helixCAM: A platform for programmable cellular assembly in bacteria and human cells.

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. We applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases for the Sexual Dimorphism Development in the Liverwort Marchantia polymorpha.

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 MpFGMYB. 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.

Massively Parallel High-Throughput Single-Cell Patterning and Large Biomolecular Delivery in Mammalian Cells Using Light Pulses.

The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100 µm with 1 cm × 1 cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100 µm micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668 Da), dextran 3000 Da, siRNA (20-24 bp), and large size enzymes (464 KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are ≈95 ± 3%, 97 ± 1%, 96 ± 1% and 94 ± 3%, with cell viability of 98 ± 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.

A digitally driven manufacturing process for high resolution patterning of cell formations.

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.

Actin-ring segment switching drives nonadhesive gap closure.

Gap closure to eliminate physical discontinuities and restore tissue integrity is a fundamental process in normal development and repair of damaged tissues and organs. Here, we demonstrate a nonadhesive gap closure model in which collective cell migration, large-scale actin-network fusion, and purse-string contraction orchestrate to restore the gap. Proliferative pressure drives migrating cells to attach onto the gap front at which a pluricellular actin ring is already assembled. An actin-ring segment switching process then occurs by fusion of actin fibers from the newly attached cells into the actin cable and defusion from the previously lined cells, thereby narrowing the gap. Such actin-cable segment switching occurs favorably at high curvature edges of the gap, yielding size-dependent gap closure. Cellular force microscopies evidence that a persistent rise in the radial component of inward traction force signifies successful actin-cable segment switching. A kinetic model that integrates cell proliferation, actin fiber fusion, and purse-string contraction is formulated to quantitatively account for the gap-closure dynamics. Our data reveal a previously unexplored mechanism in which cells exploit multifaceted strategies in a highly cooperative manner to close nonadhesive gaps.

Calcium response of spatially arranged cell networks to shear stress by confined single cell patterned microfluidic chips.

Osteoblasts in multicellular organisms are sensitive to fluid shear stress (Fss) and respond smartly with versatile patterns of intracellular calcium signal ([Ca2+]i). In this study, a spatial-single cell patterning method was developed by combining micro-contact printing (µCP) and reversible microfluidic chip mounted with vacuum together. Based on this well-defined patterning platform, it's possible to investigate calcium response to Fss modulated by spatial factors, and to characterize multiple calcium patterns quantitatively in terms of cell spacing and cell orientation. The result showed that the Fss-induced [Ca2+]i profiles revealed oscillational signal patterns in non-connected cells such as those in physical-contacted cells. Close-arrayed osteoblasts showed remarkably more [Ca2+]i oscillations than sparse-arrayed cells. The circular shape of the cells was sensitive to oscillational [Ca2+]i as a potential major cause. The consistency of cell orientation and shear stress promoted temporal homogeneity of calcium oscillations.

Programmable Electrodeposition of Janus Alginate/Poly-L-Lysine/Alginate (APA) Microcapsules for High-Resolution Cell Patterning and Compartmentalization.

Encapsulation of live cells in protective, semipermeable microcapsules is one of the kernel techniques for in vitro tissue regeneration, cell therapies, and pharmaceutical screening. Advanced fabrication techniques for cell encapsulation have been developed to meet different requirements. Existing cell encapsulation techniques place substantial constraints on the spatial patterning of live cells as well as on the compartmentalization of heterotypic cells. Alginate-Poly-L-lysine-alginate (APA) microcapsules that use sodium alginate as the polyanion and poly-L-lysine (PLL) as the polycation have been extensively employed for cell microencapsulation due to their excellent biocompatibility and biodegradability. This study proposes a novel method for developing programmable Janus APA microcapsules with variable shapes and sizes by using electrodeposition. By the versatile design of the microelectrode device, sequential electrodeposition is triggered to electro-address the cells at specific locations immobilized within a Janus APA microcapsule. The osteogenesis is evaluated by resembling cell compartmentalized and vascularized osteoblast-laden constructs. This technique allows precise spatial patterning of heterotypic cells inside the APA microcapsule, enabling the observation of cellular growth, interactions, and differentiation in a well-controlled chemical and mechanical microenvironment.

Extremely Uniform Graphene Oxide Thin Film as a Universal Platform for One-Step Biomaterial Patterning.

Graphene oxide (GO) has proven to be a highly promising material across various biomedical research avenues, including cancer therapy and stem cell-based regenerative medicine. However, creating a uniform GO coating as a thin layer on desired substrates has been considered challenging owing to the intrinsic variability of the size and shape of GO. Herein, a new method is introduced that enables highly uniform GO thin film (UGTF) fabrication on various biocompatible substrates. By optimizing the composition of the GO suspension and preheating process to the substrates, the "coffee-ring effect" is significantly suppressed. After applying a special postsmoothing process referred to as the low-oxygen concentration and low electrical energy plasma (LOLP) treatment, GO is converted to small fragments with a film thickness of up to several nanometers (≈5.1 nm) and a height variation of only 0.6 nm, based on atomic force microscopy images. The uniform GO thin film can also be generated as periodic micropatterns on glass and polymer substrates, which are effective in one-step micropatterning of both antibodies and mouse melanoma cells (B16-F10). Therefore, it can be concluded that the developed UGTF is useful for various graphene-based biological applications.

