Min-Hsien Wu

Effect of Extracellular pH on Matrix Synthesis by Chondrocytes in 3D Agarose Gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3–6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7‐day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH‐dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

Real-time and non-invasive impedimetric monitoring of cell proliferation and chemosensitivity in a perfusion 3D cell culture microfluidic chip

A perfusion three-dimensional (3D) cell culture microfluidic chip has been developed for real-time and non-invasive impedimetric monitoring of cell proliferation and chemosensitivity. In this study, human oral cancer cells (OEC-M1) were encapsulated in 3D agarose scaffold and cultured in a miniaturized chamber under perfusion of tested substance. This setting provides a more in vitro physiologically relevant microenvironment to better mimic the complex in vivo microenvironment. A pair of vertical electrodes was embedded at the opposite sidewalls of the culture chamber for the on-site impedance measurement. Cell density in the 3D construct was shown to be proportional to the impedance magnitude of the entire construct. Therefore, perfusion 3D cell culture was performed for up to 5 days and cell proliferation can be monitored by the impedimetric analysis. Moreover, real-time impedimetric monitoring of cell viability under the perfusion of anti-cancer drug in different concentrations was conducted and the impedance magnitude was directly correlated with the cell viability. From the confirmation of the endpoint cell viability assays, a concentration-dependent effect was shown; however, the response of cell viability during the drug treatment was able to be traced by the impedance measurement. The experimental results showed that cell proliferation and chemosensitivity in 3D cell culture format can be monitored by impedance measurement. This microfluidic chip has a high potential to develop a powerful analytical platform for cancer research.

Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications.

A membrane-based serpentine-shape pneumatic micropump with pumping performance modulated by fluidic resistance

This paper reports a new membrane-based pneumatic micropump with new serpentine-shape (S-shape) pneumatic channels intended for achieving high-throughput pumping in a microfluidic system at a relatively low pumping rate and a board flow rate range. The key feature of this design is the ability to modulate the pumping rates by fine-tuning the fluidic resistance of injected compressed air in the designed pneumatic microchannels and the chambers of the micropump. In the study, several S-shape pneumatic micropumps with various layouts were designed and fabricated based on thick-film photoresist lithography and polydimethylsiloxane (PDMS) replication processes. To investigate designs with a suitable pumping performance, S-shape pneumatic micropumps with varied lengths (1000, 5000 and 10 000 µm), varied widths (20, 40 and 200 µm) of the pneumatic microchannel bridging two rectangular pneumatic chambers, and different numbers of pneumatic channel bends (two and four U-shape bends) were designed and evaluated experimentally by using high-speed CCD-coupled microscopic observation of the movement of PDMS membrane pulsation and pumping rate measurements. The results revealed that under the experimental conditions studied, the layout of the S-shape pneumatic micropump with three rectangular pneumatic chambers, 5000 µm long and 40 µm wide pneumatic microchannel and four U-shape bends in the pneumatic microchannel was found to be capable of providing a broader pumping rate range from 0 to 539 µl h−1 compared to the other designs. As a whole, the experimental results demonstrate the use of fluidic resistance of injected air in a pneumatic micropump with S-shape layout to control its pumping performance, which largely expands the flexibility of its pumping application in a microfluidic system.

A microfluidic system enabling continuous characterization of specific membrane capacitance and cytoplasm conductivity of single cells in suspension

This paper presents a microfluidic system enabling continuous characterization of specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm) of single cells in suspension. In this study, cells were aspirated continuously through a constriction channel while cell elongations and impedance profiles at two frequencies (1kHz and 100kHz) were measured simultaneously using microscopy imaging and a lock-in amplifier. 1kHz impedance data were used to evaluate cellular sealing properties with constriction channel walls and 100kHz impedance data were translated to quantify equivalent membrane capacitance and cytoplasm resistance of single cells, which were further translated to Cspecific membrane and σcytoplasm. Two model cell lines (kidney tumor cell line of 786-O (n=302) and vascular smooth muscle cell line of T2 (n=216)) were used to evaluate this technique, producing Cspecific membrane of 3.67±1.00 and 4.53±1.51μF/cm(2) and σcytoplasm of 0.47±0.09 and 0.55±0.14S/m, respectively. Compared to previously reported techniques which can only collect Cspecific membrane and σcytoplasm from tens of cells, this new technique has a higher throughput, capable of collecting Cspecific membrane and σcytoplasm from hundreds of cells in 30min immediately after cell passage.

