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1.
The confined flow of red blood cells (RBCs) in microvasculature is essential for oxygen delivery to body tissues and has been extensively investigated in the literature, both in vivo and in vitro. One of the main problems still open in microcirculation is that flow resistance in microcapillaries in vivo is higher than that in vitro. This discrepancy has been attributed to the glycocalyx, a macromolecular layer lining the inner walls of vessels in vivo, but no direct experimental evidence of this hypothesis has been provided so far. Here, we investigate the flow behavior of RBCs in glass microcapillaries coated with a polymer brush (referred to as “hairy” microcapillaries as opposed to “bare” ones with no coating), an experimental model system of the glycocalyx. By high-speed microscopy imaging and image analysis, a velocity reduction of RBCs flowing in hairy microcapillaries as compared to bare ones is indeed found at the same pressure drop. Interestingly, such slowing down is larger than expected from lumen reduction due to the polymer brush and displays an on-off trend with a threshold around 70 nm of polymer brush dry thickness. Above this threshold, the presence of the polymer brush is associated with an increased RBC deformation, and RBC velocity is independent on polymer brush thickness (at the same pressure drop). In conclusion, this work provides direct support to the hypothesis that the glycocalyx is the main factor responsible of the higher flow resistance found in microcapillaries in vivo.  相似文献   

2.
The geometry of crossing structure formed by two-layer microchannels determines the axial and transverse movements of contact interface between two liquid streams, which gives us a new method for promoting the micromixers. Hence, we designed four different three-dimensional micromixers by selecting two different crossing structures as basic units (one unit is a crossing structure called “X” and the other is a reversed crossing structure called “rX”). In order to find out how the crossing-structure sequence affects the mixing performance within three-dimensional micromixers, we organized these four mixers in different ways, i.e., the first combination is X-rX-X-rX-…, the second is X-rX-rX-X-…, the third is X-X-rX-X-…, and the last one is X-X-X-X…. Consequently, quite distinct mixing phenomena are engendered. Furthermore, experiments were also conducted using the first and the last models to verify the simulation results. We infer that the last mixer is more likely to trigger chaos and convection by rotating the contact surface than the first one that merely swings the surface even when the flow rates and viscosities of the two liquid streams are increased.  相似文献   

3.
Circulating tumor cells (CTCs) shed from the primary tumor undergo significant fragmentation in the microvasculature, and very few escape to instigate metastases. Inspired by this in vivo behavior of CTCs, we report a microfluidic method to phenotype cancer cells based on their ability to arrest and fragment at a micropillar-based bifurcation. We find that in addition to cancer cell size, mechanical properties determine fragmentability. We observe that highly metastatic prostate cancer cells are more resistant to fragmentation than weakly metastatic cells, providing the first indication that metastatic CTCs can escape rupture and potentially initiate secondary tumors. Our method may thus be useful in identifying phenotypes that succumb to or escape mechanical trauma in microcirculation.  相似文献   

4.
Microvalves with different actuation methods offer great integrability and flexibility in operation of lab-on-chip devices. In this work, we demonstrate a hydrogel-based and optically controlled modular microvalve that can be easily integrated within a microfluidic device and actuated by an off-chip laser source. The microvalve is based on in-channel trapping of microgel particles, which are composed of poly(N-isopropylacrylamide) and polypyrrole nanoparticles. Upon irradiation by a near-infrared (NIR) laser, the microgel undergoes volumetric change and enables precisely localized fluid on/off switching. The response rate and the “open” duration of the microvalve can be simply controlled by adjusting the laser power and exposure time. We showed that the trapped microgel can be triggered to shrink sufficiently to open a channel within as low as ∼1–2 s; while the microgel swells to re-seal the channel within ∼6–8 s. This is so far one of the fastest optically controlled and hydrogel-based microvalves, thus permitting speedy fluidic switching applications. In this study, we successfully employed this technique to control fluidic interface between laminar flow streams within a Y-junction device. The optically triggered microvalve permits flexible and remote fluidic handling, and enables pulsatile in situ chemical treatment to cell culture in an automatic and programmed manner, which is exemplified by studies of chemotherapeutic drug induced cell apoptosis under different drug treatment strategies. We find that cisplatin induced apoptosis is significantly higher in cancer cells treated with a pulsed dose, as compared to continuous flow with a sustained dose. It is expected that our NIR-controlled valving strategy will provide a simple, versatile, and powerful alternative for liquid handling in microfluidic devices.  相似文献   

5.
Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.  相似文献   

6.
The objective of this work was to investigate the influence of hydrodynamics on the growth kinetics of surface-adhering Pseudomonas putida cells. The results showed in particular that under non substrate-limiting conditions, the early step of bacterial apparent growth rate is lower than those measured with suspended cells. Contrary to previously cited authors which explain this behavior to the different adhesive properties of the “daughter”-cells (which makes more probable the detachment of these daughter-cells), in our experimental conditions, that explanation does not hold and we show a clear dependence of growth kinetics with flow conditions, due to the formation of boundary layer concentration at low Reynolds number. These results revealed that using Monod law in the modeling of biofilm growth in fixed-biomass processes should be performed with care.  相似文献   

