Tzu-Keng Chiu

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.

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.

Development of a Microfluidic-Based Optical Sensing Device for Label-Free Detection of Circulating Tumor Cells (CTCs) Through Their Lactic Acid Metabolism

This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs), a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner.

Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model.

Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.

The Study of the Frequency Effect of Dynamic Compressive Loading on Primary Articular Chondrocyte Functions Using a Microcell Culture System

Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency (0.5, 1.0, and 2.0 Hz) effect of compressive loading (20% and 40% strain) on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% (2 Hz, 40% strain). Under the two compressive strains studied, the glycosaminoglycans (GAGs) synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation.

Circulating Tumour Cells as an Independent Prognostic Factor in Patients with Advanced Oesophageal Squamous Cell Carcinoma Undergoing Chemoradiotherapy

The role of circulating tumour cells (CTCs) in advanced oesophageal cancer (EC) patients undergoing concurrent chemoradiotherapy (CCRT) remains uncertain. A negative selection protocol plus flow cytometry was validated to efficiently identify CTCs. The CTC number was calculated and analysed for survival impact. The protocol’s efficacy in CTC identification was validated with a recovery rate of 44.6 ± 9.1% and a coefficient of variation of 20.4%. Fifty-seven patients and 20 healthy donors were enrolled. Initial staging, first response to CRT, and surgery after CRT were prognostic for overall survival, with P values of <0.0001, <0.0001, and <0.0001, respectively. The CTC number of EC patients is significantly higher (P = 0.04) than that of healthy donors. Multivariate analysis for disease-specific progression-free survival showed that surgery after response to CCRT, initial stage, and CTC number (≥21.0 cells/mL) played independent prognostic roles. For overall survival, surgery after CCRT, performance status, initial stage, and CTC number were significant independent prognostic factors. In conclusion, a negative selection plus flow cytometry protocol efficiently detected CTCs. The CTC number before CCRT was an independent prognostic factor in patients with unresectable oesophageal squamous cell carcinoma. Further large-scale prospective studies for validation are warranted.

Classification of Cells with Membrane Staining and/or Fixation Based on Cellular Specific Membrane Capacitance and Cytoplasm Conductivity

Single-cell electrical properties (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) have been regarded as potential label-free biophysical markers for the evaluation of cellular status. However, whether there exist correlations between these biophysical markers and cellular status (e.g., membrane-associate protein expression) is still unknown. To further validate the utility of single-cell electrical properties in cell type classification, Cspecific membrane and σcytoplasm of single PC-3 cells with membrane staining and/or fixation were analyzed and compared in this study. Four subtypes of PC-3 cells were prepared: untreated PC-3 cells, PC-3 cells with anti-EpCAM staining, PC-3 cells with fixation, and fixed PC-3 cells with anti-EpCAM staining. In experiments, suspended single cells were aspirated through microfluidic constriction channels with raw impedance data quantified and translated to Cspecific membrane and σcytoplasm. As to experimental results,

Isolation of label-free and viable circulating tumour cells (CTCs) from blood samples of cancer patients through a two-step process: negative selection-type immunomagnetic beads and spheroid cell culture-based cell isolation

Isolation of high-purity, label-free, and viable circulating tumour cells (CTCs) from cancer patients is crucial for subsequent analyses. To address this issue, a two-step CTC isolation scheme was proposed, wherein a spheroid cell culture was used to further purify viable CTCs after conventional negative selection-based CTC isolation methods. Our results from a cancer cell line model revealed that the survival of leukocytes in spheroid cell cultures was significantly decreased with time, whereas OECM-1 cells maintained viability and proliferated. Therefore, such a cell culture operation was expected to increase cancer cell purity in the cell spheroids. This assumption was confirmed by our results, which showed that cancer cell purities were 10.6 to 80.3-fold increased after spheroid cell culture for 8 days. In the following clinical tests, CTC-related cells were observed in 6 of 13 blood samples. Furthermore, the average purity of CTC-related cells was 34.8 ± 14.0%. By utilizing a second-step spheroid cell culture operation, the purity of CTC-related cells was greatly improved when compared with that (less than 10%) achievable by conventional negative selection-based CTC isolation. Overall, this study proposed a two-step process for the isolation of high-purity, label-free, and viable CTCs.

Development of a pneumatically driven active cover lid for multi-well microplates for use in perfusion three-dimensional cell culture

Before microfluidic-based cell culture models can be practically utilized for bioassays, there is a need for a transitional cell culture technique that can improve conventional cell culture models. To address this, a hybrid cell culture system integrating an active cover lid and a multi-well microplate was proposed to achieve perfusion 3-D cell culture. In this system, a microfluidic-based pneumatically-driven liquid transport mechanism was integrated into the active cover lid to realize 6-unit culture medium perfusion. Experimental results revealed that the flow of culture medium could be pneumatically driven in a flow-rate uniform manner. We used the system to successfully perform a perfusion 3-D cell culture of mesenchymal stem cells (MSCs) for up to 16 days. Moreover, we investigated the effects of various cell culture models on the physiology of MSCs. The physiological nature of MSCs can vary with respect to the cell culture model used. Using the perfusion 3-D cell culture format might affect the proliferation and osteogenic differentiation of MSCs. Overall, we have developed a cell culture system that can achieve multi-well microplate-based perfusion 3-D cell culture in an efficient, cost-effective, and user-friendly manner. These features could facilitate the widespread application of perfusion cell culture models for cell-based assays.

Membrane capacitance of thousands of single white blood cells

As label-free biomarkers, the electrical properties of single cells are widely used for cell type classification and cellular status evaluation. However, as intrinsic cellular electrical markers, previously reported membrane capacitances (e.g. specific membrane capacitance Cspec and total membrane capacitance Cmem) of white blood cells were derived from tens of single cells, lacking statistical significance due to low cell numbers. In this study, white blood cells were first separated into granulocytes and lymphocytes by density gradient centrifugation and were then aspirated through a microfluidic constriction channel to characterize both Cspec and Cmem. Thousands of granulocytes (ncell = 3327) and lymphocytes (ncell = 3302) from 10 healthy blood donors were characterized, resulting in Cspec values of 1.95 ± 0.22 µF cm−2 versus 2.39 ± 0.39 µF cm−2 and Cmem values of 6.81 ± 1.09 pF versus 4.63 ± 0.57 pF. Statistically significant differences between granulocytes and lymphocytes were located for both Cspec and Cmem. In addition, neural network-based pattern recognition was used to classify white blood cells, producing successful classification rates of 78.1% for Cspec and 91.3% for Cmem, respectively. These results indicate that as intrinsic bioelectrical markers, membrane capacitances may contribute to the classification of white blood cells.