Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database. Based on this analysis, we present the diversity of, and current trends in, in vitro models and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology and gene expression and note field-wide improvements over time. Finally, we discuss opportunities to collectively pursue the shared goal of hiPSC-CM model development, maturation and assessment that we believe are critical for engineering mature cardiac tissue. This Perspective analyzes the most common maturation and assessment techniques for in vitro hiPSC-derived cardiomyocyte models and makes recommendations for standardizations in this field.

The field of microrobotics has emerged as a promising area of research with significant applications in biomedicine, both in vitro and in vivo, such as targeted cargo delivery, microsurgery, and cellular manipulation. Microrobots actuated with multiple modalities have the potential for greater adaptability, robustness, and capability to perform various tasks. Modular units that can reconfigure into various shapes, create structures that may be difficult to fabricate as one whole unit, and be assembled on-site, could provide more versatility by assembly and disassembly of units on demand. Such multi-modal modular microrobots have the potential to address challenging applications. Here, we present a biocompatible cylindrical microrobot with a dome-shaped cavity. The microrobot is actuated by both magnetic and acoustic fields and forms modular microstructures of various shapes. We demonstrate the use of these microrobots for cellular manipulation by creating patterns on a surface.

Using two-photon direct laser writing, we developed a versatile platform to generate, scale, and study hiPSC-derived engineered heart tissues (EHTs) in various geometries, with the goal of promoting fiber alignment and maturation of the EHTs.

Natural tissues are composed of diverse cells and extracellular materials whose arrangements across several length scales-from subcellular lengths1 (micrometre) to the organ scale2 (centimetre)-regulate biological functions. Tissue-fabrication methods have progressed to large constructs, for example, through stereolithography3 and nozzle-based bioprinting4,5, and subcellular resolution through subtractive photoablation6-8. However, additive bioprinting struggles with sub-nozzle/voxel features9 and photoablation is restricted to small volumes by prohibitive heat generation and time10. Building across several length scales with temperature-sensitive, water-based soft biological matter has emerged as a critical challenge, leaving large classes of biological motifs-such as multiscalar vascular trees with varying calibres-inaccessible with present technologies11,12. Here we use gallium-based engineered sacrificial capillary pumps for evacuation (ESCAPE) during moulding to generate multiscalar structures in soft natural hydrogels, achieving both cellular-scale (<10 µm) and millimetre-scale features. Decoupling the biomaterial of interest from the process of constructing the geometry allows non-biocompatible tools to create the initial geometry. As an exemplar, we fabricated branched, cell-laden vascular trees in collagen, spanning approximately 300-µm arterioles down to the microvasculature (roughly ten times smaller). The same approach can micropattern the inner surface of vascular walls with topographical cues to orient cells in 3D and engineer fine structures such as vascular malformations. ESCAPE moulding enables the fabrication of multiscalar forms in soft biomaterials, paving the way for a wide range of tissue architectures that were previously inaccessible in vitro.

Microrobots, untethered miniature devices capable of performing tasks at the microscale, have gained significant attention in the fields of robotics and biomedicine. These devices hold immense potential for various industrial and scientific applications, including targeted drug delivery and cell manipulation. In this study, we present a novel magnetic rolling helical microrobot specifically designed for bio-compatible cell patterning. Our microrobot incorporates both open-loop and closed-loop control mechanisms, providing flexible, precise, and rapid control for various applications. Through experiments, we demonstrate the microrobot's ability to manipulate cells by pushing them while rolling and arranging cells into desired patterns. This result is particularly significant as it has implications for diverse biological applications such as tissue engineering and organoid development. Moreover, we showcase the effectiveness of our microrobot in a closed-loop control system, where it successfully follows a predetermined path from an origin to a destination. The combination of cellular manipulation capabilities and trajectory-tracking performance underlines the versatility and potential of our magnetic rolling helical microrobot. The ability to control and navigate the microrobot with high precision opens up new possibilities for advanced biomedical applications. These findings contribute to the growing body of knowledge in microbotics and pave the way for further research and development in the field.

