Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guérin.

Immunotherapy of bladder cancer is known to have favorable effects, although it is difficult to determine which patients will show a good response because of the different tumor microenvironments (TME). Here, we developed a bladder cancer-on-a-chip (BCOC) to mimic the TME using three-dimensional (3D) bioprinting and microfluidic technology. We fabricated a T24 and a 5637-cell line-based BCOC that also incorporated MRC-5, HUVEC, and THP-1 cells. We evaluated the effects of TME and assessed the immunologic reactions in response to different concentrations of Bacillus Calmette-Guérin (BCG) via live/dead assay and THP-1 monocytic migration, and concentrations of growth factors and cytokines. The results show that cell viability was maintained at 15% filling density in circle-shaped cell constructs at 20 µL/min microfluidic flow rate. A 3D co-culture increased the proliferation of BCOCs. We found that the appropriate time to evaluate the viability of BCOC, concentration of cytokines, and migration of monocytes was 6 h, 24 h, and three days after BGC treatment. Lastly, the immunotherapeutic effects of BCOC increased according to BCG dosage. To predict effects of immunotherapeutic agent in bladder cancer, we constructed a 3D bioprinted BCOC model. The BCOC was validated with BCG, which has been proven to be effective in the immunotherapy of bladder cancer.

Fluid dynamic simulation for cellular damage due to lymphatic flow within the anatomical arrangement of the outer hair cells in the cochlea.

Damage to the sensory hair cells in the cochlea is a major cause of hearing loss since human sensory hair cells do not regenerate naturally after damage. As these sensory hair cells are exposed to a vibrating lymphatic environment, they may be affected by physical flow. It is known that the outer hair cells (OHCs) are physically more damaged by sound than the inner hair cells (IHCs). In this study, the lymphatic flow is compared using computational fluid dynamics (CFD) based on the arrangement of the OHCs, and the effects of such flow on the OHCs is analyzed. In addition, flow visualization is used to validate the Stokes flow. The Stokes flow behavior is attributed to the low Reynolds number, and the same behavior is observed even when the flow direction is reversed. When the distance between the rows of the OHCs is large, each row is independent, but when this distance is short, the flow change in each row influences the other rows. The stimulation caused by flow changes on the OHCs is confirmed through surface pressure and shear stress. The OHCs located at the base with a short distance between the rows receive excess hydrodynamic stimulation, and the tip of the V-shaped pattern receives an excess mechanical force. This study attempts to understand the contributions of lymphatic flow to OHC damage by quantitatively suggesting stimulation of the OHCs and is expected to contribute to the development of OHC regeneration technologies in the future.

Biological and mechanical influence of three-dimensional microenvironment formed in microwell on multicellular spheroids composed of heterogeneous hair follicle stem cells.

Hair loss caused by malfunction of the hair follicle stem cells (HFSCs) and physical damage to the skin is difficult to recover from naturally. To overcome these obstacles to hair follicle (HF) regeneration, it is essential to understand the three-dimensional (3D) microenvironment and interactions of various cells within the HFs. Therefore, 3D cell culture technology has been used in HF regeneration research; specifically, multicellular spheroids have been generally adapted to mimic the 3D volumetric structure of the HF. In this study, we culture HF-derived cells, which are mainly composed of HFSCs, in the form of 3D spheroids using a microwell array and discuss the effects of the 3D cellular environment on HF morphogenesis by expression measurements of Sonic hedgehog signaling and stem cell markers in the HF spheroids. Additionally, the influences of microwell depth on HF spheroid formation and biological conditions were investigated. The biomolecular diffusion and convective flow in the microwell were predicted using computational fluid dynamics, which allows analysis of the physical stimulations occurring on the spheroid at the micro-scale. Although a simple experimental method using the microwell array was adopted in this study, the results provide fundamental insights into the physiological phenomena of HFs in the 3D microenvironment, and the numerical analysis is expected to shed light on the investigation of the geometric parameters of the microwell system.

Customized small-sized clinostat using 3D printing and gas-permeable polydimethylsiloxane culture dish.

