The multimodality cell segmentation challenge: toward universal solutions.

Cell segmentation is a critical step for quantitative single-cell analysis in microscopy images. Existing cell segmentation methods are often tailored to specific modalities or require manual interventions to specify hyper-parameters in different experimental settings. Here, we present a multimodality cell segmentation benchmark, comprising more than 1,500 labeled images derived from more than 50 diverse biological experiments. The top participants developed a Transformer-based deep-learning algorithm that not only exceeds existing methods but can also be applied to diverse microscopy images across imaging platforms and tissue types without manual parameter adjustments. This benchmark and the improved algorithm offer promising avenues for more accurate and versatile cell analysis in microscopy imaging.

Deepfake Detection Using the Rate of Change between Frames Based on Computer Vision.

Recently, artificial intelligence has been successfully used in fields, such as computer vision, voice, and big data analysis. However, various problems, such as security, privacy, and ethics, also occur owing to the development of artificial intelligence. One such problem are deepfakes. Deepfake is a compound word for deep learning and fake. It refers to a fake video created using artificial intelligence technology or the production process itself. Deepfakes can be exploited for political abuse, pornography, and fake information. This paper proposes a method to determine integrity by analyzing the computer vision features of digital content. The proposed method extracts the rate of change in the computer vision features of adjacent frames and then checks whether the video is manipulated. The test demonstrated the highest detection rate of 97% compared to the existing method or machine learning method. It also maintained the highest detection rate of 96%, even for the test that manipulates the matrix of the image to avoid the convolutional neural network detection method.

Microfluidics for the study of mechanotransduction.

Mechanical forces regulate a diverse set of biological processes at cellular, tissue, and organismal length scales. Investigating the cellular and molecular mechanisms that underlie the conversion of mechanical forces to biological responses is challenged by limitations of traditional animal models and in vitro cell culture, including poor control over applied force and highly artificial cell culture environments. Recent advances in fabrication methods and material processing have enabled the development of microfluidic platforms that provide precise control over the mechanical microenvironment of cultured cells. These devices and systems have proven to be powerful for uncovering and defining mechanisms of mechanotransduction. In this review, we first give an overview of the main mechanotransduction pathways that function at sites of cell adhesion, many of which have been investigated with microfluidics. We then discuss how distinct microfluidic fabrication methods can be harnessed to gain biological insight, with description of both monolithic and replica molding approaches. Finally, we present examples of how microfluidics can be used to apply both solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal, interstitial) to cells. Throughout the review, we emphasize the advantages and disadvantages of different fabrication methods and applications of force in order to provide perspective to investigators looking to apply forces to cells in their own research.

Piezo-Transmissive Structure Using a Multi-layered Heterogeneous Film for Optical Transmittance Modulation.

As the damage caused by the recent climate crisis increases, efforts are being made to develop low-power and high-efficiency technologies to reduce pollution for energy production worldwide. Among them, research on the mechano-responsive optical transmittance modulation technology is being actively conducted as it can be applied to various application fields for reducing energy consumption: low-power sensors and smart windows. The piezo-transmittance structure, which is one of the optical transmittance modulation structures, has fewer constraints on the installation environment; therefore, many applications have been proposed. However, it is still challenging to fabricate a piezo-transmittance structure with a large-area production, high throughput, and good tunability because of complex curing and dissolution processes. Herein, we present an efficient fabrication method for a multi-layered piezo-transmittance structure using a large-area abrasive mold and thermal imprinting process. The piezo-transmittance performance (e.g., sensitivity and relative change of transmittance) shows temperature/humidity-independent characteristics and can be designed by tuning design parameters such as the number of layers, abrasive grade, and film material. Also, the surrogate model of the performance obtained from the Monte Carlo simulation and prediction model can offer tunability for various applications. Finally, we demonstrated two energy-efficient applications: the smart window integrated with a hydraulic pump showed high thermal efficiency in indoor environment control, and the telemetry system was demonstrated to measure pressure remotely.

Multilayer microfluidic platform for the study of luminal, transmural, and interstitial flow.