Microrobots have emerged as promising tools for biomedical and in vivo applications, leveraging their untethered actuation capabilities and miniature size. Despite extensive research on diversifying multi-actuation modes for single types of robots, these tiny machines tend to have limited versatility while navigating different environments or performing specific tasks. To overcome such limitations, self-assembled microstructures with on-demand reconfiguration capabilities have gained recent attention as the future of biocompatible microrobotics, since they can address drug delivery, microsurgery, and organoid development processes. Reversible modular reconfiguration structures require specific arrangements of particles that can assume several shapes when external fields are applied. We show how magnetic interaction can be used to assemble cylindrical microrobots into modular microstructures with different shapes. The motion of the formed microstructure is actuated via an external acoustic field, which generates responsive forces in the air bubbles trapped in the inner cavity of the robots. An external magnetic field can also steer these structures. We illustrate these capabilities by assembling the robots into different shapes that can swim and be steered, showing the potential to perform biomedical applications. Furthermore, we confirm the biocompatibility of the cylindrical microrobots used as the building blocks of our microstructure. Exposing Chinese Hamster Ovary cells to our microrobots for 24 hours demonstrates cell viability when in contact with the microrobot.

High‐resolution printing afforded by two‐photon polymerization (2PP) has opened up vast design space, spanning over four orders of magnitude (sub‐μm to cm), unleashing the opportunity to fabricate architected materials with intriguing mechanical characteristics. This study seeks to exploit direct laser writing to print accordion‐shaped honeycomb lattices constituted of slender micro‐membranes to achieve in‐plane mechanical anisotropy. The deformation mechanisms governing orientation‐sensitive mechanical properties of the lattice are examined by in situ investigations in a scanning electron microscope. The microlattice displays greater flexibility, compliance, and strain hardening during in‐plane compression, whereas it is highly stiff and resistant to fatigue in the out‐of‐plane orientation. The high aspect ratio of accordion‐shaped cells imparts prominent in‐plane anisotropy, with longitudinal stiffness twice the transverse stiffness. Folding, interlocking, and compaction of micro‐walls are the major deformation mechanisms, inducing brilliant energy dissipation ability, with up to 87% mechanical work‐absorption. Digital image correlation analysis reveals that strain re‐distribution facilitated by hinges dictates the recoverability of the microlattice. This work provides useful mechanistic insights at the micrometer length scale for tuning the orientation‐sensitive mechanical response of mesostructures printed by 2PP.

Biomimetic on-chip tissue models serve as a powerful tool for studying human physiology and developing therapeutics; however, their modeling power is hindered by our inability to develop highly ordered functional structures in small length scales. Here, we demonstrate how high-precision fabrication can enable scaled-down modeling of organ-level cardiac mechanical function. We use two-photon direct laser writing (TPDLW) to fabricate a nanoscale-resolution metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of a miniaturized, induced pluripotent stem cell-derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates the ventricular fluidic function and exhibits a complete pressure-volume loop with isovolumetric phases. Together, our results demonstrate a previously unexplored application of high-precision fabrication that can be generalized to expand the accessible spectrum of organ-on-a-chip models toward structurally and biomechanically sophisticated tissue systems.

We have developed a microfluidic platform for engineering cardiac microtissues in highly-controlled microenvironments. The platform is fabricated using direct laser writing (DLW) lithography and soft lithography, and contains four separate devices. Each individual device houses a cardiac microtissue and is equipped with an integrated strain actuator and a force sensor. Application of external pressure waves to the platform results in controllable time-dependent forces on the microtissues. Conversely, oscillatory forces generated by the microtissues are transduced into measurable electrical outputs. We demonstrate the capabilities of this platform by studying the response of cardiac microtissues derived from human induced pluripotent stem cells (hiPSC) under prescribed mechanical loading and pacing. This platform will be used for fundamental studies and drug screening on cardiac microtissues.

