1,721,045 research outputs found
RECAPITULATING DENDRITIC CELL CHEMOTAXIS TOWARD LYMPHATIC VESSEL USING IN VITRO HUMAN 3D INFLAMMATION MODEL
No full textDendritic cell (DC) homing through lymphatic vessels (LVs) from peripheral tissue to lymph node (LN) plays an important role in the initial stage of immunity and inflammation. However, few in vitro models are designed to study the dynamic interactions between DCs and LVs in 3D under various inflammatory conditions. Here, we introduce a 3D LV microfluidic model compatible with the analysis of DC motility in various inflammatory conditions. Moreover, we interpret this observation focusing on the change in CCR7/CCL21 expression, a key molecular axis in DC chemotaxis. This platform shows the potential to evaluate immunomodulators for inflammatory diseases and cancers.N
High-Throughput Microfluidic 3D Cytotoxicity Assay for Cancer Immunotherapy (CACI-IMPACT Platform)
No full textAdoptive cell transfer against solid tumors faces challenges to overcome tumor microenvironment (TME), which plays as a physical barrier and provides immuno-suppressive conditions. Classical cytotoxicity assays are widely used to measure killing ability of the engineered cytotoxic lymphocytes as therapeutics, but the results cannot represent the performance in clinical application due to the absence of the TME. This paper describes a 3D cytotoxicity assay using an injection molded plastic array culture (CACI-IMPACT) device for 3D cytotoxicity assay to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Rail-based microfluidic design was integrated within a single 96-well and the wells were rectangularly arrayed in 2 x 6 to enhance the experimental throughput. The rail-based microstructures facilitate hydrogel patterning with simple pipetting so that hydrogel pre-solution aspirated with 10 mu l pipette can be patterned in 10 wells within 30s. To demonstrate 3D cytotoxicity assay, we patterned HeLa cells encapsulated by collagen gel and observed infiltration, migration and cytotoxic activity of NK-92 cells against HeLa cells in the collagen matrix. We found that 3D ECM significantly reduced migration of cytotoxic lymphocytes and access to cancer cells, resulting in lower cytotoxicity compared with 2D assays. In dense ECM, the physical barrier function of the 3D matrix was enhanced, but the cytotoxic lymphocytes effectively killed cancer cells once they contacted with cancer cells. The results implied ECM significantly influences migration and cytotoxicity of cytotoxic lymphocytes. Hence, the CACI-IMPACT platform, enabling high-throughput 3D co-culture of cytotoxic lymphocyte with cancer cells, has the potential to be used for pre-clinical evaluation of cytotoxic lymphocytes engineered for immunotherapy against solid tumors.Y
Monolithic digital patterning of polydimethylsiloxane with successive laser pyrolysis
No full text© 2020, The Author(s), under exclusive licence to Springer Nature Limited.The patterning of polydimethylsiloxane (PDMS) into complex two-dimensional (2D) or 3D shapes is a crucial step for diverse applications based on soft lithography. Nevertheless, mould replication that incorporates time-consuming and costly photolithography processes still remains the dominant technology in the field. Here we developed monolithic quasi-3D digital patterning of PDMS using laser pyrolysis. In contrast with conventional burning or laser ablation of transparent PDMS, which yields poor surface properties, our successive laser pyrolysis technique converts PDMS into easily removable silicon carbide via consecutive photothermal pyrolysis guided by a continuous-wave laser. We obtained high-quality 2D or 3D PDMS structures with complex patterning starting from a PDMS monolith in a remarkably low prototyping time (less than one hour). Moreover, we developed distinct microfluidic devices with elaborated channel architectures and a customizable organ-on-a-chip device using this approach, which showcases the potential of the successive laser pyrolysis technique for the fabrication of devices for several technological applications.N
Nanogrooved substrate promotes direct lineage reprogramming of fibroblasts to functional induced dopaminergic neurons
