Investigation of uniform sized multicellular spheroids raised by microwell arrays after the combined treatment of electric field and anti-cancer drug.

Nowadays, cancer disease is continuously identified as the leading cause of mortality worldwide. Cancer chemotherapeutic agents have been continuously developing to achieve high curative effectiveness and low side effects. However, solid tumors present the properties of low drug penetration and resistance of quiescent cells. Radiation therapy is concurrently given in some cases; but it induces different levels of adverse effects. In the current work, uniform sized multicellular spheroids were raised by microwell arrays to mimic the architecture of solid tumors. Investigation of the response of the spheroids was conducted after the treatment of alternating electric field. The result showed that the electric field could induce early apoptosis by disturbing cell membrane. Moreover, combined treatment of electric field and anti-cancer drug was applied to the spheroids. The electric field synergistically enhanced the treatment efficacy because the anti-cancer drug could permeate through the disrupted cell membrane. Significant improvement of late apoptosis was shown by the combined treatment. Because the electric field treatment induces limited side effect to the patient, lower dosage of anti-cancer drug may be applied to the patients for achieving curative effectiveness.

The influence of laser intensity activated plasmonic gold nanoparticle-generated photothermal effects on cellular morphology and viability: a real-time, long-term tracking and monitoring system.

In this study, a microfluidic apparatus embedded with microstructures was designed and aligned with a laser and dark-field microscope for real-time, long-term observation of photothermal effects on cells. Gold nanorods (AuNRs, 10 ppm) were incubated with MG-63 human osteosarcoma cells for 3 h. Then, the cells were exposed to a continuous-wave laser at a wavelength of 830 nm for 10, 20, and 30 min at 5, 9, 14, 24, and 32 W cm-2. Subsequent changes in morphology were observed. Under different conditions, cell membrane blebbing occurred at different times, indicating that actin filaments were destroyed in large quantities and apoptosis was induced. In suitable conditions, we first induced slight cell injury by causing cytoskeletal fractures with a high-energy laser; then, the cells were irradiated with a low-energy laser at 0.3 W cm-2. We found that among cells treated with the high-energy laser, cells treated additionally with a low-energy laser showed extended viability compared with cells that did not receive the additional treatment.

Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.

BACKGROUND: Local antibiotic application has been widely used in orthopedic surgery. The dose-related toxicity of antibiotics towards periosteal tissues and resulting effects on osteogenic expression are yet to be studied. METHODS: Periosteal cells harvested from the medial tibia of New Zealand White rabbits were used. A seeding density of 5 × 103 cells/cm2 was determined to be optimal for testing in the pilot study; the cells were cultured in xCELLigence 96-well plates. Microfluidic impedance analyzers were used to monitor cellular proliferation in microfluidic culture systems with exposure to three different concentrations (10 µg/mL, 100 µg/mL, and 1000 µg/mL) of cefazolin, ciprofloxacin, and vancomycin, respectively. The correlation of cell index at day 7 with optical density values from WST-1 assays using conventional cultures was evaluated by calculating the Pearson's coefficient. RNA analysis was performed to investigate the expression of osteogenic markers in the cultured cells, including core-binding factor alpha 1 (Cbfa1), osteopontin (OPN), and osteopontin promoter (OPNp), relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control. RESULTS: A significant dose-related inhibition of cell index was found for all the 3 antibiotics, whereas the WST-1 assays showed a significant dose-related inhibition of cellular proliferation only at a high dose of cefazolin (1000 µg/mL) and medium-to-high dose of ciprofloxacin (100 µg/mL and 1000 µg/mL). Pearson's coefficient analysis indicated a high correlation between the cell index and optical density values of WST-1 assays only for medium and high doses of ciprofloxacin (100 µg/mL and 1000 µg/mL); a moderate correlation was seen for cefazolin, and a low dose of ciprofloxacin (10 µg/mL). RNA analysis confirmed significant dose-related inhibition of cfba1, OPN, and OPNp expression by all three antibiotics. CONCLUSION: With optimal seeding amounts, rabbit periosteal cells can be dynamically monitored in the xCELLigence microfluidic system. Dose-related inhibition of cellular proliferation and osteogenic expression was found after exposure to cefazolin and ciprofloxacin. By providing real-time detection and exhibiting comparable correlation, microfluidic impedance-based analyzer is a feasible alternative to the conventional WST-1 assays.

