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1.
This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.  相似文献   

2.
We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 μm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 μm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (β-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 μm spheroids was hindered by the treatment with VEGF and β-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and β-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.  相似文献   

3.
Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.  相似文献   

4.
Understanding the mechanism behind cancer metastasis is a major challenge in cancer biology. Several in vitro models have been developed to mimic a cancer microenvironment by engineering cancer–endothelial cell (EC) and cancer-stromal cell interactions. It has been challenging to realistically mimic angiogenesis, intravasation, and extravasation using macro-scale approaches but recent progress in microfluidics technology has begun to yield promising results. We present a metastasis chip that produce microvessels, where EC and stromal cells can be patterned in close proximity to tumor cells. The vessels are formed following a natural morphogenic process and have smooth boundaries with proper cell-cell junctions. The engineered microvessels are perfusable and have well-defined openings toward inlet and outlet channels. The ability to introduce cancer cells into different locations bordering to the microvessel wall allowed generation and maintenance of appropriate spatial gradients of growth factors and attractants. Cancer angiogenesis and its inhibition by anti-vascular endothelial growth factor (bevacizumab) treatment were successfully reproduced in the metastasis chip. Cancer intravasation and its modulation by treatment of tumor necrosis factor-α were also modeled. Compared to other models, the unique design of the metastasis chip that engineers a clear EC-cancer interface allows precise imaging and quantification of angiogenic response as well as tumor cell trans-endothelial migration. The metastasis chip presented here has potential applications in the investigation of fundamental cancer biology as well as in drug screening.  相似文献   

5.
Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.  相似文献   

6.
The majority of cancer deaths are linked to tumor spread, or metastasis, but 3D in vitro metastasis models relevant to the tumor microenvironment (including interstitial fluid flow) remain an area of unmet need. Microfluidics allows us to introduce controlled flow to an in vitro cancer model to better understand the relationship between flow and metastasis. Here, we report new hybrid spheroid-on-chip in vitro models for the impact of interstitial fluid flow on cancer spread. We designed a series of reusable glass microfluidic devices to contain one spheroid in a microwell under continuous perfusion culture. Spheroids derived from established cancer cell lines were perfused with complete media at a flow rate relevant to tumor interstitial fluid flow. Spheroid viability and migratory/invasive capabilities were maintained on-chip when compared to off-chip static conditions. Importantly, using flow conditions modeled in vitro, we are the first to report flow-induced secretion of pro-metastatic factors, in this case cytokines vascular endothelial growth factor and interleukin 6. In summary, we have developed a new, streamlined spheroid-on-chip in vitro model that represents a feasible in vitro alternative to conventional murine in vivo metastasis assays, including complex tumor environmental factors, such as interstitial fluid flow, extracellular matrices, and using 3D models to model nutrient and oxygen gradients. Our device, therefore, constitutes a robust alternative to in vivo early-metastasis models for determination of novel metastasis biomarkers as well as evaluation of therapeutically relevant molecular targets not possible in in vivo murine models.  相似文献   

7.
Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the host''s vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells.  相似文献   

8.
Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.  相似文献   

9.
Lee K  Kim C  Young Yang J  Lee H  Ahn B  Xu L  Yoon Kang J  Oh KW 《Biomicrofluidics》2012,6(1):14114-141147
We propose a simple method for forming massive and uniform three-dimensional (3-D) cell spheroids in a multi-level structured microfluidic device by gravitational force. The concept of orienting the device vertically has allowed spheroid formation, long-term perfusion, and retrieval of the cultured spheroids by user-friendly standard pipetting. We have successfully formed, perfused, and retrieved uniform, size-controllable, well-conditioned spheroids of human embryonic kidney 293 cells (HEK 293) in the gravity-oriented microfluidic device. We expect the proposed method will be a useful tool to study in-vitro 3-D cell models for the proliferation, differentiation, and metabolism of embryoid bodies or tumours.  相似文献   