7.
In this work, we conduct a computational study on the loading of cryoprotective agents into cells in preparation for cryopreservation. The advantages of microfluidics in cryopreserving cells include control of fluid flow parameters for reliable cryoprotectant loading and reproducible streamlined processing of samples. A 0.25 m long, three inlet T-junction microchannel serves as an idealized environment for this process. The flow field and concentration distribution are determined from a computational fluid dynamics study and cells are tracked as inert particles in a Lagrangian frame. These particles are not confined to streamlines but can migrate laterally due to the Segre-Sildeberg effect for particles in a shear flow. During this tracking, the local concentration field surrounding the cell is monitored. This data are used as input into the Kedem-Katchalsky equations to numerically study passive solute transport across the cell membrane. As a result of the laminar flow, each cell has a unique pathline in the flow field resulting in different residence times and a unique external concentration field along its path. However, in most previous studies, the effect of a spatially varying concentration field on the transport across the cell membrane is ignored. The dynamics of this process are investigated for a population of cells released from the inlet. Using dimensional analysis, we find a governing parameter α, which is the ratio of the time scale for membrane transport to the average residence time in the channel. For α <  = 0.224, cryoprotectant loading is completed to within 5% of the target concentration for all of the cells. However, for α > 0.224, we find the population of cells does not achieve complete loading and there is a distribution of intracellular cryoprotective agent concentration amongst the population. Further increasing α beyond a value of 2 leads to negligible cryoprotectant loading. These simulations on populations of cells may lead to improved microfluidic cryopreservation protocols where more consistent cryoprotective agent loading and freezing can be achieved, thus increasing cell survival.  相似文献   

8.
In the 70ies of the last century, ther term “preanalytical phase” was introduced in the literature. This term describes all actions and aspects of the “brain to brain circle” of the medical laboratory diagnostic procedure happening before the analytical phase. The author describes his personal experiences in the early seventies and the following history of increasing awareness of this phase as the main cause of “laboratory errors”. This includes the definitions of influence and interference factors as well as the first publications in book, internet, CD-Rom and recent App form over the past 40 years. In addition, a short summary of previous developments as prerequesits of laboratory diagnostic actions is described from the middle age matula for urine collection to the blood collection tubes, anticoagulants and centrifuges. The short review gives a personal view on the possible causes of missing awareness of preanalytical causes of error and future aspects of new techniques in regulation of requests to introduction of quality assurance programs for preanalytical factors.  相似文献   

9.
For cancer patients, the enumeration of rare circulating tumor cells (CTCs) in peripheral blood is a strong prognostic indicator of the severity of the cancer; for the general population, the capture of CTCs is needed for use as a clinical tool for cancer screening, early detection, and treatment assessment. Here, we present a fast, high-purity (∼90%) and high-efficiency (>90%) method for the segregation and undamaged recovery of CTCs using a spatially gradated microfluidic chip. Further, by lysing the red blood cells we achieved not only a significant reduction in the overall processing time but also mitigated the blood clogging problem commonly encountered in microfluidic-based CTC isolation systems. To clinically validate the chip, we employed it to detect and capture CTCs from 10 liver cancer patients. Positive CTC enumeration was observed in all the blood samples, and the readings ranged from a low of 1–2 CTCs (1 patient) to a high of >20 CTCs (2 patients) with the balance having 3–20 CTCs per 3-ml blood sample. The work here indicates that our system can be developed for use in cancer screening, metastatic assessment, and chemotherapeutic response and for pharmacological and genetic evaluation of single CTCs.  相似文献   

10.
Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.  相似文献   

11.
Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy.  相似文献   

12.
There is currently a growing interest in control of stretching of DNA inside nanoconfined regions due to the possibility to analyze and manipulate single biomolecules for applications such as DNA mapping and barcoding, which are based on stretching the DNA in a linear fashion. In the present work, we couple Finite Element Methods and Monte Carlo simulations in order to study the conformation of DNA molecules confined in nanofluidic channels with neutral and charged walls. We find that the electrostatic forces become more and more important when lowering the ionic strength of the solution. The influence of the nanochannel cross section geometry is also studied by evaluating the DNA elongation in square, rectangular, and triangular channels. We demonstrate that coupling electrostatically interacting walls with a triangular geometry is an efficient way to stretch DNA molecules at the scale of hundreds of nanometers. The paper reports experimental observations of λ-DNA molecules in poly(dimethylsiloxane) nanochannels filled with solutions of different ionic strength. The results are in good agreement with the theoretical predictions, confirming the crucial role of the electrostatic repulsion of the constraining walls on the molecule stretching.  相似文献   

