Kin Fong Lei

Development of graphene-based sensors on paper substrate for the measurement of pH value of analyte

With excellent properties of graphene material, a number of graphene-based devices have been developed with excellent performance in the past decades. However, difficulty of processing graphene material is a hurdle for its practical realization. In this work, we propose to use vacuum filtration process as the major technique to fabricate graphene-based devices. This technique is simple and effective to practically realize graphene-based sensors on paper substrate, which are equipped with the advantages of low cost, simplicity, flexibility, and disposability. In order to show the functionality of the sensor, determination of pH value of analyte was demonstrated by direct measurement of the resistance across the sensor. Results showed that the sensor has the sensitivity of 30.8 Ω/pH and high linearity (R2=0.9282). Since graphene material can be functionalized by various molecules, it is expected that the graphene-based sensor can be further developed to realize more chemical and biological assays with high sensitivity and specificity for remote environment.

Real-time and label-free impedimetric analysis of the formation and drug testing of tumor spheroids formed via the liquid overlay technique

To achieve high clinical relevance of drug testing, a spheroid culture model is generally used for a standard assay of early tumors. Currently, the quantification of tumor spheroids is generally based on imaging techniques and conventional bioassays. However, additional processing and sophisticated instrumentation are required, which makes high throughput screening infeasible. In this study, tumor spheroids were formed on a thin layer of non-adherent surface (agarose hydrogel), and their responses were monitored by impedance measurements across the interdigitated electrodes fabricated below the hydrogel. The impedance magnitude change was defined as an index to describe the summation effect of the number and size of tumor spheroids. Real-time and label-free impedimetric analysis of the formation and chemosensitivity of tumor spheroids was demonstrated. The technique has the simplicity of device fabrication and experimental processing. Real-time and quantitative assessment could be realized for a routine tool of a spheroid culture.

Towards a high throughput impedimetric screening of chemosensitivity of cancer cells suspended in hydrogel and cultured in a paper substrate

In order to achieve high predictive value of cell chemosensitivity test for clinical efficacy, cancer cells were suggested to be encapsulated and cultured in hydrogel to mimic the natural microenvironment of tumors. However, handling 3D cells/hydrogel culture construct is tedious and cellular response is difficult to be quantitatively analyzed. In the current study, a novel platform for conducting 3D cell culture and analyzing cell viability has been developed towards a high throughput drug screening. Cells encapsulated in the hydrogel were cultured in the microwells of a paper substrate. The substrate was then immersed in the culture medium containing drug for 2 days. At 24 and 48h during the culture course, the paper substrate was placed on the measurement electrodes for conducting the impedance measurement in order to quantify the cell viability in the hydrogel. Cell viability of two human hepatoma cell lines (Huh7 and Hep-G2) was quantitatively investigated under the treatment of two drugs (doxorubicin and etoposide). The results represented by IC50 values revealed that Huh7 cells had a higher drug resistance than Hep-G2 cells and doxorubicin had a higher efficacy than etoposide for treating hepatocellular carcinoma. The current work has demonstrated a high throughput, convenient, and quantitative platform for the investigation of chemosensitivity of cells cultured in the 3D environment.

Development of a co-culture device for the study of human tenocytes in response to the combined stimulation of electric field and platelet rich plasma (PRP)

One of the objectives of rotator cuff repairs is to achieve biological healing and recovery in the tendon-bone zone. Some clinical evaluations reported the feasibility of tendon healing based on the stimulations of electric field and platelet-rich plasma (PRP). However, because of lack of appropriate tool for in vitro primary culture under complicated conditions, the efficacy and standard protocol of these healing approaches are still controversial among clinical experts. In this study, a novel co-culture device was developed for the study of tenocytes proliferation under single and combined stimulations of electric field and PRP. The device was a culture well divided into three sub-chambers separated by a barrier and embedded with a pair of parallel plate electrodes. Tenocytes and PRP gel could be respectively loaded into the sub-chambers and cultured with interlinked medium. Hence, tenocytes could concurrently receive a uniform electric field and platelet-derived growth factors by diffusion. Results revealed that the proliferation of tenocytes could be significantly enhanced by these stimulations. The device provides a precise and practical approach for the in vitro study of tendon healing, especially for PRP study. Moreover, optimization of the conditions of electric field and PRP could be determined by in vitro screening procedure before surgery to provide a personalized therapy.

