RESUMO
Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials for treating injury or disease.
Assuntos
Células Endoteliais , Hipóxia , Humanos , Cápsulas , Células Endoteliais/metabolismo , Materiais Biocompatíveis , Hidrogéis , Oxigênio/metabolismoRESUMO
The practical application of microfluidic liver models for in vitro drug testing is partly hampered by their reliance on human primary hepatocytes, which are limited in number and have batch-to-batch variation. Human stem cell-derived hepatocytes offer an attractive alternative cell source, although their 3D differentiation and maturation in a microfluidic platform have not yet been demonstrated. We develop a pump-free microfluidic 3D perfusion platform to achieve long-term and efficient differentiation of human liver progenitor cells into hepatocyte-like cells (HLCs). The device contains a micropillar array to immobilize cells three-dimensionally in a central cell culture compartment flanked by two side perfusion channels. Constant pump-free medium perfusion is accomplished by controlling the differential heights of horizontally orientated inlet and outlet media reservoirs. Computational fluid dynamic simulation is used to estimate the hydrostatic pressure heads required to achieve different perfusion flow rates, which are experimentally validated by micro-particle image velocimetry, as well as viability and functional assessments in a primary rat hepatocyte model. We perform on-chip differentiation of HepaRG, a human bipotent progenitor cell, and discover that 3D microperfusion greatly enhances the hepatocyte differentiation efficiency over static 2D and 3D cultures. However, HepaRG progenitor cells are highly sensitive to the time-point at which microperfusion is applied. Isolated HepaRG cells that are primed as static 3D spheroids before being subjected to microperfusion yield a significantly higher proportion of HLCs (92%) than direct microperfusion of isolated HepaRG cells (62%). This platform potentially offers a simple and efficient means to develop highly functional microfluidic liver models incorporating human stem cell-derived HLCs. Biotechnol. Bioeng. 2017;114: 2360-2370. © 2017 Wiley Periodicals, Inc.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Diferenciação Celular/fisiologia , Hepatócitos/fisiologia , Dispositivos Lab-On-A-Chip , Técnicas de Cultura de Órgãos/instrumentação , Perfusão/instrumentação , Células-Tronco/fisiologia , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Hepatócitos/citologia , Humanos , Técnicas de Cultura de Órgãos/métodos , Células-Tronco/citologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
Osteoarthritis (OA) is a debilitating degenerative disease affecting multiple joint tissues, including cartilage, bone, synovium, and adipose tissues. OA presents diverse clinical phenotypes and distinct molecular endotypes, including inflammatory, metabolic, mechanical, genetic, and synovial variants. Consequently, innovative technologies are needed to support the development of effective diagnostic and precision therapeutic approaches. Traditional analysis of bulk OA tissue extracts has limitations due to technical constraints, causing challenges in the differentiation between various physiological and pathological phenotypes in joint tissues. This issue has led to standardization difficulties and hindered the success of clinical trials. Gaining insights into the spatial variations of the cellular and molecular structures in OA tissues, encompassing DNA, RNA, metabolites, and proteins, as well as their chemical properties, elemental composition, and mechanical attributes, can contribute to a more comprehensive understanding of the disease subtypes. Spatially resolved biology enables biologists to investigate cells within the context of their tissue microenvironment, providing a more holistic view of cellular function. Recent advances in innovative spatial biology techniques now allow intact tissue sections to be examined using various -omics lenses, such as genomics, transcriptomics, proteomics, and metabolomics, with spatial data. This fusion of approaches provides researchers with critical insights into the molecular composition and functions of the cells and tissues at precise spatial coordinates. Furthermore, advanced imaging techniques, including high-resolution microscopy, hyperspectral imaging, and mass spectrometry imaging, enable the visualization and analysis of the spatial distribution of biomolecules, cells, and tissues. Linking these molecular imaging outputs to conventional tissue histology can facilitate a more comprehensive characterization of disease phenotypes. This review summarizes the recent advancements in the molecular imaging modalities and methodologies for in-depth spatial analysis. It explores their applications, challenges, and potential opportunities in the field of OA. Additionally, this review provides a perspective on the potential research directions for these contemporary approaches that can meet the requirements of clinical diagnoses and the establishment of therapeutic targets for OA.
Assuntos
Osteoartrite , Humanos , Osteoartrite/diagnóstico , Membrana Sinovial/metabolismo , Metabolômica , Fenótipo , ProteômicaRESUMO
Microfluidic devices with open lattice structures, equivalent to a type of porous media, allow for the manipulation of fluid transport processes while having distinct structural, mechanical, and thermal properties. However, a fundamental understanding of the design principles for the solid structure in order to achieve consistent and desired flow patterns remains a challenge, preventing its further development and wider applications. Here, through quantitative and mechanistic analyses of the behavior of multi-phase phenomena that involve gas-liquid-solid interfaces, we present a design framework for microfluidic devices containing porous architectures (referred to as poroFluidics) for deterministic control of multi-phase fluid transport processes. We show that the essential properties of the fluids and solid, including viscosity, interfacial tension, wettability, as well as solid manufacture resolution, can be incorporated into the design to achieve consistent flow in porous media, where the desired spatial and temporal fluid invasion sequence can be realized. Experiments and numerical simulations reveal that different preferential flow pathways can be controlled by solid geometry, flow conditions, or fluid/solid properties. Our design framework enables precise, multifunctional, and dynamic control of multi-phase transport within engineered porous media.