Over the past few decades, research on life in space has increased. Owing to the expensive nature of and the challenges associated with conducting experiments in real space, clinostats, which continuously randomize the gravity vector by using motors, have been used to generate simulated microgravity (SMG) on Earth. Herein, by using a 3D printing method, we develop a customized small-sized clinostat (CS clinostat) that is easy to manufacture, inexpensive, and robust. Moreover, we develop and fabricate a gas-permeable polydimethylsiloxane culture dish that fits inside the CS clinostat. To validate SMG generation, ovarian cancer cells (OV- 90, TOV-21G, and Caov-3) were applied to demonstrate a significant reduction in caveolin-1 expression, a biomarker of SMG, indicating SMG generation. The proposed CS clinostat system has good accessibility for SMG research, which makes it useful as a tool for biologists, who are unfamiliar with conventional clinostat equipment, to conduct preliminary studies in the space environment.

Fluid dynamic design for mitigating undesired cell effects and its application to testis cell response testing to endocrine disruptors.

Microfluidic devices have emerged as powerful tools for cell-based experiments, offering a controlled microenvironment that mimic the conditions within the body. Numerous cell experiment studies have successfully utilized microfluidic channels to achieve various new scientific discoveries. However, it has been often overlooked that undesired and unnoticed propagation of cellular molecules in such bio-microfluidic channel systems can have a negative impact on the experimental results. Thus, more careful designing is required to minimize such unwanted issues through deeper understanding and careful control of chemically and physically predominant factors at the microscopic scale. In this paper, we introduce a new approach to improve microfluidic channel design, specifically targeting the mitigation of the aforementioned challenges. To minimize the occurrence of undesired cell positioning upstream from the main test section where a concentration gradient field locates, an additional narrow port structure was devised between the microfluidic upstream channel and each inlet reservoir. This port also functioned as a passive lock that hold the flow at rest via fluid-air surface tension, which facilitated manual movement of the device even when cell attachment was not achieved completely. To demonstrate the practicability of the system, we conducted experiments and diffusion simulations on the effect of endocrine disruptors on germ cells. To this end, a bisphenol-A (BPA) concentration gradient was generated in the main channel of the system at BPA concentrations ranging from 120.8 µM to 79.3 µM, and the proliferation of GC-1 cells in the BPA gradient environment was quantitatively evaluated. The features and concepts of the introduced design is to minimize unexpected and ignored error sources, which will be one of the issues to be considered in the development of microfluidic systems to explore extremely delicate cellular phenomena.

Microfluidic automation using elastomeric valves and droplets: reducing reliance on external controllers.

This paper gives an overview of elastomeric valve- and droplet-based microfluidic systems designed to minimize the need of external pressure to control fluid flow. This Concept article introduces the working principle of representative components in these devices along with relevant biochemical applications. This is followed by providing a perspective on the roles of different microfluidic valves and systems through comparison of their similarities and differences with transistors (valves) and systems in microelectronics. Despite some physical limitation of drawing analogies from electronic circuits, automated microfluidic circuit design can gain insights from electronic circuits to minimize external control units, while implementing high-complexity and high-throughput analysis.

Proliferative effects of nanobubbles on fibroblasts.

In recent years, the potential of nanobubbles (NBs) for biological activation has been actively investigated. In this study, we investigated the proliferative effects of nitrogen NBs (N-NBs) on fibroblast cells using cell assays with image analysis and flow cytometry. A high concentration of N-NBs (more than 4 × 108 NBs/mL) was generated in Dulbecco's modified Eagle's medium (DMEM) using a gas-liquid mixing method. In image analysis, the cells were counted and compared, which showed an 11% increase in cell number in the culture medium with N-NBs. However, in two further cell cytometry analyses, the effect of nanobubbles on cell division was found to be insignificant (approximately 2%); as there is insufficient evidence that N-NB is involved in cell division mechanism, further studies are needed to determine whether NB affects other cellular mechanisms such as apoptosis. This study presents the first successful attempt of directly generating and quantifying N-NBs in a culture medium for cell culture. The findings suggest that the N-NBs in the culture medium can potentially facilitate cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-022-00242-y.

DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the ß-Catenin-P-Glycoprotein Signaling Pathway.

Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan-Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while ß-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active ß-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

Novel microwell with a roof capable of buoyant spheroid culture.

