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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. [ABSTRACT FROM AUTHOR]

The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are not well known. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high-molecular-weight protein(s) in fetal bovine serum that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and in the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as 3 days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting and micropatterning techniques. Impact statement Bioprinting is a potential strategy to achieve microvascularization in engineered tissues. However, the controlled self-organization of patterned endothelial cells into perfusable microvasculature remains challenging. We used DNA Programmed Assembly of Cells to create cell-dense, capillary-sized cords of endothelial cells with complete control over their structure. We optimized the matrix and media conditions to promote self-organization and maturation of these endothelial cords into stable and perfusable microvascular networks.

Cell‐laden hydrogel fibers/tubules are one of the fundamentals of tissue engineering. They have been proven as a promising method for constructing biomimetic tissues, such as muscle fibers, nerve conduits, tendon and vessels, etc. However, current hydrogel fiber/tubule production methods have limitations in ordered cell arrangements, thus impeding the biomimetic configurations. Acoustic cell patterning is a cell manipulation method that has good biocompatibility, wide tunability, and is contact‐free. However, there are few studies on acoustic cell patterning for fiber production, especially on the radial figure cell arrangements, which mimic many native tissue‐like cell arrangements. Here, an acoustic cell patterning system that can be used to produce hydrogel fibers/tubules with tunable cell patterns is shown. Cells can be pre‐patterned in the liquid hydrogel before being extruded as cross‐linked hydrogel fibers/tubules. The radial patterns can be tuned with different complexities based on the acoustic resonances. Cell viability assays after 72 h confirm good cell viability and proliferation. Considering the biocompatibility and reliability, the present method can be further used for a variety of biomimetic fabrications. [ABSTRACT FROM AUTHOR]

A technology that can effectively distinguish between various odorants with high sensitivity and selectivity has numerous applications ranging from water quality testing to disease diagnosis. Here, we report a cell-based odorant-sensing display that utilizes Sf21 cells expressing odorant receptors, co-receptors, and a calcium-sensitive fluorescent protein as the sensing elements for detecting multiple odorants. Integrated micropatterns of the sensor cells in a few hundred micrometer-size patterns were fabricated on photoactivatable cell-anchoring surfaces consisting of photo-responsive polymeric materials. In the microfluidic system equipped with the sensing display, the injection of two model odorants, such as Bombykal and 1-octen-3-ol, at micro-molar concentrations resulted in selective and rapid fluorescence emission from the corresponding sensor cell patterns. Furthermore, when both odorants were injected together, the fluorescence from each corresponding sensor cell could be observed simultaneously. This study provides the proof of principle that the current cell patterning system enables the discrimination of odors, including multiple odorants, through a finely patterned sensing display on the device.

Abstract In vitro models are necessary to study the pathophysiology of the disease and the development of effective, tailored treatment methods owing to the complexity and heterogeneity of breast cancer and the large population affected by it. The cellular connections and tumor microenvironments observed in vivo are often not recapitulated in conventional two-dimensional (2D) cell cultures. Therefore, developing 3D in vitro models that mimic the complex architecture and physiological circumstances of breast tumors is crucial for advancing our understanding of the illness. A 3D scaffold-free in vitro disease model mimics breast cancer pathophysiology by allowing cells to self-assemble/pattern into 3D structures, in contrast with other 3D models that rely on artificial scaffolds. It is possible that this model, whether applied to breast tumors using patient-derived primary cells (fibroblasts, endothelial cells, and cancer cells), can accurately replicate the observed heterogeneity. The complicated interactions between different cell types are modelled by integrating critical components of the tumor microenvironment, such as the extracellular matrix, vascular endothelial cells, and tumor growth factors. Tissue interactions, immune cell infiltration, and the effects of the milieu on drug resistance can be studied using this scaffold-free 3D model. The scaffold-free 3D in vitro disease model for mimicking tumor pathophysiology in breast cancer is a useful tool for studying the molecular basis of the disease, identifying new therapeutic targets, and evaluating treatment modalities. It provides a more physiologically appropriate high-throughput platform for screening large compound library in a 96–384 well format. We critically discussed the rapid development of personalized treatment strategies and accelerated drug screening platforms to close the gap between traditional 2D cell culture and in vivo investigations. Graphical Abstract

