Microinstrumentation for Brain Organoids.

Brain organoids are three-dimensional aggregates of self-organized differentiated stem cells that mimic the structure and function of human brain regions. Organoids bridge the gaps between conventional drug screening models such as planar mammalian cell culture, animal studies, and clinical trials. They can revolutionize the fields of developmental biology, neuroscience, toxicology, and computer engineering. Conventional microinstrumentation for conventional cellular engineering, such as planar microfluidic chips; microelectrode arrays (MEAs); and optical, magnetic, and acoustic techniques, has limitations when applied to three-dimensional (3D) organoids, primarily due to their limits with inherently two-dimensional geometry and interfacing. Hence, there is an urgent need to develop new instrumentation compatible with live cell culture techniques and with scalable 3D formats relevant to organoids. This review discusses conventional planar approaches and emerging 3D microinstrumentation necessary for advanced organoid-machine interfaces. Specifically, this article surveys recently developed microinstrumentation, including 3D printed and curved microfluidics, 3D and fast-scan optical techniques, buckling and self-folding MEAs, 3D interfaces for electrochemical measurements, and 3D spatially controllable magnetic and acoustic technologies relevant to two-way information transfer with brain organoids. This article highlights key challenges that must be addressed for robust organoid culture and reliable 3D spatiotemporal information transfer.

Untethered shape-changing devices in the gastrointestinal tract.

INTRODUCTION: Advances in microfabrication, automation, and computer engineering seek to revolutionize small-scale devices and machines. Emerging trends in medicine point to smart devices that emulate the motility, biosensing abilities, and intelligence of cells and pathogens that inhabit the human body. Two important characteristics of smart medical devices are the capability to be deployed in small conduits, which necessitates being untethered, and the capacity to perform mechanized functions, which requires autonomous shape-changing. AREAS COVERED: We motivate the need for untethered shape-changing devices in the gastrointestinal tract for drug delivery, diagnosis, and targeted treatment. We survey existing structures and devices designed and utilized across length scales from the macro to the sub-millimeter. These devices range from triggerable pre-stressed thin film microgrippers and spring-loaded devices to shape-memory and differentially swelling structures. EXPERT OPINION: Recent studies demonstrate that when fully enabled, tether-free and shape-changing devices, especially at sub-mm scales, could significantly advance the diagnosis and treatment of GI diseases ranging from cancer and inflammatory bowel disease (IBD) to irritable bowel syndrome (IBS) by improving treatment efficacy, reducing costs, and increasing medication compliance. We discuss the challenges and possibilities associated with ensuring safe, reliable, and autonomous operation of these smart devices.

Toward Single Cell Tattoos: Biotransfer Printing of Lithographic Gold Nanopatterns on Live Cells.

Lithographic nanopatterning techniques such as photolithography, electron-beam lithography, and nanoimprint lithography (NIL) have revolutionized modern-day electronics and optics. Yet, their application for creating nanobio interfaces is limited by the cytotoxic and two-dimensional nature of conventional fabrication methods. Here, we present a biocompatible and cost-effective transfer process that leverages (a) NIL to define sub-300 nm gold (Au) nanopattern arrays, (b) amine functionalization of Au to transfer the NIL-arrays from a rigid substrate to a soft transfer layer, (c) alginate hydrogel as a flexible, degradable transfer layer, and (d) gelatin conjugation of the Au NIL-arrays to achieve conformal contact with live cells. We demonstrate biotransfer printing of the Au NIL-arrays on rat brains and live cells with high pattern fidelity and cell viability and observed differences in cell migration on the Au NIL-dot and NIL-wire printed hydrogels. We anticipate that this nanolithography-compatible biotransfer printing method could advance bionics, biosensing, and biohybrid tissue interfaces.

Swelling characteristics of DNA polymerization gels.