Dissociated effect and Chemosensitive enhancement of tumor spheroids influenced by an electric field in a microdevice.

The use of electric field for cancer therapy has been proposed for a novel non-invasive cancer therapeutic approach that provides better quality of life for patients. However, argument of the efficacy hampers the therapeutic development for various cancer diseases. More scientific evidences are necessary to be addressed by basic research. The current in vitro cell culture study reports the responses of tumor spheroids after the application of an alternating electric field. Human hepatocarchinoma cells suspended in soft hydrogel were cultured in a cell culture device embedded with stimulating electrodes. Tumor spheroids gradually formed and alternating electric field was then applied during the culture course. Investigation of cell viability and cell cycle were conducted to optimize the treatment conditions. The results showed that the electric potential of 1.0 Vpp and frequency of 130 kHz was the minimal effective conditions for inhibiting tumor spheroids. Importantly, dissociation of tumor spheroids was observed after the treatment. The effectiveness of chemotherapeutic agents was shown to be enhanced while the electric filed was simultaneously applied to the tumor spheroids. These results provided solid foundation for developing the effective therapeutic strategies.

Proliferation arrest, selectivity, and chemosensitivity enhancement of cancer cells treated by a low-intensity alternating electric field.

Elimination of serious side effects is a desired feature of cancer therapy. Alternating electric field treatment is one approach to the non-invasive treatment of cancer. The efficacy and safety of this novel therapy are confirmed for the treatment of glioblastoma multiforme. In the current study, we co-cultured cancer cells and normal cells to investigate the selectivity and chemosensitivity enhancement of an electric field treatment. Cancer cells (cell line: HeLa and Huh7) and fibroblasts (cell line: HEL299) were cultured in an in-house-developed cell culture device embedded with stimulating electrodes. A low-intensity alternating electric field was applied to the culture. The field significantly induced proliferation arrest of the cancer cells, while had limited influence on the fibroblasts. Moreover, in combination with the anti-cancer drug, damage to the cancer cells was enhanced by the electric field. Thus, a lower dosage of the drug could be applied to achieve the same treatment effectiveness. This study provides evidence that low-intensity electric field treatment selectively induced proliferation arrest and enhanced the chemosensitivity of the cancer cells. This electro-chemotherapy could be developed and applied as a regional cancer therapy with minimal side effects.

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.

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.

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

To 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. Graphical Abstract High 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.

Development of a multi-band photoacoustic tomography imaging system based on a capacitive micromachined ultrasonic transducer array.

Photoacoustic tomography (PAT) as a hybrid technology combines the high optical contrast and high acoustic resolution in a single imaging modality. However, most of the available PAT systems cannot comprehensively or accurately characterize biological systems at multiple length scales due to the use of narrow bandwidth commercial ultrasonic transducers. In this study, we fabricated a novel multi-band capacitive micromachined ultrasonic transducer (CMUT) array, and first developed a CMUT-based multi-band photoacoustic tomography (MBPAT) imaging system. The MBPAT imaging system was examined by the phantom experiment, and then was successfully applied to image the zebrafish in vivo. The imaging results indicated that CMUT-array-based MBPAT can provide a more comprehensive and accurate characterization of biological tissues, which exhibit the potential of MBPAT/CMUT in various areas of biomedical imaging.

Development of a Flexible Non-Metal Electrode for Cell Stimulation and Recording.