In vitro models are necessary to study the pathophysiology of the disease and the development of effective, tailored treatment methods owing to the complexity and heterogeneity of breast cancer and the large population affected by it. The cellular connections and tumor microenvironments observed in vivo are often not recapitulated in conventional two-dimensional (2D) cell cultures. Therefore, developing 3D in vitro models that mimic the complex architecture and physiological circumstances of breast tumors is crucial for advancing our understanding of the illness. A 3D scaffold-free in vitro disease model mimics breast cancer pathophysiology by allowing cells to self-assemble/pattern into 3D structures, in contrast with other 3D models that rely on artificial scaffolds. It is possible that this model, whether applied to breast tumors using patient-derived primary cells (fibroblasts, endothelial cells, and cancer cells), can accurately replicate the observed heterogeneity. The complicated interactions between different cell types are modelled by integrating critical components of the tumor microenvironment, such as the extracellular matrix, vascular endothelial cells, and tumor growth factors. Tissue interactions, immune cell infiltration, and the effects of the milieu on drug resistance can be studied using this scaffold-free 3D model. The scaffold-free 3D in vitro disease model for mimicking tumor pathophysiology in breast cancer is a useful tool for studying the molecular basis of the disease, identifying new therapeutic targets, and evaluating treatment modalities. It provides a more physiologically appropriate high-throughput platform for screening large compound library in a 96–384 well format. We critically discussed the rapid development of personalized treatment strategies and accelerated drug screening platforms to close the gap between traditional 2D cell culture and in vivo investigations. [ABSTRACT FROM AUTHOR]

Modularity is one of the important structural properties that affect information processing and other functionalities of neuronal networks. Researchers have developed in-vitro clustered network models for reproducing the modularity, but it is still challenging to control the segregation and integration of several sub-populations of them. We cultured clustered networks with alginate patterning and collected the electrophysiological signals to investigate the changes in functional properties during the development. We built inter-connected neuronal clusters using alginate micro-patterning with a circular shape on the surface of the micro-electrode array. The neuronal clusters were enabled to be connected at 3 or 10 days-in-vitro (DIV) by removing the barrier. The neuronal signals from different types of networks were collected from 16 to 34 DIV, and functional characteristics were examined. Connectivity and burst motif analysis were carried out to find out the relation between the structure and function of the networks. Neuronal networks with clustered structure showed different activity properties from the random networks along the development. The clustered networks had more short-range connections compared to the random networks. In the network burst motif analysis, the clustered networks showed more various patterns and a slower propagation of the activation patterns. In this study, we successfully cultured neuronal networks with clustered structure, and the structure affected the functional properties. The network model suggested in this study will be a good solution for observing the effect of structure on function during their development. [ABSTRACT FROM AUTHOR]

Abstract Cell‐laden hydrogel fibers/tubules are one of the fundamentals of tissue engineering. They have been proven as a promising method for constructing biomimetic tissues, such as muscle fibers, nerve conduits, tendon and vessels, etc. However, current hydrogel fiber/tubule production methods have limitations in ordered cell arrangements, thus impeding the biomimetic configurations. Acoustic cell patterning is a cell manipulation method that has good biocompatibility, wide tunability, and is contact‐free. However, there are few studies on acoustic cell patterning for fiber production, especially on the radial figure cell arrangements, which mimic many native tissue‐like cell arrangements. Here, an acoustic cell patterning system that can be used to produce hydrogel fibers/tubules with tunable cell patterns is shown. Cells can be pre‐patterned in the liquid hydrogel before being extruded as cross‐linked hydrogel fibers/tubules. The radial patterns can be tuned with different complexities based on the acoustic resonances. Cell viability assays after 72 h confirm good cell viability and proliferation. Considering the biocompatibility and reliability, the present method can be further used for a variety of biomimetic fabrications.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization. [ABSTRACT FROM AUTHOR]

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana , those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator Mp FGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha. [ABSTRACT FROM AUTHOR]

Antibody arrays have great implications in many biomedical settings. However, commonly used patterning methods have difficulties in generating antibody arrays with both high resolution and multiplexity, limiting their applications. Here, a convenient and versatile technique for the patterning of multiple antibodies with resolution down to 20 µm is reported using micropillar‐focused droplet printing and microcontact printing. Droplets of antibody solutions are first printed and stably confined on the micropillars of a stamp, and then the antibodies absorbed on the micropillars are contact‐printed to the target substrate, generating antibody patterns faithfully replicating the micropillar array. The effect of different parameters on the patterning results is investigated, including hydrophobicity of the stamps, override time of the droplet printing, incubation time, and the diameters of the capillary tips and micropillars. To demonstrate the utility of the method, multiplex arrays of anti‐EpCAM and anti‐CD68 antibodies is generated to capture breast cancer cells and macrophages, respectively, on the same substrate, and successful capturing of individual cell types and enrichment among the cells are achieved. It is envision that this method would serve as a versatile and useful protein patterning tool for biomedical applications. [ABSTRACT FROM AUTHOR]

