Chien-Yu Fu

A microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis

Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.

Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX

Colorectal cancer (CRC) is the most frequently diagnosed cancer around the world, causing about 700,000 deaths every year. It is clear now that a small fraction of CRC, named colorectal cancer stem cells (CSCs) exhibiting self-renewal and extensive proliferative activities, are hard to be eradicated. Unfortunately, highly specific biomarkers for colorectal CSC (CR-CSCs) are lacking that prohibits the development of effective therapeutic strategies. This study designed and manufactured a novel microfluidic system capable of performing a fully automated cell-based, systematic evolution of ligands by exponential enrichment (SELEX) process. Eight CR-CSC/CRC-specific aptamers were successfully selected using the microfluidic chip. Three of the aptamers showed high affinities towards their respective target cells with a dissociation constant of 27.4, 28.5 and 12.3 nM, which are comparable to that of antibodies.

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

A microfluidic device for antimicrobial susceptibility testing based on a broth dilution method.

Bacterial resistance to antimicrobial compounds is increasing at a faster rate than the development of new antibiotics; this represents a critical challenge for clinicians worldwide. Normally, the minimum inhibitory concentration of an antibiotic, the dosage at which bacterial growth is thwarted, provides an effective quantitative measure for antimicrobial susceptibility testing, and determination of minimum inhibitory concentration is conventionally performed by either a serial broth dilution method or with the commercially available Etest® (Biomerieux, France) kit. However, these techniques are relatively labor-intensive and require a significant amount of training. In order to reduce human error and increase operation simplicity, a simple microfluidic device that can perform antimicrobial susceptibility testing automatically via a broth dilution method to accurately determine the minimum inhibitory concentration was developed herein. As a proof of concept, wild-type (ATCC 29212) and vancomycin-resistant Enterococcus cells were incubated at five different vancomycin concentrations on-chip, and the sample injection, transport, and mixing processes occurred within five reaction chambers and three reagent chambers via the chips automatic dispensation and dilution functions within nine minutes. The minimum inhibitory concentration values measured after 24h of antibiotic incubation were similar to those calculated using Etest®. With its high flexibility, reliability, and portability, the developed microfluidic device provides a simple method for antimicrobial susceptibility testing in an automated format that could be implemented for clinical and point-of-care applications.

High-efficiency rare cell identification on a high-density self-assembled cell arrangement chip

Detection of individual target cells among a large amount of blood cells is a major challenge in clinical diagnosis and laboratory protocols. Many researches show that two dimensional cells array technology can be incorporated into routine laboratory procedures for continuously and quantitatively measuring the dynamic behaviours of large number of living cells in parallel, while allowing other manipulations such as staining, rinsing, and even retrieval of targeted cells. In this study, we present a high-density cell self-assembly technology capable of quickly spreading over 300 000 cells to form a dense mono- to triple-layer cell arrangement in 5 min with minimal stacking of cells by the gentle incorporation of gravity and peripheral micro flow. With this self-assembled cell arrangement (SACA) chip technology, common fluorescent microscopy and immunofluorescence can be utilized for detecting and analyzing target cells after immuno-staining. Validated by experiments with real human peripheral blood samples, the SACA chip is suitable for detecting rare cells in blood samples with a ratio lower than 1/100 000. The identified cells can be isolated and further cultured in-situ on a chip for follow-on research and analysis. Furthermore, this technology does not require external mechanical devices, such as pump and valves, which simplifies operation and reduces system complexity and cost. The SACA chip offers a high-efficient, economical, yet simple scheme for identification and analysis of rare cells. Therefore, potentially SACA chip may provide a feasible and economical platform for rare cell detection in the clinic.

Integration of organic opto-electrowetting and poly(ethylene) glycol diacrylate (PEGDA) microfluidics for droplet manipulation

This paper reports a fabrication technology which integrates organic opto-electrowetting (OEW) with PEGDA-based microfluidics between stacked ITO glass chips for droplet generation and manipulation. Organic OEW can be realized by using titanium oxide phthalocyanine (TiOPc) as a photoconductive layer. Optical images can be projected on an organic OEW area to induce the local virtual electrodes which reduce the surface energy. Low-molecular-weight (LMW) PEGDA material is used to form stable and biocompatible microchannels between the stacked ITO glass chips by UV photopolymerization process. PEGDA-based microfluidics provides a simpler way to enhance droplet-in-oil applications in organic OEW. This technology requires only spin-coating and photolithography and is more cost-effective than previous methods.

Microfluidic platforms for rapid screening of cancer affinity reagents by using tissue samples

Cancer is the most serious disease worldwide, and ovarian cancer (OvCa) is the second most common type of gynecological cancer. There is consequently an urgent need for early-stage detection of OvCa, which requires affinity reagent biomarkers for OvCa. Systematic evolution of ligands by exponential enrichment (SELEX) and phage display technology are two powerful technologies for identifying affinity reagent biomarkers. However, the benchtop protocols for both screening technologies are relatively lengthy and require well-trained personnel. We therefore developed a novel, integrated microfluidic system capable of automating SELEX and phage display technology. Instead of using cancer cell lines, it is the first work which used tissue slides as screening targets, which possess more complicated and uncovered information for affinity reagents to recognize. This allowed for the identification of aptamer (nucleic acid) and peptide probes specific to OvCa cells and tissues. Furthermore, this developed system could be readily modified to uncover affinity reagents for diagnostics or even target therapy of other cancer cell types in the future.

Toxicity analysis of poly(sodium-4-styrenesulfonate) coated graphene on HMEC-1 cells under dynamic conditions mimicking blood flow

Graphene has been proven to have great potential in medical applications. The goal of this study is to evaluate the toxicity of graphene on human microvascular endothelial cells (HMEC-1) under flowing conditions mimicking blood circulation. Graphene was prepared by the electrochemical exfoliation method and conjugated with poly(sodium-4-styrenesulfonate) (PSS) to improve its hydrophilicity and dispersion behavior. Under static cell growth conditions, 80 mg ml−1 of graphene reduced cell viability to 60% of the control. Graphene was observed to aggregate on the cell surface by interacting with filopodia. In contrast, graphene applied to cells under flowing conditions showed no significant negative influence on the cells regardless of graphene concentration, incubation time, and the exerted shear force. Graphene also showed lower toxicity towards cells in a flowing culture than multi-walled carbon nanotubes (MWCNTs) and carbon black. Together, these results suggest that the prolonged interaction of graphene with the cell surface is critical in causing cell damage. The lower circulatory toxicity of graphene compared to MWCNTs and carbon black also suggests that graphene is more suitable for use in drug delivery systems.

A 3-D capillary-endothelium-mimetic microfluidic chip for studying the extravasation behaviour of neutrophils

Inflammation is a fundamental biology problem with daily threats to human health. In early inflammatory stage, the most important cell type involved is neutrophil, which represents the most massive leukocyte population. In this paper, we report a 3D reusable microfluidic chip which mimics the capillary endothelial lining and we have made an attempt to imitate the hemodynamic-factor in order to study the extravasation behavior of neutrophils. This microsystem is a continuous flow system which mimics the dynamic, three dimensional micro environment in the blood vessel.