Guidelines for Manufacturing and Application of Organoids: Kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

A Perforated Plate-Based Cell Showering Device for Uniform Cell Distribution over Various Culture Substrates.

Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell distribution, the seeding process of in vitro cell culture still highly relies on the traditional method using manual pipetting. Because manual pipetting cannot ensure a uniform cell distribution and has the possibility of compromising experimental reproducibility, an accurate and systemic seeding method that enables uniform cell seeding over versatile culture substrates is required. Here, we developed a perforated plate-based cell seeding device called the CellShower, which enabled uniform cell seeding over a large area of cell culture substrates. The working principles of the CellShower are based on the laminar filling flow and capillary force in microfluidics, and the design of the CellShower was optimized with numerical simulations. The versatility of the CellShower in view of uniform cell seeding was demonstrated by applying it to various types of culture substrates from a conventional culture dish to culture substrates having nanotopography, porous structures, and 3D concave structures. The CellShower and its operating principles are expected to contribute to enhancing the accuracy and reproducibility of biological experiments.

Overexpression of Dock180 and Elmo1 in Melanoma is Associated with Cell Survival and Migration.

BACKGROUND: Melanoma is one of the most aggressive and metastatic skin cancers. Although overexpression of Dock180 and Elmo1 has been identified in various cancers, including glioma, ovarian cancer, and breast cancer, their expression and functions in melanoma remain unknown. OBJECTIVE: This study aims to confirm the expression of Dock180 and Elmo1, their underlying mechanisms, and roles in melanoma. METHODS: Both immunohistochemical staining and Western blotting were used to confirm expression of Dock180 and Elmo1 in human melanoma. To identify roles of Dock180 and Elmo1 in cell survival, apoptosis and migration, downregulation of Dock180 or Elmo1 in melanoma cells with small interfering RNA (siRNA) was performed. RESULTS: We identified overexpression of Dock180 and Elmo1 in human melanoma compared to normal skin ex vivo. Inhibition of Dock180 or Elmo1 following siRNA in melanoma cells reduced cell viability and increased apoptosis as supported by increased proportion of cells with Annexin V-PE (+) staining and sub-G0/G1 peak in cell cycle analysis. Moreover, inhibition of Dock180 or Elmo1 regulated apoptosis-related proteins, showing downregulation of Bcl-2, caspase-3, and PARP and upregulation of Bax, PUMA, cleaved caspase-3, and cleaved PARP. Furthermore, knockdown of Dock180 and Elmo1 in melanoma cells reduced cell migration and changed cellular signaling pathways including ERK and AKT. Vemurafenib decreased cell viability in concentration-dependent manner, while transfection with Dock180- or Elmo1-specific siRNA in melanoma cells significantly reduced cell viability. CONCLUSION: Our results suggest that both Dock180 and Elmo1 may be associated with cancer progression, and can be potential targets for treatment of melanoma.

Recent Progress in Fabrication of Electrospun Nanofiber Membranes for Developing Physiological In Vitro Organ/Tissue Models.

Nanofiber membranes (NFMs), which have an extracellular matrix-mimicking structure and unique physical properties, have garnered great attention as biomimetic materials for developing physiologically relevant in vitro organ/tissue models. Recent progress in NFM fabrication techniques immensely contributes to the development of NFM-based cell culture platforms for constructing physiological organ/tissue models. However, despite the significance of the NFM fabrication technique, an in-depth discussion of the fabrication technique and its future aspect is insufficient. This review provides an overview of the current state-of-the-art of NFM fabrication techniques from electrospinning techniques to postprocessing techniques for the fabrication of various types of NFM-based cell culture platforms. Moreover, the advantages of the NFM-based culture platforms in the construction of organ/tissue models are discussed especially for tissue barrier models, spheroids/organoids, and biomimetic organ/tissue constructs. Finally, the review concludes with perspectives on challenges and future directions for fabrication and utilization of NFMs.

Curcumin Enhances the Anticancer Effects of Binimetinib on Melanoma Cells by Inducing Mitochondrial Dysfunction and Cell Apoptosis with Necroptosis.