The development of biomolecular stimuli-responsive hydrogels is important for biomimetic structures, soft robots, tissue engineering, and drug delivery. DNA polymerization gels are a new class of soft materials composed of polymer gel backbones with DNA duplex crosslinks that can be swollen by sequential strand displacement using hairpin-shaped DNA strands. The extensive swelling can be tuned using physical parameters such as salt concentration and biomolecule design. Previously, DNA polymerization gels have been used to create shape-changing gel automata with a large design space and high programmability. Here we systematically investigate how the swelling response of DNA polymerization gels can be tuned by adjusting the design and concentration of DNA crosslinks in the hydrogels or DNA hairpin triggers, and the ionic strength of the solution in which swelling takes place. We also explore the effect hydrogel size and shape have on the swelling response. Tuning these variables can alter the swelling rate and extent across a broad range and provide a quantitative connection between biochemical reactions and macroscopic material behaviour.

Toward single cell tattoos: Biotransfer printing of lithographic gold nanopatterns on live cells.

Lithographic nanopatterning techniques like photolithography, electron-beam lithography, and nanoimprint lithography (NIL) have revolutionized modern-day electronics and optics. Yet, their application for creating nano-bio interfaces is limited by the cytotoxic and two-dimensional nature of conventional fabrication methods. Here, we present a biocompatible and cost-effective transfer process that leverages (a) NIL to define sub-300 nm gold (Au) nanopattern arrays, (b) amine functionalization of Au to transfer the NIL-arrays from a rigid substrate to a soft transfer layer, (c) alginate hydrogel as a flexible, degradable transfer layer, and (d) gelatin conjugation of the Au NIL-arrays to achieve conformal contact with live cells. We demonstrate biotransfer printing of the Au NIL-arrays on rat brains and live cells with high pattern fidelity and cell viability and observed differences in cell migration on the Au NIL-dot and NIL-wire printed hydrogels. We anticipate that this nanolithography-compatible biotransfer printing method could advance bionics, biosensing, and biohybrid tissue interfaces.

Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function.

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.

First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Untethered unidirectionally crawling gels driven by asymmetry in contact forces.

Reversible thermoresponsive hydrogels, which swell and shrink (deswell) in the temperature range of 30° to 60°C, provide an attractive material class for operating untethered soft robots in human physiological and ambient conditions. Crawling has been demonstrated previously with thermoresponsive hydrogels but required patterned or constrained gels or substrates to break symmetry for unidirectional motion. Here, we demonstrate a locomotion mechanism for unidirectionally crawling gels driven by spontaneous asymmetries in contact forces during swelling and deswelling of segmented active thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) and passive polyacrylamide (pAAM) bilayers with suspended linkers. Actuation studies demonstrate the consistent unidirectional movement of these gel crawlers across multiple thermal cycles on flat, unpatterned substrates. We explain the mechanism using finite element simulations and by varying experimental parameters such as the linker stiffness and the number of bilayer segments. We elucidate design criteria and validate experiments using image analysis and finite element models. We anticipate that this mechanism could potentially be applied to other shape-changing locomotors.

Autonomous Untethered Microinjectors for Gastrointestinal Delivery of Insulin.

The delivery of macromolecular drugs via the gastrointestinal (GI) tract is challenging as these drugs display low stability as well as poor absorption across the intestinal epithelium. While permeation-enhancing drug delivery methods can increase the bioavailability of low molecular weight drugs, the effective delivery of high molecular weight drugs across the tight epithelial cell junctions remains a formidable challenge. Here, we describe autonomous microinjectors that are deployed in the GI tract, then efficiently penetrate the GI mucosa to deliver a macromolecular drug, insulin, to the systemic circulation. We performed in vitro studies to characterize insulin release and assess the penetration capability of microinjectors and we measured the in vivo release of insulin in live rats. We found that the microinjectors administered within the luminal GI tract could deliver insulin transmucosally to the systemic circulation at levels similar to those with intravenously administered insulin. Due to their small size, tunability in sizing and dosing, wafer-scale fabrication, and parallel, autonomous operation, we anticipate that these microinjectors will significantly advance drug delivery across the GI tract mucosa to the systemic circulation in a safe manner.

Shell microelectrode arrays (MEAs) for brain organoids.

