Novel Toilet Paper-Based Point-Of-Care Test for the Rapid Detection of Fecal Occult Blood: Instrument Validation Study.

BACKGROUND: Colorectal cancer screening by fecal occult blood testing has been an important public health test and shown to reduce colorectal cancer-related mortality. However, the low participation rate in colorectal cancer screening by the general public remains a problematic public health issue. This fact could be attributed to the complex and unpleasant operation of the screening tool. OBJECTIVE: This study aimed to validate a novel toilet paper-based point-of-care test (ie, JustWipe) as a public health instrument to detect fecal occult blood and provide detailed results from the evaluation of the analytic characteristics in the clinical validation. METHODS: The mechanism of fecal specimen collection by the toilet-paper device was verified with repeatability and reproducibility tests. We also evaluated the analytical characteristics of the test reagents. For clinical validation, we conducted comparisons between JustWipe and other fecal occult blood tests. The first comparison was between JustWipe and typical fecal occult blood testing in a central laboratory setting with 70 fecal specimens from the hospital. For the second comparison, a total of 58 volunteers were recruited, and JustWipe was compared with the commercially available Hemoccult SENSA in a point-of-care setting. RESULTS: Adequate amounts of fecal specimens were collected using the toilet-paper device with small day-to-day and person-to-person variations. The limit of detection of the test reagent was evaluated to be 3.75 µg of hemoglobin per milliliter of reagent. Moreover, the test reagent also showed high repeatability (100%) on different days and high reproducibility (>96%) among different users. The overall agreement between JustWipe and a typical fecal occult blood test in a central laboratory setting was 82.9%. In the setting of point-of-care tests, the overall agreement between JustWipe and Hemoccult SENSA was 89.7%. Moreover, the usability questionnaire showed that the novel test tool had high scores in operation friendliness (87.3/100), ease of reading results (97.4/100), and information usefulness (96.1/100). CONCLUSIONS: We developed and validated a toilet paper-based fecal occult blood test for use as a point-of-care test for the rapid (in 60 seconds) and easy testing of fecal occult blood. These favorable characteristics render it a promising tool for colorectal cancer screening as a public health instrument.

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.

Circulating epithelial cell enumeration facilitates the identification and follow-up of a patient with early stage papillary thyroid microcarcinoma: A case report.

BACKGROUND: This study examines whether the measurement of circulating epithelial cells (CECs) facilitates the identification and follow-up of a patient with thyroid cancer. METHODS: A 29-y-old woman with no cancer history was enrolled as a healthy control in a CEC study. CECs were enriched from the peripheral blood by the negative selection system PowerMag. Various medical examinations were performed on the patient to establish the diagnosis and to follow-up her disease status during treatment. RESULTS: This patient had unexpectedly high CEC counts that were sustained for more than two weeks. Thyroid gland ultra-sonography revealed lesions in the left lobe that could not be confirmed as cancer by magnetic resonance imaging, (18)F-fludeoxyglucose-positron emission tomography-computed tomography or cytopathological analysis, but were histologically confirmed after thyroidectomy as papillary thyroid microcarcinoma. Both the CEC count and serum thyroglobulin (Tg) concentration were significantly decreased after thyroidectomy, and they and the patient's disease status were correlated during remnant ablation therapy. The CEC count returned to normal when the patient was disease-free 10 months after thyroidectomy. CONCLUSIONS: CEC testing facilitates the identification of individuals at risk for cancer. Longitudinal follow-up of the CEC count may complement serum Tg testing for monitoring the status of patients with thyroid cancer.

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.

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.

A pneumatically-driven microfluidic system for size-tunable generation of uniform cell-encapsulating collagen microbeads with the ultrastructure similar to native collagen.

This study reports a microfluidic system for high throughput, uniform, and size-tunable generation of cell-containing collagen microbeads. The principle is based on two pneumatically-driven mechanisms to achieve multi-channel mixture suspension transportation, and to actuate the spotting actions of micro-vibrators that continuously generate tiny collagen micro-droplets into a thin oil layer and then a sterile Pluronic® F127 surfactant solution located below. The temporarily formed collagen microdroplets are then thermally gelatinized. By regulating the feeding rate of cells/collagen suspension, and the spotting frequency of micro-vibrator, the size of the collagen microbeads can be manipulated. One of the key technical features is its capability to generate uniform collagen microbeads (coefficient of variation: 5.4-8.6 %) with sizes ranging from 73.9 to 349.3 µm in diameter. This is currently difficult to achieve using the existing methods particularly the generation of cell-encapsulating collagen microbeads with diameter less than 100 µm. Another advantageous trait is that the ultrastructure of the generated collagen microbeads is similar to that found in native collagen. In this study, moreover, the use of the proposed device for the microencapsulation of 3T3 cells in collagen microbeads has been successfully demonstrated showing that the encapsulated cells maintained high cell viability (96 ± 2 %). Furthermore, a reasonable proliferative capability of the encapsulated cells was observed during 7 days culture. As a whole, the proposed device has opened up a new route to generate cell-containing collagen microbeads, which is found particularly meaningful for biomedical applications.

