for example in treatment of diabetes or Parkinson's disease, and provide new ways to study and control behavior of individual cells both inside and outside our bodies," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard SEAS.


News Article | February 22, 2017
Site: www.eurekalert.org

Working with human breast cancer cells and mice, researchers at Johns Hopkins say they have identified a biochemical pathway that triggers the regrowth of breast cancer stem cells after chemotherapy. The regrowth of cancer stem cells is responsible for the drug resistance that develops in many breast tumors and the reason that for many patients, the benefits of chemo are short-lived. Cancer recurrence after chemotherapy is frequently fatal. "Breast cancer stem cells pose a serious problem for therapy," says lead study investigator Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine, director of the Vascular Biology Program at the Johns Hopkins Institute for Cell Engineering and a member of the Johns Hopkins Kimmel Cancer Center. "These are the cells that can break away from a tumor and metastasize; these are the cells you most want to kill with chemotherapy. Paradoxically, though, cancer stem cells are quite resistant to chemotherapy." Semenza says previous studies have shown that resistance to chemotherapy arises from the hardy nature of cancer stem cells, which are often found in the centers of tumors, where oxygen levels are quite low. Their survival is made possible through proteins known as hypoxia-inducible factors (HIFs), which turn on genes that help the cells survive in a low-oxygen environment. In this new study, described Feb. 21 in Cell Reports, Semenza and his colleagues conducted gene expression analysis of multiple human breast cancer cell lines grown in the laboratory after exposure to chemotherapy drugs, like carboplatin, which stops tumor growth by damaging cancer cell DNA. The team found that the cancer cells that survived tended to have higher levels of a protein known as glutathione-S-transferase O1, or GSTO1. Experiments showed that HIFs controlled the production of GSTO1 in breast cancer cells when they were exposed to chemotherapy; if HIF activity was blocked in these lab-grown cells, GSTO1 was not produced. Semenza notes that GSTO1 and related GST proteins are antioxidant enzymes, but GSTO1's role in chemotherapy resistance did not require its antioxidant activity. Instead, following exposure to chemotherapy, GSTO1 binds to a protein called the ryanodine receptor 1, or RYR1, that triggers the release of calcium, which causes a chain reaction that transforms ordinary breast cancer cells into cancer stem cells. To more directly assess the role of GSTO1 and RYR1 in the breast tumor response to chemotherapy, the researchers injected human breast cancer cells into the mammary gland of mice and then treated the mice with carboplatin after tumors had formed. In addition to using normal breast cancer cells in the experiments, the team also used cancer cells that had been genetically engineered to lack either GSTO1 or RYR1. Loss of either GSTO1 or RYR1, the researchers report, decreased the number of cancer stem cells in the primary tumor, blocked metastasis of cancer cells from the primary tumor to the lungs, decreased the duration of chemotherapy required to induce remission and increased the duration of time after chemotherapy was stopped that the mice remained tumor-free. Although the study showed that blocking the production of GSTO1 may improve the efficacy of chemotherapy drugs, such as carboplatin, GSTO1 is only one of many proteins that are produced under the control of HIFs in breast cancer cells that have been exposed to chemotherapy. The Semenza lab is working to develop drugs that can block the action of HIFs, with the hope that HIF inhibitors will make chemotherapy more effective. Other authors of the report include Haquin Li, Ivan Chen, Larissa Shimoda, Youngrok Park, Chuanzhao Zhang, Linh Tran and Huimin Zhang of the Johns Hopkins University School of Medicine. This work was supported by an Impact Award from the Department of Defense (grant number W81XWH-12-1-0464) and a Research Professor Award from the American Cancer Society.