Brain organoids are important models for mimicking some three-dimensional (3D) cytoarchitectural and functional aspects of the brain. Multielectrode arrays (MEAs) that enable recording and stimulation of activity from electrogenic cells offer notable potential for interrogating brain organoids. However, conventional MEAs, initially designed for monolayer cultures, offer limited recording contact area restricted to the bottom of the 3D organoids. Inspired by the shape of electroencephalography caps, we developed miniaturized wafer-integrated MEA caps for organoids. The optically transparent shells are composed of self-folding polymer leaflets with conductive polymer-coated metal electrodes. Tunable folding of the minicaps' polymer leaflets guided by mechanics simulations enables versatile recording from organoids of different sizes, and we validate the feasibility of electrophysiology recording from 400- to 600-µm-sized organoids for up to 4 weeks and in response to glutamate stimulation. Our studies suggest that 3D shell MEAs offer great potential for high signal-to-noise ratio and 3D spatiotemporal brain organoid recording.

Directing Multicellular Organization by Varying the Aspect Ratio of Soft Hydrogel Microwells.

Multicellular organization with precise spatial definition is essential to various biological processes, including morphogenesis, development, and healing in vascular and other tissues. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of 3D geometry of soft hydrogels are less well defined. Here, the discovery of a new mode of endothelial cell self-organization guided by combinatorial effects of stiffness and geometry, independent of protein or chemical patterning, is described. Endothelial cells in 2 kPa microwells are found to be ≈30 times more likely to migrate to the edge to organize in ring-like patterns than in stiff 35 kPa microwells. This organization is independent of curvature and significantly more pronounced in 2 kPa microwells with aspect ratio (perimeter/depth) < 25. Physical factors of cells and substrates that drive this behavior are systematically investigated and a mathematical model that explains the organization by balancing the dynamic interaction between tangential cytoskeletal tension, cell-cell, and cell-substrate adhesion is presented. These findings demonstrate the importance of combinatorial effects of geometry and stiffness in complex cellular organization that can be leveraged to facilitate the engineering of bionics and integrated model organoid systems with customized nutrient vascular networks.

Label-Free Spectroscopic SARS-CoV-2 Detection on Versatile Nanoimprinted Substrates.

Widespread testing and isolation of infected patients is a cornerstone of viral outbreak management, as underscored during the ongoing COVID-19 pandemic. Here, we report a large-area and label-free testing platform that combines surface-enhanced Raman spectroscopy and machine learning for the rapid and accurate detection of SARS-CoV-2. Spectroscopic signatures acquired from virus samples on metal-insulator-metal nanostructures, fabricated using nanoimprint lithography and transfer printing, can provide test results within 25 min. Not only can our technique accurately distinguish between different respiratory and nonrespiratory viruses, but it can also detect virus signatures in physiologically relevant matrices such as human saliva without any additional sample preparation. Furthermore, our large area nanopatterning approach allows sensors to be fabricated on flexible surfaces allowing them to be mounted on any surface or used as wearables. We envision that our versatile and portable label-free spectroscopic platform will offer an important tool for virus detection and future outbreak preparedness.

Magnetic Resonance Guided Navigation of Untethered Microgrippers.

Microsurgical tools offer a path to less invasive clinical procedures with improved access, reduced trauma, and better recovery outcomes. There are a variety of rigid and flexible endoscopic devices that have significantly advanced diagnostics and microsurgery. However, they rely on wires or tethers for guidance and operation of small end-effector tools. While untethered physiologically responsive microgrippers have been previously shown to excise tissue from deep gastrointestinal locations in animal models, there are challenges associated with guiding them along paths and moving them to specific locations. In this communication, the magnetic dipole moment of untethered thermally responsive grippers is optimized for efficient coupling to external magnetic resonance (MR) fields. Gripper encapsulation in a millimeter sized wax pellet reduces the friction with the surrounding tissue and MR Navigation (MRN) of a 700 µm sized microgripper is realized within narrow channels in tissue phantoms and in an ex vivo porcine esophagus. The results show convincing proof-of-concept evidence that it is possible to sequentially image, move, and guide a submillimeter functional microsurgical tool in tissue conduits using a commercial preclinical MR system, and when combined with prior demonstrations of physiologically responsive in vivo biopsy are an important step towards the clinical translation of untethered microtools.