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.

High-purity and label-free isolation of circulating tumor cells (CTCs) in a microfluidic platform by using optically-induced-dielectrophoretic (ODEP) force.

Negative selection-based circulating tumor cell (CTC) isolation is believed valuable to harvest more native, and in particular all possible CTCs without biases relevant to the properties of surface antigens on the CTCs. Under such a cell isolation strategy, however, the CTC purity is normally compromised. To address this issue, this study reports the integration of optically-induced-dielectrophoretic (ODEP) force-based cell manipulation, and a laminar flow regime in a microfluidic platform for the isolation of untreated, and highly pure CTCs after conventional negative selection-based CTC isolation. In the design, six sections of moving light-bar screens were continuously and simultaneously exerted in two parallel laminar flows to concurrently separate the cancer cells from the leukocytes based on their size difference and electric properties. The separated cell populations were further partitioned, delivered, and collected through the two flows. With this approach, the cancer cells can be isolated in a continuous, effective, and efficient manner. In this study, the operating conditions of ODEP for the manipulation of prostate cancer (PC-3) and human oral cancer (OEC-M1) cells, and leukocytes with minor cell aggregation phenomenon were first characterized. Moreover, performances of the proposed method for the isolation of cancer cells were experimentally investigated. The results showed that the presented CTC isolation scheme was able to isolate PC-3 cells or OEC-M1 cells from a leukocyte background with high recovery rate (PC-3 cells: 76-83%, OEC-M1 cells: 61-68%), and high purity (PC-3 cells: 74-82%, OEC-M1 cells: 64-66%) (set flow rate: 0.1 µl min(-1) and sample volume: 1 µl). The latter is beyond what is currently possible in the conventional CTC isolations. Moreover, the viability of isolated cancer cells was evaluated to be as high as 94 ± 2%, and 95 ± 3% for the PC-3, and OEC-M1 cells, respectively. Furthermore, the isolated cancer cells were also shown to preserve their proliferative capability. As a whole, this study has presented an ODEP-based microfluidic platform that is capable of isolating CTCs in a continuous, label-free, cell-friendly, and particularly highly pure manner. All these traits are found particularly meaningful for exploiting the harvested CTCs for the subsequent cell-based, or biochemical assays.

An integrated microfluidic cell culture system for high-throughput perfusion three-dimensional cell culture-based assays: effect of cell culture model on the results of chemosensitivity assays.

Although microfluidic cell culture systems are versatile tools for cellular assays, their use has yet to set in motion an evolutionary shift away from conventional cell culture methods. This situation is mainly due to technical hurdles: the operational barriers to the end-users, the lack of compatible detection schemes capable of reading out the results of a microfluidic-based cellular assay, and the lack of fundamental data to bridge the gap between microfluidic and conventional cell culture models. To address these issues, we propose a high-throughput, perfusion, three-dimensional (3-D) microfluidic cell culture system encompassing 30 microbioreactors. This integrated system not only aims to provide a user-friendly cell culture tool for biologists to perform assays but also to enable them to obtain precise data. Its technical features include (i) integration of a heater chip based on transparent indium tin oxide glass, providing stable thermal conditions for cell culturing; (ii) a microscale 3-D culture sample loading scheme that is both efficient and precise; (iii) a non-mechanical pneumatically driven multiplex medium perfusion mechanism; and (iv) a microplate reader-compatible waste medium collector array for the subsequent high throughput bioassays. In this study, we found that the 3-D culture sample loading method provided uniform sample loading [coefficient of variation (CV): 3.2%]. In addition, the multiplex medium perfusion mechanism led to reasonably uniform (CV: 3.6-6.9%) medium pumping rates in the 30 microchannels. Moreover, we used the proposed system to perform a successful cell culture-based chemosensitivity assay. To determine the effects of cell culture models on the cellular proliferation, and the results of chemosensitivity assays, we compared our data with that obtained using three conventional cell culture models. We found that the nature of the cell culture format could lead to different evaluation outcomes. Consequently, when establishing a cell culture model for in vitro cell-based assays, it might be necessary to investigate the fundamental physiological variations of the cultured cells in different culture systems to avoid any misinterpretation of data. As a whole, we have developed an integrated microfluidic cell culture system that overcomes several technical hurdles commonly encountered in the practical application of microfluidic cell culture systems, and we have obtained fundamental information to reconcile differences found with data acquired using conventional methods.