This study presents a method of producing flexible electrodes for potentially simultaneously stimulating and measuring cellular signals in retinal cells. Currently, most multi-electrode applications rely primarily on etching, but the metals involved have a certain degree of brittleness, leaving them prone to cracking under prolonged pressure. This study proposes using silver chloride ink as a conductive metal, and polydimethysiloxane (PDMS) as the substrate to provide electrodes with an increased degree of flexibility to allow them to bend. This structure is divided into the electrode layer made of PDMS and silver chloride ink, and a PDMS film coating layer. PDMS can be mixed in different proportions to modify the degree of rigidity. The proposed method involved three steps. The first segment entailed the manufacturing of the electrode, using silver chloride ink as the conductive material, and using computer software to define the electrode size and micro-engraving mechanisms to produce the electrode pattern. The resulting uniform PDMS pattern was then baked onto the model, and the flow channel was filled with the conductive material before air drying to produce the required electrode. In the second stage, we tested the electrode, using an impedance analyzer to measure electrode cyclic voltammetry and impedance. In the third phase, mechanical and biocompatibility tests were conducted to determine electrode properties. This study aims to produce a flexible, non-metallic sensing electrode which fits snugly for use in a range of measurement applications.

The Structure Design of Piezoelectric Poly(vinylidene Fluoride) (PVDF) Polymer-Based Sensor Patch for the Respiration Monitoring under Dynamic Walking Conditions.

This study reports a piezoelectric poly(vinylidene fluoride) (PVDF) polymer-based sensor patch for respiration detections in dynamic walking condition. The working mechanism of respiration signal generation is based on the periodical deformations on a human chest wall during the respiratory movements, which in turn mechanically stretch the piezoelectric PVDF film to generate the corresponding electrical signals. In this study, the PVDF sensing film was completely encapsulated within the sensor patch forming a mass-spring-damper mechanical system to prevent the noises generated in a dynamic condition. To verify the design of sensor patch to prevent dynamic noises, experimental investigations were carried out. Results demonstrated the respiration signals generated and the respiratory rates measured by the proposed sensor patch were in line with the same measurements based on a commercial respiratory effort transducer both in a static (e.g., sitting) or dynamic (e.g., walking) condition. As a whole, this study has developed a PVDF-based sensor patch which is capable of monitoring respirations in a dynamic walking condition with high fidelity. Other distinctive features include its small size, light weight, ease of use, low cost, and portability. All these make it a promising sensing device to monitor respirations particularly in home care units.

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.

Protein binding reaction enhanced by bi-directional flow driven by on-chip thermopneumatic actuator.

A microfluidic immunoassay system was developed for the study of the enhancement of protein binding reaction. The system mainly consisted of a thermopneumatic actuator and a reaction chamber. Reagent was pre-installed in the on-chip reservoir and manipulated by the actuator. Such design could eliminate the external tubing connections in order to reduce the waste of reagent and improve the portability. The on-chip actuator could manipulate the reagent bi-directionally to induce vortexes in the chamber. Enhancement of protein binding reaction was demonstrated by the protein model pair, i.e., mouse IgG and anti-mouse IgG. By such bi-directional fluid motion, more binding opportunities between suspended protein and its surface-immobilized counterpart were generated to improve the performance of immunoassay. It showed that an 83.74 % enhancement of the binding reaction was achieved, compared with the static situation. As a whole, the proposed microfluidic system is highly integrated and can enhance the protein binding efficiency using such novel design. The developed system can be easily extended to multi-reagents immunoassay protocols and provides a useful platform for point-of-care applications.

Bubble-driven mixer integrated with a microfluidic bead-based ELISA for rapid bladder cancer biomarker detection.

In this study, fine bubbles were successfully generated and used as a simple, low-cost driving force for mixing fluids in an integrated microfluidic bead-based enzyme-linked immunosorbent assay (ELISA) to rapidly and quantitatively detect apolipoprotein A1 (APOA1), a biomarker highly correlated with bladder cancer. A wooden gas diffuser was embedded underneath a microfluidic chip to refine injected air and generate bubbles of less than 0.3 mm. The rising bubbles caused disturbances and convection in the fluid, increasing the probability of analyte interaction. This setup not only simplifies the micromixer design but also achieves rapid mixing with a small airflow as a force. We used this bubble-driven micromixer in a bead-based ELISA that targeted APOA1. The results indicate that this micromixer reduced the time for each incubation from 60 min in the conventional assay to 8 min with the chip, resulting in a reduction of total ELISA reaction time from 3-4 h to 30-40 min. Furthermore, the concentration detection limit was 9.16 ng/mL, which was lower than the detection cut-off value (11.16 ng/mL) for bladder cancer diagnosis reported in the literature. Therefore, this chip can be used to achieve rapid low-cost bladder cancer detection and may be used in point-of-care cancer monitoring.