BACKGROUND: Recent studies suggest that MEK1/2 inhibitors, including binimetinib, significantly improve malignant melanoma (MM) patient survival. Growing evidence suggests that phytochemicals, especially curcumin, can overcome drug resistance in cancer cells through a variety of mechanisms. OBJECTIVE: This study aims to examine curcumin's efficacy in vitro combined with binimetinib in human MM cells. METHODS: We used 2D monolayer and 3D spheroid human epidermal melanocyte culture models, HEMn-MP (human epidermal melanocytes, neonatal, moderately pigmented), and two human MM cell lines, G361 and SK-MEL-2, to evaluate cell viability, proliferation, migration, death, and reactive oxygen species (ROS) production following single therapy treatment, with either curcumin or binimetinib, or a combination of both. RESULTS: Compared to MM cells treated with single therapy, those with combination therapy showed significantly decreased cell viability and increased ROS production. We observed apoptosis following both single and combination therapies. However only those who had had combination therapy had necroptosis. CONCLUSION: Collectively, our data demonstrates that curcumin exerts significant synergistic anticancer effects on MM cells by inducing ROS and necroptosis when combined with binimetinib. Therefore, a strategy of adding curcumin to conventional anticancer agents holds promise for treating MM.

Facile and adhesive-free method for bonding nanofiber membrane onto thermoplastic polystyrene substrate to fabricate 3D cell culture platforms.

Nanofiber (NF) membranes have been highlighted as functional materials for biomedical applications owing to their high surface-to-volume ratios, high permeabilities, and extracellular matrix-like biomimetic structures. Because many in vitro platforms for biomedical applications are made of thermoplastic polymers (TP), a simple and leak-free method for bonding NF membranes onto TP platforms is essential. Here, we propose a facile but leak-free localized thermal bonding method for integrating 2D or 3D-structured NF membrane onto a TP supporting substrate while preserving the pristine nanofibrous structure of the membrane, based on localized preheating of the substrate. A methodology for determining the optimal preheating temperature was devised based on a numerical simulation model considering the melting temperature of the NF material and was experimentally validated by evaluating bonding stability and durability under cell culture conditions. The thermally-bonded interface between the NF membrane and TP substrate was maintained stably for 3 weeks allowing the successful construction of an intestinal barrier model. The applicability of the localized thermal bonding method was also demonstrated on various combinations of TP materials (e.g., polystyrene and polymethylmethacrylate) and geometries of the supporting substrate, including a culture insert and microfluidic chip. We expect the proposed localized thermal bonding method to contribute toward broadening and realizing the practical applications of functional NF membranes in various biomedical fields.

A Liquid Triboelectric Series.

The triboelectric series is a generally accepted method for describing the triboelectric effect. It provides a way to control the double face of the ubiquitous triboelectric effect: causes of unpredictable accidents and the resultant surface charge as energy sources. However, previous studies have been biased in solids despite being observed in liquids (liquid-solid contact electrification). Therefore, a liquid triboelectric series is necessary to be established to manipulate the liquid triboelectric effect according to the appropriate goal. In this study, a liquid triboelectric series is first established to describe the triboelectric properties of each liquid when contact electrification occurs with a solid surface. The liquid triboelectric series covers electrolytes, organic solvents, oxidants, and higher sugar alcohols. Common chemical groups can be derived from the liquid triboelectric series that hydroxyl groups enhance, and benzene groups suppress the liquid triboelectric effect. The results are demonstrated by the amplified efficiency of an energy harvester and particle contamination after surface washing. This study will play a pivotal role in understanding the liquid-solid contact electrification phenomenon and providing new perspectives on the applications of the liquid triboelectric effect.

Human iPS-derived blood-brain barrier model exhibiting enhanced barrier properties empowered by engineered basement membrane.

The basement membrane (BM) of the blood-brain barrier (BBB), a thin extracellular matrix (ECM) sheet underneath the brain microvascular endothelial cells (BMECs), plays crucial roles in regulating the unique physiological barrier function of the BBB, which represents a major obstacle for brain drug delivery. Owing to the difficulty in mimicking the unique biophysical and chemical features of BM in in vitro systems, current in vitro BBB models have suffered from poor physiological relevance. Here, we describe a highly ameliorated human BBB model accomplished by an ultra-thin ECM hydrogel-based engineered basement membrane (nEBM), which is supported by a sparse electrospun nanofiber scaffold that offers in vivo BM-like microenvironment to BMECs. BBB model reconstituted on a nEBM recapitulates the physical barrier function of the in vivo human BBB through ECM mechano-response to physiological relevant stiffness (∼500 kPa) and exhibits high efflux pump activity. These features of the proposed BBB model enable modelling of ischemic stroke, reproducing the dynamic changes of BBB, immune cell infiltration, and drug response. Therefore, the proposed BBB model represents a powerful tool for predicting the BBB permeation of drugs and developing therapeutic strategies for brain diseases.

