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.

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.

Hydrogel-Assisted Electrospinning for Fabrication of a 3D Complex Tailored Nanofiber Macrostructure.

Electrospinning has shown great potential in tissue engineering and regenerative medicine due to a high surface-area-to-volume ratio and an extracellular matrix-mimicking structure of electrospun nanofibers, but the fabrication of a complex three-dimensional (3D) macroscopic configuration with electrospun nanofibers remains challenging. In the present study, we developed a novel hydrogel-assisted electrospinning process (GelES) to fabricate a 3D nanofiber macrostructure with a 3D complex but tailored configuration by utilizing a 3D hydrogel structure as a grounded collector instead of a metal collector in conventional electrospinning. The 3D hydrogel collector was discovered to effectively concentrate the electric field toward itself similar to the metal collector, thereby depositing electrospun nanofibers directly on its exterior surface. Synergistic advantages of the hydrogel (e.g., biocompatibility and thermally reversible sol-gel transition) and the 3D nanofiber macrostructure (e.g., mechanical robustness and high permeability) provided by the GelES process were demonstrated in a highly permeable tubular tissue graft and a robust drug- or cell-encapsulation construct. GelES is expected to broaden potential applications of electrospinning to not only provide in vivo drug/cell delivery and tissue regeneration but also an in vitro drug testing platform by increasing the degree of freedom in the configuration of the 3D nanofiber macrostructure.

Collagen immobilization on ultra-thin nanofiber membrane to promote in vitro endothelial monolayer formation.

The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

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.

Development of an in vitro 3D choroidal neovascularization model using chemically induced hypoxia through an ultra-thin, free-standing nanofiber membrane.

Choroidal neovascularization (CNV) is the pathological growth of new blood vessels in the sub-retinal pigment epithelial (RPE) space from the choroid through a break in the Bruch's membrane (BM). Despite its importance in studying biological processes and drug discovery, the development of an in vitro CNV model that achieves the physiological structures of native RPE-BM-choroidal capillaries (CC) is still challenging. Here, we develop a novel 3D RPE-BM-CC complex biomimetic system on an ultra-thin, free-standing nanofiber membrane. The thickness of the pristine nanofiber membrane is 2.17 ±â€¯0.81 µm, and the Matrigel-coated nanofiber membrane attains a permeability coefficient of 2.95 ±â€¯0.25 × 10-6 cm/s by 40 kDa FITC-dextran, which is similar to the physiological value of the native BM. On the in vitro 3D RPE-BM-CC complex system, we demonstrate endothelial cell invasion across the 3D RPE-BM-CC complex and the mechanism of the invasion by imposing a hypoxic condition, which is thought to be the major pathological cause of CNV. Furthermore, alleviation of the invasion is achieved by treating with chrysin and anti-VEGF antibody. Thus, the in vitro 3D RPE-BM-CC complex biomimetic system can recapitulate essential features of the pathophysiological environment and be employed for the screening of drug candidates to reduce the number of costly and time-consuming in vivo tests or clinical trials.

Ultra-thin, aligned, free-standing nanofiber membranes to recapitulate multi-layered blood vessel/tissue interface for leukocyte infiltration study.

Leukocyte infiltration plays critical roles in tissue inflammation for pathogen clearance and tumor eradication. This process is regulated by complex microenvironments in blood vessels, including inflamed endothelium, blood flow, and perivascular components. The role of perivascular components in leukocyte infiltration has not been systematically investigated until recently mostly due to lack of technology. In this work, we developed a three-dimensional multi-layered blood vessel/tissue model with a nanofiber membrane, enabling real-time visualization of dynamic leukocyte infiltration and subsequent interaction with perivascular macrophages. We directly fabricated a highly aligned, free-standing nanofiber membrane with an ultra-thin thickness of ∼1 µm in microfluidic systems. Coating the nanofiber membrane with matrigel showed synergetic topographical and biochemical effects on the reconstitution of a well-aligned endothelial monolayer on the membrane. Our 3D multi-layered blood vessel/tissue model will offer a powerful and versatile tool for investigating the mechanism of leukocyte tissue infiltration and subsequent immune responses.

Role of Grounded Liquid Collectors in Precise Patterning of Electrospun Nanofiber Mats.

Liquid collectors are applicable as ground collectors in electrospinning, which fabricates complex nanofiber architectures. However, the influence of the electrical properties of liquid collectors on the controlled deposition of electrospun nanofiber mats has received little attention. Here, we prepare two types of liquid collectors (electrolyte solutions and dielectric liquids) and newly scrutinize their roles in the patterning of electrospun nanofiber mats in experiments and in numerical simulations. By simulating the concentrations of the electric fields around the liquid collectors, we indirectly evaluated the patternability of the collectors. The patternability trends were verified by the patterning of nanofiber mats on line-array-shaped liquid collectors fabricated by electrospinning. The deposition accuracy of the electrolyte solution collector was very high, equivalent to that of a conventional metal collector even at low salt concentrations (e.g., 0.01 M KCl). However, the nanofiber mats fabricated by electrospinning with the dielectric liquid collector showed retarded patternability.