RESUMO
The fabrication of microfluidic devices has progressed from cleanroom manufacturing to replica molding in polymers, and more recently to direct manufacturing by subtractive (e.g., laser machining) and additive (e.g., 3D printing) techniques, notably digital light processing (DLP) photopolymerization. However, many methods require technical expertise and DLP 3D printers remain expensive at a cost â¼15-30 K USD with â¼8 M pixels that are 25-40 µm in size. Here, we introduce (i) the use of low-cost (â¼150-600 USD) liquid crystal display (LCD) photopolymerization 3D printing with â¼8-58 M pixels that are 18-35 µm in size for direct microfluidic device fabrication, and (ii) a poly(ethylene glycol) diacrylate-based ink developed for LCD 3D printing (PLInk). We optimized PLInk for high resolution, fast 3D printing and biocompatibility while considering the illumination inhomogeneity and low power density of LCD 3D printers. We made lateral features as small as 75 µm, 22 µm-thick embedded membranes, and circular channels with a 110 µm radius. We 3D printed microfluidic devices previously manufactured by other methods, including an embedded 3D micromixer, a membrane microvalve, and an autonomous capillaric circuit (CC) deployed for interferon-γ detection with excellent performance (limit of detection: 12 pg mL-1, CV: 6.8%). We made PLInk-based organ-on-a-chip devices in 384-well plate format and produced 3420 individual devices within an 8 h print run. We used the devices to co-culture two spheroids separated by a vascular barrier over 5 days and observed endothelial sprouting, cellular reorganization, and migration. LCD 3D printing together with tailored inks pave the way for democratizing access to high-resolution manufacturing of ready-to-use microfluidic and organ-on-a-chip devices by anyone, anywhere.
Assuntos
Dispositivos Lab-On-A-Chip , Cristais Líquidos , Impressão Tridimensional , Cristais Líquidos/química , Humanos , Polietilenoglicóis/química , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas MicrofisiológicosRESUMO
Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.
Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Cromatografia em Gel , Proteínas/análise , Ultracentrifugação/métodosRESUMO
Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80 µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink are tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility is assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D-printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering and 3D cell culture as demonstrated here, and possibly in OoC, implants, wearables, and soft robotics.
Assuntos
Elastômeros , Engenharia Tecidual , Elastômeros/química , Impressão TridimensionalRESUMO
Constant matrix remodeling and cellular heterogeneity in cancer are key contributors to its development and can profoundly alter treatment efficacy. Developingin-vitromodels containing relevant features that can recapitulate these aspects of the tumor microenvironment and that are well characterized can circumvent the limitations of conventional 2D cultures and animal models. Automated fabrication methods combined with biomimetic biomaterials have provided the opportunity to create platforms that can potentially incorporate a heterogeneous population of cells in a 3D environment that allows cell-cell and cell-ECM interactions with reproducibility. This study used 3D extrusion bioprinting and a composite bioink containing a reinforced decellularized extracellular matrix (ECM) hydrogel to fabricate a head and neck cancerin-vitromodel. The constituents of this model included fibroblasts and active ECM proteins to represent the stroma, along with HNSCC cells to represent the tumor component. The topographical characterization of the bioink showed a fibrous network with nanometer-sized pores. After cell encapsulation and model fabrication, we observed spheroid development and growth over time with cancer cells in the core and fibroblasts in the periphery. Our model is compatible with matrix metalloproteinase (MMP) quantification techniques and showed significant differences in the presence of MMP-9 and MMP-10 compared to the control groups. This characterized model is proposed as a tool for further translational and drug discovery applications since it provides a biomimetic scenario that allows the study of the tumor microenvironmentin-vitrousing nondestructive longitudinal monitoring over time.
Assuntos
Bioimpressão , Neoplasias , Animais , Alicerces Teciduais , Engenharia Tecidual/métodos , Matriz Extracelular Descelularizada , Reprodutibilidade dos Testes , Matriz Extracelular/metabolismo , Neoplasias/metabolismo , Bioimpressão/métodos , Impressão Tridimensional , Microambiente TumoralRESUMO
Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.