Microwells are used in studies to mimic the in vivo environment through an in vitro environment by generating three-dimensional cell spheroids. These microwells have been fabricated in various shapes using different methods according to the research purpose. However, because all microwells up to now have an open top, it has been difficult to culture spheroids of floating cells due to their low density, such as human adipose-derived stem cells (hASCs) that differentiate into adipocytes. Therefore, the labor-intensive hanging droplet method has been mainly used for the study of adipocytes. Here, we introduce a sigma-well, which is a microwell in the shape of the Greek letter sigma (σ) with a roof. Because of its unique shape, the sigma-well is advantageous for the culture of floating cells by reducing cell loss and external interference. The sigma-well was fabricated using the principle of surface tension of polydimethylsiloxane as well as air trapping and thermal expansion. Unlike conventional microwells, because the center of the bottom surface and the inlet of the sigma-well are not located on the same line and have a difference of approximately 218 µm, the spheroids are cultured more stably and may not escape the cavity. In this study, hASC and adipocyte spheroids differentiated using these sigma-wells were successfully cultured. In addition, through cytokine diffusion simulation, it was confirmed that the diffusion and mass transfer in the sigma-well was lower than that in the conventional microwell. It is expected that the morphological features of the sigma-well, which cannot be easily obtained by other methods, can be beneficial for the study of buoyant cell types such as adipocytes.

Engineered Microsystems for Spheroid and Organoid Studies.

3D in vitro model systems such as spheroids and organoids provide an opportunity to extend the physiological understanding using recapitulated tissues that mimic physiological characteristics of in vivo microenvironments. Unlike 2D systems, 3D in vitro systems can bridge the gap between inadequate 2D cultures and the in vivo environments, providing novel insights on complex physiological mechanisms at various scales of organization, ranging from the cellular, tissue-, to organ-levels. To satisfy the ever-increasing need for highly complex and sophisticated systems, many 3D in vitro models with advanced microengineering techniques have been developed to answer diverse physiological questions. This review summarizes recent advances in engineered microsystems for the development of 3D in vitro model systems. The relationship between the underlying physics behind the microengineering techniques, and their ability to recapitulate distinct 3D cellular structures and functions of diverse types of tissues and organs are highlighted and discussed in detail. A number of 3D in vitro models and their engineering principles are also introduced. Finally, current limitations are summarized, and perspectives for future directions in guiding the development of 3D in vitro model systems using microengineering techniques are provided.

Coarsening behavior of bulk nanobubbles in water.

In recent years, minuscule gas bubbles called bulk nanobubbles (BNBs) have drawn increasing attention due to their unique properties and broad applicability in various technological fields, such as biomedical engineering, water treatment, and nanomaterials. However, questions remain regarding the stability and behavior of BNBs. In the present work, BNBs were generated in water using a gas-liquid mixing method. NB analysis was performed using a nanoparticle tracking analysis (NTA) method to investigate the coarsening behavior of BNBs in water over time. The diameters of the BNBs increased, and their cubic radii increased linearly (r3 ~ t) over time. While the concentration of BNBs decreased, the total volume of BNBs remained the same. The size distribution of the BNBs broadened, and the concentration of larger BNBs increased over time. These results indicate that relatively small BNBs disappeared due to dissolution and larger BNBs grew through mass transfer between BNBs instead of coalescence. In other words, BNBs underwent Ostwald ripening: gas molecules from smaller BNBs diffused through the continuous phase to be absorbed into larger BNBs.

Electrically-driven hydrogel actuators in microfluidic channels: fabrication, characterization, and biological application.

The utility of electro-responsive smart materials has been limited by bubble generation (hydrolysis) during application of electrical fields and by biocompatibility issues. Here we describe the design of a device that overcomes these limitations by combining material properties, new design concepts, and microtechnology. 4-hydroxybutyl acrylate (4-HBA) was used as a backbone hydrogel material, and its actuating behavior, bending force, and elasticity were extensively characterized as a function of size and acrylic acid concentration. To prevent bubble generation, the system was designed such that the hydrogel actuator could be operated at low driving voltages (<1.2 V). A microfluidic channel with an integrated electroactive hydrogel actuator was developed for sorting particles. This device could be operated in cell culture media, and the sorting capabilities were initially assessed by sorting droplets in an oil droplet emulsion. Biocompatibility was subsequently tested by sorting mouse embryoid bodies (mEBs) according to size. The sorted and collected mEBs maintained pluripotency, and selected mEBs successfully differentiated into three germ layers: endoderm, mesoderm, and ectoderm. The electroactive hydrogel device, integrated into a microfluidic system, successfully demonstrated the practical application of smart materials for use in cell biology.