Engineering porous membranes mimicking in vivo basement membrane for organ-on-chips applications.

Porous membrane-based microfluidic chips are frequently used for developing in vitro tissue-barrier models, the so-called tissue barriers-on-chips (TBoCs). The porous membrane in a TBoC plays a crucial role as an alternative to an in vivo basement membrane (BM). To improve the physiological relevance of an artificial porous membrane, it should possess complex BM-like characteristics from both biophysical and biochemical perspectives. For practical use, artificial membranes should have high mechanical robustness, and their fabrication processes should be conducive to mass production. There have been numerous approaches to accomplishing these requirements in BM-like porous membranes. Extracellular matrix (ECM) hydrogels have emerged as physiologically relevant materials for developing artificial BMs; they remarkably improve the phenotypes and functions of both cells and their layers when compared to previous synthetic porous membranes. However, for practical use, the poor mechanical robustness of ECM membranes needs to be improved. Recently, an advanced ECM membrane reinforced with a nanofiber scaffold has been introduced that possesses both BM-like characteristics and practical applicability. This advanced ECM membrane is expected to promote not only in vivo-like cellular functions but also cellular responses to drugs, which in turn further facilitates the practical applications of TBoCs.

Sutureless transplantation ofin vivopriming human mesenchymal stem cell sheet promotes the therapeutic potential for cardiac repair.

The heart, contrary to its small size, vigorously pumps oxygen and nutrients to our entire body indeterminably; and thus, its dysfunction could be devastating. Until now, there ave been several major obstacles to applying a cardiac patch for the treatment for myocardial infarction, including poor integration and low engraftment rates, due to the highly-curved surface of the heart and its dynamic nature. Here, we demonstrate a novel way for a comprehensive cardiac repair achieved by the sutureless transplantation of a highly integrablein vivopriming bone marrow mesenchymal stem cell (BMSC) sheet based on the utilization of a highly aligned thermoresponsive nanofiber membrane. Moreover, we developed a BMSC sheet specialized for vascular regeneration through 'in-vivopriming' using human umbilical vein endothelial cells. A prolonged secretion of multiple angiogenic cytokines, such as vascular endothelial growth factor, angiopoietin-1, insulin-like growth factor-1, which was observedin vitrofrom the specialized BMSC sheet seemed to lead a significant improvement in the cardiac function, including intrinsic contractibility and remodeling. In this study, we provide strong evidence thatin vivopriming of a human BMSC sheet develops the therapeutic potential for cardiac repair.

Intratumoral immunotherapy using a TLR2/3 agonist, L-pampo, induces robust antitumor immune responses and enhances immune checkpoint blockade.

BACKGROUND: Toll-like receptors (TLRs) are critical innate immune sensors that elicit antitumor immune responses in cancer immunotherapy. Although a few TLR agonists have been approved for the treatment of patients with early-stage superficial cancers, their therapeutic efficacy is limited in patient with advanced invasive cancers. Here, we identified the therapeutic role of a TLR2/3 agonist, L-pampo (LP), which promotes antitumor immunity and enhances the immune checkpoint blockade. METHODS: We generated LP by combining a TLR2 agonist, Pam3CSK4, with a TLR3 agonist, Poly (I:C). Immune responses to stimulation with various TLR agonists were compared. Tumor-bearing mice were intratumorally treated with LP, and their tumor sizes were measured. The antitumor effects of LP treatment were determined using flow cytometry, multiplexed imaging, and NanoString nCounter immune profiling. The immunotherapeutic potential of LP in combination with α-programmed cell death protein-1 (PD-1) or α-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) was evaluated in syngeneic MC38 colon cancer and B16F10 melanoma. RESULTS: The LP treatment induced a potent activation of T helper 1 (Th1) and 2 (Th2)-mediated immunity, tumor cell apoptosis, and immunogenic tumor cell death. Intratumoral LP treatment effectively inhibited tumor progression by activating tumor-specific T cell immunity. LP-induced immune responses were mediated by CD8+ T cells and interferon-γ, but not by CD4+ T cells and CD25+ T cells. LP simultaneously activated TLR2 and TLR3 signaling, thereby extensively changing the immune-related gene signatures within the tumor microenvironment (TME). Moreover, intratumoral LP treatment led to systemic abscopal antitumor effects in non-injected distant tumors. Notably, LP treatment combined with ɑPD-1 and ɑCTLA-4 further enhanced the efficacy of monotherapy, resulting in complete tumor regression and prolonged overall survival. Furthermore, LP-based combination immunotherapy elicited durable antitumor immunity with tumor-specific immune memory in colon cancer and melanoma. CONCLUSIONS: Our study demonstrated that intratumoral LP treatment improves the innate and adaptive antitumor immunity within the TME and enhances the efficacy of αPD-1 and αCTLA-4 immune checkpoint blockade.