Integrating solid-state sensor and microfluidic devices for glucose, urea and creatinine detection based on enzyme-carrying alginate microbeads

A solid-state sensor embedded microfluidic chip is demonstrated for the detection of glucose, urea and creatinine in human serum. In the presented device, magnetic powder-containing enzyme-carrying alginate microbeads are immobilized on the surface of an electrolyte-insulator-semiconductor (EIS) sensor by means of a step-like obstacle in the microchannel and an external magnetic force. The sample is injected into the microchannel and reacts with the enzyme contained within the alginate beads; prompting the release of hydrogen ions. The sample concentration is then evaluated by measuring the resulting change in the voltage signal of the EIS sensor. The reaction time and alginate bead size are optimized experimentally using a standard glucose solution. The experimental results show that the device has a detection range of 2-8mM, 1-16mM and 10(-2)-10mM for glucose, urea and creatinine, respectively. Furthermore, it is shown that the device is capable of sequentially measuring all three indicators in a human serum sample. Finally, it is shown that the measured values of the glucose, urea and creatinine concentrations obtained using the device deviate from those obtained using a commercial kit by just 5.17%, 6.22% and 13.53%, respectively. This method can be extended to sequentially measure multiple blood indicators in the sample chip by replacing different types of enzyme in alginate bead and can address the enzyme preservation issue in the microfluidic device. Overall, the results presented in this study indicate that the microfluidic chip has significant potential for blood monitoring in point-of-care applications.

Application of indium tin oxide (ITO)-based microheater chip with uniform thermal distribution for perfusion cell culture outside a cell incubator

This study reports a transparent indium tin oxide (ITO)-based microheater chip and its applicability for perfusion cell culture outside a cell incubator. The attempt of the proposed ITO microheater is to take the role of conventional bulky incubator for cell culture in order to improve integratability with the experimental setup for continuous/perfusion cell culture, to facilitate microscopic observation or other online monitoring activities during cell culture, or even to provide portability of cell culture operation. In this work, numerical simulation and experimental evaluation have been conducted to justify that the presented device is capable of providing a spatially uniform thermal environment and precise temperature control with a mild deviation of ±0.2°C, which is suitable for a general cell culture practice. Besides, to testify that the thermal environment generated by the presented device is well compatible with conventional cell incubator, chondrocyte perfusion culture was carried out. Results demonstrated that the physiology of the cultured chondrocytes on the developed ITO microheater chip was consistent with that of an incubator. All these not only demonstrate the feasibility of using the presented ITO microheater as a thermal control system for cell culture outside a cell incubator but also reveal its potential for other applications in which excellent thermal control is required.

A microfluidic system for cell type classification based on cellular size-independent electrical properties