Diagnostic testing that facilitates containment, surveillance, and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or future respiratory viruses, depends on a sample collection device that efficiently collects nasopharyngeal tissue and that can be manufactured on site when an outbreak or public health emergency is declared by a government. Here two novel stereolithography-based three-dimensional (3D)-printed nasopharyngeal swabs are reported which are made using a biocompatible and sterilizable photoresist. Such swabs are readily manufactured on-site and on-demand to ensure availability, if supply chain shortages emerge. Additionally, the 3D-printed swabs easily adapt to current workflow and testing procedures in hospital clinical laboratories to allow for effortless scaling up of test kits. Finally, the 3D-printed nasopharyngeal swabs demonstrate concordant SARS-CoV-2 testing results between the 3D-printed swabs and the COPAN commercial swabs, and enable detection of SARS-CoV-2 in clinical samples obtained from autopsies.

Resolution and field-of-view often represent a fundamental tradeoff in microscopy. Atomic force microscopy (AFM), in which a cantilevered probe deflects under the influence of local forces as it scans across a substrate, is a key example of this tradeoff with high resolution imaging being largely limited to small areas. Despite the tremendous impact of AFM in fields including materials science, biology, and surface science, the limitation in imaging area has remained a key barrier to studying samples with intricate hierarchical structure. Here, we show that massively parallel AFM with >1000 probes is possible through the combination of a cantilever-free probe architecture and a scalable optical method for detecting probe–sample contact. Specifically, optically reflective conical probes on a comparatively compliant film are found to comprise a distributed optical lever that translates probe motion into an optical signal that provides sub-10 nm vertical precision. The scalability of this approach makes it well suited for imaging applications that require high resolution over large areas.

Direct laser writing (DLW) via two-photon polymerization is an emerging highly precise technique for the fabrication of intricate cellular scaffolds. Despite recent progress in using two-photon-polymerized scaffolds to probe fundamental cell behaviors, new methods to direct and modulate microscale cell alignment and selective cell adhesion using two-photon-polymerized microstructures are of keen interest. Here, a DLW-fabricated 2D and 3D hydrogel microstructures, with alternating soft and stiff regions, for precisely controlled cell alignment are reported. The use of both cell-adhesive and cell-repellent hydrogels allows selective adhesion and alignment of human mesenchymal stem cells within the printed structure. Importantly, DLW patterning enables cell alignment on flat surfaces as well as irregular and curved 3D microstructures, which are otherwise challenging to pattern with cells.

Iridium has been implanted at 1MeV and 500keV in the dose range 1 to 3x1017/cm2 at 525°C to form a buried Ir-rich region ∼2500Å below the surface in (111)Si. Samples were annealed at temperatures in the range 600°C to 1200°C to form buried IrSi3 layers. Rutherford backscattering and channeling analysis in conjunction with transmission electron microscopy were used to study the formation and the alignment of the IrSi3 layer to the silicon host as a function of annealing. For a dose of 2x1017 Ir/cm2, stoichiometric IrSi3 formed beneath ∼ 2000Å of Si after annealing to 1100°C. The IrSi3 layer was aligned with the Si substrate with χmin ∼ 12%, but a band of precipitates remained either side of the main layer.

The nascent field of bioelectronic medicine seeks to decode and modulate peripheral nervous system signals to obtain therapeutic control of targeted end organs and effectors. Current approaches rely heavily on electrode-based devices, but size scalability, material and microfabrication challenges, limited surgical accessibility, and the biomechanically dynamic implantation environment are significant impediments to developing and deploying peripheral interfacing technologies. Here, we present a microscale implantable device – the nanoclip – for chronic interfacing with fine peripheral nerves in small animal models that begins to meet these constraints. We demonstrate the capability to make stable, high signal-to-noise ratio recordings of behaviorally-linked nerve activity over multi-week timescales. In addition, we show that multi-channel, current-steering-based stimulation within the confines of the small device can achieve multi-dimensional control of a small nerve. These results highlight the potential of new microscale design and fabrication techniques for realizing viable devices for long-term peripheral interfacing.