Gastrointestinal-resident, shape-changing microdevices extend drug release in vivo.

Extended-release gastrointestinal (GI) luminal delivery substantially increases the ease of administration of drugs and consequently the adherence to therapeutic regimens. However, because of clearance by intrinsic GI motility, device gastroretention and extended drug release over a prolonged duration are very challenging. Here, we report that GI parasite-inspired active mechanochemical therapeutic grippers, or theragrippers, can reside within the GI tract of live animals for 24 hours by autonomously latching onto the mucosal tissue. We also observe a notable sixfold increase in the elimination half-life using theragripper-mediated delivery of a model analgesic ketorolac tromethamine. These results provide first-in-class evidence that shape-changing and self-latching microdevices enhance the efficacy of extended drug delivery.

Multicomponent DNA Polymerization Motor Gels.

Hydrogels with the ability to change shape in response to biochemical stimuli are important for biosensing, smart medicine, drug delivery, and soft robotics. Here, a family of multicomponent DNA polymerization motor gels with different polymer backbones is created, including acrylamide-co-bis-acrylamide (Am-BIS), poly(ethylene glycol) diacrylate (PEGDA), and gelatin-methacryloyl (GelMA) that swell extensively in response to specific DNA sequences. A common mechanism, a polymerization motor that induces swelling is driven by a cascade of DNA hairpin insertions into hydrogel crosslinks. These multicomponent hydrogels can be photopatterned into distinct shapes, have a broad range of mechanical properties, including tunable shear moduli between 297 and 3888 Pa and enhanced biocompatibility. Human cells adhere to the GelMA-DNA gels and remain viable during ≈70% volumetric swelling of the gel scaffold induced by DNA sequences. The results demonstrate the generality of sequential DNA hairpin insertion as a mechanism for inducing shape change in multicomponent hydrogels, suggesting widespread applicability of polymerization motor gels in biomaterials science and engineering.

3D Printing of an In Situ Grown MOF Hydrogel with Tunable Mechanical Properties.

Due to their precisely modifiable microporosity and chemical functionality, Metal-Organic Frameworks (MOFs) have revolutionized catalysis, separations, gas storage, drug delivery, and sensors. However, because of their rigid and brittle powder morphology, it is challenging to build customizable MOF shapes with tunable mechanical properties. Here, we describe a new three-dimensional (3D) printing approach to create stretchable and tough MOF hydrogel structures with tunable mechanical properties. We formulate a printable ink by combining prepolymers of a versatile double network (DN) hydrogel of acrylamide and alginate, a shear-thinning agent, and MOF ligands. Importantly, by simultaneous cross-linking of alginate and in situ growth of the HKUST-1 using copper ions, we are able to create composites with high MOF dispersity in the DN hydrogel matrix with high pore accessibility. We extensively characterize the inks and uncover parameters to tune modulus, strength, and toughness of the 3D prints. We also demonstrate the excellent performance of the MOF hydrogels for dye absorption. Our approach incorporates all of the advantageous attributes of 3D printing while offering a rational approach to merge stretchable hydrogels and MOFs, and our findings are of broad relevance to wearables, implantable and flexible sensors, chemical separations, and soft robotics.

Untethered Single Cell Grippers for Active Biopsy.

Single cell manipulation is important in biosensing, biorobotics, and quantitative cell analysis. Although microbeads, droplets, and microrobots have been developed previously, it is still challenging to simultaneously excise, capture, and manipulate single cells in a biocompatible manner. Here, we describe untethered single cell grippers, that can be remotely guided and actuated on-demand to actively capture or excise individual or few cells. We describe a novel molding method to micropattern a thermally responsive wax layer for biocompatible motion actuation. The multifingered grippers derive their energy from the triggered release of residual differential stress in bilayer hinges composed of silicon oxides. A magnetic layer enables remote guidance through narrow conduits and fixed tissue sections ex vivo. Our results provide an important advance in high-throughput single cell scale biopsy tools important to lab-on-a-chip devices, microrobotics, and minimally invasive surgery.