Abstract The regulation of cell behavior is crucial for understanding many fundamental biological processes and designing novel medical devices. Nano/micropatterning has been developed as a means of controlling cellular properties and behaviors. Using cinnamoyl groups that have undergone photodimerization, we have prepared facile photopatterning films with adjustable surface properties for cell patterning via simple steps. Our strategy for controlling surface properties uses the decrease in free volume induced by photocrosslinking. This paper describes the surface‐patterning properties of photocrosslinkable polymer films and the cell adhesion to their micropatterned surfaces. Upon culturing cells on micropatterned surfaces, preferential cell adhesion, alignment and proliferation are observed in the unexposed regions. Additionally, diverse surface patterns of photocrosslinkable polymer films have been fabricated using various photomasks, enabling effective control of cell adhesion and alignment. The ability to regulate cellular behavior via microfabricated surfaces of photocrosslinkable polymer films enables a new and facile approach for designing scaffolds for the development of novel advanced materials, tissue engineering, and regenerative medicine.

This paper presents the engineering and validation of an enabling technology that facilitates new capabilities in in vitro cell models for high-throughput screening and tissue engineering applications. This is conducted through a computerized system that allows the design and deposition of high-fidelity microscale patterned coatings that selectively alter the chemical and topographical properties of cell culturing surfaces. Significantly, compared to alternative methods for microscale surface patterning, this is a digitally controlled and automated process thereby allowing scientists to rapidly create and explore an almost infinite range of cell culture patterns. This new capability is experimentally validated across six different cell lines demonstrating how the precise microscale deposition of these patterned coatings can influence spatiotemporal growth and movement of endothelial, fibroblast, neuronal and macrophage cells. To further demonstrate this platform, more complex patterns are then created and shown to guide the behavioral response of colorectal carcinoma cells. [ABSTRACT FROM AUTHOR]

Acoustofluidics has been a promising approach using sound waves to manipulate particles and actuate fluids in biomedical applications. It usually generates acoustic radiation force and acoustic streaming to initiate diffraction, reflection and interference, building up a pressure distribution to facilitate accurate manipulation of micro- or nano-scale particles and fluids. Owing to its remarkable contact-free and biocompatible advantages, acoustofluidics has been used in high-throughput cell analysis, size-controllable organoid structures, and functional tissue mimics. We enumerate the basic concepts and the sufficient research of acoustofluidics in precise patterning and tissue engineering in this review, including the design and function of four typical acoustofluidic devices, various forms of cell patterning and 3D tissue engineering. Meanwhile, we outlined current challenges and future directions of acoustofluidics in biomedicine and tissue engineering.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Organs-on-chips (OoCs) are microfluidic devices that contain bioengineered tissues or parts of natural tissues or organs and can mimic the crucial structures and functions of living organisms. They are designed to control and maintain the cell- and tissue-specific microenvironment while also providing detailed feedback about the activities that are taking place. Bioprinting is an emerging technology for constructing artificial tissues or organ constructs by combining state-of-the-art 3D printing methods with biomaterials. The utilization of 3D bioprinting and cells patterning in OoC technologies reinforces the creation of more complex structures that can imitate the functions of a living organism in a more precise way. Here, we summarize the current 3D bioprinting techniques and we focus on the advantages of 3D bioprinting compared to traditional cell seeding in addition to the methods, materials, and applications of 3D bioprinting in the development of OoC microsystems. [ABSTRACT FROM AUTHOR]

Fabrication of three-dimensional tissues using living cells is a promised approach for drug screening experiment and in vitro disease modeling. To study a physiological neuronal function, three-dimensional cell patterning and construction of neuronal cell network were required. In this study, we proposed a three-dimensional cell drawing methodology in hydrogel to construct the three-dimensional neuronal cell network. PC-12 cells, which were used as neuronal cell differentiation model, were dispensed into a collagen hydrogel using a micro injector with a three-dimensional position control. To maintain the three-dimensional position of cells, atelocollagen was kept at sol-gel transition state during cell dispensing. As the results, PC-12 cells were patterned in the atelocollagen gel to form square pattern with different depth. In the patterned cellular lines, PC-12 cells elongated neurites and form a continuous cellular network in the atelocollagen gel. It was suggested that our three-dimensional cell drawing technology has potentials to reconstruct three-dimensional neuronal networks for an investigation of physiological neuronal functions. [ABSTRACT FROM AUTHOR]