Toward the Development of an Artificial Brain on a Micropatterned and Material-Regulated Biochip by Guiding and Promoting the Differentiation and Neurite Outgrowth of Neural Stem/Progenitor Cells

An in vitro model mimicking the in vivo environment of the brain must be developed to study neural communication and regeneration and to obtain an understanding of cellular and molecular responses. In this work, a multilayered neural network was successfully constructed on a biochip by guiding and promoting neural stem/progenitor cell differentiation and network formation. The biochip consisted of 3 × 3 arrays of cultured wells connected with channels. Neurospheroids were cultured on polyelectrolyte multilayer (PEM) films in the culture wells. Neurite outgrowth and neural differentiation were guided and promoted by the micropatterns and the PEM films. After 5 days in culture, a 3 × 3 neural network was constructed on the biochip. The function and the connections of the network were evaluated by immunocytochemistry and impedance measurements. Neurons were generated and produced functional and recyclable synaptic vesicles. Moreover, the electrical connections of the neural network were confirmed by measuring the impedance across the neurospheroids. The current work facilitates the development of an artificial brain on a chip for investigations of electrical stimulations and recordings of multilayered neural communication and regeneration.

Comparison between xCELLigence biosensor technology and conventional cell culture system for real-time monitoring human tenocytes proliferation and drugs cytotoxicity screening

BackgroundLocal injections of anesthetics, NSAIDs, and corticosteroids for tendinopathies are empirically used. They are believed to have some cytotoxicity toward tenocytes. The maximal efficacy dosages of local injections should be determined. A commercial 2D microfluidic xCELLigence system had been developed to detect real-time cellular proliferation and their responses to different stimuli and had been used in several biomedical applications. The purpose of this study is to determine if human tenocytes can successfully proliferate inside xCELLigence system and the result has high correlation with conventional cell culture methods in the same condition.MethodsFirst passage of human tenocytes was seeded in xCELLigence and conventional 24-well plates. Ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone with different concentrations (100, 50, and 10% diluted of clinical usage) were exposed in both systems. Gene expression of type I collagen, type III collagen, tenascin-C, decorin, and scleraxis were compared between two systems.ResultsHuman tenocytes could proliferate both in xCELLigence and conventional cell culture systems. Cytotoxicity of each drug revealed dose-dependency when exposed to tenocytes in both systems. Significance was found between groups. All the four drugs had comparable cytotoxicity in their 100% concentration. When 50% concentration was used, betamethasone had a relatively decreased cytotoxicity among them in xCELLigence but not in conventional culture. When 10% concentration was used, betamethasone had the least cytotoxicity. Strong and positive correlation was found between cell index of xCELLigence and result of WST-1 assay (Pearson’s correlation [r]xa0=xa00.914). Positive correlation of gene expression between tenocytes in xCELLigence and conventional culture was also observed. Type I collagen: [r]u2009=u20090.823; type III collagen: [r]u2009=u20090.899; tenascin-C: [r]u2009=u20090.917; decorin: [r]u2009=u20090.874; and scleraxis: [r]u2009=u20090.965.ConclusionsHuman tenocytes could proliferate inside xCELLigence system. These responses varied when tenocytes were exposed to different concentrations of ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone. The result of cell proliferation and gene expression of tenocytes in both xCELLigence and conventional culture system is strongly correlated.Clinical relevancexCELLigence culture system may replace conventional cell culture, which made real-time tenocyte proliferation monitoring possible.