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.

Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays.

This study reports a microfluidic cell culture chip consisting of 48 microbioreactors for high-throughput perfusion 3-dimensional (3-D) cell culture-based assays. Its advantages include the capability for multiplexed and backflow-free medium delivery, and both efficient and high-throughput micro-scale, 3-D cell culture construct loading. In this work, the microfluidic cell culture chip is fabricated using two major processes, specifically, a computer-numerical-controlled (CNC) mold machining process and a polydimethylsiloxane (PDMS) replication process. The chip is composed of micropumps, microbioreactors, connecting microchannels and a cell/agarose scaffold loading mechanism. The performance of the new pneumatic micropumps and the cell/agarose scaffold loading mechanism has been experimentally evaluated. The experimental results show that this proposed multiplexed medium-pumping design is able to provide a uniform pumping rate ranging from 1.5 to 298.3 µl hr(-1) without any fluid backflow and the resultant medium contamination. In addition, the simple cell/agarose loading method has been proven to be able to load the 3-D cell culture construct uniformly and efficiently in all 48 microbioreactors investigated. Furthermore, a micro-scale, perfusion, 3-D cell culture-based assay has been successfully demonstrated using this proposed cell culture chip. The experimental results are also compared to a similar evaluation using a conventional static 3-D cell culture with a larger scale culture. It is concluded that the choice of a cell culture format can influence assay results. As a whole, because of the inherent advantages of a miniaturized perfusion 3-D cell culture assay, the cell culture chip not only can provide a stable, well-defined and more biologically-meaningful culture environment, but it also features a low consumption of research resources. Moreover, due to the integrated medium pumping mechanism and the simple cell/agarose loading method, this chip is economical and time efficient. All of these traits are particularly useful for high-precision and high-throughput 3-D cell culture-based assays.

Microfluidic cell culture systems for drug research.

In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

A microfluidic cell culture platform for real-time cellular imaging.

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 mum, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.

A high throughput perfusion-based microbioreactor platform integrated with pneumatic micropumps for three-dimensional cell culture.

This study reports a new perfusion-based, micro three-dimensional (3-D) cell culture platform for high-throughput cell culture using enabling microfluidic technologies. In this work, the micro 3-D cell culture platform is fabricated based on SU-8 lithography and polydimethylsiloxane replication processes. The micro cell culture platform can maintain homogenous and stable culture environments, as well as provide pumping of multiple mediums and efficient cell/agarose (scaffold) loading functions, which allows realization of more precise and high-throughput cell culture-based assays. In this study, the design of a high-throughput medium pumping mechanism was especially highlighted. A new serpentine-shaped pneumatic micropump was used to provide the required medium pumping mechanism. Pneumatic microchannels with a varied length and U-shape bending corners were designed to connect three rectangular pneumatic chambers such that one can fine-tune the pumping rate of the S-shape micropump by using the fluidic resistance. To achieve a high-throughput medium pumping function, a pneumatic tank was designed to simultaneously activate all of the 30 pneumatic micropumps with a uniform pumping rate. Results show that the pumping rates of the 30 integrated micropumps were statistically uniform with a flow rate ranging from 8.5 to 185.1 microl h(-1), indicating the present multiple medium pumping mechanism is feasible for high-throughput medium delivery purposes. Furthermore, as a demonstration case study, 3-D culture of oral cancer cell was successfully performed, showing that the cell viability remained as high as 95% - 98% during the 48 h cell culture. As the result of miniaturization, this perfusion-based 3-D cell culture platform not only provides a well-defined and stable culture condition, but also greatly reduces the sample/reagent consumption and the need for human intervention. Moreover, due to the integrated capability for multiple medium pumping, high-throughput research work can be achieved. These traits are found particularly useful for high-precision and high-throughput, 3-D cell culture-based assay.