Successful differentiation of neural stem/progenitor cells cultured on electrically adjustable indium tin oxide (ITO) surface.

In order to control differentiation of neural cells and guide the developed neurites to targets, polyelectrolyte multilayer (PEM) films were used because of their capability of modulation of electrical charged characteristics, thickness, and stiffness. In this work, we suggested that indium tin oxide (ITO) is an alternative surface to achieve the above-mentioned objectives. A microfluidic system with four culture chambers was developed and each chamber consisted of parallel ITO surfaces for the application of adjustable electrical field. Neural stem/progenitor cells (NSPCs) were respectively cultured on the ITO surfaces with and without PEM film, constructed by alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA). Analyses of cell morphology, cytotoxicity, process outgrowth, differentiated cell types, and neuron functionality were compared between both surfaces. In this study, NSPCs successfully differentiated on ITO surface with electrical stimulation. The optimal electrical potential was found to be 80 mV that could stimulate the longest process, i.e., >300 µm, after 3 days culture. Cell differentiation, process development, and functionality of differentiated neuron on ITO surface were shown to be strongly controlled by the electrical stimulation that can be simply adjusted by external equipment. The electrically adjustable cell differentiation reported here could potentially be applied to neurochip for the study of neural signal transmission in a well-constructed network.

Investigation of Stem Cell-Like Characteristics and Immune Cell Interaction of Tumor Cells Survived from Continuous Shear Flow Environment.

When tumor cells are released from a primary tumor into the bloodstream or lymphatic circulation system, they are exposed to a continuous shear flow environment. This environment exerts physical stresses on the tumor cells, which can activate apoptotic pathways. However, certain tumor cells have the ability to adapt to these mechanical stresses, enhancing their likelihood of survival and promoting metastasis. In this study, these tumor cells survived from shear flow environment are examined and revealed to closely link to stem cell-like characteristics. Higher gene expression levels of self-renewal and differentiation markers and enhanced abilities of migration, spheroid formation, and colony formation are shown. Moreover, the interaction between immune cells and the surviving cells is investigated. The results show that the surviving cells possess immune escape capabilities, implying their ability to evade immune surveillance. Additionally, these surviving cells display characteristics reminiscent of stem cells. This study holds great importance in advancing the understanding of tumor biology. By comprehending the behavior and properties of these surviving cells, new therapeutic strategies can be developed to specifically target circulating tumor cells (CTCs) and enhance cancer treatment outcomes.

Study of cell migration trajectory on two-dimensional continuous stiffness gradient surface edited by grayscale photopolymerization.

In native tissues, cells encounter a diverse range of stiffness, which can significantly affect their behavior and function. The ability of cells to sense and respond to these mechanical cues is essential for various physiological processes, including cell migration. Cell migration is a complex process influenced by multiple factors, with substrate stiffness emerging as a critical determinant. This study developed a technique to edit the stiffness of polyacrylamide (PAA) hydrogel substrates by adjusting the grayscale level of a photomask during photopolymerization. By analyzing cell morphologies on the hydrogel, we confirmed the development of a single PAA hydrogel substrate with continuous stiffness gradients. This method was used to explore the correlation between substrate stiffness and cell migration dynamics. The study found that cells typically migrated from softer to stiffer surfaces. When the cells initially located on stiffer surfaces, they were able to travel longer distances. Additionally, a continuous 2D stiffness gradient surface was fabricated to explore how cells migrate on smoother versus steeper stiffness gradients. The results showed that cells tended to migrate more readily on smoother stiffness gradient surfaces compared to steeper ones. This study provides valuable insights into cell migration dynamics on substrates with varying stiffness gradients. The results underscore the importance of the mechanical environment in cancer cell migration and offer promising directions for developing interventions to prevent cancer spread.

Development of a Folding Paper System To Enable the Analysis of Gene Profile of Short- and Long-Distance Cancer Cell Migration.