Efficient delivery of oxygen and nutrients to tissues requires an intricate balance of blood, lymphatic, and interstitial fluid pressures (IFPs), and gradients in fluid pressure drive the flow of blood, lymph, and interstitial fluid through tissues. While specific fluid mechanical stimuli, such as wall shear stress, have been shown to modulate cellular signaling pathways along with gene and protein expression patterns, an understanding of the key signals imparted by flowing fluid and how these signals are integrated across multiple cells and cell types in native tissues is incomplete due to limitations with current assays. Here, we introduce a multi-layer microfluidic platform (MµLTI-Flow) that enables the culture of engineered blood and lymphatic microvessels and independent control of blood, lymphatic, and IFPs. Using optical microscopy methods to measure fluid velocity for applied input pressures, we demonstrate varying rates of interstitial fluid flow as a function of blood, lymphatic, and interstitial pressure, consistent with computational fluid dynamics (CFD) models. The resulting microfluidic and computational platforms will provide for analysis of key fluid mechanical parameters and cellular mechanisms that contribute to diseases in which fluid imbalances play a role in progression, including lymphedema and solid cancer.

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 µm microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

Fabrication of omega-shaped microwell arrays for a spheroid culture platform using pins of a commercial CPU to minimize cell loss and crosstalk.

A cell spheroid culture has the benefit of simulating in vivo three-dimensional cell environments. Microwell systems have been developed to mass-produce large quantities of uniform spheroids, and are frequently used in research areas, such as cell biology, anticancer drug development, and regenerative therapy. Recently reported concave-bottomed microwell systems have delivered more benefits in producing spheroids of higher quality and facilitating more effective research. However, microwell fabrication methods are often complicated or expensive, and there are inherent limitations in the functions and characteristics of existing microwells. Therefore, further studies on concave microwell systems are required. In this study, we fabricate spherical microwells with funnel-shaped entrance structures for spheroid culture; the shape is an upside-down omega ([Formula: see text]), and is thus named 'Omega-well'. The Omega-well array is fabricated using the capillary action of liquid polymer on the pins of a computer central processing unit, which is accomplished without requiring expensive materials or difficult procedures. Various characteristic analyses are performed by experiments and computer simulation. It is demonstrated that cell loss is minimized during cell seeding, a produced spheroid does not easily escape, and that crosstalk between microwells is significantly reduced. The novel fabrication method and Omega-well platform proposed in this study are highly practical, and thus will be useful tools in biology and pharmaceutical labs.

Hypergravity-induced multicellular spheroid generation with different morphological patterns precisely controlled on a centrifugal microfluidic platform.

In living tissue, cells exist in three-dimensional (3D) microenvironments with intricate cell-cell interactions. To model these cellular environments, numerous techniques for generating cell spheroids have been proposed and improved. However, previously reported methods still have limitations in uniformity, reproducibility, scalability, throughput, etc. Here, we present a centrifugal microfluidic-based spheroid (CMS) formation method for generating both co-culture and mono-culture 3D spheroids in a highly controlled manner. We designed circularly arrayed microwells to allow the even distribution of cells introduced at the center of a rotating platform and to provide identical hypergravity conditions at each well by the centrifugal forces generated. Compared with conventional well plate-based spheroid formation, the CMS formation method significantly promotes sphericity and consistency in both size and shape with high production yields. In addition to mono-culture spheroids, we successfully generated co-culture spheroids in concentric, Janus, and sandwich shapes using human adipose-derived stem cells and human lung fibroblasts, demonstrating the versatility of our CMS formation method. We believe that our new method for generating 3D spheroids will become one of the essential technologies in the field of 3D cell culture. We also expect that we are providing an innovative means to assess cellular responses, including cell motility under different hypergravity conditions.

Networked concave microwell arrays for constructing 3D cell spheroids.

