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.

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.

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 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.

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.

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.

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.

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.

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.

Three-dimensional cartilage tissue regeneration system harnessing goblet-shaped microwells containing biocompatible hydrogel.

Differentiation of stem cells into chondrocytes has been studied for the engineering of cartilage tissue. However, stem cells cultured two-dimensionally have limited ability to differentiate into chondrocytes, which led to the development of three-dimensional culture systems. A recently developed microtechnological method uses microwells as a tool to form uniformly sized spheroids. In this study, we fabricated an array (10 × 10) of goblet-shaped microwells based on polydimethylsiloxane for spheroid culture. A central processing unit (CPU) was used to form holes, and metallic beads were used to form hemispherical microwell geometry. The holes were filled with Pluronic F-127 to prevent cells from sinking through the holes and allowing the cells to form spheroids. Viability and chondrogenic differentiation of human adipose-derived stem cells were assessed. The fabrication method using a micro-pin mold and metallic beads is easy and cost-effective. Our three-dimensional spheroid culture system optimizes the efficient differentiation of cells and has various applications, such as drug delivery, cell therapy, and tissue engineering.

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.

Floating-on-water Fabrication Method for Thin Polydimethylsiloxane Membranes.

Polydimethylsiloxane (PDMS) membranes are used in various applications, such as microvalves, micropumps, microlenses, and cell culture substrates, with various thicknesses from microscale to nanoscale. In this study, we propose a simple fabrication method for PDMS membranes on a water surface, referred to as the floating-on-water (FoW) method. FoW can be used to easily fabricate PDMS membranes with thicknesses of a few micrometers (minimum 3 µm) without special equipment. In addition, as the membrane is fabricated on the water surface, it can be easily handled without damage. In addition, alternative membrane structures were demonstrated, such as membrane-on-pins and droplet-shaped membranes. FoW can be widely used in various applications that require PDMS membranes with microscale thicknesses.

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.

Effect of off-plane bifurcation angles of primary bronchi on expiratory flows in the human trachea.

BACKGROUND: The human airway is exposed to the development of diverse flow patterns based on differences in its morphological/geometrical parameters across individuals. Although effects of the asymmetry between the right and left main bronchi on airway flows have been investigated in the past, there exists a paucity in terms of studies that focus on the role of stronger physiological asymmetric features, such as off-plane bifurcation angles of primary bronchi, in expiratory flows. METHOD: Computational fluid dynamic techniques have been used to demonstrate presence of Dean-type secondary flows and vortices in the bifurcation region. Formation of a distinctive pattern was observed corresponding to an increase in the off-plane branching angle. An experiment involving 3D printed airways and smoke was also performed to visualize flow patterns and verify simulation results. RESULTS: Good agreement was observed between computational and experimental results. Furthermore, it was revealed that the predicted wall shear stress distribution demonstrated significant changes (with a maximum shear stress increase of 30.7%) compared to conventional airway models that adopt symmetric bifurcation angles. The overall flow demonstrated a swerving motion, which was characterized by tracking the vortex cores (maximum accumulated radial movement of 72.6°) when they ascended towards the trachea inlet in off-plane airway models. CONCLUSIONS: It was confirmed that off-plane bifurcations in human trachea significantly alter the flow characteristics in expiratory flows. It is expected that the results of this study will provide useful information regarding increasingly advanced patient-specific treatments for respiratory diseases in the trachea.

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.

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.

Responses of human adipose-derived stem cells to interstitial level of extremely low shear flows regarding differentiation, morphology, and proliferation.

Human cells encounter a range of shear stress levels in situ and this natural variability in shear stress implies that realistic investigations of cell type characteristics may depend on nontrivial shear stress models. Human adipose-derived stem cells (hASCs) differentiate near the blood capillary vessels where interstitial flows predominate. However, the effects of interstitial levels of shear on hASCs are not fully understood. In this study, we propose a microfluidic shear generation system, in which a gradient distribution of the interstitial level of shear flow is created to investigate the effects of interstitial-level shear flow on hASCs. To generate such a gradient profile of interstitial-level shear stress, we fabricated a semicircle-shaped microfluidic channel, and generated an extremely low flow using an osmosis-driven pump. Changes to hASC morphology, proliferation, and differentiation were observed under shear stresses of 1.8 × 10-3-2.4 × 10-3 Pa. At higher shear stresses, we found higher proliferation rates, stronger actin structures, and lower differentiation. We also conducted computational simulations of a monolayer culture, which showed that the shear stress level even on a single cell varies owing to the change of the cell thickness between the pseudopodia and the nucleus. We found that hASCs detectably respond to extremely low levels of shear flow, above a threshold of ∼2.0 × 10-3 Pa. Our microplatform may be useful for quantitating biological responses and function changes of other stem cells and cancer cells to interstitial-level shear flows.

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.