Stiffness-tunable substrate fabrication by DMDbased optical projection lithography for cancer cell invasion studies.

OBJECTIVE: Cancer cell invasion is a critical cause of fatality in cancer patients. Physiologically relevant tumor models play a key role in revealing the mechanisms underlying the invasive behavior of cancer cells. However, most existing models only consider interactions between cells and extracellular matrix (ECM) components while neglecting the role of matrix stiffness in tumor invasion. Here, we propose an effective approach that can construct stiffness-tunable substrates using digital mirror device (DMD)-based optical projection lithography to explore the invasion behavior of cancer cells. The printability, mechanical properties, and cell viability of three-dimensional (3D) models can be tuned by the concentration of prepolymer and the exposure time. The invasion trajectories of gastric cancer cells in tumor models of different stiffness were automatically detected and tracked in real-time using a deep learning algorithm. The results show that tumor models of different mechanical stiffness can yield distinct regulatory effects. Moreover, owing to the biophysical characteristics of the 3D in vitro model, different cellular substructures of cancer cells were induced. The proposed tunable substrate construction method can be used to build various microstructures to achieve simulation of cancer invasion and antitumor screening, which has great potential in promoting personalized therapy.

Nanomechanical Analysis of Living Small Extracellular Vesicles to Identify Gastric Cancer Cell Malignancy Based on a Biomimetic Peritoneum.

Gastric cancer is one of the most prevalent digestive malignancies. The lack of effective in vitro peritoneal models has hindered the exploration of the potential mechanisms behind gastric cancer's peritoneal metastasis. An accumulating body of research indicates that small extracellular vesicles (sEVs) play an indispensable role in peritoneal metastasis of gastric cancer cells. In this study, a biomimetic peritoneum was constructed. The biomimetic model is similar to real peritoneum in internal microstructure, composition, and primary function, and it enables the recurrence of peritoneal metastasis process in vitro. Based on this model, the association between the mechanical properties of sEVs and the invasiveness of gastric cancer was identified. By performing nanomechanical analysis on sEVs, we found that the Young's modulus of sEVs can be utilized to differentiate between malignant clinical samples (ascites) and nonmalignant clinical samples (peritoneal lavage). Furthermore, patients' ascites-derived sEVs were verified to stimulate the mesothelial-to-mesenchymal transition, thereby promoting peritoneal metastasis. In summary, nanomechanical analysis of living sEVs could be utilized for the noninvasive diagnosis of malignant degree and peritoneal metastasis of gastric cancer. This finding is expected to contribute future treatments.

Micromixing within microfluidic devices: Fundamentals, design, and fabrication.

As one of the hot spots in the field of microfluidic chip research, micromixers have been widely used in chemistry, biology, and medicine due to their small size, fast response time, and low reagent consumption. However, at low Reynolds numbers, the fluid motion relies mainly on the diffusive motion of molecules under laminar flow conditions. The detrimental effect of laminar flow leads to difficulties in achieving rapid and efficient mixing of fluids in microchannels. Therefore, it is necessary to enhance fluid mixing by employing some external means. In this paper, the classification and mixing principles of passive (T-type, Y-type, obstructed, serpentine, three-dimensional) and active (acoustic, electric, pressure, thermal, magnetic field) micromixers are reviewed based on the presence or absence of external forces in the micromixers, and some experiments and applications of each type of micromixer are briefly discussed. Finally, the future development trends of micromixers are summarized.

An aquatic biomimetic butterfly soft robot driven by deformable photo-responsive hydrogel.

Taking inspiration from the locomotor behaviors of a butterfly, we have developed an underwater soft robot that imitates its movements. This biomimetic robot is constructed using a deformable photo-responsive material that exhibits high biological compatibility and impressive deformation capabilities in response to external stimuli. First, we investigate composite materials consisting of poly-N-isopropylacrylamide (PNIPAM) and multi-walled carbon nanotubes (MWCNTs). Then, using photocuring printing technology, we successfully fabricate a biomimetic butterfly soft robot utilizing these composite materials. The robot is driven by visible light, enabling it to achieve periodic wing movement and fly upward at an average speed of 3.63 mm s-1. In addition, the robot achieves additional functionalities such as flying over obstacles and carrying small objects during the ascending flight. These outcomes have a significant impact on the advancement of flexible biomimetic robots and offer valuable insights for the research of biomimetic robots driven by visible light.

