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1.
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   

2.
Hepatoprotectant is critical for the treatment of liver disease. This study first reported the application of a liver chip in the hepatoprotective effect assessment. We first established a biomimetic sinusoid-on-a-chip by laminating four types of hepatic cell lines (HepG2, HUVEC, LX-2, and U937 cells) in a single microchannel with the help of laminar flow in the microchannel and some micro-fences. This chip was straightforward to fabricate and operate and was able to be long-term cultured. It also demonstrated better hepatic activity (cell viability, albumin synthesis, urea secretion, and cytochrome P450 enzyme activities) over the traditional planar cell culture model. Then, we loaded three hepatoprotectants (tiopronin, bifendatatum, and glycyrrhizinate) into the chip followed by the addition of acetaminophen as a toxin. We successfully observed the hepatoprotective effect of these hepatoprotectants in the chip, and we also found that bifendatatum predominantly reduced alanine transaminase secretion, tiopronin predominantly reduced lactate dehydrogenase secretion, and glycyrrhizinate predominantly reduced aspartate transaminase secretion, which revealed the different mechanisms of these hepatoprotectants and provided a clue for following molecular biological study of the protecting mechanism.  相似文献   

3.
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   

4.
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   

5.
The aim of the present study was to evaluate the antidiabetic and ameliorative potential of aqueous extract of Ficus bengalensis bark in streptozotocin induced diabetic rats. The effect of oral administration of aqueous extract of F. bengalensis bark on blood glucose, serum electrolytes, serum glycolytic enzymes, liver microsomal protein, hepatic cytochrome P-450 dependent monooxygenase enzymes and lipid peroxidation in liver and kidney of streptozotocin -induced diabetic rats was studied. Oral administration of Ficus bengalensis to fed, fasted and glucose loaded diabetic rats significantly [F > 0.05 (ANOVA) and P< 0.05 (DMRT)] decreased the blood glucose level at 5 hrs and restored the levels of serum electrolytes, glycolytic enzymes and hepatic cytochrome P-450 dependent enzyme systems and decreased the formation of liver and kidney lipid peroxides at the end of 12 weeks. Further, the aqueous extract of Ficus bengalensis at a dose of 500mg/kg/day exhibits significant antidiabetic and ameliorative activity as evidenced by histological studies in normal and Ficus bengalensis treated streptozotocin induced diabetic rats. On the basis of our findings, it could be used as an antidiabetic and ameliorative agent for better management of diabetes mellitus.  相似文献   

6.
Droplet microfluidic technology has the potential to significantly reduce reagent use, and therefore, lower costs of assays employed in drug discovery campaigns. In addition to the reduction in costs, this technology can also reduce evaporation and contamination which are often problems seen in miniaturized microtitre plate formats. Despite these advantages, we currently advise caution in the use of these microfluidic approaches as there remains a lack of understanding of the artefacts of the systems such as reagent partitioning from droplet to carrier oil and interaction of the biological reagents with the water-oil interface. Both types of artefact can lead to inaccurate and misleading data. In this paper, we present a study of the partitioning of a number of drug-like molecules in a range of oils and evidence of protein binding at the water-oil interface which results in reduced activity of a cytochrome P450 enzyme. Data presented show that the drug-like molecules partitioned the least into fluorocarbon oils and the interaction of the 1A2 cytochrome at the water-oil interface resulted in a lower or complete absence of enzyme activity. This loss of activity of cytochrome 1A2 could be restored by the use of secondary blocking proteins although changes in the pharmacology of known 1A2 inhibitors were observed. The artefacts described here due to reagents partitioning into the carrier oil or protein binding at the water-oil interface significantly impact the potential use of these microfluidic systems as a means to carry out miniaturized biological assays, and further work is needed to understand the impact and reduction of these phenomena.  相似文献   

7.
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   

8.
In this study, we propose a microfluidic cell culture device mimicking the microscopic structure in liver tissue called hepatic cords. The cell culture area of the device was designed to align hepatocytes in two lines in a similar way to hepatic cords. Thanks to the structural design together with a cell seeding procedure, rat primary hepatocytes were successfully aligned in two lines and cultured under perfusion condition. It is shown that aligned hepatocytes gradually self-organize and form bile canaliculi along the hepatic cord-like structure. The present technique to culture hepatocytes with functional bile canaliculi could be used as an alternative to animal testing in the field of drug discovery and toxicological studies, and also be beneficial to tissue engineering applications.  相似文献   

9.
The wound-healing assay is an easy and economical way to quantify cell migration under diverse stimuli. Traditional assays such as scratch assays and barrier assays are widely and commonly used, but neither of them can represent the complicated condition when a wound occurs. It has been suggested that wound-healing is related to electric fields, which were found to regulate wound re-epithelialization. As a wound occurs, the disruption of epithelial barrier short-circuits the trans-epithelial potential and then a lateral endogenous electric field is created. This field has been proved invitro as an important cue for guiding the migration of fibroblasts, macrophages, and keratinocytes, a phenomenon termed electrotaxis or galvanotaxis. In this paper, we report a microfluidic electrical-stimulated wound-healing chip (ESWHC) integrating electric field with a modified barrier assay. This chip was used to study the migration of fibroblasts under different conditions such as serum, electric field, and wound-healing-promoting drugs. We successfully demonstrate the feasibility of ESWHC to effectively and quantitatively study cell migration during wound-healing process, and therefore this chip could be useful in drug discovery and drug safety tests.  相似文献   