News Article | January 26, 2016
Site: www.biosciencetechnology.com

Researchers in the field of mechanobiology are evolving our understanding of health by revealing new insights into how the body's physical forces and mechanics impact development, physiological health, and prevention and treatment of disease. At the Wyss Institute for Biologically Inspired Engineering at Harvard University, engineers and biomedical scientists have assembled to form collaborative teams that are helping to drive this exciting area of research forward toward real-world applications. Now, a new study suggests mechanically-driven therapies that promote skeletal muscle regeneration through direct physical stimulation could one day replace or enhance drug and cell-based regenerative treatments. Discovered by a team at the Wyss Institute and the Harvard School of Engineering and Applied Sciences, the finding was published on January 25 in the journal Proceedings of the National Academy of Sciences. "Chemistry tends to dominate the way we think about medicine, but it has become clear that physical and mechanical factors play very critical roles in regulating biology," said Harvard bioengineer David Mooney, Ph.D., senior author on the new study, who is a Wyss Institute Core Faculty member and the Robert P. Pinkas Family Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS). "The results of our new study demonstrate how direct physical and mechanical intervention can impact biological processes and can potentially be exploited to improve clinical outcomes. " The multi-disciplinary team spanning the Wyss Institute's Programmable Nanomaterials and Bioinspired Robotics platforms was led by Mooney and also included soft roboticist Conor Walsh, Ph.D., who is a Wyss Core Faculty member, Associate Professor of Mechanical and Biomedical Engineering at Harvard SEAS and Founder of the Harvard Biodesign Lab, and biomechanical engineer Georg Duda, Ph.D., who is a Wyss Associate Core Faculty member, Vice-Director of the Berlin-Brandenburg Center for Regenerative Therapies (BCRT) and the Director of the Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration at Charité-Universitätsmedizin Berlin. In humans, up to half of body mass is made up of skeletal muscle, which plays a key role in locomotion, posture, and breathing. Although skeletal muscles can overcome minor tears and bruising without intervention, major injuries commonly caused by motor vehicle accidents, other traumas, or nerve damage can lead to extensive scarring, fibrous tissue, and loss of muscle function. The team applied combined murine models of muscle injury and hind limb ischemia to investigate two potential mechanotherapies: an implanted magnetic biocompatible gel and an external, soft robotic pressurized cuff. To alleviate severe muscle injuries, the team implanted a magnetized gel called a "biphasic ferrogel" so that it would be in direct contact with the damaged tissue. Another experimental group of mice did not receive the ferrogel implant, but instead were fitted with a soft robotic, non-invasive pressurized cuff over the injured leg. Then, the ferrogel was subjected to magnetic pulses to apply cyclic stimulation to the muscle, while pulses of air allowed the cuff to cyclically massage the hind leg. Both groups received two weeks of localized mechanical perturbation using the two distinct methods. The researchers discovered that cyclic mechanical stimulation provided by either magnetized gel or robotic cuff both resulted in a two-and-a-half-fold improvement in muscle regeneration and reduced tissue scarring over the course of two weeks, ultimately leading to an improvement in regained muscle function and an exciting new finding that mechanical stimulation of muscle alone can foster regeneration. To their surprise, the ferrogel implant and pressurized cuff also resulted in very similar levels of regeneration, suggesting that the use of non-invasive pressurized cuffs or devices could one day help heal patients suffering from severe muscle injuries. "Until now most approaches to muscle regeneration have been biologic, relying on the use of drugs or cells," said Christine Cezar, Ph.D., lead author on the study who completed her doctoral research at the Wyss Institute and Harvard SEAS. "Our finding that mechanical stimulation alone is enough to enhance muscle repair could open the door to new non-biologic therapies, or even combinatorial therapies that employ both mechanical and biological interventions to treat severely damaged skeletal muscles." The direct stimulation of muscle tissue increases the transport of oxygen, nutrients, fluids and waste removal from the site of the injury, which are all vital components of muscle health and repair. And according to Mooney, one of the most exciting aspects of this research is that its translation to the clinic in the form of a stimulatory device could be relatively rapid as compared to drug or cell therapies. Down the road, the principle of using mechanical stimulation to enhance regeneration or reduce formation of scarring or fibrosis could also be applied to a wide range of medical devices that interface mechanical components with body tissues. Currently, clinical devices are often plagued by the formation of thickened tissue capsules that form at the intersection of machine and man. The team plans to explore how the findings can make the jump from the laboratory to the clinic. "This work clearly demonstrates that mechanical forces are as important biological regulators as chemicals and genes, and it shows the immense potential of developing mechanotherapies to treat injury and disease," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is a pioneer and leader in the field of mechanobiology. Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School (HMS) and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard SEAS. "The challenge now is to advance this new mechanotherapeutic approach from the bench to bedside, where the real impact on human lives can occur."