Organ-on-a-chip technology fosters the potential of in vitro models of cardiac function. Nevertheless, modeling the ventricular ejection has been challenging due to the lack of 3D scaffolds of ventricular-like geometry that can accommodate the tissue while providing structural stability and contractile directionality. Using two-photon direct laser writing, we fabricate a concave, 1mm 3 scaffold that is then embedded in a microfluidic, organ-on-a-chip system. We seed the system with iPSC-derived cardiac tissue and demonstrate the ability of the scaffold to preserve the 3D concave tissue structure, promote cardiomyocyte alignment and generate significant ejection fractions. The presented system sets the foundation for novel in vitro studies on ventricular function and remodeling.

The construction of a complex, 3D optical metamaterial challenges conventional nanofabrication techniques. These metamaterials require patterning of both a deformable mechanical substrate and an optically-active structure with ∼200 nm resolution and precision. The soft nature of the deformable mechanical materials often precludes the use of resist-based techniques for patterning. Furthermore, FIB deposition approaches produce metallic structures with considerable disorder and impurities, impairing their optical response. In this paper we discuss a novel solution to this nanofabrication challenge - the integration of direct laser writing and MEMS stencil patterning. We demonstrate a variety of methods that enable this integration and then show how one can produce optically-active, 3D metamaterials. We present optical characterization data on one of these metamaterials to demonstrate the viability of our nanofabrication approach.

Direct laser writing via two-photon polymerization (2PP) is an emerging micro- and nanofabrication technique to prepare predetermined and architecturally precise hydrogel scaffolds with high resolution and spatial complexity. As such, these scaffolds are increasingly being evaluated for cell and tissue engineering applications. This article first discusses the basic principles and photoresists employed in 2PP fabrication of hydrogels, followed by an in-depth introduction of various mechanical and biological characterization techniques used to assess the fabricated structures. The design requirements for cell and tissue related applications are then described to guide the engineering, physicochemical, and biological efforts. Three case studies in bone, cancer, and cardiac tissues are presented that illustrate the need for structured materials in the next generation of clinical applications. This paper concludes by summarizing the progress to date, identifying additional opportunities for 2PP hydrogel scaffolds, and discussing future directions for 2PP research.

Cell migration, critical to numerous biological processes, can be guided by surface topography. Studying the effects of topography on cell migration is valuable for enhancing our understanding of directional cell migration and for functionally engineering cell behavior. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined waveforms with curvature and length scales that are similar to those found in physiological settings, such as the luminal walls of blood vessels that endothelial cells migrate along. We find that endothelial cells migrate fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreases as sinusoid wavelength increases. Interestingly, inhibition of Rac1 decreases directional migration on sine wave topographies but not on flat surfaces with micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate the effects and mechanisms of topography on cell migration by fabricating a wide array of physiologically-relevant surfaces with curvatures that are challenging to fabricate using conventional manufacturing techniques.

Atomic force microscope (AFM) probes and AFM imaging by extension are the product of exceptionally refined silicon micromachining, but are also restricted by the limitations of these fabrication techniques. Here, the nanoscale additive manufacturing technique direct laser writing is explored as a method to print monolithic cantilevered probes for AFM. Not only are 3D printed probes found to function effectively for AFM, but they also confer several advantages, most notably the ability to image in intermittent contact mode with a bandwidth approximately ten times larger than analogous silicon probes. In addition, the arbitrary structural control afforded by 3D printing is found to enable programming the modal structure of the probe, a capability that can be useful in the context of resonantly amplifying nonlinear tip-sample interactions. Collectively, these results show that 3D printed probes complement those produced using conventional silicon micromachining and open the door to new imaging techniques.