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

While many tools exist to study immune‐cell chemotaxis in vitro, current methods often lack desirable features. Using fluid‐walled microfluidics, circuits are built around primary murine macrophages deposited in pre‐defined patterns on Petri dishes or microplates. Concentration gradients of complement component 5a (C5a) are established in flow‐free or flowing environments, cell migration imaged, and cell directionality and velocity correlated to calculated local C5a concentrations. In flow‐free circuits built around patterned macrophages, only cells nearest the C5a source migrate regardless of local attractant concentration. Conversely, in flowing circuits free from intercellular signaling and attractant degradation, only cells distant from the source migrate. In both systems, cells respond to lower C5a concentrations than previously reported (≈0.1 pM). Finally, macrophages follow instantly‐shifted gradients better than slowly‐shifting ones, suggesting that migration depends on both spatial and temporal responses to concentration. [ABSTRACT FROM AUTHOR]

Many mammalian tissues have a specific cellular arrangement that enables their unique function. For example, parallel alignment of myofibers enables uniaxial muscle contraction. To engineer structured tissues ex vivo, it is critical to recapitulate this cellular arrangement. Conventional 3D encapsulation often fails to recapitulate this complexity, motivating the need for advanced patterning approaches. In this work, an acoustofluidic device to continuously pattern mammalian cells within hydrogel fibers is engineered. Contactless acoustofluidic forces are used to control the spacing between parallel lines of cells. To enable continuous extrusion of cell‐laden hydrogel fibers, a low friction Teflon tube is integrated into the device. A photopolymerizable hydrogel allows triggering gelation externally with light once the cells are under the influence of the acoustic field, setting the patterned cells within the hydrogel fiber. Using this device, the muscle progenitor cells (myoblasts) within the hydrogel are patterned in parallel lines to mimic the structure of skeletal muscle. The increased formation of myotubes and spontaneous twitching of the myotubes in patterned samples are observed. This approach combining continuous fabrication with the tunability of acoustofluidics can create complex 3D tissues to engineer skeletal muscles as well as tendons, ligaments, vascular networks, or combinations thereof in the future. [ABSTRACT FROM AUTHOR]

Cell migration refers to the directional movement of cells to the surrounding cell-free zone in response to chemical and mechanical stimuli. A cell migration assay is an essential device for studying pharmaceutical and medical problems. In this paper, we present a novel approach to a cell migration assay on a chip with two merits, namely (i) simultaneous creation of many cell samples on the same condition and (ii) cells migrating while being stressed in a fluidic environment. The first merit has addressed the problem of poor reproducibility in experimental studies for medical problems such as wound healing, and the second merit has made the cell migration device, which is an in vitro environment, more biomimetic. The two merits are attributed to a novel mechanical method to simultaneously create many cell-free zones and to the design of a microfluidic process to create shear stress in cells uniformly. Two applications were studied on our device to explore its effectiveness. The first application is regarding the combination chemotherapy of cisplatin and doxorubicin (Adriamycin) on cervical cancer cells (HeLa). The second application is regarding inhibiting the migration of endothelial cells (HUVEC) in the process of anti-angiogenesis. [ABSTRACT FROM AUTHOR]

Bacteria have evolved as intelligent microorganisms that can colonize and form highly structured and cooperative multicellular communities with sophisticated singular and collective behaviors. The initial stages of colony formation and intercellular communication are particularly important to understand and depend highly on the spatial organization of cells. Controlling the distribution and growth of bacterial cells at the nanoscale is, therefore, of great interest in understanding the mechanisms of cell-cell communication at the initial stages of colony formation. Staphyloccocus aureus, a ubiquitous human pathogen, is of specific clinical importance due to the rise of antibiotic resistant strains of this species, which can cause life-threatening infections. Although several methods have attempted to pattern bacterial cells onto solid surfaces at single cell resolution, no study has truly controlled the 3D architectures of growing colonies. Herein, we present a simple, low-cost method to pattern S. aureus bacterial colonies and control the architecture of their growth. Using the wetting properties of micropatterened poly(dimethyl siloxane) platforms, with help from the physiological activities of the S. aureus cells, we fabricated connected networks of bacterial microcolonies of various sizes. Unlike conventional heterogeneous growth of biofilms on surfaces, the patterned S. aureus microcolonies in this work grow radially from nanostrings of a few bacterial cells, to form micrometer-thick rods when provided with a nutrient rich environment. This simple, efficient, and low-cost method can be used as a platform for studies of cell-cell communication phenomena, such as quorum sensing, horizontal gene transfer, and metabolic cross-feeding especially during initial stages of colony formation.