The engineered three-dimensional (3D) cell cultivation system for the production of multicellular spheroids has attracted considerable attention due to its improved in vivo relevance to cellular communications compared with the traditional two-dimensional (2D) cell culture platform. The formation and maintenance of cell spheroids in a healthy condition is the critical factor for tissue engineering applications such as the repair of damaged tissues, the development of organ replacement parts and preclinical drug tests. However, culturing spheroids in conventional isolated single wells shows limited yield and reduced maintenance periods due to the lack of proper supplies of nutrition as well as intercellular chemical signaling. Here, we develop novel networked concave microwell arrays for the effective construction of 3D multi-cellular spheroids. The proposed method provides a suitable structure for the diffusion of oxygen, water-soluble nutrients and cytokines for cell-cell interactions between the spheroids in neighboring microwells. We have further demonstrated that hepatocyte spheroid cultured networked concave microwells show enhanced cell viability and albumin secretion compared to the un-networked control group over two weeks. Our results reveal that multi-cellular functionality can be tuned up by networking individual 3D spheroids without supplying additional chemicals or biological supplements. We anticipate our result to be useful in high-throughput cellular screening platforms to study cell-cell interactions, in response to diverse chemical stimuli as well as the development of the in vivo mimicking of the customized 3D tissue culture system.

Magnetic force-assisted self-locking metallic bead array for fabrication of diverse concave microwell geometries.

Spheroid cell culture is very useful for further understanding cellular behavior including motility and biochemical reaction since it mimics three-dimensional (3D) in vivo organ tissue. Among previously proposed various methods for spheroid production, such as hanging drop and spinner flask, microwell is a recently developed method harnessing microtechnology to produce uniform-sized spheroids. Although soft-lithography has been popular for creating microwell arrays, a 3D spherical geometry has been regarded as difficult to fabricate using conventional methods, or often requires complex fabrication processes and expensive equipment. Here, we propose a new method for fabricating concave microwells for cell spheroid production and culture. To demonstrate this method, we fabricated a 30 × 30 microwell array in 3 × 3 cm plates, utilizing metal beads, a through-hole array, and an assembly of small magnets. The spherical metal beads were used as a mold for the microwell, naturally creating the desired 3D concave microwell geometry. One of the key ideas was to place and hold each metal bead in the designated through-hole using the small magnet array. We also performed computational simulation of the magnetostatic force to design and observe the magnetic force field in detail. In addition, to provide a practical demonstration of the proposed system in cell biology, we created and cultured adipose-derived stem cell spheroids for 14 days for chondrogenic differentiation. This method allows further variations in microwell geometry that will enhance the method's applicability as a helpful tool for various studies in cell biology, cancer research, and tissue engineering.

Features of Microsystems for Cultivation and Characterization of Stem Cells with the Aim of Regenerative Therapy.

Stem cells have infinite potential for regenerative therapy thanks to their advantageous ability which is differentiable to requisite cell types for recovery and self-renewal. The microsystem has been proved to be more helpful to stem cell studies compared to the traditional methods, relying on its advantageous feature of mimicking in vivo cellular environments as well as other profitable features such as minimum sample consumption for analysis and multiprocedures. A wide variety of microsystems were developed for stem cell studies; however, regenerative therapy-targeted applications of microtechnology should be more emphasized and gain more attractions since the regenerative therapy is one of ultimate goals of biologists and bioengineers. In this review, we introduce stem cell researches harnessing well-known microtechniques (microwell, micropattern, and microfluidic channel) in view point of physical principles and how these systems and principles have been implemented appropriately for characterizing stem cells and finding possible regenerative therapies. Biologists may gain information on the principles of microsystems to apply them to find solutions for their current challenges, and engineers may understand limitations of the conventional microsystems and find new chances for further developing practical microsystems. Through the well combination of engineers and biologists, the regenerative therapy-targeted stem cell researches harnessing microtechnology will find better suitable treatments for human disorders.

Microplatforms for gradient field generation of various properties and biological applications.

Well-designed microfluidic platforms can be excellent tools to eliminate bottleneck problems or issues that have arisen in biological fields by providing unprecedented high-resolution control of mechanical and chemical microenvironments for cell culture. Among such microtechnologies, the precise generation of biochemical concentration gradients has been highly regarded in the biorelated scientific fields; even today, the principles and mechanisms for gradient generation continue to be refined, and the number of applications for this technique is growing. Here, we review the current status of the concentration gradient generation technologies achieved in various microplatforms and how they have been and will be applied to biological issues, particularly those that have arisen from cancer research, stem cell research, and tissue engineering. We also provide information about the advances and future challenges in the technological aspects of microscale concentration gradient generation.