A multi-stimulus-responsive bionic fish microrobot for remote intelligent control applications.

In nature, all creatures have their unique characteristics that allow them to adapt to the complex and changeable living environments. In recent years, bionic fish has received increased attention from the research community, and many fish-like microrobots driven by the Marangoni effect have been developed. They are generally characterized by easy operation and rapid driving. However, traditional fish-like microrobots can only be driven by a single stimulus and move on two-dimensional (2D) gas-liquid interfaces, which greatly limits their ability in obstacle avoidance and transportation. In this article, we propose a multi-stimulus-responsive bionic fish microrobot, which is made of temperature-responsive hydrogel poly(N-isopropylacrylamide) (pNIPAM). This microrobot is impregnated with carbon nanotubes (CNTs) and Fe3O4 and therefore has magnetic and photothermal conversion properties. Under the action of optical, magnetic or ethanol molecules, the microrobot can perform complex programmable translational motion on 2D surfaces and controllable rising and sinking, while realizing motion simulation and obstacle avoidance. The microrobot is expected to be used for a wide range of applications in intelligent control systems.

Upconversion optogenetics-driven biohybrid sensor for infrared sensing and imaging.

Living organisms are far superior to state-of-the-art devices in visual perception as they have evolved a wide number of capabilities that encompass our most advanced technologies. By leveraging the performance of living organisms and directly interfacing them with artificial components, it can use the intricacy and metabolic efficiency of biological visual sensing within artificial machines. Inspired by the molecular basis (transient receptor potential, TRP) for infrared detection of pit-bearing organisms, we propose a TRP-like biohybrid sensor by integrating upconversion nanoparticles (UCNP) and optogenetically engineered cells on a graphene transistor for infrared sensing and imaging. The UCNP converts infrared light irradiation into blue light, the blue light activates the cells expressed with channelrhodopsin-2 (ChR2) and induces transmembrane photocurrent, and the photocurrent is detected by a biocompatible graphene transistor. Stepwise and overall experimental results show that, upon infrared light irradiation, the UCNP can rapidly mediate cellular photocurrents, which further translates into the extra output current of the graphene transistor. More notably, the response speed of the biohybrid sensor is 1∼3 orders of magnitude faster than those of TRPs heterologously expressed in cell lines in the literature, which confirms the response time advantage of the combination of UCNP and ChR2 within the sensor in place of TRPs. The biohybrid sensor can successfully image infrared targets, proving the feasibility of developing bionic infrared sensing devices by biohybrid integration of nonliving nanomaterials and biological components. This work opens up an avenue for biohybrid sensors to develop the bionic infrared vision that promisingly reproduces the functional superiority of natural organisms. STATEMENT OF SIGNIFICANCE: Infrared sensing and imaging have a wide range of military and civilian applications. Organisms have evolved excellent infrared vision with the molecular basis, transient receptor potential (TRP), and the performance is superior to existing state-of-the-art infrared devices. Inspired by this, a TRP-like biohybrid sensor based on upconversion optogenetics and a 2D material-based device is developed for infrared sensing and imaging. The biohybrid sensor has a relatively fast response speed that is 1∼3 orders of magnitude faster than that of the heterologously expressed TRPs, which enables its capability of infrared imaging with a single pixel-based method. This work broadens the spectrum of biohybrid sensing based on engineered cells to infrared, advancing the process of reproducing the excellent infrared detection of organisms.

A Multifunctional Light-Driven Swimming Soft Robot for Various Application Scenarios.