Continuous monitoring of cell transfection efficiency with micropatterned substrates.

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.

User-friendly cell patterning methods using a polydimethylsiloxane mold with microchannels.

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.

Fluorescein isothiocyanate and blue light irradiation alter cell-adhesiveness of cross-linked albumin films for cell patterning.

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.

Click Decoration of Bombyx mori Silk Fibroin for Cell Adhesion Control.

Silk fibroin produced by the domesticated silkworm, Bombyx mori, has been studied widely as a substrate for tissue engineering applications because of its mechanical robustness and biocompatibility. However, it is often difficult to precisely tune silk fibroin's biological properties due to the lack of easy, reliable, and versatile methodologies for decorating it with functional molecules such as those of drugs, polymers, peptides, and enzymes necessary for specific applications. In this study we applied an azido-functionalized silk fibroin, AzidoSilk, produced by a state-of-the-art biotechnology, genetic code expansion, to produce silk fibroin decorated with cell-repellent polyethylene glycol (PEG) chains for controlling the cell adhesion property of silk fibroin film. Azido groups can act as selective handles for chemical reactions such as a strain-promoted azido-alkyne cycloaddition (SPAAC), known as a click chemistry reaction. We found that azido groups in AzidoSilk film were selectively decorated with PEG chains using SPAAC. The PEG-decorated film demonstrated decreased cell adhesion depending on the lengths of the PEG chains. Azido groups in AzidoSilk can be decomposed by UV irradiation. By partially decomposing azido groups in AzidoSilk film in a spatially controlled manner using photomasks, cells could be spatially arranged on the film. These results indicated that SPAAC could be an easy, reliable, and versatile methodology to produce silk fibroin substrates having adequate biological properties.

TWI1 regulates cell-to-cell movement of OSH15 to control leaf cell fate.

Cell pattern formation in plant leaves has attracted much attention from both plant biologists and breeders. However, in rice, the molecular mechanism remains unclear. Here, we describe the isolation and functional characterization of TWISTED-LEAF1 (TWI1), a critical gene involved in the development of the mestome sheath, vascular bundle sheath, interveinal mesophyll and sclerenchyma in rice leaves. Mutant twi1 plants have twisted leaves which might be caused by the compromised development and disordered patterning of bundle sheath, sclerenchyma and interveinal mesophyll cells. Expression of TWI1 can functionally rescue these mutant phenotypes. TWI1 encodes a transcription factor binding protein that interacts with OSH15, a class I KNOTTED1-like homeobox (KNOX) transcription factor. The cell-to-cell trafficking of OSH15 is restricted through its interaction with TWI1. Knockout or knockdown of OSH15 in twi1 rescues the twisted leaf phenotype. These studies reveal a key factor controlling cell pattern formation in rice leaves.

The nuclear hormone receptor gene Nr2c1 (Tr2) is a critical regulator of early retina cell patterning.

Nuclear hormone receptors play a major role in the development of many tissues. This study uncovers a novel role for testicular receptor 2 (Tr2, Nr2c1) in defining the early phase of retinal development and regulating normal retinal cell patterning and topography. The mammalian retina undergoes an overlapping yet biphasic period of development to generate all seven retinal cell types. We discovered that Nr2c1 expression coincides with development of the early retinal cells. Loss of Nr2c1 causes a severe vision deficit and impacts early, but not late retina cell types. Retinal cone cell topography is disrupted with an increase in displaced amacrine cells. Additionally, genetic background significantly impacts phenotypic outcome of cone photoreceptor cells but not amacrine cells. Chromatin-IP experiments reveal NR2C1 regulates early cell transcription factors that regulate retinal progenitor cells during development, including amacrine (Satb2) and cone photoreceptor regulators thyroid and retinoic acid receptors. This study supports a role for Nr2c1 in defining the biphasic period of retinal development and specifically influencing the early phase of retinal cell fate.

microRNA-31 modulates skeletal patterning in the sea urchin embryo.

MicroRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. microRNA-31 (miR-31) is known to play a role in cancer, bone formation and lymphatic development. However, studies to understand the function of miR-31 in embryogenesis have been limited. We examined the regulatory role of miR-31 in early development using the sea urchin as a model. miR-31 is expressed at all stages of development and its knockdown (KD) disrupts the patterning and function of primary mesenchyme cells (PMCs), which form the embryonic skeleton spicules. We identified that miR-31 directly represses Pmar1, Alx1, Snail and VegfR7 within the PMC gene regulatory network using reporter constructs. Further, blocking the miR-31-mediated repression of Alx1 and/or VegfR7 in the developing embryo resulted in defects in PMC patterning and skeletogenesis. The majority of the mislocalized PMCs in miR-31 KD embryos did not express VegfR10, indicating that miR-31 regulates VegfR gene expression within PMCs. In addition, miR-31 indirectly suppresses Vegf3 expression in the ectoderm. These results indicate that miR-31 coordinately suppresses genes within the PMCs and in the ectoderm to impact PMC patterning and skeletogenesis. This study identifies the novel function and molecular mechanism of miR-31-mediated regulation in the developing embryo.