A biofabrication strategy for creating planar multiscale protein, hydrogel, and cellular patterns, and simultaneously generating microscale topographical features is developed that laterally confines the patterned cells and direct their growth in cell permissive hydrogels.

A biofabrication strategy for creating planar multiscale protein, hydrogel, and cellular patterns, and simultaneously generating microscale topographical features is developed that laterally confines the patterned cells and direct their growth in cell permissive hydrogels.

This study demonstrates the rapid fabrication and utility of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film for cell patterning. The film was obtained on a cell culture surface by microcasting MPC polymer ethanol solution into a degassed polydimethylsiloxane mold with a desired pattern. After removal of the mold, 293AD cells were cultured on the surface of the polymer film with the patterned microstructures. Patterned cell adhesion restricted by the film was successfully maintained during at least a 168-h cultivation. The microcast MPC polymer film can be prepared rapidly and used for efficient long-term cell confinement.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Organs-on-chips (OoCs) are microfluidic devices that contain bioengineered tissues or parts of natural tissues or organs and can mimic the crucial structures and functions of living organisms. They are designed to control and maintain the cell- and tissue-specific microenvironment while also providing detailed feedback about the activities that are taking place. Bioprinting is an emerging technology for constructing artificial tissues or organ constructs by combining state-of-the-art 3D printing methods with biomaterials. The utilization of 3D bioprinting and cells patterning in OoC technologies reinforces the creation of more complex structures that can imitate the functions of a living organism in a more precise way. Here, we summarize the current 3D bioprinting techniques and we focus on the advantages of 3D bioprinting compared to traditional cell seeding in addition to the methods, materials, and applications of 3D bioprinting in the development of OoC microsystems.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in the C. elegans zygote that the feedback relies on aPKC phosphorylation of Cdc42 at serine 71. The turnover of CDC-42 phosphorylation ensures optimal aPKC asymmetry and activity throughout polarisation by tuning Par6/aPKC association with Par3 and Cdc42. Moreover, turnover of Cdc42 phosphorylation regulates actomyosin cortex dynamics that are known to drive aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust control of polarisation in many cell types., Competing Interests: Declaration of interest The authors declare no competing interest

Fabrication of three-dimensional tissues using living cells is a promised approach for drug screening experiment and in vitro disease modeling. To study a physiological neuronal function, three-dimensional cell patterning and construction of neuronal cell network were required. In this study, we proposed a three-dimensional cell drawing methodology in hydrogel to construct the three-dimensional neuronal cell network. PC-12 cells, which were used as neuronal cell differentiation model, were dispensed into a collagen hydrogel using a micro injector with a three-dimensional position control. To maintain the three-dimensional position of cells, atelocollagen was kept at sol-gel transition state during cell dispensing. As the results, PC-12 cells were patterned in the atelocollagen gel to form square pattern with different depth. In the patterned cellular lines, PC-12 cells elongated neurites and form a continuous cellular network in the atelocollagen gel. It was suggested that our three-dimensional cell drawing technology has potentials to reconstruct three-dimensional neuronal networks for an investigation of physiological neuronal functions.

The effect of cell–cell contact on gene transfection is mainly unknown. Usually, transfection is carried out in batch cell cultures without control over cellular interactions, and efficiency analysis relies on complex and expensive protocols commonly involving flow cytometry as the final analytical step. Novel platforms and cell patterning are being studied to control cellular interactions and improve quantification methods. In this study, we report the use of surface patterning of fibronectin for the generation of two types of mesenchymal stromal cell patterns: single‐cell patterns without cell‐to‐cell contact, and small cell‐colony patterns. Both scenarios allowed the integration of the full transfection process and the continuous monitoring of thousands of individualized events by fluorescence microscopy. Our results showed that cell‐to‐cell contact clearly affected the transfection, as single cells presented a maximum transfection peak 6 h earlier and had a 10% higher transfection efficiency than cells with cell‐to‐cell contact. [ABSTRACT FROM AUTHOR]