Active matter therapeutics.

Nanotherapies based on micelles, liposomes, polymersomes, nanocapsules, magnetic nanoparticles, and noble metal nanoparticles have been at the forefront of drug delivery in the past few decades. Some of these nanopharmaceuticals have been commercially applied to treat a wide range of diseases, from dry eye syndrome to cancer. However, the majority involve particles that are passive, meaning that they do not change shape, and they lack motility; the static features can limit their therapeutic efficacy. In this review, we take a critical look at an emerging field that seeks to utilize active matter for therapeutics. In this context, active matter can be broadly referred to as micro or nanosized constructs that energetically react with their environment or external fields and translate, rotate, vibrate or change shape. Essentially, the recent literature suggests that such particles could significantly augment present-day drug delivery, by enhancing transport and increasing permeability across anatomical barriers by transporting drugs within solid tumor microenvironments or disrupting cardiovascular plaque. We discuss examples of such particles and link the transport and permeability properties of active matter to potential therapeutic applications in the context of two major diseases, namely cancer and heart disease. We also discuss potential challenges, opportunities, and translational hurdles.

Biomimetic human small muscular pulmonary arteries.

Changes in structure and function of small muscular arteries play a major role in the pathophysiology of pulmonary hypertension, a burgeoning public health challenge. Improved anatomically mimetic in vitro models of these microvessels are urgently needed because nonhuman vessels and previous models do not accurately recapitulate the microenvironment and architecture of the human microvascular wall. Here, we describe parallel biofabrication of photopatterned self-rolled biomimetic pulmonary arterial microvessels of tunable size and infrastructure. These microvessels feature anatomically accurate layering and patterning of aligned human smooth muscle cells, extracellular matrix, and endothelial cells and exhibit notable increases in endothelial longevity and nitric oxide production. Computational image processing yielded high-resolution 3D perspectives of cells and proteins. Our studies provide a new paradigm for engineering multicellular tissues with precise 3D spatial positioning of multiple constituents in planar moieties, providing a biomimetic platform for investigation of microvascular pathobiology in human disease.

3D printing and characterization of a soft and biostable elastomer with high flexibility and strength for biomedical applications.

Recent advancements in 3D printing have revolutionized biomedical engineering by enabling the manufacture of complex and functional devices in a low-cost, customizable, and small-batch fabrication manner. Soft elastomers are particularly important for biomedical applications because they can provide similar mechanical properties as tissues with improved biocompatibility. However, there are very few biocompatible elastomers with 3D printability, and little is known about the material properties of biocompatible 3D printable elastomers. Here, we report a new framework to 3D print a soft, biocompatible, and biostable polycarbonate-based urethane silicone (PCU-Sil) with minimal defects. We systematically characterize the rheological and thermal properties of the material to guide the 3D printing process and have determined a range of processing conditions. Optimal printing parameters such as printing speed, temperature, and layer height are determined via parametric studies aimed at minimizing porosity while maximizing the geometric accuracy of the 3D-printed samples as evaluated via micro-CT. We also characterize the mechanical properties of the 3D-printed structures under quasistatic and cyclic loading, degradation behavior and biocompatibility. The 3D-printed materials show a Young's modulus of 6.9 ± 0.85 MPa and a failure strain of 457 ± 37.7% while exhibiting good cell viability. Finally, compliant and free-standing structures including a patient-specific heart model and a bifurcating arterial structure are printed to demonstrate the versatility of the 3D-printed material. We anticipate that the 3D printing framework presented in this work will open up new possibilities not only for PCU-Sil, but also for other soft, biocompatible and thermoplastic polymers in various biomedical applications requiring high flexibility and strength combined with biocompatibility, such as vascular implants, heart valves, and catheters.