In cancer metastasis, where mortality rates remain high despite advancements in medical treatments, understanding the molecular pathways and cellular dynamics underlying tumor spread is critical for devising more effective therapeutic strategies. Here, a folding paper system was proposed and developed to mimic native tumor microenvironment. This system, composed of 7 stacked layers of paper enclosed in a holder, allows for the culture of cancer cells under conditions mimicking those found in solid tumors, including limited oxygen and nutrients. Because of the migratory capabilities of cancer cells, the cells in the center layer could migrated to outer layers of the paper stack, enabling the differentiation of cells based on their migratory potential. Subsequent gene expression analysis, conducted through RT-PCR and RNA sequencing, revealed significant correlations between cancer cell migration distance and the expression of genes associated with hypoxia, metabolism, ATP production, and cellular process. Moreover, our study identified cells with aggressive phenotypic traits from the outer layers of the paper stack, highlighting the potential of this system for enabling the study of aggressive cancer cell characteristics. Validation of the folding paper system against clinical carcinoma tissue demonstrated its ability to faithfully mimic the native tumor microenvironment. Overall, our findings underscore the utility of the folding paper system as a valuable tool for investigating and identifying critical molecular pathways involved in cancer metastasis.

Bundled carbon nanotube-based sensor on paper-based microfluidic device.

Bundled carbon nanotube (CNT)-based sensor has been fabricated on paper substrate for chemical sensing applications. Integration of the sensor and fluidic channel was demonstrated for the potential development of a paper-based microfluidic device. In this work, electrical pH measurement of analyte solution was presented to show the functionality of the device. The device with the functions of fluidic transportation and chemical sensing was fabricated on a single paper. The bundled CNT-based sensor was first formed on a sheet of paper by vacuum filtration process. Hence, the hydrophilic channel across the sensor was defined by the application of polydimethylsiloxane (PDMS) material. Therefore, aqueous solution, e.g., sample, can be passively transported along the channels by wicking through the hydrophilic fibers of paper. The pH value of the solution can be electrically measured by the sensor. Determination of the pH value from 3 to 11 of the solutions was demonstrated by measuring the resistance change of the sensor. Because the proposed device is low cost, simple, flexible, and disposable, it is suitable for the development of the analytical device for the developing countries and harsh environments. Moreover, because CNT has excellent properties and can be functionalized by various molecules, the proposed paper-based microfluidic device has potential to realize more chemical and biological assays on paper with high sensitivity and specificity.

Analysis of Cellular Crosstalk and Molecular Signal between Periosteum-Derived Precursor Cells and Peripheral Cells During Bone Healing Process Using a Paper-Based Osteogenesis-On-A-Chip Platform.

Periosteum-derived progenitor cells (PDPCs) are highly promising cell sources that are indispensable in the bone healing process. Adipose-derived stem cells (ADSCs) are physiologically close to periosteum tissue and release multiple growth factors to promote the bone healing process. Co-culturing PDPCs and ADSCs can construct periosteum-bone tissue microenvironments for the study of cellular crosstalk and molecular signal in the bone healing process. In the current work, a paper-based osteogenesis-on-a-chip platform was successfully developed to provide an in vitro three-dimensional coculture model. The platform was a paper substrate sandwiched between PDPC-hydrogel and ADSC-hydrogel suspensions. Cell secretion could be transferred through the paper substrate from one side to another side. Growth factors including BMP2, TGF-ß, POSTN, Wnt proteins, PDGFA, and VEGFA were directly analyzed by a paper-based immunoassay. Cellular crosstalk was studied by protein expression on the paper substrate. Moreover, osteogenesis of PDPCs was investigated by examining the mRNA expressions of PDPCs after culture. Neutralizing and competitive assays were conducted to understand the correlation between growth factors secreted from ADSCs and the osteogenesis of PDPCs. In vitro periosteum-bone tissue microenvironment was established by the paper-based osteogenesis-on-a-chip platform. The proposed approach provides a promising assay of cellular crosstalk and molecular signal in 3D coculture microenvironment that may potentially lead to the development of effective bone regeneration therapy.