Differentiation of neural progenitor cells in a microfluidic chip-generated cytokine gradient.

In early embryonic development, spatial gradients of diffusible signaling molecules play important roles in controlling differentiation of cell types or arrays in diverse tissues. Thus, the concentration of exogenous cytokines or growth factors at any given time is crucial to the formation of an enriched population of a desired cell type from primitive stem cells in vitro. Microfluidic technology has proven very useful in the creation of cell-friendly microenvironments. Such techniques are, however, currently limited to a few cell types. Improved versatility is required if these systems are to become practically applicable to stem cells showing various plasticity ranges. Here, we built a microfluidic platform in which cells can be exposed to stable concentration gradients of various signaling molecules for more than a week with only minimal handling and no external power source. To maintain stability of the gradient concentration, the osmotic pumping performance was optimized by balancing the capillary action and hydraulic pressure in the inlet reagent reservoirs. We cultured an enriched population of neural progenitors derived from human embryonic stem cells in our microfluidic chamber for 8 days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor 8, and bone morphogenetic protein 4). Neural progenitors successfully differentiated into neurons, generating a complex neural network. The average numbers of both neuronal cell body clusters and neurite bundles were directly proportional to sonic hedgehog concentrations in the gradient chip. The system was shown to be useful for both basic and translational research, with straightforward mechanisms and operational schemes.

Fabrication of three-dimensional microarray structures by controlling the thickness and elasticity of poly(dimethylsiloxane) membrane.

In this paper, we propose a method to construct three-dimensional curved microstructures with easy control of the size, position and shape, by exploiting the elasticity of poly(dimethylsiloxane) (PDMS) membranes and basic physics. For this end, we developed the method to handle thin PDMS membrane safely, and to replicate PDMS microstructure from the PDMS mold. Using this method, we demonstrated two potential applications: (1) the use of concave well for the formation of embryoid body (EB) to differentiate into neuronal cells, and (2) the fabrication of SU-8 and hydrogel microparticles having diverse curved shapes. The curved structures were successfully fabricated with simple process, and EBs were formed in the concave well and differentiated into the neuronal cells. Microparticles with diverse shapes were fabricated from a range of materials for potential use as drug carrier and pH responsive micro-actuator elements.

Membrane-bottomed microwell array added to Transwell insert to facilitate non-contact co-culture of spermatogonial stem cell and STO feeder cell.

In vivo cells express their characteristics in three-dimensional (3D) microenvironments via cell-cell interactions through autocrine, contact-dependent, paracrine, and synaptic signaling, often between heterologous cell types. Various in vitro 3D microwell-based culture methods have been proposed to further identify cellular characteristics by recreating cellular environments, typically in the form of spheroids and organoids, thereby realizing contact-based cell-cell interactions. However, in vivo cells generally exhibit multiple cellular interaction modes that have not been completely evaluated using existing microwell-based methods. This has led to a demand for more advanced and comprehensive methods. This study introduces a novel apparatus, the membrane-bottomed microwell (MBM) for non-contact co-cultures and 3D cell cultures. The MBM is a combination of a Transwell and a microwell array; these have previously been utilized to facilitate heterologous cell co-culturing and spheroid 3D cell culturing, respectively. In the Transwell insert, the lower part of the MBM is immersed in the culture media in which the cells are being two-dimensionally (2D) cultured, and the spheroids of the MBM are affected by the 2D cultured cells via the membrane at the bottom of the microwell. Here, we describe the methods for manufacturing the MBM in detail and elucidate the results of simulations of diffusion through the bottom of the membrane. We validate the proposed MBM for the spheroid culture of spermatogonial stem cells (SSCs), which had previously been 2D co-cultured with Sandos inbred mouse (SIM)-derived 6-thioguanine- and ouabain-resistant (STO; a mouse embryonic feeder cell line) feeder cells. The proposed system is shown to facilitate successful SSC spheroid culturing with paracrine signaling of STOs through an apparatus that simplifies both the loading and the evaluation processes; therefore, we believe that our findings will enable a more comprehensive understanding of SSCs and associated phenomena and that our system can be applied to various in vitro cell and tissue experiments.

