The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels, relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into Non-obese Diabetic/Severe Combined Immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D-culture technique can promote effective generation of niche-independent single stem cell-based 3D organoids.

Due to the recent demand for high-throughput cellular assays, a lot of efforts have been made on miniaturization of cell-based biosensors by preparing cell microarrays. Various microfabrication technologies have been used to generate cell microarrays, where cells of different phenotypes are immobilized either on a flat substrate (positional array) or on particles (solution or suspension array) to achieve multiplexed and high-throughput cell-based biosensing. After introducing the fabrication methods for preparation of the positional and suspension cell microarrays, this review discusses the applications of the cell microarray including toxicology, drug discovery and detection of toxic agents.

Porous hydroxyapatite (HAp) scaffolds are commonly used for hard tissue regeneration because of their biocompatibility and osteoconduction properties, but they are limited in terms of bioactivity and osteoinduction. This study investigated the fabrication of HAp scaffolds coated with poly(lactic-co-glycolic acid)/45S5 bioactive glass (PLGA/BG) composite microfibers using the sponge replica method and electrospinning process for improved bioactivity and osteoinductivity during osteogenesis. Characterization of the HAp/PLGA/BG (HPB) scaffold was carried out by examining morphology and ion release. The biological evaluation of HPB scaffolds was carried out by assessing cytotoxicity, cell proliferation, and cell differentiation using MC3T3-E1 preosteoblasts. The results showed that all HPB scaffolds exhibited a high porosity of 89.2% and had porous structures coated with a layer of composite containing BG ions (Si–Ca–Na–P). These scaffolds enabled controlled release of Si, Ca, Na, and P ions for up to 28 days. There was no significant difference in cytotoxicity between the scaffolds. Cell proliferation on HPB scaffolds was increased from day 1 to 3. In addition, cell viability on the HPB scaffolds was confirmed with LIVE/DEAD assay. Cell differentiation, as shown by alkaline phosphatase activity and western blot analyses, indicated that HPB scaffolds with 20 and 30 min of coating induced higher levels of osteogenesis-related markers compared to other scaffolds. Furthermore, immunocytochemistry indicated osteopontin expression. Alizarin red staining indicated that HPB scaffolds with 20 min of coating were more effective than the HAp scaffold in terms of mineralization. In conclusion, HAp scaffolds coated with PLGA/BG for 20 min are promising materials for osteogenic activity and may be a potential bone substitute for tissue engineering.

Three-dimensional (3D) cultures recapitulate the microenvironment of tissue-resident stem cells and enable them to modulate their properties. We determined whether salivary gland-resident stem cells (SGSCs) are primed by a 3D spheroid culture prior to treating irradiation-induced salivary hypofunction using in-vitro coculture and in-vivo transplant models. 3D spheroid-derived SGSCs (SGSCs3D) were obtained from 3D culture in microwells consisting of a nanofiber bottom and cell-repellent hydrogel walls, and were examined for salivary stem or epithelial gene/protein expression, differentiation potential, and paracrine secretory function compared with monolayer-cultured SGSCs (SGSCs2D) in vitro and in vivo. SGSCs3D expressed increased salivary stem cell markers (LGR5 and THY1) and pluripotency markers (POU5F1 and NANOG) compared with SGSCs2D. Also, SGSCs3D exhibited enhanced potential to differentiate into salivary epithelial cells upon differentiation induction and increased paracrine secretion as compared to SGSCs2D. Wnt signaling was activated by 3D spheroid formation in the microwells and suppression of the Wnt/β-catenin pathway led to reduced stemness of SGSCs3D. Enhanced radioprotective properties of SGSCs3D against radiation-induced salivary hypofunction was confirmed by an organotypic 3D coculture and in-vivo transplantation experiments. The 3D spheroid culture of SGSCs in nanofibrous microwells promotes stem cell properties via activation of Wnt signaling. This may contribute to SGSC priming prior to regenerative therapy to restore salivary hypofunction after radiotherapy.