10.
The 3D multicellular spheroids with intact cell–cell junctions have major roles in biological research by virtue of their unique advantage of mimicking the cellular physiological environments. In this work, a durable superamphiphobic silica aerogel surface (SSAS) has been fabricated for the upward culture of 3D multicellular spheroids. Poly(3,4-ethylenedioxythiophene) (PEDOT) was first electrodeposited on a conductive steel mesh as a first template for porous silica coating. Soot particles were then applied as a second template to construct a cauliflower-like silica aerogel nanostructure. After fluorination, a hierarchical structure with re-entrant curvature was finally fabricated as a durable superamphiphobic surface. This superamphiphobic surface also presented excellent antifouling towards biomacromolecules and cells, which has been demonstrated by the successful upward culture of cell spheroids. The upward culture makes the observation of cellular behavior in situ possible, holding great potential for 3D cellular evaluation in vitro.  相似文献   

11.
Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.  相似文献   

12.
Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.  相似文献   

13.
The aorta, with ascending, arch, thoracic and abdominal segments, responds to the heartbeat, senses metabolites and distributes blood to all parts of the body. However, the heterogeneity across aortic segments and how metabolic pathologies change it are not known. Here, a total of 216 612 individual cells from the ascending aorta, aortic arch, and thoracic and abdominal segments of mouse aortas under normal conditions or with high blood glucose levels, high dietary salt, or high fat intake were profiled using single-cell RNA sequencing. We generated a compendium of 10 distinct cell types, mainly endothelial (EC), smooth muscle (SMC), stromal and immune cells. The distributions of the different cells and their intercommunication were influenced by the hemodynamic microenvironment across anatomical segments, and the spatial heterogeneity of ECs and SMCs may contribute to differential vascular dilation and constriction that were measured by wire myography. Importantly, the composition of aortic cells, their gene expression profiles and their regulatory intercellular networks broadly changed in response to high fat/salt/glucose conditions. Notably, the abdominal aorta showed the most dramatic changes in cellular composition, particularly involving ECs, fibroblasts and myeloid cells with cardiovascular risk factor-related regulons and gene expression networks. Our study elucidates the nature and range of aortic cell diversity, with implications for the treatment of metabolic pathologies.  相似文献   

14.
Wound healing is an essential physiological process for tissue homeostasis, involving multiple types of cells, extracellular matrices, and growth factor/chemokine interactions. Many in vitro studies have investigated the interactions between cues mentioned above; however, most of them only focused on a single factor. In the present study, we design a wound healing device to recapitulate in vivo complex microenvironments and heterogeneous cell situations to investigate how three types of physiologically related cells interact with their microenvironments around and with each other during a wound healing process. Briefly, a microfluidic device with a micropillar substrate, where diameter and interspacing can be tuned to mimic the topographical features of the 3D extracellular matrix, was designed to perform positional cell loading on the micropillar substrate, co-culture of three types of physiologically related cells, keratinocytes, dermal fibroblasts, and human umbilical vein endothelial cells, as well as an investigation of their interactions during wound healing. The result showed that cell attachment, morphology, cytoskeleton distribution, and nucleus shape were strongly affected by the micropillars, and these cells showed collaborative response to heal the wound. Taken together, these findings highlight the dynamic relationship between cells and their microenvironments. Also, this reproducible device may facilitate the in vitro investigation of numerous physiological and pathological processes such as cancer metastasis, angiogenesis, and tissue engineering.  相似文献   

15.
Cell migration is an essential process involved in the development and maintenance of multicellular organisms. Electric fields (EFs) are one of the many physical and chemical factors known to affect cell migration, a phenomenon termed electrotaxis or galvanotaxis. In this paper, a microfluidics chip was developed to study the migration of cells under different electrical and chemical stimuli. This chip is capable of providing four different strengths of EFs in combination with two different chemicals via one simple set of agar salt bridges and Ag/AgCl electrodes. NIH 3T3 fibroblasts were seeded inside this chip to study their migration and reactive oxygen species (ROS) production in response to different EF strengths and the presence of β-lapachone. We found that both the EF and β-lapachone level increased the cell migration rate and the production of ROS in an EF-strength-dependent manner. A strong linear correlation between the cell migration rate and the amount of intracellular ROS suggests that ROS are an intermediate product by which EF and β-lapachone enhance cell migration. Moreover, an anti-oxidant, α-tocopherol, was found to quench the production of ROS, resulting in a decrease in the migration rate.  相似文献   