No full textThe generation of dopaminergic (DA) neurons via direct lineage reprogramming can potentially provide a novel therapeutic platform for the study and treatment of Parkinson's disease. Here, we showed that nanoscale biophysical stimulation can promote the direct lineage reprogramming of somatic fibroblasts to induced DA (iDA) neurons. Fibroblasts that were cultured on flat, microgrooved, and nanogrooved substrates responded differently to the patterned substrates in terms of cell alignment. Subsequently, the DA marker expressions, acquisition of mature DA neuronal phenotypes, and the conversion efficiency were enhanced mostly on the nanogrooved substrate. These results may be attributed to specific histone modifications and transcriptional changes associated with mesenchymal-to-epithelial transition. Taken together, these results suggest that the nanopatterned substrate can serve as an efficient stimulant for direct lineage reprogramming to iDA neurons, and its effectiveness confirms that substrate nanotopography plays a critical role in the cell fate changes during direct lineage reprogramming. (C) 2014 Elsevier Ltd. All rights reserved.N
All-in-one microfluidic design to integrate vascularized tumor spheroid into high-throughput platform
No full textThe development of a scalable and highly reproducible in vitro tumor microenvironment (TME) platform still sheds light on new insights into cancer metastasis mechanisms and anticancer therapeutic strategies. Here, we present an all-in-one injection molded plastic array three-dimensional culture platform (All-in-One-IMPACT) that integrates vascularized tumor spheroids for highly reproducible, high-throughput experimentation. This device allows the formation of self-assembled cell spheroids on a chip by applying the hanging drop method to the cell culture channel. Then, when the hydrogel containing endothelial cells and fibroblasts is injected, the spheroid inside the droplet can be patterned together in three dimensions along the culture channel. In just two steps above, we can build a vascularized TME within a defined area. This process does not require specialized user skill and minimizes error-inducing steps, enabling both reproducibility and high throughput of the experiment. We have successfully demonstrated the process, from spheroid formation to tumor vascularization, using patient-derived cancer cells (PDCs) as well as various cancer cell lines. Furthermore, we performed combination therapies with Taxol (paclitaxel) and Avastin (bevacizumab), which are used in standard care for metastatic cancer. The All-in-One IMPACT is a powerful tool for establishing various anticancer treatment strategies through the development of a complex TME for use in high-throughput experiments.N
A microphysiological system-based potency bioassay for the functional quality assessment of mesenchymal stromal cells targeting vasculogenesis
No full textMesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculo-genesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a sig-nificant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.Y
High-Throughput Microfluidic Platform for Real-time Investigation of Lipid Droplet Accumulation in Microalgae
Full text link학위논문 (석사)-- 서울대학교 대학원 : 기계항공공학부, 2014. 2. Jeon Noo Li.Microalgae offer great promise to contribute a significant portion of the renewable fuels that is required by the Renewable Fuels Standard. Algal biofuels is based mainly on the high lipid content of the algal cells and thus would be an ideal feedstock for high energy density transportation fuels, such as biodiesel, green diesel, green jet fuel and green gasoline. With high lipid productivity of dominant, fast-growing algae is a major prerequisite for commercial production of microalgae oil-derived biodiesel. However, under optimal growth conditions, large amounts of algal biomass are produced, but with relatively low lipid contents. Meanwhile, species with high lipid contents are typically slow growing. Currently, the single cells observation and quantification of lipid accumulation after the stationary growth phase under various stress conditions is still a challenge. To solve this issue, we have conducted the microfluidic platform to investigate the development of lipid droplet in individual microalgae, Chlamydomonas reinhardtii by immobilizing monolayer cells on the glass surface coated with gelatin. In addition, our novel platform able to eliminate the absorption of BODIPY fluorescence into the polydimethylsiloxane (PDMS) microchannel and also the media can be changed easily. In the end, the lipid droplet accumulation was observed in-real-time at the single cell resolution under different conditions of light and nutrient, allowing the correlations among lipid trigger conditions and lipid production, as evidenced with BODIPY 505/515 fluorescence lipid staining.
