Collagen Immobilization on Ultra-thin Nanofiber Membrane to Promote In Vitro Endothelial Monolayer Formation

The endothelialization on the poly (epsilon-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (epsilon-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (epsilon-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 (epsilon-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (epsilon-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 (epsilon-caprolactone) nanofibers and the maintenance of the original structure of poly (epsilon-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (epsilon-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 (epsilon-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (epsilon-caprolactone) nanofiber scaffolds.11Ysciescopu

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 4X faster electro-spinning 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.11Nsciescopu

Electrospun random/aligned hybrid nanofiber mat for development of multi-layered cardiac muscle patch

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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.11Nsciescopu

One-step fabrication of a tunable nanofibrous well insert via electrolyte-assisted electrospinning

1110Ysciescopu

Fabrication of an align-random distinct, heterogeneous nanofiber mat endowed with bifunctional properties for engineered 3D cardiac anisotropy

The development of multilayered anisotropic scaffolds with a 3D anisotropy similar to that of native cardiac tissue with layer-specific oriented multiple cell layers is a major challenge in the reconstruction of engineered cardiac tissue in vitro. Herein, we present an electrospun align-random distinct, heterogeneous nanofiber (NF) mat to facilitate the construction of a multilayered anisotropic scaffold with different orientations, which recreates the cardiac anisotropy via hollow patterned electrolyte-assisted electrospinning (HP-ELES). Based on the HP-ELES process, a distinct but heterogeneous polycaprolactone (PCL) NF mat, which comprised an ultra-thin (<10 mu m) aligned NF membrane at the core and a thick random NF mat at the peripheral rim, was readily fabricated. The fabricated heterogeneous NF mat was endowed with bifunctional properties of not only uniaxially aligned topographical cues to align the cardiomyocytes (CMs) followed by creating a uniaxial contractile motion but also mechanical stability to enable manual manipulation provided by the aligned NF membrane and random NF mat, respectively. Intriguingly, the in vitro cell culture of CMs possessing spontaneous contraction on the heterogeneous NF mat demonstrated cell alignment and subsequent uniaxial contraction along with aligned NFs. The stacking triple layers also exhibited multiaxial contraction, which potentially simulates the squeezing force of the heart tissue. HP-ELES is highly expected to increase the use of ultra-thin aligned NF membranes for the development of in vitro anisotropic organ models and for in vivo tissue regeneration requiring multilayered anisotropic scaffolds owing to the ease of the manual manipulation of the membrane.11Nsciescopu

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&apos;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 +/- 0.81 mu m, and the Matrigel-coated nanofiber membrane attains a permeability coefficient of 2.95 +/- 0.25 x 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.11Nsciescopu