The locomotor behavior of creatures in nature can bring a lot of inspiration for the fabrication of soft actuators. In this paper, we fabricated a bionic light-driven swimming soft robot that can perform grasping of tiny objects and achieve the task of object transfer. By adding carbon nanotubes (CNTs), the temperature-sensitive hydrogels can be endowed with light-responsive properties. The fabricated composite hydrogel structure can control the contraction and expansion of volume by light, which is similar to the contraction and diastole behavior of muscles. The oscillation of the fish tail and the grasping action of the normally closed micromanipulator can be achieved by the control of the irradiation of the xenon light source. The bending of the bionic arm can be controlled by the irradiation of a near-infrared (NIR) laser, which transforms the spatial position and posture of the micromanipulator. The proposed scheme is feasible for miniaturized fabrication and application of flexible actuators. This work provides some important insights for the study of light-driven microrobots and light-driven flexible actuators.

Advances in precise single-cell capture for analysis and biological applications.

Cells are the basic structural and functional units of living organisms. However, conventional cell analysis only averages millions of cell populations, and some important information is lost. It is essential to quantitatively characterize the physiology and pathology of single-cell activities. Precise single-cell capture is an extremely challenging task during cell sample preparation. In this review, we summarize the category of technologies to capture single cells precisely with a focus on the latest development in the last five years. Each technology has its own set of benefits and specific challenges, which provide opportunities for researchers in different fields. Accordingly, we introduce the applications of captured single cells in cancer diagnosis, analysis of metabolism and secretion, and disease treatment. Finally, some perspectives are provided on the current development trends, future research directions, and challenges of single-cell capture.

Recent advances in optically induced di-electrophoresis and its biomedical applications.

The development of the micro/nano science and technology has promoted the evolvement of human civilization tremendously. The advancement of the micro/nano science and technology highly depends on the progress of the micro/nano manipulation techniques, and the micro/nano-scaled manipulation level is the critical sign of the micro/nano science and technology. This review, aimed at the demand and the challenge of the micro/nano material and biomedical fields and related to the scientific issues and implementation techniques of the optically induced di-electrophoresis (ODEP). We explained its working principle, manipulating method, and influencing factors of ODEP force to a certain extent. A number of application fields based-ODEP technology and specific applications so far are summarized and reviewed. Finally, some perspectives are provided on current development trends, future research directions, and challenges of ODEP.

Recent advances in acoustic microfluidics and its exemplary applications.

Acoustic-based microfluidics has been widely used in recent years for fundamental research due to its simple device design, biocompatibility, and contactless operation. In this article, the basic theory, typical devices, and technical applications of acoustic microfluidics technology are summarized. First, the theory of acoustic microfluidics is introduced from the classification of acoustic waves, acoustic radiation force, and streaming flow. Then, various applications of acoustic microfluidics including sorting, mixing, atomization, trapping, patterning, and acoustothermal heating are reviewed. Finally, the development trends of acoustic microfluidics in the future were summarized and looked forward to.

Customized construction of microscale multi-component biostructures for cellular applications.

In vitro biofabrication is employed in fields such as biomedicine and those using biomimetic materials. However, it suffers from drawbacks such as low resolution, applicability on a limited range of components, and difficulty in purposefully depositing specific cells in three-dimensional space. Hence, this paper proposes a digital micromirror device-based optical projection lithography (DOPL) system for producing multi-component microstructures with resolutions of tens of microns and explores the behavior of cells with these structures. The printability and mechanical properties of these microstructures were investigated to assess their reproduction quality and the ability to control their structural characteristics. The results show that when DOPL is used with polyethylene glycol dimethacrylate (PEGDMA) hydrogel, an array of micropits can be fabricated within a few minutes. Furthermore, uniform cell spheroids form rapidly with high throughput when they are seeded into the micropits. Additionally, PEGDMA and gelatin methacryloyl (GelMA) were used to construct multi-component microstructures, and it was demonstrated that cells with various morphologies selectively adhere to the heterogeneous interface. In addition, DOPL could enable deposition of various cells for constructing microenvironments and for drug screening. Finally, a biomimetic peritoneal model was constructed. Overall, this work demonstrates the versatility of this system and its potential in cellular applications such as cell behavior research, drug screening, and tissue engineering.

