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1.
The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane  相似文献   

2.
In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells).  相似文献   

3.
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   

4.
Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications.  相似文献   

5.
Cancer cell migration through tissue pores and tracks into the bloodstream is a critical biological step for cancer metastasis. Although in vivo studies have shown that expression of vimentin can induce invasive cell lines, its role in cell cytoskeleton reorganization and cell motility under in vitro physical confinement remains unknown. Here, a microfluidic device with cell culture chamber and collagen-coated microchannels was developed as an in vitro model for physiological confinement environments. Using this microchannel assay, we demonstrated that the knockdown of vimentin decreases 3T3 fibroblast cell directional migration speed in confined microchannels. Additionally, as cells form dynamic membranes that define the leading edge of motile cells, different leading edge morphologies of 3T3 fibroblast and 3T3 vimentin knockdown cells were observed. The leading edge morphology change under confinement can be explained by the effect of vimentin on cytoskeletal organization and focal adhesion. The microfluidic device integrated with a time-lapse microscope provided a new approach to study the effect of vimentin on cell adhesion, migration, and invasiveness.  相似文献   

6.
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   

7.
Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.  相似文献   

8.
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   

9.
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   

10.
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   

11.
Extracellular matrix (ECM) proteins are required for cell culture. In this paper, we report the use of O(2) plasma bonding to fabricate a perfusion culture microchamber array chip with identical-size ECM spots in the isolated microchambers. The chip was fabricated by assembly of two poly(dimethylsiloxane) (PDMS) layers, a microfluidic network layer, and an ECM array layer, which were aligned and then bonded by O(2) plasma oxidation with protection of the ECM microarray with a physical mask made from PDMS. We successfully cultivated Chinese hamster ovary K1 cells in the microchambers with fibronectin. In the fibronectin microchambers, the cells adhered and extended after 12 h of static culture and then grew over the course of 1 d of perfusion culture.  相似文献   

12.
Spatially and temporally resolved delivery of soluble factors is a key feature for pharmacological applications. In this framework, microfluidics coupled to multisite electrophysiology offers great advantages in neuropharmacology and toxicology. In this work, a microfluidic device for biochemical stimulation of neuronal networks was developed. A micro-chamber for cell culturing, previously developed and tested for long term neuronal growth by our group, was provided with a thin wall, which partially divided the cell culture region in two sub-compartments. The device was reversibly coupled to a flat micro electrode array and used to culture primary neurons in the same microenvironment. We demonstrated that the two fluidically connected compartments were able to originate two parallel neuronal networks with similar electrophysiological activity but functionally independent. Furthermore, the device allowed to connect the outlet port to a syringe pump and to transform the static culture chamber in a perfused one. At 14 days invitro, sub-networks were independently stimulated with a test molecule, tetrodotoxin, a neurotoxin known to block action potentials, by means of continuous delivery. Electrical activity recordings proved the ability of the device configuration to selectively stimulate each neuronal network individually. The proposed microfluidic approach represents an innovative methodology to perform biological, pharmacological, and electrophysiological experiments on neuronal networks. Indeed, it allows for controlled delivery of substances to cells, and it overcomes the limitations due to standard drug stimulation techniques. Finally, the twin network configuration reduces biological variability, which has important outcomes on pharmacological and drug screening.  相似文献   

13.
Cholesterol and phospholipids were estimated in serum and bile from hepatic duct and gallbladder of twenty five patients of gallstone with functioning gallbladder (Group-I) and an equal number of patients having diseases other than of hepatobiliary system acting as control (Group-II). Group-I patients showed high serum cholesterol and low serum phospholipid levels as compared to those of Group-II. Cholesterol levels in hepatic duct and gallbladder bile were higher in Group-I than in Group-II whereas the phospholipid levels in the bile of Group-I were lower than in Group-II. The phospholipid: cholesterol ratios in hepatic duct and gallbladder bile of Group-I were 2.76 and 3.03 respectively as compared to 5.62 and 5.92 in Group-II.  相似文献   

