Human mesenchymal stromal cells (hMSCs) are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress, cell aggregation, and substrate changes encountered in 3D dynamic bioreactor cultures. However, little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles (EVs) including the subset, “exosomes”, which contain therapeutic proteins, nucleic acids, and lipids from the parent cells. In this study, bone marrow–derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25, 40, and 64 RPM (0.1–0.3 dyn/cm²). The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture. The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10, 19a, 19b, 21, 132, and 377. EV protein cargo was characterized by proteomics analysis, showing upregulation of metabolic, autophagy and ROS-related proteins comparing with 2D cultured EVs. In addition, the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor, showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor. This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke, Alzheimer's disease, and multiple sclerosis.

The blood–brain barrier (BBB) is a vital structure for maintaining homeostasis between the blood and the brain in the central nervous system (CNS). Biomolecule exchange, ion balance, nutrition delivery, and toxic molecule prevention rely on the normal function of the BBB. The dysfunction and the dysregulation of the BBB leads to the progression of neurological disorders and neurodegeneration. Therefore, in vitro BBB models can facilitate the investigation for proper therapies. As the demand increases, it is urgent to develop a more efficient and more physiologically relevant BBB model. In this review, the development of the microfluidics platform for the applications in neuroscience is summarized. This article focuses on the characterizations of in vitro BBB models derived from human stem cells and discusses the development of various types of in vitro models. The microfluidics-based system and BBB-on-chip models should provide a better platform for high-throughput drug-screening and targeted delivery.

Retinal organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that mimic the retina’s spatial and temporal differentiation, making them useful as in vitro retinal development models. Retinal organoids can be assembled with brain organoids, the 3D self-assembled aggregates derived from hPSCs containing different cell types and cytoarchitectures that resemble the human embryonic brain. Recent studies have shown the development of optic cups in brain organoids. The cellular components of a developing optic vesicle-containing organoids include primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. The importance of retinal organoids in ocular diseases such as age-related macular degeneration, Stargardt disease, retinitis pigmentosa, and diabetic retinopathy are described in this review. This review highlights current developments in retinal organoid techniques, and their applications in ocular conditions such as disease modeling, gene therapy, drug screening and development. In addition, recent advancements in utilizing extracellular vesicles secreted by retinal organoids for ocular disease treatments are summarized.

The significant roles of extracellular vesicles (EVs) as intracellular mediators, disease biomarkers, and therapeutic agents, make them a scientific hotspot. In particular, EVs secreted by human stem cells show significance in treating neurological disorders, such as Alzheimer’s disease and ischemic stroke. However, the clinical applications of EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration. Superparamagnetic iron oxide (SPIO) nanoparticles are biocompatible and have been shown to improve the targeting ability of EVs. In particular, ultrasmall SPIO (USPIO, <50 nm) are more suitable for labeling nanoscale EVs due to their small size. In this study, induced forebrain neural progenitor cortical organoids (iNPCo) were differentiated from human induced pluripotent stem cells (iPSCs), and the iNPCo expressed FOXG1, Nkx2.1, α-catenin, as well as β-tubulin III. EVs were isolated from iNPCo media, then loaded with USPIOs by sonication. Size and concentration of EV particles were measured by nanoparticle tracking analysis, and no significant changes were observed in size distribution before and after sonication, but the concentration decreased after labeling. miR-21 and miR-133b decreased after sonication. Magnetic resonance imaging (MRI) demonstrated contrast visualized for the USPIO labeled EVs embedded in agarose gel phantoms. Upon calculation, USPIO labeled EVs exhibited considerably shorter relaxation times, quantified as T₂ and T₂^* values, reducing the signal intensity and generating higher MRI contrast compared to unlabeled EVs and gel only. Our study demonstrated that USPIO labeling was a feasible approach for in vitro tracking of brain organoid-derived EVs, which paves the way for further in vivo examination.

The choroid plexus (ChP) is a complex structure in the human brain that is responsible for the secretion of cerebrospinal fluid (CSF) and forming the blood–CSF barrier (B-CSF-B). Human-induced pluripotent stem cells (hiPSCs) have shown promising results in the formation of brain organoids in vitro; however, very few studies to date have generated ChP organoids. In particular, no study has assessed the inflammatory response and the extracellular vesicle (EV) biogenesis of hiPSC-derived ChP organoids. In this study, the impacts of Wnt signaling on the inflammatory response and EV biogenesis of ChP organoids derived from hiPSCs was investigated. During days 10–15, bone morphogenetic protein 4 was added along with (+/−) CHIR99021 (CHIR, a small molecule GSK-3β inhibitor that acts as a Wnt agonist). At day 30, the ChP organoids were characterized by immunocytochemistry and flow cytometry for TTR (~72%) and CLIC6 (~20%) expression. Compared to the −CHIR group, the +CHIR group showed an upregulation of 6 out of 10 tested ChP genes, including CLIC6 (2-fold), PLEC (4-fold), PLTP (2–4-fold), DCN (~7-fold), DLK1 (2–4-fold), and AQP1 (1.4-fold), and a downregulation of TTR (0.1-fold), IGFBP7 (0.8-fold), MSX1 (0.4-fold), and LUM (0.2–0.4-fold). When exposed to amyloid beta 42 oligomers, the +CHIR group had a more sensitive response as evidenced by the upregulation of inflammation-related genes such as TNFα, IL-6, and MMP2/9 when compared to the −CHIR group. Developmentally, the EV biogenesis markers of ChP organoids showed an increase over time from day 19 to day 38. This study is significant in that it provides a model of the human B-CSF-B and ChP tissue for the purpose of drug screening and designing drug delivery systems to treat neurological disorders such as Alzheimer’s disease and ischemic stroke.

Background: Choroid plexus is a complex structure in the human brain that is responsible for the secretion of extracellular vesicles (EVs) in cerebrospinal fluid. Few studies to date have generated choroid plexus (ChP) organoids differentiated from human induced pluripotent stem cells (hiPSCs) and analyzed their secreted EVs. The scalable Vertical-Wheel bioreactors (VWBRs) provide low shear stress and a controlled environment. Methods: This study utilized VWBRs for the differentiation of hiPSCs into ChP organoids and generation of the secreted EVs compared to a static culture. Additionally, this study loaded curcumin into ChP organoid-derived EVs, performed EV lyophilization, and determined the ability of the re-hydrated EVs to alleviate neuro-inflammation. Results: The results demonstrated that the VWBR culture exhibited more aerobic metabolism and active glucose and glutamine consumption than the static control. Consequently, the ChP markers and Endosomal Sorting Complexes Required for Transport-dependent and -independent EV biogenesis genes were significantly upregulated (2–3-fold) in the VWBR, producing four-fold-higher EVs per mL media than the static control. The EVs retained similar size and zeta potential after lyophilization and re-hydration. The cells exposed to amyloid beta 42 oligomers and treated with the curcumin-loaded re-hydrated EVs showed high viability and the reduced inflammatory response determined by TNF-α and IL-6 expression. Conclusions: This study demonstrates a scalable bioreactor system to promote ChP organoid differentiation and generation of EV-based cell-free therapeutics to treat neural inflammation in various neurological disorders.