Enhanced Differentiation Capacity and Transplantation Efficacy of Insulin-Producing Cell Clusters from Human iPSCs Using Permeable Nanofibrous Microwell-Arrayed Membrane for Diabetes Treatment.

Although pancreatic islet transplantation is a potentially curative treatment for insulin-dependent diabetes, a shortage of donor sources, low differentiation capacity, and transplantation efficacy are major hurdles to overcome before becoming a standard therapy. Stem cell-derived insulin-producing cells (IPCs) are a potential approach to overcoming these limitations. To improve the differentiation capacity of the IPCs, cell cluster formation is crucial to mimic the 3D structure of the islet. This study developed a biodegradable polycaprolactone (PCL) electrospun nanofibrous (NF) microwell-arrayed membrane permeable to soluble factors. Based on the numerical analysis and experimental diffusion test, the NF microwell could provide sufficient nutrients, unlike an impermeable PDMS (polydimethylsiloxane) microwell. The IPC clusters in the NF microwells showed higher gene expression of insulin and PDX1 and insulin secretion than the PDMS microwells. The IPC clusters in the NF microwell-arrayed membrane could be directly transplanted. Transplanted IPC clusters in the microwells survived well and expressed PDX1 and insulin. Additionally, human c-peptide was identified in the blood plasma at two months after transplantation of the membranes. The NF microwell-arrayed membrane can be a new platform promoting IPC differentiation capacity and realizing an in situ transplantation technique for diabetic patients.

Thin and stretchable extracellular matrix (ECM) membrane reinforced by nanofiber scaffolds for developingin vitrobarrier models.

An extracellular matrix (ECM) membrane made up of ECM hydrogels has great potentials to develop a physiologically relevant organ-on-a-chip because of its biochemical and biophysical similarity toin vivobasement membranes (BMs). However, the limited mechanical stability of the ECM hydrogels makes it difficult to utilize the ECM membrane in long-term and dynamic cell/tissue cultures. This study proposes a thin but robust and transparent ECM membrane reinforced with silk fibroin (SF)/polycaprolactone (PCL) nanofibers, which is achieved byin situself-assembly throughout a freestanding SF/PCL nanofiber scaffold. The SF/PCL nanofiber-reinforced ECM (NaRE) membrane shows biophysical characteristics reminiscent of native BMs, including small thickness (<5µm), high permeability (<9 × 10-5cm s-1), and nanofibrillar architecture (∼10-100 nm). With the BM-like characteristics, the nanofiber reinforcement ensured that the NaRE membrane stably supported the construction of various types ofin vitrobarrier models, from epithelial or endothelial barrier models to complex co-culture models, even over two weeks of cell culture periods. Furthermore, the stretchability of the NaRE membrane allowed emulating the native organ-like cyclic stretching motions (10%-15%) and was demonstrated to manipulate the cell and tissue-level functions of thein vitrobarrier model.

Perichondrium-inspired permeable nanofibrous tube well promoting differentiation of hiPSC-derived pellet toward hyaline-like cartilage pellet.