News Article | January 25, 2016
Site: phys.org

"This work represents an elegant advance in programmable materials assembly, made possible by a multidisciplinary approach," said Jennifer Lewis, Sc.D., senior author on the new study. "We have now gone beyond integrating form and function to create transformable architectures." Lewis is a Core Faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Hansjörg Wyss Professor of Biologically Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Science (SEAS). L. Mahadevan, Ph.D., a Wyss Core Faculty member as well as the Lola England de Valpine Professor of Applied Mathematics, Professor of Organismic and Evolutionary Biology, and Professor of Physics at Harvard University and Harvard SEAS, is a co-author on the study. Their team also includes co-author, Ralph Nuzzo, Ph.D., the G.L. Clark Professor of Chemistry at the University of Illinois at Urbana-Champaign. In nature, flowers and plants have tissue composition and microstructures that result in dynamic morphologies that change according to their environments. Mimicking the variety of shape changes undergone by plant organs such as tendrils, leaves, and flowers in response to environmental stimuli like humidity and/or temperature, the 4D-printed hydrogel composites developed by Lewis and her team are programmed to contain precise, localized swelling behaviors. Importantly, the hydrogel composites contain cellulose fibrils that are derived from wood and are similar to the microstructures that enable shape changes in plants. Reported on January 25 in a new study in Nature Materials, the 4D printing advance combined materials science and mathematics through the involvement of the study's co-lead authors A. Sydney Gladman, who is a graduate research assistant advised by Lewis and specializing in the printing of polymers and composites at the Wyss Institute and SEAS, and Elisabetta Matsumoto, Ph.D., who is a postdoctoral fellow at the Wyss and SEAS advised by Mahadevan and specializing in condensed matter and material physics. By aligning cellulose fibrils during printing, the hydrogel composite ink is encoded with anisotropic swelling and stiffness, which can be patterned to produce intricate shape changes. The anisotropic nature of the cellulose fibrils gives rise to varied directional properties that can be predicted and controlled. Just like wood, which can be split easier along the grain rather than across it. Likewise, when immersed in water, the hydrogel-cellulose fibril ink undergoes differential swelling behavior along and orthogonal to the printing path. Combined with a proprietary mathematical model developed by the team that predicts how a 4D object must be printed to achieve prescribed transformable shapes, the new method opens up many new and exciting potential applications for 4D printing technology including smart textiles, soft electronics, biomedical devices, and tissue engineering. "Using one composite ink printed in a single step, we can achieve shape-changing hydrogel geometries containing more complexity than any other technique, and we can do so simply by modifying the print path," said Gladman. "What's more, we can interchange different materials to tune for properties such as conductivity or biocompatibility." The composite ink that the team uses flows like liquid through the printhead, yet rapidly solidifies once printed. A variety of hydrogel materials can be used interchangeably resulting in different stimuli-responsive behavior, while the cellulose fibrils can be replaced with other anisotropic fillers of choice, including conductive fillers. "Our mathematical model prescribes the printing pathways required to achieve the desired shape-transforming response," said Matsumoto. "We can control the curvature both discretely and continuously using our entirely tunable and programmable method." Specifically, the mathematical modeling solves the "inverse problem", which is the challenge of being able to predict what the printing toolpath must be in order to encode swelling behaviors toward achieving a specific desired target shape. "It is wonderful to be able to design and realize, in an engineered structure, some of nature's solutions," said Mahadevan, who has studied phenomena such as how botanical tendrils coil, how flowers bloom, and how pine cones open and close. "By solving the inverse problem, we are now able to reverse-engineer the problem and determine how to vary local inhomogeneity, i.e. the spacing between the printed ink filaments, and the anisotropy, i.e. the direction of these filaments, to control the spatiotemporal response of these shapeshifting sheets. " "What's remarkable about this 4D printing advance made by Jennifer and her team is that it enables the design of almost any arbitrary, transformable shape from a wide range of available materials with different properties and potential applications, truly establishing a new platform for printing self-assembling, dynamic microscale structures that could be applied to a broad range of industrial and medical applications," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children's Hospital and Professor of Bioengineering at Harvard SEAS.