13.
Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 106 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.  相似文献   

14.
In this paper, we present a low cost and equipment-free blood filtration device capable of producing plasma from blood samples with mL-scale capacity and demonstrate its clinical application for hepatitis B diagnosis. We report the results of in-field testing of the device with 0.8–1 ml of undiluted, anticoagulated human whole blood samples from patients at the National Hospital for Tropical Diseases in Hanoi, Vietnam. Blood cell counts demonstrate that the device is capable of filtering out 99.9% of red and 96.9% of white blood cells, and the plasma collected from the device contains lower red blood cell counts than plasma obtained from a centrifuge. Biochemistry and immunology testing establish the suitability of the device as a sample preparation unit for testing alanine transaminase (ALT), aspartate transaminase (AST), urea, hepatitis B “e” antigen (HBeAg), hepatitis B “e” antibody (HBe Ab), and hepatitis B surface antibody (HBs Ab). The device provides a simple and practical front-end sample processing method for point-of-care microfluidic diagnostics, enabling sufficient volumes for multiplexed downstream tests.  相似文献   

15.
We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.  相似文献   

16.
Prostate specific antigen is considered to be a tumour marker having maximum utility and specificity for prostate cancer since decades. After the discovery of methods to quantify different molecular fractions of prostate specific antigen (PSA), its usefulness in diagnosing early prostate cancer cases has increased tremendously. The “specificity” of PSA, is now challenged by many studies which proved that PSA, once believed to be secreted exclusively by prostatic epithelium, is also present in females. The exact biological role of extraprostatic PSA is still debatable though many theories substantiated by in vitro evidence has been put forward. With the advent of ultrasensitive analytical techniques, PSA is now quantifiable in female serum in its various molecular forms and this has led to many assumptions of it being useful as a marker in female breast cancers. In a similar scenario to prostate cancer, the ratio of free to total PSA is shown to be useful in detecting early breast cancer cases. It is also shown to be a good prognostic indicator and a predictor of response to therapy and recurrence. Apart from its role in breast cancer, it has been advocated to be a marker of hyper androgenic states in women like hirsutism and polycystic ovarian syndrome. Conflicting reports regarding the role of extra prostatic PSA is accumulating but it has been proven beyond doubt that PSA is no longer specific and confined to prostate gland. Various studies have registered that PSA is an ubiquitous molecule, secreted by hormone responsive organs and its synthesis is stimulated by androgens and progesterone but not oestrogens. In this article, a review of various literatures is done about the presence of extra prostatic PSA, its probable role in those sites as well as its utility as a tumour marker in breast cancer.  相似文献   

17.
We describe a microfluidic device for on-chip chemical processing, such as staining, and subsequent washing of cells. The paper introduces “separator walls” to increase the on-chip incubation time and to improve the quality of washing. Cells of interest are concentrated into a treatment stream of chemical reagents at the first separator wall for extended on-chip incubation without causing excess contamination at the output due to diffusion of the unreacted treatment chemicals, and then are directed to the washing stream before final collections. The second separator wall further reduces the output contamination from diffusion to the washing stream. With this approach, we demonstrate on-chip leukocyte staining with Rhodamine 6G and washing. The results suggest that other conventional biological and analytical processes could be replaced by the proposed device.  相似文献   

18.
Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235–1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.  相似文献   

19.
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20.
Chemotherapy drugs, used for prevention of uncontrolled cell proliferation in certain tissues as well as inducing apoptosis in tumor cells, are important candidates for treatment of cancer. The synthesized 2-amino-4H-chromene-3-carbonitrile derivatives effective on cancerous cells resistant to other drugs such as Paclitaxel were used due to their ability in induction of apoptosis. The growth inhibitory and inducing apoptosis activities were determined. In order to make it target-oriented, the best compound was conjugated with gold nanoparticles (NPs) by aspartic acid with chemical reduction method. Cytotoxicity effect of 2-amino-4H-chromene-3-carbonitrile derivatives against the T47D breast cancer cell line was determined by MTT assay. The synthesis of gold NPs was confirmed by transmission electron microscopy, UV–Vis and dynamic light scattering. To assess the effects of compounds on the process of apoptosis, staining methods with acridine orange–ethidium bromide and Hoechst staining by fluorescence microscopy and DNA fragmentation by the diphenylamine method were used. The synthesized compounds containing two NH2 groups on benzene rings, demonstrated more cytotoxicity effect. The effect of conjugation with gold NPs and the induction of apoptosis were studied with the best compound. The cytotoxicity effects of the synthesized 2-amino-4H-chromene-3-carbonitrile compounds were changed by replacement of NO2 group on thiol ring with different chemical groups on the benzene ring. Analyses of treated cell lines by conjugated and non-conjugated forms of compounds verified their ability in inducing apoptosis while conjugated form demonstrated higher apoptosis.  相似文献   

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