14.
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   

15.
We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.  相似文献   

16.
In this paper, we demonstrate for the first time the technique to using microfluidics to fabricate tissue engineering scaffolds with uniform pore sizes. We investigate both the bubble generation of the microfluidic device and the application of foam as a tissue engineering scaffold. Our microfluidic device consists of two concentric tapered channels, which are made by micropipettes. Nitrogen gas and aqueous alginate solution with Pluronic® F127 surfactant are pumped through the inner and the outer channels, respectively. We observe rich dynamic patterns of bubbles encapsulated in the liquid droplets. The size of the bubble depends linearly on the gas pressure and inversely on the liquid flow rate. In addition, monodisperse bubbles self-assemble into crystalline structures. The liquid crystalline foams are further processed into open-cell solid foams. The novel foam gel was used as a scaffold to culture chondrocytes.  相似文献   

17.
In this paper, we use a spiral channel inertial focusing device for isolation and purification of chromosomes, which are highly asymmetric. The method developed is proposed as a sample preparation process for transchromosomic research. The proposed microfluidics-based chromosome separation approach enables rapid, label-free isolation of bioactive chromosomes and is compatible with chromosome buffer. As part of this work, particle force analysis during the separation process is performed utilizing mathematic models to estimate the expected behavior of chromosomes in the channel and the model validated with experiments employing fluorescent beads. The chromosome sample is further divided into subtypes utilizing fluorescent activated cell sorting , including small condensed chromosomes, single chromosomes, and groups of two chromosomes (four sister chromatids). The separation of chromosome subtypes is realized based on their shape differences in the spiral channel device under high flow rate conditions. When chromosomes become aligned in the shear flow, the balance between the inertial focusing force and the Dean flow drag force is determined by the chromosome projection area and aspect ratio, or shape difference, leading to different focusing locations in the channel. The achieved results indicate a new separation regime in inertial microfluidics that can be used for the separation of non-spherical particles based on particle aspect ratios, which could potentially be applied in fields such as bacteria subtype separation and chromosome karyotyping.  相似文献   

18.
Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.  相似文献   

19.
In this paper, we develop a microfluidic device capable of generating nitric oxide (NO) gradients for cell culture using spatially controlled chemical reactions. NO plays an essential role in various biological activities, including nervous, immune, and cardiovascular systems. The device developed in this paper can control NO gradients without utilizing expensive and hazardous high purity NO gas sources or direct addition of NO donors. Consequently, the device provides an efficient, cost-effective, robust, and stable platform to generate NO gradients for cell culture studies. In the experiments, NO gradients are first characterized using a NO-sensitive fluorescence dye, and cell experiments using aortic smooth muscle cells are conducted. The results demonstrate that the device can alter the intracellular NO concentrations and further affect the Ca2+ concentration oscillation for the cells. The device developed in this paper provides a powerful platform for researchers better study the biological roles of NO and its spatial distribution using in vitro cell models with minimal instrumentation.  相似文献   

20.
Arsenic a metalloid and environmental contaminated has been found to be associated with public health problems in the affected areas. It is naturally occurred in groundwater and its accumulation in plant and animals leads to toxicity in several tissues most notably hepatic organ. Arsenic exposures (3 mg/kg body weight/day for 30 days) in mice exhibited increased arsenic and Zn levels in hepatocytes associated with enhanced oxidative stress in hepatocytes while there were no significantly changes were observed in Cu level. An increase in the lipid peroxidation and decrease in the levels of reduced glutathione and activity of superoxide dismutase, catalase, and glutathione peroxidase were observed in arsenic treated mice as compared to controls. Arsenic exposure in mice also caused a significant change in serum biomarkers in the SGOT, SGPT and creatinine as compared to the controls. There were no significant changes in the serum levels of total protein in these mice. Co-administration of arsenic and fruit extract of amla (500 mg/kg body weight/day for 30 days) caused a significant reduction of arsenic transference associated with significantly decreases hepatic arsenic levels and balanced the antioxidant enzyme and levels of serum hepatic enzymes like SGOT and SGPT. The results of the present study clearly demonstrate the antioxidant property of amla that could be responsible for its protective efficacy in arsenic induced hepatic toxicity.  相似文献   

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