16.
Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 μm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a “pinball machine-like” array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling.  相似文献   

17.
Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.  相似文献   

18.
Whilst laboratory-on-chip cell separation systems using dielectrophoresis are increasingly reported in the literature, many systems are afflicted by factors which impede “real world” performance, chief among these being cell loss (in dead spaces, attached to glass and tubing surfaces, or sedimentation from flow), and designs with large channel height-to-width ratios (large channel widths, small channel heights) that make the systems difficult to interface with other microfluidic systems. In this paper, we present a scalable structure based on 3D wells with approximately unity height-to-width ratios (based on tubes with electrodes on the sides), which is capable of enriching yeast cell populations whilst ensuring that up to 94.3% of cells processed through the device can be collected in tubes beyond the output.  相似文献   

19.
IntroductionEpithelial cells (ECs) are structures regularly observed during urine microscopy analysis. The correct identification of EC subtypes can be useful since renal tubular epithelial cells (RTECs) are clinically relevant. We investigate the urinary ECs report and the judgement of its clinical importance by Brazilian laboratories.Materials and methodsA survey with four questions was made available to participants of the Urinalysis External Quality Assessment Program (EQAP) from Controllab. Laboratories composed 3 groups: (1) differentiating ECs subtypes: “squamous”, “transitional” and “RTECs”; (2) differentiating ECs subtypes: “squamous” or “non-squamous” cells; (3) without ECs subtype identification. Participants did not necessarily answer to all questions and the answers were evaluated both within the same laboratory’s category and within different categories of laboratories.ResultsA total of 1336 (94%) laboratories answered the survey; Group 1, 119/140 (85%) reported that ECs differentiation is important to the physician and 62% want to be evaluated by EQAP, while in Group 3, 455/1110 (41%) reported it is useful to them, however only 25% want be evaluated by EQAP. Group 2 laboratories 37/51 (73%) reported that the information is important, but only 13/52 (25%) are interested in an EQAP with differentiation of the 3 ECs subtypes.ConclusionMost of the laboratories do not differentiate ECs in the three subtypes, despite the clinical importance of RTECs. Education of laboratory staff about the clinical significance of urinary particles should be considered a key priority.  相似文献   

20.
IntroductionAn appropriate management of anaemia laboratory tests is crucial for a correct diagnosis and treatment. A non-sequential “shotgun” approach (where every anaemia related test is ordered) causes workload and cost increases and could be potentially harmful. We have implemented a Decision Support System through our laboratory information system (LIMS) based on reflexive algorithms and automatic generation of interpretative reports specifically in diagnosis of anaemia for primary care patients.Materials and methodsWhen a request contained an “Anaemia Suspicion Study” profile, more than twenty automatic reflexive rules were activated in our LIMS based upon laboratory results. These rules normally involved the addition of reflexive tests. A final report was automatically generated for each interpretation which was always reviewed for their validity by two staff pathologists. We measured the impact of this system in the ordering of most common anaemia related tests and if a proper treatment was established based on the interpretive report.ResultsFrom all the studies performed, only 12% were positive being “iron deficiency” and “anaemia of chronic disease” the most frequent causes, 62% and 17%, respectively. Proper treatment was established in 88% of these anaemic patients. Total iron, transferrin, ferritin, folate and vitamin B12 demand decreased substantially after implementation representing a cost reduction of 40% only for these five tests.ConclusionsOur system has easily improved patient outcomes, advising on individual clinical cases. We have also noticeably reduced the number of over-requested tests and laboratory costs.  相似文献   

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