Particles and cells can be patterned and moved (i.e., manipulated) precisely using acoustically driven techniques. To date, application of acoustic particle manipulation has been limited to plain surfaces. There is much potential for applying acoustic manipulation techniques to surfaces with microfabricated structures for high-throughput sensing. But adding thin film structures could alter manipulation characteristics compared to a plain surface. Using a two-chip setup that allows the wave generating device to be reused, we study the feasibility of acoustofluidic micro-manipulation on a surface-micromachined silicon (SMS) chip. The SMS chip is a complex superstrate with generic thin-film structures fabricated by patterning and etching multiple layers of thin films, with properties meant to represent a broad range of microfabricated devices. We report notable alterations in the particle separation distances on the SMS chip compared to a bare silicon superstrate, which we attribute to a change in wave type through a comparison of different superstrates prepared. We demonstrate a high cell viability after acoustic manipulation of live cells on the SMS chip. The results herein demonstrate the possibility of integrating a suite of microfabricated sensors on a chip with acoustically driven manipulation capabilities for multiplexed sensing and analysis for bio-applications. [ABSTRACT FROM AUTHOR]

Collective cell migration of epithelial tumor cells is one of the important factors for elucidating cancer metastasis and developing novel drugs for cancer treatment. Especially, new roles of E-cadherin in cancer migration and metastasis, beyond the epithelial-mesenchymal transition, have recently been unveiled. Here, we quantitatively examined cell motility using micropatterned free edge migration model with E-cadherin re-expressing EC96 cells derived from adenocarcinoma gastric (AGS) cell line. EC96 cells showed increased migration features such as the expansion of cell islands and straightforward movement compared to AGS cells. The function of tight junction proteins known to E-cadherin expression were evaluated for cell migration by knockdown using sh-RNA. Cell migration and straight movement of EC96 cells were reduced by knockdown of ZO-1 and claudin-7, to a lesser degree. Analysis of the migratory activity of boundary cells and inner cells shows that EC96 cell migration was primarily conducted by boundary cells, similar to leader cells in collective migration. Immunofluorescence analysis showed that tight junctions (TJs) of EC96 cells might play important roles in intracellular communication among boundary cells. ZO-1 is localized to the base of protruding lamellipodia and cell contact sites at the rear of cells, indicating that ZO-1 might be important for the interaction between traction and tensile forces. Overall, dynamic regulation of E-cadherin expression and localization by interaction with ZO-1 protein is one of the targets for elucidating the mechanism of collective migration of cancer metastasis. [ABSTRACT FROM AUTHOR]

Cell migration refers to the directional movement of cells to the surrounding cell-free zone in response to chemical and mechanical stimuli. A cell migration assay is an essential device for studying pharmaceutical and medical problems. In this paper, we present a novel approach to a cell migration assay on a chip with two merits, namely (i) simultaneous creation of many cell samples on the same condition and (ii) cells migrating while being stressed in a fluidic environment. The first merit has addressed the problem of poor reproducibility in experimental studies for medical problems such as wound healing, and the second merit has made the cell migration device, which is an in vitro environment, more biomimetic. The two merits are attributed to a novel mechanical method to simultaneously create many cell-free zones and to the design of a microfluidic process to create shear stress in cells uniformly. Two applications were studied on our device to explore its effectiveness. The first application is regarding the combination chemotherapy of cisplatin and doxorubicin (Adriamycin) on cervical cancer cells (HeLa). The second application is regarding inhibiting the migration of endothelial cells (HUVEC) in the process of anti-angiogenesis.

Conventional cell patterning methods are mainly based on hydrophilic/hydrophobic differences or chemical coating for cell adhesion/non-adhesion with wavering strength as it varies with the substrate surface conditions, including the cell type and the extracellular matrix components (ECMs) coating; thus, the versatility and stability of cell patterning methods must be improved. In this study, we propose a new cell patterning method using a light-responsive gas-generating polymer (LGP) and a conventional fluorescence microscope. Herein, cells and cellular tissues are easily released from the substrate surface by the nitrogen gas bubbles generated from LGP by the excitation light for fluorescence observation without harming the cells. The LGP-implanted chip was fabricated by packing LGP into a polystyrene (PS) microarray chip with a concave pattern. HeLa cells were spread on the LGP-implanted chips coated with three different ECMs (fibronectin, collagen, and poly-D-lysine), and all HeLa cells on the three LGP patterns were released. The pattern error between the LGP pattern and the remaining HeLa cells was 8.81 ± 4.24 μm, less than single-cell size. In addition, the LGP-implanted chip method can be applied to millimeter-scale patterns, with less than 30 s required for cell patterning. Therefore, the proposed method is a simple and rapid cell patterning method with high cell patterning accuracy of less than the cell size error, high scalability, versatility, and stability unaffected by the cell type or the ECM coating.