10.
The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlateTM (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte''s functions, MatrigelTM-embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes'' responses to external stimuli.  相似文献   

11.
The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 × 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ∼1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies.  相似文献   

12.
Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.  相似文献   

13.
Ota H  Kodama T  Miki N 《Biomicrofluidics》2011,5(3):34105-3410515
Spheroids that are formed from aggregated cells have enhanced biological function compared to individual cells. In particular, hetero-spheroids composed of different types of cells, such as hepatocytes and endothelial cells, express tissue specific functions at a high level, which is advantageous for more precise drug screening and biological research. In this study, we propose rapid formation of size-controlled three-dimensional hetero-cell aggregates consisting of hepatocytes and endothelial cells using micro-rotation flow. Based on previous data, these aggregates are expected to ultimately become hetero-spheroids. The hepatocytes are coated with collagen gel films less than 200 nm thick, which were experimentally verified to increase adhesion strength between hepatocytes and endothelial cells. Gel-coated hepatocytes and endothelial cells are collected in an array by micro-rotational flow, thereby forming hetero-cell aggregates within 2 min. This array allowed the size of the three-dimensional cell aggregates to be hydrodynamically controlled, with standard deviations of less than 19%, by varying the cell density of the medium without altering the device geometry. Endothelial cells were successfully and uniformly dispersed in the aggregates. The proposed microfluidic device, with its capability of rapidly forming size-controlled hetero-cell aggregates, will offer an efficient experimental platform for future hetero-spheroid study that will contribute to drug screening and regenerative medicine.  相似文献   

14.
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   

15.
Robust bubble-free priming of complex microfluidic chips represents a critical, yet often unmet prerequisite to enable their practical and widespread application. Towards this end, the usage of a network of capillary stop valves as a generic design feature is proposed. Design principles, numerical simulations, and their application in the development of a microfluidic cell culture device are presented. This chip comprises eight parallel chambers for the assembly and cultivation of human hepatocytes and endothelial cells. The inlet channel divides into cell chambers, after which the flows are reunited to a single chip outlet. Dimensions and geometry of channels and cell chambers are designed to yield capillary burst pressures sequentially increasing towards the chip outlet. Thus, progress of liquid flow through the device is predefined by design and enclosure of air bubbles inside the microfluidic structures is efficiently avoided. Capillary stop valves were designed using numerical simulations. Devices were fabricated in cyclic olefin polymer. Pressure during filling was determined experimentally and is in good agreement with data obtained from simulation.  相似文献   

16.
Chen H  Li J  Zhang H  Li M  Rosengarten G  Nordon RE 《Biomicrofluidics》2011,5(4):44117-4411713
Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 μl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis.  相似文献   

17.
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   

18.
The serum and hepatic enzymes of rats were studied after exposed to country made liquor (CML) along with two chelating agents (glutathione and Selenium). There was a significant increase in several serum enzyme levels (viz., aspartate transaminase, alanine transaminase, alkaline phosphatase, sorbitol dehydrogenase, glutamate dehydrogenase, bilirubin) and decrease in various hepatic enzymes (Succinic dehydrogenase, Glucose 6-phosphatase, 5'Nucleotiease, Acid phosphatase, Acid ribonuclease, Cytochrome P-450) due to repeated administration of CML (2ml/100g of body weight). Results of this study revealed that the GSH and Se could give a significant protective action in serum and hepatic enzymes of CML exposed rats.  相似文献   

19.
Ma D  Chen H  Li Z  He Q 《Biomicrofluidics》2010,4(4):44107
Cell culture and harvest are the most upstream operation for a completely integrated cell assay chip. In our previous work, thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) was successfully grafted onto polydimethylsiloxane (PDMS) surface via benzophenone-initiated photopolymerization. In the present work, the PNIPAAm-grafted-PDMS (PNIPAAm-g-PDMS) surface was explored for thermomodulated cell culture and noninvasive harvest in microfluidic channels. Using COS 7 fibroblast from African green monkey kidney as the model cells, the thermomodulated adhering and detaching behaviors of the cells on the PNIPAAm-g-PDMS surfaces were optimized with respect to PNIPAAm-grafting yields and gelatin modification. The viability of the cells cultured on and harvested from the PNIPAAm-g-PDMS surface with the thermomodulated noninvasive protocol was estimated against the traditional cell culture∕harvest method involving trypsin digestion. The configuration of the microchannel on the PNIPAAm-g-PDMS chip was evaluated for static cell culture. Using a pipette-shaped PNIPAAm-g-PDMS microchannel, long-term cell culture could be achieved at 37 °C with periodic change of the culture medium every 12 h. After moving the microchip from the incubator set at 37 °C to the room temperature, the proliferated cells could be spontaneously detached from the PNIPAAm-g-PDMS surface of the upstream chamber and transferred by a gentle fluid flow to the downstream chamber, wherein the transferred cells could be subcultured. The thermomodulated cell culture, harvest, and passage operations on the PNIPAAm-g-PDMS microfluidic channels were demonstrated.  相似文献   

20.
We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.  相似文献   

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