Bubble-based microrobots enable digital assembly of heterogeneous microtissue modules.

The specific spatial distribution of tissue generates a heterogeneous micromechanical environment that provides ideal conditions for diverse functions such as regeneration and angiogenesis. However, to manufacture microscale multicellular heterogeneous tissue modulesin vitroand then assemble them into specific functional units is still a challenging task. In this study, a novel method for the digital assembly of heterogeneous microtissue modules is proposed. This technique utilizes the flexibility of digital micromirror device-based optical projection lithography and the manipulability of bubble-based microrobots in a liquid environment. The results indicate that multicellular microstructures can be fabricated by increasing the inlets of the microfluidic chip. Upon altering the exposure time, the Young's modulus of the entire module and different regions of each module can be fine-tuned to mimic normal tissue. The surface morphology, mechanical properties, and internal structure of the constructed bionic peritoneum were similar to those of the real peritoneum. Overall, this work demonstrates the potential of this system to produce and control the posture of modules and simulate peritoneal metastasis using reconfigurable manipulation.

Characterization of interconnectivity of gelatin methacrylate hydrogels using photoacoustic imaging.

Hydrogels can provide a three-dimensional microenvironment for cells and thus serve as an extracellular matrix in a biofabrication process. The properties of hydrogels, such as their porosity and mechanical properties, significantly influence the cell growth. However, there is still a lack of effective methods for characterizing the hydrogel structure noninvasively. Herein, a photoacoustic (PA) imaging-based method is proposed for the characterization of gelatin methacrylate (GelMA) hydrogels. Owing to their high PA contrast, red blood cells (RBCs) are included as mediators in the GelMA hydrogel to analyze its pore distribution. The interconnectivity of the pores is further analyzed through the lysis of RBCs. The diffusion of the RBC lysis buffer in the GelMA is consistent with the trend observed in simulations. The analyzed vitality of HEK293 cells in different GelMA hydrogels reveals that understanding the diffusion of solutes (i.e., nutrients) is a potential strategy to optimize the hydrogel parameters during biofabrication.

Density Regulation and Localization of Cell Clusters by Self-Assembled Femtosecond-Laser-Fabricated Micropillar Arrays.

Tumor cell clusters of varying sizes and densities have different metastatic potentials. Three-dimensional (3D) patterned structures with rational topographical and mechanical properties are capable of guiding the 3D clustering of tumor cells. In this study, single femtosecond laser pulses were used to fabricate individual high-aspect-ratio micropillars via two-photon polymerization (TPP). By combining this approach with capillary-force self-assembly, complex 3D microstructure patterns were constructed with a high efficiency. The microstructures were able to regulate the formation of cell clusters at different cell seeding densities and direct self-guided 3D assembly of cell clusters of various sizes and densities. Localization of cell clusters was achieved using grid-indexed samples to address individual cell clusters, which holds great promise for in situ cell cluster culture and monitoring and for applications such as RNA sequencing of cell clusters.

Visible Light-Driven Jellyfish-like Miniature Swimming Soft Robot.

Soft actuators that exhibit large deformation and can move at a fast speed in response to external stimuli have been in high demand for biomimetic applications. In this paper, we propose a convenient approach to fabricate a reversible and thermal-responsive composite hydrogel. Under the irradiation of visible light, the striped hydrogel can bend at a speed of up to 65.72°/s with carbon nanotubes loaded at a concentration of 3 mg/mL. A jellyfish-like miniature soft robot is made using this hydrogel. When driven by visible light, the robot can move at a maximum speed of 3.37 mm/s. Besides swimming, other motion modes, including walking and jumping, are also achieved by the robot. In addition, the robot can perform directional transportation of tiny objects. As a new actuation approach for the research of jellyfish-like miniature soft robots, this work is of great significance to the development of flexible bionic robots. Moreover, this work also offers some important insights into the research of biomimetic robots driven by visible light.

