The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, ...the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm
2
(± 0.9 Ω.cm
2
) and 28.2 Ω.cm
2
(± 1.3 Ω.cm
2
) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm
2
. Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm
2
. This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and ...real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.
Abstract Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell ...proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.
In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic ...cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated.
HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI.
Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage.
Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.
•RhZrO2 catalyst for H2 production via low temperature ATR of methane is proposed.•Synthesis and characterization of RhZrO2 catalyst are outlined.•Its high stability and low carbon formation at low ...temperatures are demonstrated.•Nickel kinetic laws of SMR and ATR hold for Rhodium catalyst too.•The catalytic activity of RhZrO2 is 7 times higher than the one of NiAl2O3 catalyst.
A Rh-based catalyst for low temperature hydrogen generation in membrane microreactor applications has been developed and characterized. A RhZrO2 catalyst with 1.4wt% Rh was prepared by incipient wetness impregnation and was tested for both methane reforming and autothermal reforming at temperatures interesting for membrane reactor applications (i.e. temperatures below 700°C and steam-to-carbon ratio of 2). The kinetic parameters to describe the reaction rate of both methane steam reforming (SMR) and auto-thermal reforming (ATR) over the RhZrO2 catalyst have been determined using a 1D heterogeneous packed bed reactor model to properly account for mass and heat transfer resistances.
The experimental results demonstrate that the RhZrO2 catalyst is extremely active for ATR and resistant to coke formation at much lower temperatures and steam-to-carbon ratios compared with conventional Ni-based catalysts. This makes the new catalyst especially suitable for integration in a Pd-based membrane microreactor with a maximum allowable operation temperature of about 650°C dictated by the membrane stability.
A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and ...microfluidic devices have the potential to facilitate high‐throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three‐port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF‐based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high‐throughput live cell detection and differentiation without the need for cell‐specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single‐cell analysis.
This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to ...the fact that no labelling is required.
Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer.
Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used.
Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.
Objectives Fulvestrant is an estrogen receptor (ER) antagonist that binds, blocks and degrades the estrogen receptor and is currently used in adjuvant treatment in postmenopausal women with ...ER-positive breast cancer as an alternative for tamoxifen. As an antagonist, it may induce or aggravate climacteric symptoms. In order to alleviate these symptoms, one could consider hormone therapy. The objective of this study was to analyze the effect of fulvestrant alone or in combination with different steroids in human breast cancer cells in vitro, and to demonstrate whether these steroids will compromise the efficacy of fulvestrant in ER-positive breast cancer cells.
Methods We performed experiments in vitro with various hormone therapy preparations (estradiol (E2), dihydrodydrogesterone (DHD) and tibolone) at a concentration of 10−6 mol l alone or combined with fulvestrant in different breast cancer cell lines, ER-positive and ER-negative. After an incubation of 144 h, proliferation and apoptosis were measured. The first was measured by quantification of the expression of cyclin D1 mRNA, the latter by the Nicoletti fragmentation assay.
Results This in vitro study revealed clear differences in results when various hormone therapy preparations, alone or combined with fulvestrant, are added to ER-positive and ER-negative breast cancer cell lines.
Conclusions Our study demonstrated that fulvestrant, an ER antagonist used in the treatment of ER-positive breast cancer, combined with E2 and DHD or in combination with tibolone, is not compromised in its efficacy in inducing apoptosis in ER-positive breast cancer cell lines in vitro.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
PDF