RESUMO
Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment surrounding the joint tissue. Recent development in microfluidic organ-on-chip (OoC) systems have demonstrated various techniques to mimic and modulate tissue physiological environments. Adaptations of these techniques have demonstrated success in capturing a joint tissue's tissue physiology for studying the mechanism of OA. Adapting these techniques and strategies can help create human-specific in vitro models that recapitulate the cellular processes involved in OA. This review aims to comprehensively summarise various demonstrations of microfluidic platforms in mimicking joint microenvironments for future platform design iterations.
Assuntos
Osteoartrite , Qualidade de Vida , Animais , Humanos , Microfluídica/métodos , Modelos AnimaisRESUMO
Organs-on-a-chip, or OoCs, are microfluidic tissue culture devices with micro-scaled architectures that repeatedly achieve biomimicry of biological phenomena. They are well positioned to become the primary pre-clinical testing modality as they possess high translational value. Current methods of fabrication have facilitated the development of many custom OoCs that have generated promising results. However, the reliance on microfabrication and soft lithographic fabrication techniques has limited their prototyping turnover rate and scalability. Additive manufacturing, known commonly as 3D printing, shows promise to expedite this prototyping process, while also making fabrication easier and more reproducible. We briefly introduce common 3D printing modalities before identifying two sub-types of vat photopolymerization - stereolithography (SLA) and digital light processing (DLP) - as the most advantageous fabrication methods for the future of OoC development. We then outline the motivations for shifting to 3D printing, the requirements for 3D printed OoCs to be competitive with the current state of the art, and several considerations for achieving successful 3D printed OoC devices touching on design and fabrication techniques, including a survey of commercial and custom 3D printers and resins. In all, we aim to form a guide for the end-user to facilitate the in-house generation of 3D printed OoCs, along with the future translation of these important devices.
Assuntos
Sistemas Microfisiológicos , Impressão Tridimensional , Estereolitografia , Microtecnologia , Dispositivos Lab-On-A-ChipRESUMO
Inter-patient and intra-tumour heterogeneity (ITH) have prompted the need for a more personalised approach to cancer therapy. Although patient-derived xenograft (PDX) models can generate drug response specific to patients, they are not sustainable in terms of cost and time and have limited scalability. Tumour Organ-on-Chip (OoC) models are in vitro alternatives that can recapitulate some aspects of the 3D tumour microenvironment and can be scaled up for drug screening. While many tumour OoC systems have been developed to date, there have been limited validation studies to ascertain whether drug responses obtained from tumour OoCs are comparable to those predicted from patient-derived xenograft (PDX) models. In this study, we established a multiplexed tumour OoC device, that consists of an 8 × 4 array (32-plex) of culture chamber coupled to a concentration gradient generator. The device enabled perfusion culture of primary PDX-derived tumour spheroids to obtain dose-dependent response of 5 distinct standard-of-care (SOC) chemotherapeutic drugs for 3 colorectal cancer (CRC) patients. The in vitro efficacies of the chemotherapeutic drugs were rank-ordered for individual patients and compared to the in vivo efficacy obtained from matched PDX models. We show that quantitative correlation analysis between the drug efficacies predicted via the microfluidic perfusion culture is predictive of response in animal PDX models. This is a first study showing a comparative framework to quantitatively correlate the drug response predictions made by a microfluidic tumour organ-on-chip (OoC) model with that of PDX animal models.
RESUMO
Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.
RESUMO
Multi-organ perfusion systems offer the unique opportunity to mimic different physiological systemic interactions. However, existing multi-organ culture platforms have limited flexibility in specifying the culture conditions, device architectures, and fluidic connectivity simultaneously. Here, we report a modular microfluidic platform that addresses this limitation by enabling easy conversion of existing microfluidic devices into tissue and fluid control modules with self-aligning magnetic interconnects. This enables a 'stick-n-play' approach to assemble planar perfusion circuits that are amenable to both bioimaging-based and analytical measurements. A myriad of tissue culture and flow control TILE modules were successfully constructed with backward compatibility. Finally, we demonstrate applications in constructing recirculating multi-organ systems to emulate liver-mediated bioactivation of nutraceuticals and prodrugs to modulate their therapeutic efficacies in the context of atherosclerosis and cancer. This platform greatly facilitates the integration of existing organs-on-chip models to provide an intuitive and flexible way for users to configure different multi-organ perfusion systems.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Técnicas de Cultura de Órgãos , Suplementos Nutricionais , Humanos , Fígado/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/instrumentação , Pró-Fármacos/farmacologiaRESUMO
The advent of 3D printing technologies promises to make microfluidic organ-on-chip technologies more accessible for the biological research community. To date, hydrogel-encapsulated cells have been successfully incorporated into 3D printed microfluidic devices. However, there is currently no 3D printed microfluidic device that can support multicellular spheroid culture, which facilitates extensive cell-cell contacts important for recapitulating many multicellular functional biological structures. Here, we report a first instance of fabricating a 3D printed microfluidic cell culture device capable of directly immobilizing and maintaining the viability and functionality of 3D multicellular spheroids. We evaluated the feasibility of two common 3D printing technologies i.e. stereolithography (SLA) and PolyJet printing, and found that SLA could prototype a device comprising of cell immobilizing micro-structures that were housed within a microfluidic network with higher fidelity. We have also implemented a pump-free perfusion system, relying on gravity-driven flow to perform medium perfusion in order to reduce the complexity and footprint of the device setup, thereby improving its adaptability into a standard biological laboratory. Finally, we demonstrated the biological performance of the 3D printed device by performing pump-free perfusion cultures of patient-derived parental and metastatic oral squamous cell carcinoma tumor and liver cell (HepG2) spheroids with good cell viability and functionality. This paper presents a proof-of-concept in simplifying and integrating the prototyping and operation of a microfluidic spheroid culture device, which will facilitate its applications in various drug efficacy, metabolism and toxicity studies.