Microstructured Photo-Crosslinked Poly(Trimethylene Carbonate) for Use in Soft Lithography Applications: A Biodegradable Alternative for Poly(Dimethylsiloxane).

Photo-crosslinkable poly(trimethylene carbonate) (PTMC) macromers were used to fabricate microstructured surfaces. Microstructured PTMC surfaces were obtained by hot embossing the macromer against structured silicon masters and subsequent photo-crosslinking, resulting in network formation. The microstructures of the master could be precisely replicated, limiting the shrinkage. Microstructured PTMC was investigated for use in two different applications: as stamping material to transfer a model protein to another surface and as structured substrate for cell culture. Using the flexible and elastic materials as stamps, bovine serum albumin labelled with fluorescein isothiocyanate was patterned on glass surfaces. In cell culture experiments, the behavior of human mesenchymal stem cells on nonstructured and microstructured PTMC surfaces was investigated. The cells strongly adhered to the PTMC surfaces and proliferated well. Compared to poly(dimethylsiloxane) (PDMS), which is commonly used in soft lithography, the PTMC networks offer significant advantages. They show better compatibility with cells, are biodegradable, and have much better mechanical properties. Both materials are transparent, flexible, and elastic at room temperature, but the tear resistance of PTMC networks is much higher than that of PDMS. Thus, PTMC might be an alternative material to PDMS in the fields of biology, medicine, and tissue engineering, in which microfabricated devices are increasingly being applied.

Time Sequential Single-Cell Patterning with High Efficiency and High Density.

Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm², which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.

CRR1 encoding callose synthase functions in ovary expansion by affecting vascular cell patterning in rice.

The ovary of rice undergoes rapid expansion immediately after fertilization, and this process determines the final sink strength potential of caryopses. To date, work on rice grain development has mainly focused on endosperm filling, whereas information on the essential elements for ovary expansion remains limited. We report here a functional analysis of the ovary expansion retarded mutant crr1 in rice. Map-based cloning revealed that CRR1 encodes a protein homologous to the Arabidopsis callose synthases AtGSL8 and AtGSL10. Point mutation in crr1 resulted in alternative splicing, which led to the formation of the truncated crr1 protein without the ß-glucan synthase domain. Iodine staining showed that there were few starch granules and these were unevenly distributed in the pericarp of crr1, and a 5,6-carboxyfluorescein diacetate transport assay revealed that carbohydrates were less efficiently unloaded from the lateral vasculature into the developing caryopsis. CRR1 transcripts were detected in all plant organs, with the highest level found in receptacles, which are mainly composed of vascular tissues. Analysis of pCRR1::GUS transgenic plants showed that CRR1 was specifically expressed in vascular bundle cells. Consistently, loss of function of CRR1 led to disordered patterns of vascular cells in the ovaries and receptacles of the mutant. Furthermore, a small portion of cells in the vascular bundles of crr1 showed defective cell wall formation, and callose deposition was specifically reduced at the plasmodesmata (PD) of cells with aberrant walls. Our results suggest that CRR1 performs a pivotal role in determining initial ovary expansion in rice, possibly via the PD-mediated permeability of cell fate determinants for vascular cell differentiation.

Laser-fabricated cell patterning stencil for single cell analysis.

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.

Engineered bone scaffolds with Dielectrophoresis-based patterning using 3D printing.

Patterning of cells into a specific pattern is an important procedure in tissue engineering to facilitate tissue culture and ingrowth. In this paper, a new type of 3D-printed scaffold utilizing dielectrophoresis (DEP) for active cell seeding and patterning was proposed. This scaffold adopted a concentric-ring design that is similar to native bone tissues. The scaffold was fabricated with a commercial three-dimensional (3D) printer. Polylactic Acid (PLA) was selected as the material for the printer and the fabricated scaffold was coated with gold to enhance the conductivity for DEP manipulation. Simulation from COMSOL confirmed that non-uniform electric fields were successfully generated under a voltage input. The properties of the scaffold were first characterized through a series of experiments. Then, preosteoblast MC3T3-E1 cells were seeded onto the coated scaffold and multiple cellular rings were observed under the microscope. The biocompatibility of the material was also examined and mineralized bone nodules were detected using Alizarin Red S Staining after 28 days of culture. The proposed scaffold design can enable formation of multiple ring patterns via DEP and the properties of the scaffold are suitable for bone tissue culture. This new type of 3D-printed scaffold with cell seeding mechanism offers a new and rapid approach for fabricating engineered scaffolds that can arrange cells into different patterns for various tissue engineering applications.