Alignment and patterning of neurons have great importance in some research fields, especially for regenerative medicine and for the formation of artificial neural networks. Alignment of neurons on quartz grooves and parylene cues on quartz substrate was evaluated in this work. The neurons' alignment on quartz grooves is considered to be topographical alignment, while the neurons' alignment on parylene cues on quartz substrate is considered to be chemical alignment. Both quartz grooves' and parylene cues' widths were fabricated in a range from 2 µm to 8 µm; quartz grooves' heights were in a range from 0.25 µm to 4 µm, while parylene cues' heights were only 0.25 µm. Neurons were dissociated hippocampal neurons from rat E18. Neurons were cultivated on test substrates for 7 days before alignment evaluation. As expected, neurons aligned according to the direction of grooves and cues; however, transversal growth direction was also observed with much smaller tendency. Chemical alignment was found to be more effective than topographical alignment. If parylene cues are thin and distanced enough, then neurons have a tendency to follow the direction of individual parylene cues; however, neurons on quartz grooves have a tendency just to follow a preferable direction than individual quartz grooves. [ABSTRACT FROM AUTHOR]

In large-volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC's natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin-streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We preposition MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

Laser cell patterning is a distinctly effective technique for creating cell arrangements in culture that replicate in vivo tissue structure for studying contact-mediated cell-cell interactions. Conventional laser-based single-cell-manipulation techniques are limited by their inability to pattern irregularly shaped cells, such as rod-shaped cardiomyocytes. We report use of a spatial light modulator loaded with a computer-generated phase map to shape a single laser source into multiple laser-guidance beams distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes in an end-to-end connected alignment to replicate the in vivo heart muscle cell connection without use of substrate surface modifications, which can interfere with in vivo-like cell-cell and cell-extracellular matrix interactions. Our research demonstrates that two-stage multiple beam laser guidance is effective: 1) A dual-beam configuration horizontally translates a cell from the outlet of the microfluidic cell delivery channel to a position above the cell deposition site and 2) A quad-beam configuration rapidly propels the cell axially through the suspension medium to the culture substrate with vertical movement of the cell patterning chamber. Our study reveals that 90% of the patterned cells maintained end-to-end connection 30 min after patterning, and mechanical junctions could be reinstalled between laser connected cells after overnight incubation. This demonstrates that multiple beam laser patterning is an outstanding tool for in vitro studying contact-mediated cell-cell interactions among irregularly shaped cells.

High-resolution cell patterning technology is a versatile tool for studying tissue engineering and in vitro preclinical research, but simple methods that can be implemented easily are lacking. Here, we demonstrate a simple electrochemical approach for arranging cells into various patterns, enabling rapid and accurate localization for cell array formation. A voltage pulse is applied directly to the pre-patterned conducting substrate, causing cellular damage and rapidly forming live/dead cell arrays. The array platform, which focuses on assay miniaturization, is applicable to cell-based drug screening as well as high-content cell culture experiments.

Three-dimensional (3D) in vitro tissue models are superior to two-dimensional (2D) cell cultures in replicating natural physiological/pathological conditions by recreating the cellular and cell-matrix interactions more faithfully. Nevertheless, current 3D models lack either the rich multicellular environment or fail to provide appropriate biophysical stimuli both of which are required to properly recapitulate the dynamic in vivo microenvironment of tissues and organs. Here, we describe the rapid construction of multicellular, tubular tissue constructs termed Tissue-in-a-Tube using self-assembly process in tubular molds with the ability to incorporate a variety of biophysical stimuli such as electrical field, mechanical deformation, and shear force of the fluid flow. Unlike other approaches, this method is simple, requires only oxygen permeable silicone tubing that molds the tissue construct and thin stainless-steel pins inserted in it to anchor the construct and could be used to provide electrical and mechanical stimuli, simultaneously. The annular region between the tissue construct and the tubing is used for perfusion. Highly stable, macroscale, and robust constructs anchored to the pins form as a result of self-assembly of the extracellular matrix (ECM) and cells in the bioink that is filled into the tubing. We demonstrate patterning of grafts containing cell types in the constructs in axial and radial modes with clear interface and continuity between the layers. Different environmental factors affecting cell behavior such as compactness of the structure and size of the constructs can be controlled through parameters such as initial cell density, ECM content, tubing size, as well as the distance between anchor pins. Using connectors, network of tubing can be assembled to create complex macrostructured tissues (centimeters length) such as fibers that are bifurcated or columns with different axial thicknesses which can then be used as building blocks for biomimetic constructs or tissue regeneration. The method is versatile and compatible with various cell types including endothelial, epithelial, skeletal muscle cells, osteoblast cells, and neuronal cells. As an example, long mature skeletal muscle and neuronal fibers as well as bone constructs were fabricated with cellular alignment dictated by the applied electrical field. The versatility, speed, and low cost of this method is suited for widespread application in tissue engineering and regenerative medicine.