The pellet formation has been regarded as a golden standard forin vitrochondrogenic differentiation. However, a spatially inhomogeneous chondrogenic microenvironment around a pellet resulted from the use of a traditional impermeable narrow tube, such as the conical tube, undermines the differentiation performance and therapeutic potential of differentiated cartilage pellet in defective articular cartilage treatment. To address this drawback, a perichondrium-inspired permeable nanofibrous tube (PINaT) well with a nanofibrous wall permeable to gas and soluble molecules is proposed. The PINaT well was fabricated with a micro deep drawing process where a flat thin nanofibrous membrane was transformed to a 3.5 mm deep tube well with a ∼50µm thick nanofibrous wall. Similar toin vivoperichondrium, the PINaT well was found to allow oxygen and growth factor diffusion required for chondrogenic differentiation across the entire nanofibrous wall. Analyses of gene expressions (COL2A1, COL10A1, ACAN, and SOX9), proteins (type II and X collagen), and glycosaminoglycans contents were conducted to assess the differentiation performance and clinical efficacy of differentiated cartilage pellet. The regulated spatially homogeneous chondrogenic microenvironment around the human induced pluripotent stem cell-derived pellet (3 × 105cells per pellet) in the PINaT well remarkably improved the quality of the differentiated pellet toward a more hyaline-like cartilage pellet. Furthermore, an accelerated chondrogenic differentiation process of the pellet produced by the PINaT well was achieved for 14 days, demonstrating a hyaline cartilage-specific marker similar to the control pellet differentiated for 20 days. Finally, the enhanced clinical efficacy of the hyaline-like cartilage pellet was confirmed using an osteochondral defect rat model, with the repaired tissue resembling hyaline cartilage rather than fibrous cartilage after 8 weeks of regeneration.

Multiplex recreation of human intestinal morphogenesis on a multi-well insert platform by basolateral convective flow.

Here, we report a multiplex culture system that enables simultaneous recreation of multiple replications of the three-dimensional (3D) microarchitecture of the human intestinal epithelium in vitro. The "basolateral convective flow-generating multi-well insert platform (BASIN)" contains 24 nano-porous inserts and an open basolateral chamber applying controllable convective flow in the basolateral compartment that recreates a biomimetic morphogen gradient using a conventional orbital shaker. The mechanistic approach by which the removal of morphogen inhibitors in the basolateral medium can induce intestinal morphogenesis was applied to manipulate the basolateral convective flow in space and time. In a multiplex BASIN, we successfully regenerated a 3D villi-like intestinal microstructure using the Caco-2 human intestinal epithelium that presents high barrier function with minimal insert-to-insert variations. The enhanced cytodifferentiation and proliferation of the 3D epithelial layers formed in the BASIN were visualized with markers of absorptive (villin) and proliferative cells (Ki67). The paracellular transport and efflux profiles of the microengineered 3D epithelial layers in the BASIN confirmed its reproducibility, robustness, and scalability for multiplex biochemical or pharmaceutical studies. Finally, the BASIN was used to investigate the effects of dextran sodium sulfate on the intestinal epithelial barrier and morphology to validate its practical applicability for investigating the effects of external chemicals on the intestinal epithelium and constructing a leaky-gut model. We envision that the BASIN may provide an improved multiplex, scalable, and physiological intestinal epithelial model that is readily accessible to researchers in both basic and applied sciences.

A deep and permeable nanofibrous oval-shaped microwell array for the stable formation of viable and functional spheroids.

Despite the potential of a nanofibrous (NF) microwell array as a permeable microwell array to improve the viability and functions of spheroids, thanks to the superior permeability to both gases and solutes, there have still been difficulties regarding the stable formation of spheroids in the NF microwell array due to the low aspect ratio (AR) and the large interspacing between microwells. This study proposes a nanofibrous oval-shaped microwell array, named the NOVA microwell array, with both a high AR and a high well density, enabling us to not only collect cells in the microwell with a high cell seeding efficiency, but also to generate multiple viable and functional spheroids in a uniform and stable manner. To realize a deep NOVA microwell array with a high aspect ratio (AR = 0.9) and a high well density (494 wells cm-2), we developed a matched-mold thermoforming process for the fabrication of both size- and AR-controllable NOVA microwell arrays with various interspacing between microwells while maintaining the porous nature of the NF membrane. The human hepatocellular carcinoma (HepG2) cell spheroids cultured on the deep NOVA microwell array not only had uniform size and shape, with a spheroid circularity of 0.80 ± 0.03 at a cell seeding efficiency of 94.29 ± 9.55%, but also exhibited enhanced viability with a small fraction of dead cells and promoted functionality with increased albumin secretion, compared with the conventional impermeable microwell array. The superior characteristics of the deep NOVA microwell array, i.e. a high AR, a high well density, and a high permeability, pave the way to the production of various viable and functional spheroids and even organoids in a scalable manner.

