Versatile Fabrication of Size- and Shape-Controllable Nanofibrous Concave Microwells for Cell Spheroid Formation

Abstract

Although the microfabrication techniques for microwells enabled to guide physiologically relevant three-dimensional cell spheroid formation, there have been substantial interests to more closely mimic nano/microtopographies of in vivo cellular microenvironment. Here, we developed a versatile fabrication process for nanofibrous concave microwells (NCMs) with a controllable size and shape. The key to the fabrication process was the use of an array of hemispherical convex electrolyte solution drops as the grounded collector for electrospinning, which greatly improved the degree of freedom of the size, shape, and curvature of an NCM. A polymer substrate with through-holes was prepared for the electrolyte solution to come out through the hole and to naturally form a convex shape because of surface tension. Subsequent electrolyte-assisted electrospinning process enabled to achieve various arrays of NCMs of triangular, rectangular, and circular shapes with sizes ranging from 1000 μm down to 250 μm. As one example of biomedical applications, the formation of human hepatoma cell line (HepG2) spheroids was demonstrated on the NCMs. The results indicated that the NCM enabled uniform, size-controllable spheroid formation of HepG2 cells, resulting in 1.5 times higher secretion of albumin from HepG2 cells on the NCM on day 14 compared with those on a nanofibrous flat microwell as a control.

The Tumor-on-Chip: Recent Advances in the Development of Microfluidic Systems to Recapitulate the Physiology of Solid Tumors

“…Adhesion can be prevented by coating the culture surface with agar [153], agarose [120,130,152,154], poly (2-hydroxyethyl methacrylate) (polyHEMA) [125], or tri-block co-polymers, such as Pluronic (F108) [26]. Other materials have been confirmed as suitable non-adherent surfaces due to their topography [155] or high hydrophilicity. For example, the commercially available Ultra Low Attachment (ULA) microwell culture plates have become a popular resource for fabricating spheroids [20,126,156], as has hydrophilic filter paper, which enables the spontaneous formation of prostate cancer spheroids and a significant enrichment of the cancer stem cell (CSC) population using regular cell culture medium [131].…”

Flipped Well-Plate Hanging-Drop Technique for Growing Three-Dimensional Tumors

“…, handing drop insertion ( Tung et al, 2011 ; Zhao et al, 2019 ; Fu et al, 2021 ; Liu et al, 2021 ), including magnetic levitation ( Kim et al, 2013 ; Tseng et al, 2015 ; Ge et al, 2018 ), and nonadhesive surface ( Friedrich et al, 2009 ; Liao et al, 2019 )] were developed to address several applications. Products such as the ultra-low attachment (ULA) plates are available ( Vinci et al, 2012 ; Raghavan et al, 2016 ) along with other techniques for 3D spheroidal culture development, for example, microfabrication ( Lee J. M et al, 2018 ; Park et al, 2018 ; Ganguli et al, 2021 ; Sun et al, 2021 ), soft lithography ( Cha et al, 2017 ; Kuo et al, 2017 ), and floating methods ( Vadivelu et al, 2015 ; Kim et al, 2021 ) among others. Despite the widespread interest, the current culture methods have several limitations, including nonuniformity of spheroid formation, irregular sphericity, short-term cultures, evaporative loss of culture media, and limited volume (size of spheroids) of culture ( Shi et al, 2018 ; De Souza Moraes et al, 2020 ; Han et al, 2021 ).…”

Conformal Fabrication of an Electrospun Nanofiber Mat on a 3D Ear Cartilage-Shaped Hydrogel Collector Based on Hydrogel-Assisted Electrospinning

“…Electrospinning is an effective and versatile technique for fabricating nanofibers and their assemblies, which has been extensively studied over the past decades [1]. Due to their unique properties such as high porosity, high surface-to-volume ratio, and extracellular matrix-mimicking structure [2], extraordinarily electromagnetism, electrospun nanofibers, and their assemblies have been created substantial interests from various research fields, including clothing [3], environmental filter [4][5][6], battery [7], and tissue-engineered scaffolds [8][9][10]. However, the chaotic motion of electrospun nanofibers due to bending instability has hampered the accurate and precise control in constructing nanofiber assemblies and generally created randomly interwoven 2D nanofiber mat [11].…”