Protein micropatterned substrates have been used to control cell size, shape, and cell–cell contacts, characteristics that influence a range of cell behaviors from early cell differentiation to late stages of maturation. Knowing the initial island cell seeding density is important to interpreting results and understanding downstream cell behavior. While studies routinely report the intended or target cell seeding density, they do not report the actual cell seeding density on the islands. As cells proliferate, differences in initial cell seeding density could compound and may lead to misinterpretation of results. In this work, we present a cell seeding simulation and apply it to 100s of islands with a range of geometries (sizes and shapes) to explore how island cell seeding density relates to the target or unpatterned cell seeding density. We first experimentally validate the simulation and then show that normalized island cell seeding density depends on island size, shape, and spacing, but can be predicted solely from island area to perimeter ratio, A2P, via a power law relationship for a wide range of island geometries. Interestingly, normalized island cell seeding density is the same as the normalized unpatterned cell seeding density for A2P ≥ 17 µm. This simulation will help to design micropatterned substrates and to have more accurate representation of the island cell seeding density at the start of experiments. By knowing the island cell seeding density, we can more easily reproduce results across research groups to understand the roles of cell–cell contact and cell size and shape on cell behavior. We present a cell seeding simulation on protein-micropatterned substrates and use it to simulate seeding across 100s of island geometries (size, shape, and spacing) covering two orders of magnitude in size. The simulation shows that island cell density varies significantly with island geometry compared to the target seeding density. However, island cell density can be predicted from one geometric parameter – the island's area to perimeter ratio. Results will help direct researchers on how to achieve uniform cell density across all island geometries. Since cell density and island shape both influence cell behaviors, such as differentiation, this simulation may help to isolate these factors, facilitate micropatterned substrate design, and provide a mechanism for more reproduceable results across studies. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Cross-linked albumin films, which are non-cell adhesive, were altered to be cell-adhesive by the combination of varied concentrations of fluorescein isothiocyanate and blue light irradiation. In this system, cell patterning was developed with two different cell lines by sequential seeding. Abbreviations: BSA: bovine serum albumin; EGDE: ethylene glycol diglycidyl ether; PBS: Dulbecco's phosphate buffered saline [ABSTRACT FROM AUTHOR]

Techniques for partitioning cell adhesion are useful tools in biological and medical experiments. However, conventional cell patterning methods require special apparatus, special materials or high‐level skills. Therefore, we have developed a new cell patterning methodology which can be easily carried out in biological laboratories. Non‐cell adhesive material including hydrogel or gas patterns to restrict cell adhesion on a culture dish or glass substrates can be constructed by exploiting a polydimethylsiloxane (PDMS) mold with microchannels. The PDMS molds suck non‐adhesive materials into microchannels from the inlet of the microchannels and the materials are immobilized onto the substrates with a desired pattern. High resolution under a few micrometers and long‐term stability can be realized. This method has been used for analysis of stem cells, muscle cells, neuron development and other cells in collaboration with many biological researchers. Several examples to use this technique are introduced in this review. [ABSTRACT FROM AUTHOR]

Abstract Precise spatial positioning and isolation of mammalian cells is a critical component of many single cell experimental methods and biological engineering applications. Although a variety of cell patterning methods have been demonstrated, many of these methods subject cells to high stress environments, discriminate against certain phenotypes, or are a challenge to implement. Here, we demonstrate a rapid, simple, indiscriminate, and minimally perturbing cell patterning method using a laser fabricated polymer stencil. The stencil fabrication process requires no stencil-substrate alignment, and is readily adaptable to various substrate geometries and experiments.