Keywords: Microalgae, Microfluidic, Single cell immobilization, Lipids accumulation, Real–time observation, Glass-PDMS sandwich deviceAbstract i
Contents iii
List of Figures v
1. Introduction 1
1.1 Microfluidics and cell biology 1
1.2 Current applications of microfluidic in microalgae research 2
1.3 Motivation and objective 5
2. Methods and Materials 6
2.1 Device design and simulation 6
2.2 Photolithography 6
2.3 Chemicals and materials 7
2.4 Chlamydomonas reinhardtii culture 8
2.5 Surface immobilization of C.reinhardtii 9
2.6 Viability assay 10
3. Microalgae dynamic assay for lipid accumulation study 20
3.1 Glass-PDMS sandwich device fabrication 20
3.2 System installation 21
3.3 Lipid body accumulation 23
3.4 Lipid droplet staining dye and solution preparation25
3.5 BODIPY absorption into PDMS 26
3.6 Cell imaging and data analysis 27
4. Results and Discussion 28
5. Conclusions 32
References 34
Abstract (Korean)37
Acknowledgement 39Maste
3D Microphysiological System-Inspired Scalable Vascularized Tissue Constructs for Regenerative Medicine
No full textMicrophysiological systems (MPSs), based on microfabrication technologies and cell culture, can faithfully recapitulate the complex physiology of various tissues. However, 3D tissues formed using MPS have limitations in size and accessibility; their use in regenerative medicine is, therefore, still challenging. Here, an MPS-inspired scale-up vascularized engineered tissue construct that can be used in regenerative medicine is designed. Endothelial cell-laden hydrogels are sandwiched between two through-hole membranes. The microhole array in the through-hole membranes enables the molecular transport across the hydrogel layer, allowing long-term cell culture. Furthermore, the time-controlled delamination of through-hole membranes enables the harvesting of cell-cultured hydrogel constructs without damaging the capillary network. Importantly, when the tissue constructs are implanted in a mouse ischemic model, they protect against necrosis and promoted functional recovery to a greater extent than implanted cells, hydrogels, and simple gel-cell mixtures.N
Enhanced bone repair by guided osteoblast recruitment using topographically defined implant
No full textThe rapid recruitment of osteoblasts in bone defects is an essential prerequisite for efficient bone repair. Conventionally, osteoblast recruitment to bone defects and subsequent bone repair has been achieved using growth factors. Here, we present a methodology that can guide the recruitment of osteoblasts to bone defects with topographically defined implants (TIs) for efficient in vivo bone repair. We compared circular TIs that had microgrooves in parallel or radial arrangements with nonpatterned implants for osteoblast migration and in vivo bone formation. In vitro, the microgrooves in the TIs enhanced both the migration and proliferation of osteoblasts. Especially, the microgrooves with radial arrangement demonstrated a much higher efficiency of osteoblast recruitment to the implants than did the other types of implants, which may be due to the efficient guidance of cell migration toward the cell-free area of the implants. The expression of the intracellular signaling molecules responsible for the cell migration was also upregulated in osteoblasts on the microgrooved TIs. In vivo, the TI with radially defined topography demonstrated much greater bone repair in mouse calvarial defect models than in the other types of implants. Taken together, these results indicate that implants with physical guidance can enhance tissue repair by rapid cell recruitment.Y
Biocompatible Cost‐Effective Electrophysiological Monitoring with Oxidation‐Free Cu–Au Core–Shell Nanowire
No full textIn spite of its excellent electrical, mechanical, and low-cost characteristics, copper nanowire has fatal issues in the oxidation problem and the lack of biological compatibility, which occasionally outweighs its advantages and limits its usage as electronics or biodevice applications. In this study, a novel wet chemical synthesis method is developed for the oxidation-free Cu-Au core-shell nanowire based on the prepared Cu nanowire with alkylamine-mediated synthesis and ligand exchange. The synthesized Cu-Au core-shell nanowire exhibits improved electrical stability against thermal oxidation under the harsh environment of 80 degrees C and 80% relative humidity. Additionally, to substantiate suitability for the biomedical application, the enhanced chemical stability and biocompatibility are investigated by utilizing the artificial perspiration and the cell culture. As a proof-of-concept demonstration, high performance wearable electromyogram (EMG), electrocardiogram (ECG) sensors for electrophysiological monitoring with the Cu-Au core-shell nanowire electrode are demonstrated with superior oxidation-resistance and biocompatibility even after the harsh environment test. The Cu-Au core-shell nanowire can provide promising, cost-effective electrode materials for various wearable electronics applications.Y
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