This paper presents a microfluidic system enabling cell type classification based on continuous characterization of size-independent electrical properties (e.g., specific membrane capacitance (C(specific membrane)) and cytoplasm conductivity (σ(cytoplasm)). In this study, cells were aspirated continuously through a constriction channel, while cell elongation and impedance profiles at two frequencies (1 kHz and 100 kHz) were measured simultaneously. Based on a proposed distributed equivalent circuit model, 1 kHz impedance data were used to evaluate cellular sealing properties with constriction channel walls and 100 kHz impedance data were translated to C(specific membrane) and σ(cytoplasm). Two lung cancer cell lines of CRL-5803 cells (n(cell) = 489) and CCL-185 cells (n(cell) = 487) were used to evaluate this technique, producing a C(specific membrane) of 1.63 ± 0.52 μF cm(-2) vs. 2.00 ± 0.60 μF cm(-2), and σ(cytoplasm) of 0.90 ± 0.19 S m(-1)vs. 0.73 ± 0.17 S m(-1). Neural network-based pattern recognition was used to classify CRL-5803 and CCL-185 cells, producing success rates of 65.4% (C(specific membrane)), 71.4% (σ(cytoplasm)), and 74.4% (C(specific membrane) and σ(cytoplasm)), suggesting that these two tumor cell lines can be classified based on their electrical properties.

Structural properties and sensing performance of high-k Nd2TiO5 thin layer-based electrolyte-insulator-semiconductor for pH detection and urea biosensing.

For high sensitive pH sensing, an electrolyte-insulator-semiconductor (EIS) device with Nd(2)TiO(5) thin layers fabricated on Si substrates by means of reactive sputtering and the subsequent post-deposition annealing (PDA) treatment was proposed. In this work, the effect of thermal annealing (600, 700, 800, and 900 degrees C) on the structural characteristics of Nd(2)TiO(5) thin layer was investigated by X-ray diffraction, X-ray photoelectron spectroscopy, and atomic force microscopy. The observed structural properties were then correlated with the resulting pH sensing performances. For enzymatic field-effect-transistors-based urea biosensing, a hybrid configuration of the proposed Nd(2)TiO(5) thin layer with urease-immobilized alginate film attached was established. Within the experimental conditions investigated, the EIS device with the Nd(2)TiO(5) thin layer annealed at 800 degrees C exhibited a higher pH detection sensitivity of 57.2 mV/pH, a lower hysteresis voltage of 2.33 mV, and a lower drift rate of 1.80 mV/h compared to those at other annealing temperatures. These results are attributed to the formation of a thinner low-k interfacial layer at the oxide/Si interface and the higher surface roughness occurred at this annealing temperature. Furthermore, the presented urea biosensor was also proved to be able to detect urea with good linearity (R(2)=0.99) and reasonable sensitivity of 9.52 mV/mM in the urea concentration range of 3-40 mM. As a whole, the present work has provided some fundamental data for the use of Nd(2)TiO(5) thin layer for EIS-based pH detection and the extended application for biosensing.

Application of high throughput perfusion micro 3-D cell culture platform for the precise study of cellular responses to extracellular conditions -effect of serum concentrations on the physiology of articular chondrocytes

Mammalian cells are sensitive to extracellular microenvironments. In order to faithfully explore the physiological responses of cells to extracellular conditions, a steady, homogenous, and three-dimensional (3-D) culture environment is required because it can provide a more quantifiable and biologically-relevant culture condition. To achieve this, this study reports a perfusion micro cell culture platform encompassing 22 microbioreactor units for high throughput 3-D cell culture. The cell culture platform structurally consisting of a plug and a microbioreactor chamber module was simply fabricated by replica molding of polydimethylsiloxane (PDMS) polymer. The platform features in the proposed plug module with multiple molds incorporated, facilitating the preparation of cell encapsulated 3-D hydrogel constructs in a precise and efficient manner. This trait is found particularly useful for high-precision and high-throughput micro 3-D cell culture-based assay. In this study, the real value of the proposed platform to maintain a stable and homogenous culture condition was discussed. Besides, the application of the presented platform for precisely investigating the effect of serum concentration on the metabolic activities and biosynthetic abilities of articular chondrocytes was also demonstrated. As a whole, the proposed device has paved an alternative route to carry out high throughput micro-scale 3-D perfusion cell culture in a simple, cost-effective and precise manner. The promising applications include 3-D cell culture-based high throughput drug or toxicity testing/screening, or other investigations on the cell biology, where the precise quantification of the links between the cellular responses and extracellular conditions is required.