High throughput and automatic colony formation assay based on impedance measurement technique

AbstractTo predict the response of in vivo tumors, in vitro culture of cell colonies was suggested to be a standard assay to achieve high clinical relevance. To describe the responses of cell colonies, the most widely used quantification method is to count the number and size of cell colonies under microscope. That makes the colony formation assay infeasible to be high throughput and automated. In this work, in situ analysis of cell colonies suspended in soft hydrogel was developed based on impedance measurement technique. Cell colonies cultured between a pair of parallel plate electrodes were successfully analyzed by coating a layer of base hydrogel on one side of electrode. Real-time and label-free monitoring of cell colonies was realized during the culture course. Impedance magnitude and phase angle respectively represented the summation effect of colony responses and size of colonies. In addition, dynamic response of drug-treated colonies was demonstrated. High throughput and automatic colony formation assay was realized to facilitate more objective assessments in cancer research.n Graphical AbstractHigh throughput and automatic colony formation assay was realized by in situ impedimetric analysis across a pair of parallel plate electrodes in a culture chamber. Cell colonies suspended in soft hydrogel were cultured under the tested substance and their dynamic response was represented by impedance data.

Co-Culturing Cancer Cells and Normal Cells in a Biochip under Electrical Stimulation

The ideal cancer therapeutic strategy is to inhibit the tumor with minimal influence on the normal tissue. Recently, applying an alternating electric field for inhibiting tumor was developed; but, it has not been adopted to be one of the regular therapeutic options. More basic scientific evidence is needed to clarify the efficacy and safety. In the current study, co-culturing cancer cells and normal cells under the electrical stimulation was conducted to provide evidence of this novel cancer therapy. A microfluidic cell culture biochip has been developed and consisted of nine culture chambers incorporating with stimulating electrodes. Cells cultured in the chamber received uniform electric field and cell viability was studied during the culture course. The electric field perturbs cell division and the correlation between cell proliferation rate and inhibition effect was studied among five cell lines, i.e., Huh7, HeLa, TW06, BM1, and HEL299. The results confirmed that cells with higher proliferation rate responded to a higher inhibition. In addition, co-culturing cancer cells and normal cells was conducted to mimic in vivo microenvironment that consists of both cancer and stromal cells. The cancer cells and normal cells were respectively transduced with green fluorescent protein and red fluorescent protein in order to differentiate the cells in a same culture chamber. During the culture course, the electric field was applied to the culture chamber and both cells simultaneously received the field. The results indicated that the growth of the cancer cells were inhibited while the normal cells were maintained. These results provided the evidence of the therapeutic efficacy and safety. Moreover, the microfluidic cell culture biochip could be used for the systematic and precise investigations of the cellular responses under the electrical stimulation.

Investigation of osteogenic activity of primary rabbit periosteal cells stimulated by multi-axial tensile strain

Periosteum–derived cells was indicated to respond to mechanical force and have stem cell potential capable of differentiating into multiple tissue. Investigation of osteogenic activity under mechanical stimulation is important to understand the therapeutic conditions of fracture healing. In this work, a cell culture platform was developed for respectively providing isotropic and anisotropic axial strain. Primary rabbit periosteal cells were isolated and cultured in the chamber. Multi-axial tensile strain was received and osteogenic activity was investigated by mRNA expressions of CBFA1 and OPN. The highest mRNA expression was found in moderate strain (5-8%) under anisotropic axial strain. These results provided important foundation for further in vivo studies and development of tailor-made stretching rehabilitation equipment.

Development of a 2-chamber culture system for impedimetric monitoring of cell-cell interaction

In cancer research, study of cell-cell interaction is important to understand tumor initiation, progression, metastasis, and therapeutic resistance. Conventionally, transwell system was adopted and cell proliferation was quantified by end-point bio-assays. The operations are labor-intensive and time-consuming while studying of the dynamic cellular responses of cell-cell interaction. Although impedance measurement was suggested to be a promising technique to monitor cellular responses, electrodes cannot be integrated into the transwell for the measurement purpose. In this work, a 2-chamber culture system incorporated with impedance measurement technique was developed to quantitatively study cell-cell interaction. The chamber was composed of 2 sub-chambers separated with a barrier. By this design, two types of cells could be independently cultured and concurrently monitored under common medium supplied. Cell-cell interaction was demonstrated by aberrant cell proliferation induced by the EGF secreted from the transfected cells cultured on another sub-chamber. Real-time and non-invasive monitoring of cell-cell interaction was successfully demonstrated. This work provides a practical solution for monitoring the dynamic cellular responses of cell-cell interaction during the culture course. It is a reliable and convenient platform and facilitate more quantitative assessments in cancer research.