Improved chondrogenic performance with protective tracheal design of Chitosan membrane surrounding 3D-printed trachea.

In recent tracheal tissue engineering, limitations in cartilage reconstruction, caused by immature delivery of chondrocyte-laden components, have been reported beyond the complete epithelialization and integration of the tracheal substitutes with the host tissue. In an attempt to overcome such limitations, this article introduces a protective design of tissue-engineered trachea (TraCHIM) composed of a chitosan-based nanofiber membrane (CHIM) and a 3D-printed biotracheal construct. The CHIM was created from chitosan and polycaprolactone (PCL) using an electrospinning process. Upon addition of chitosan to PCL, the diameter of electrospun fibers became thinner, allowing them to be stacked more closely, thereby improving its mechanical properties. Chitosan also enhances the hydrophilicity of the membranes, preventing them from slipping and delaminating over the cell-laden bioink of the biotracheal graft, as well as protecting the construct. Two weeks after implantation in Sprague-Dawley male rats, the group with the TraCHIM exhibited a higher number of chondrocytes, with enhanced chondrogenic performance, than the control group without the membrane. This study successfully demonstrates enhanced chondrogenic performance of TraCHIM in vivo. The protective design of TraCHIM opens a new avenue in engineered tissue research, which requires faster tissue formation from 3D biodegradable materials, to achieve complete replacement of diseased tissue.

Arterial Internal Elastic Lamina-Inspired Membrane for Providing Biochemical and Structural Cues in Developing Artery-on-a-Chip.

In vitro artery models constructed on a membrane-based microfluidic chip, called an artery-on-a-chip, have been spotlighted as a powerful platform for studying arterial physiology. However, due to the use of a flat and porous membrane that cannot mimic the in vivo internal elastic lamina (IEL), the physiological similarity in the phenotypes and the arrangements of the endothelial cells (ECs) and aortic smooth muscle cells (AoSMCs) has been limited in the previously developed artery-on-a-chips. Herein, we developed an innovative membrane mimicking the structures of IEL by utilizing electrospun aligned silk fibroin/polycaprolactone nanofiber membranes. An arterial IEL-mimicking (AIM) membrane was about 5 µm thick and composed of orthogonally aligned nanofibers with a diameter of around 400 nm, which were highly comparable to the IEL. Such structural similarity was found to induce the ECs and SMCs to be elongated and orthogonally aligned as in the in vivo artery. In particular, the SMCs cultured on the AIM membrane maintained a healthy state showing increased αSMA mRNA expression, which was easily lost on the conventional membrane. We constructed an AIM membrane-integrated artery-on-a-chip having an orthogonal arrangement of ECs and SMCs, which was desirable but difficult to be realized with the previous artery-on-a-chip.

Facile Fabrication of Electrospun Nanofiber Membrane-Integrated PDMS Microfluidic Chip via Silver Nanowires-Uncured PDMS Adhesive Layer.

Although direct electrospinning has been frequently utilized to develop a nanofiber membrane-integrated microfluidic chip, the dielectric substrate material retards the deposition of electrospun nanofibers on the substrate, and the rough surface formed by deposited nanofibers hinders the successful sealing. In this study we introduce a facile fabrication process of an electrospun nanofiber membrane-integrated polydimethylsiloxane (PDMS) microfluidic chip, called a NFM-PDMS chip, by applying the functional layer. The functional layer consists of a silver nanowires (AgNWs)-embedded uncured PDMS adhesive layer (SNUP), which not only effectively concentrates the electric field toward the PDMS substrate, but also provides a smooth surface for robust sealing. The AgNWs in the SNUP play a crucial role as a grounded collector and enable approximately 4× faster electrospinning than the conventional method, forming a free-standing nanofiber membrane. The uncured PDMS adhesive layer in the SNUP maintains the smooth surface after electrospinning and allows the rapid and leakage-free bonding of the NFM-PDMS chip using plasma treatment. A practical application of the NFM-PDMS chip is demonstrated by culturing the human keratinocyte cell line, HaCaT cells. The HaCaT cells are well grown on the free-standing nanofiber membrane under dynamic flow conditions, maintaining good viability over 95% for 7 days of culture.