Simultaneous generation of chemical concentration and mechanical shear stress gradients using microfluidic osmotic flow comparable to interstitial flow.

Cells are very sensitive to various microenvironmental cues, including mechanical stress and chemical gradients. Therefore, physiologically relevant models of cells should consider how cells sense and respond to microenvironmental cues. This can be accomplished by using microfluidic systems, in which fluid physics can be realized at a nanoliter scale. Here we describe a simple and versatile method to study the generation of chemical concentration and mechanical shear stress gradients in a single microfluidic chip. Our system uses an osmotic pump that produces very slow (

Study of cellular behaviors on concave and convex microstructures fabricated from elastic PDMS membranes.

Cells respond to geometrical cues, as well as to biochemical and mechanical stimuli. Recent progress in micro- and nano-technology has allowed researchers to create microbeads, micro-circular islands, and microposts, that can be used to examine the effect of geometrical cues on cellular behavior. Knowledge of changes in cell mechanics and morphology in response to geometric cues is important for understanding the basic behavior of cells during development and pathological processes. Most previous research in this area has focused on cell responses to two-dimensional planar or rectilinear structures. Very few studies have examined cell responses to three-dimensional curved structures because of the difficulty of fabricating such microstructures. Here we describe a novel method for the fabrication of convex and concave microstructures by use of a thin poly(dimethylsiloxane) (PDMS) membrane, SU-8 shadow mask, and negative air pressure without using any complicated silicon processes. We successfully fabricated concave and convex microstructures, with base diameters of 200-300 microm and depth (or height) of 50-150 microm (aspect ratios up to 1 : 0.5), and used these microstructures to study the responses of cultured L929 mouse fibroblast cells and human mesenchymal stem cells. These cells clearly sensed the three-dimensional microscale curvature and actively "escaped" from concave patterns, but not from those which were convex. Thus, it appears that microscale concave structures suppress cell adhesion and proliferation. We hypothesized that this might relate to deformation of the plasma membrane and subsequent opening of membrane channels. We anticipate that our system will be useful for various bio-MEMS (micro electro mechanical system) applications, including formation of uniformly-sized embryoid bodies, embryonic stem cell differentiation, and the fabrication of cell docking devices, microbioreactors, and microlenses as well as cell mechanics study.

DNA-enrichment microfluidic chip for chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency.

Ice-lithographic fabrication of concave microwells and a microfluidic network.

We describe a novel method to produce concave microwells utilizing solid-liquid phase change. This method, named 'ice-lithography', does not require any lithographic processes and consists of a few simple steps that yield multiple concave microwells. We demonstrated that the shape and size of the microwells can be controlled by varying substrates and vapor-collection time. Patterned wells with sizes in the range of 10 microm to several millimeters in diameter could be produced. Additionally, we fabricated a uniformly aligned concave microwell pattern and a microfluidic network. Ice-lithography has potential biological and biomedical applications in areas such as the fabrication of cell docking devices and microbioreactors as well as the formation of uniformly sized embryoid bodies.

Quantitative analysis of pulsatile flow contribution to ultrafiltration.

We evaluated the quantitative contribution of pulsatile flow to ultrafiltration (UF) in terms of fluid power, membrane stretch, and reduction of membrane layering. An in vitro comparison of the UF rate using pulsatile and roller pumps was performed with distilled water and bovine whole blood. The mean transmembrane pressure (TMPm) and UF rate were higher with the pulsatile pump for the same mean flow rate: 6.6 mm Hg and 21.1 mL/min higher on average for distilled water and 34.2 mm Hg and 31.4 mL/min higher on average for blood. The average UF rate was 8.4 mL/min higher with the pulsatile pump for the same TMPm with bovine blood. However, the relationship between the UF rate and the TMPm was independent of the flow configuration for distilled water. We showed that the higher UF rate in the pulsatile pump is mainly due to greater fluid power and reduction of membrane layering, while the membrane stretch was not an important factor.