News Article | January 28, 2016
Site: phys.org

But now, a team of researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) led by George Church, Ph.D., has developed a new method for engineering a broad range of biosensors to detect and signal virtually any desired molecule using living eukaryotic cells. Church, who is a Wyss Core Faculty member and the Robert Winthrop Professor of Genetics at HMS, and his team reported their findings in the journal eLife. To test their new method, the team experimentally engineered yeast, plant, and mammalian cells to contain customizable ligand-binding domains (LBDs), which are receptors for hormones and other types of small molecules. These custom LBDs are tailored so that they only bind and "detect" a specific molecule of interest, such as the human hormone progesterone or the drug digoxin. Once the LBD binds to the target molecule, a secondary "signal" component fused to the LBD can be programmed to emit fluorescence or regulate gene expression. The components of this biosensor—the LBD in combination with the fluorescent or genetic signal—degrade and fade away if the target molecule is not identified. Strikingly, the team successfully engineered Arabidopsis plants to act as multicellular botanical biosensors, containing a custom LBD to recognize the drug digoxin and a luminescent signal protein to emit light when digoxin is "detected". These Arabidopsis biosensors gave off fluorescence when the plants were exposed to digoxin, proving that whole organisms can visually light up to signal detection of an arbitrary molecule. "Like many eukaryotic organisms, plants are full of diverse hormones that make it challenging to sense and respond to a specific hormone of interest," said Dan Mandell, Ph.D., the study's co-first author and a Wyss Institute Technology Development Fellow and Postdoctoral Research Fellow at HMS. "But using our strategy, the Arabidopsis plants we engineered exhibited a 50-fold increase in luminescence in the presence of digoxin—very easily visualized—which could inspire exciting future applications involving trees or plants that detect harmful environmental pollutants or toxins and give off a visible indicator." "Biosensors that can tell you about their environment are extremely useful for a broad range of applications," said Church. "You can imagine if they were used in agricultural plants, they can tell you about the condition of the soil, the presence of toxins or pests that are bothering them." The team not only demonstrated its novel methodology in plants but also described its efficacy in turning yeast and mammalian cells into precise biosensors, which could one day be leveraged for use in industries that rely on the productivity of yeast or livestock, or for use as medical sensors. Overall, the method is extremely tunable and portable, meaning it can be used in a wide variety of organisms to detect a broad range of small molecules. An additional capability of the new biosensing methodology is the ability to connect it to gene regulators instead of fluorescent proteins. Such biosensors could precisely regulate gene transcription in order to improve yields of small molecules in organisms used for industrial bioproduction. Yeast, for example, could therefore be engineered to produce a desired molecule from a renewable feedstock, and furthermore programmed to self-identify the most efficient individuals within a population of producers so that only the highest producing yeast would survive. In this way, a population of organisms leveraged for bioproduction of pharmaceuticals or other valuable molecules could quickly self-evolve to become extremely efficient and productive. The team in fact used this strategy to evolve yeast that can produce the hormone progesterone with several-fold higher yield. The biosensors could have a direct impact on human health as well, given that the team also used their method to tightly regulate the gene editing mechanism CRISPR-Cas9 inside living human cells, a step forward towards preventing unintended changes to the genome during gene therapy. "These new reprogramming capabilities developed by the Church team open up an entirely new realm where ordinary organisms can be transformed into extraordinary living cellular devices that can sense specific signals and produce appropriate responses, whether its enhancing production of biofuels or secreting a therapeutic when the cells sense inflammation or infection. It's another great enabling capability that will undoubtedly advance the entire field of synthetic biology.," said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences. Explore further: New 'magnetic yeast' marks step toward harnessing Nature's magnetic capabilities

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