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.

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.

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.

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.

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.

Human kidney organoids model the tacrolimus nephrotoxicity and elucidate the role of autophagy.

BACKGROUND/AIMS: Tacrolimus has been used as an immunosuppressive agent in organ transplantation. Despite the therapeutic benefits, tacrolimus's use is limited due to its nephrotoxicity. To reduce tacrolimus nephrotoxicity, effective humanized experimental models may be helpful. Here, we modeled tacrolimus nephrotoxicity using kidney organoids derived from human inducible pluripotent stem cells (iPSCs) in vitro. METHODS: Kidney organoids were differentiated from the CMC11 iPSC cell line, re-seeded in 96-well plates, and treated with tacrolimus at doses of 0, 30, or 60 µM for 24 hours. This in vitro model was compared to a mouse model of tacrolimus nephrotoxicity and the associated mechanisms were investigated. RESULTS: The size of the kidney organoids and cell viability decreased in dose-dependent manners after treatment with tacrolimus. The number of tubular cells decreased with a loss of polarity, similar to the effects seen in mouse tacrolimus nephrotoxicity. Ultrastructural analysis showed numerous vacuoles in the proximal tubular cells of the kidney organoids treated with tacrolimus. Tacrolimus treatment induced oxidative stress and mitochondrial dysfunction, and autophagic activity was enhanced in the kidney organoids. Rapamycin, an autophagy inducer, accelerated cell death in the kidney organoid model of tacrolimus nephrotoxicity, which was attenuated by treatment with 3-methyladenine, an autophagy inhibitor. These findings indicate that the augmentation of autophagy by rapamycin treatment accelerated tacrolimus nephrotoxicity. CONCLUSION: Our data suggest that human kidney organoids are an effective in vitro model of tacrolimus nephrotoxicity and that autophagy plays a critical role in tacrolimus nephrotoxicity.

Grayscale mask-assisted photochemical crosslinking for a dense collagen construct with stiffness gradient.

Despite the potential of a collagen construct with a stiffness gradient for investigating cell-extracellular matrix (ECM) stiffness interaction or recapitulating an in vivo tissue interface, it has been developed in a limited way due to the low and poorly controllable mechanical properties of the collagen. This study proposes a novel fabrication process to achieve a compressed collagen construct with a stiffness gradient, named COSDIENT, at a level of ~ 1 MPa while maintaining in vivo ECM-like dense collagen fibrillar structures. The COSDIENT was fabricated by collagen compression followed by grayscale mask-assisted UV-riboflavin crosslinking. The collagen compression process enabled the remarkable increase in the stiffness of the collagen gel from ~ 1-10 kPa to ~ 1 MPa by physical compaction. The subsequent UV-riboflavin crosslinking with a continuous-tone grayscale mask could simply generate a gradual change of UV irradiation followed by modulating riboflavin-mediated crosslinking, thereby resulting in a continuous stiffness gradient with a range of 1.16-4.38 MPa in the single compressed collagen construct. The suggested grayscale mask-assisted photochemical crosslinking had no effect on the physical and optical properties of the original compressed collagen construct, while inducing gradual changes of chemical bonds among collagen fibrils. A skin wound healing assay with epidermal keratinocytes was finally applied as an application example of the COSDIENT to examine the effect of stiffness on the skin keratinocyte behavior.

A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function.

Herein, a collagen gel-coated and aligned nanofiber membrane named Col-ANM is developed, which remarkably improves endothelial barrier function by providing biochemical and topographical cues simultaneously. Col-ANM is fabricated by collagen gel coating process on an aligned polycaprolactone (PCL) nanofiber membrane, which is obtained by a simple electrospinning process adopting a parallel electrode collector. Human umbilical vein endothelial cells (HUVECs) cultured on Col-ANM exhibit remarkably enhanced endothelial barrier function with high expression levels of intercellular junction proteins of ZO-1 and VE-cadherin, a high TEER, and a cellular permeability compared with the artificial porous membranes in commercial cell culture well inserts. The enhanced endothelial barrier function is conjectured to be attributed to the synergistic effects of topographical and biochemical cues provided by the aligned PCL nanofibers and collagen gel in the Col-ANM, respectively. Finally, the reactive oxygen species is applied to the HUVEC monolayer formed on the Col-ANM to destroy the tight junctions between HUVECs. The destruction of the tight junctions is demonstrated by the decreased TEER value over time. Results indicate the potential of Col-ANM in modeling endothelial barrier dysfunction-related diseases.