Self-assembled microcage fabrication for manipulating and selectively capturing microparticles and cells.

Single-cell-scale selective manipulation and targeted capture play a vital role in cell behavior analysis. However, selective microcapture has primarily been performed in specific circumstances to maintain the trapping state, making the subsequent in situ characterization and analysis of specific particles or cells difficult and imprecise. Herein, we propose a novel method that combines femtosecond laser two-photon polymerization (TPP) micromachining technology with the operation of optical tweezers (OTs) to achieve selective and targeted capture of single particles and cells. Diverse ordered microcages with different shapes and dimensions were self-assembled by micropillars fabricated via TPP. The micropillars with high aspect ratios were processed by single exposure, and the parameters of the micropillar arrays were investigated to optimize the capillary-force-driven self-assembly process of the anisotropic microcages. Finally, single microparticles and cells were selectively transported to the desired microcages by manipulating the flexibly of the OTs in a few minutes. The captured microparticles and cells were kept trapped without additional forces.

Engineering Biological Tissues from the Bottom-Up: Recent Advances and Future Prospects.

Tissue engineering provides a powerful solution for current organ shortages, and researchers have cultured blood vessels, heart tissues, and bone tissues in vitro. However, traditional top-down tissue engineering has suffered two challenges: vascularization and reconfigurability of functional units. With the continuous development of micro-nano technology and biomaterial technology, bottom-up tissue engineering as a promising approach for organ and tissue modular reconstruction has gradually developed. In this article, relevant advances in living blocks fabrication and assembly techniques for creation of higher-order bioarchitectures are described. After a critical overview of this technology, a discussion of practical challenges is provided, and future development prospects are proposed.

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens.

Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.

Biomimetic construction of peritoneum to imitate peritoneal metastasis using digital micromirror device-based optical projection lithography.

Currently, the mechanisms underlying the peritoneal metastasis of gastric cancer cells and the function of mesothelial cells during this process are unclear, primarily due to the absence of an effective in vitro peritoneal model. In this study, we constructed a biomimetic peritoneal model using a digital micromirror device-based optical projection lithography system. This model enabled the simulation of a damaged peritoneum, which allowed for a comparison of the characteristics of an undamaged peritoneum, such as porosity, mechanical properties, and surface morphology, with those of a damaged peritoneum. Biological inertness and removability of the polyethylene glycol dimethacrylate hydrogel were exploited to fabricate an arrayed heterogeneous interface that imitated a damaged human peritoneum. The porous structure of the peritoneum was achieved by adjusting the ratio of collagen I to gelatin methacryloyl; this structure of the peritoneum might contribute to its shock absorption property. Atomic force microscopy characterization showed that the outermost layers of the model peritoneum and real peritoneum were similar in surface morphology and mechanical properties. Furthermore, we reproduced the process of peritoneal metastasis in vitro. The numbers of gastric cancer cells that adhered to the heterogeneous interface were different, and mesothelial cells played an essential role in peritoneal metastasis. Our findings indicate that this model can be utilized in preclinical drug screening and personalized therapy.

Facile Method for Fabricating Microfluidic Chip Integrated with Microwell Arrays for Cell Trapping.

With the development of biomedical technology, personalized diagnosis and treatment at the single-cell level are becoming more important in the medical field. As one of the most powerful tools, microfluidic chips have shown significant potential for various applications related to cell separation, cell proliferation, and cell behavior analysis. However, fabricating microfluidic devices requires complicated procedures and high-cost equipment. In this study, an optofluidic maskless lithography method was proposed for rapid fabrication of microfluidic devices integrated with microwells. Through the use of this approach, microwells can be on-line designed and the exposure patterns can be modulated. Single or multi polystyrene microspheres were successfully trapped by using the designed microwells. The capture of MCF-7 cells and cell arrays indicated that the microfluidic devices fabricated in the present study can be applied for cell research.