Integrating Oxygen and 3D Cell Culture System: A Simple Tool to Elucidate the Cell Fate Decision of hiPSCs.

Oxygen, as an external environmental factor, plays a role in the early differentiation of human stem cells, such as induced pluripotent stem cells (hiPSCs). However, the effect of oxygen concentration on the early-stage differentiation of hiPSC is not fully understood, especially in 3D aggregate cultures. In this study, we cultivated the 3D aggregation of hiPSCs on oxygen-permeable microwells under different oxygen concentrations ranging from 2.5 to 20% and found that the aggregates became larger, corresponding to the increase in oxygen level. In a low oxygen environment, the glycolytic pathway was more profound, and the differentiation markers of the three germ layers were upregulated, suggesting that the oxygen concentration can function as a regulator of differentiation during the early stage of development. In conclusion, culturing stem cells on oxygen-permeable microwells may serve as a platform to investigate the effect of oxygen concentration on diverse cell fate decisions during development.

Correction: Liquid marble-based digital microfluidics - fundamentals and applications.

Correction for 'Liquid marble-based digital microfluidics - fundamentals and applications' by Chin Hong Ooi et al., Lab Chip, 2021, DOI: .

Liquid marble-based digital microfluidics - fundamentals and applications.

Liquid marbles are droplets with volume typically on the order of microliters coated with hydrophobic powder. Their versatility, ease of use and low cost make liquid marbles an attractive platform for digital microfluidics. This paper provides the state of the art of discoveries in the physics of liquid marbles and their practical applications. The paper first discusses the fundamental properties of liquid marbles, followed by the summary of different techniques for the synthesis of liquid marbles. Next, manipulation techniques for handling liquid marbles are discussed. Applications of liquid marbles are categorised according to their use as chemical and biological reactors. The paper concludes with perspectives on the future development of liquid marble-based digital microfluidics.

Inertial Microfluidic Purification of Floating Cancer Cells for Drug Screening and Three-Dimensional Tumor Models.

Floating cancer cells can survive the programmed death anoikis process after detaching from the extracellular matrix for the anchorage-dependent cells. Purification of viable floating cancer cells is essential for many biomedical studies, such as drug screening and cancer model development. However, the floating cancer cells are mixed with dead cells and debris in the medium supernatant. In this paper, we developed an inertial microfluidic device with sinusoidal microchannels to continuously remove dead cells and debris from viable cells. First, we characterized the differential inertial focusing properties of polystyrene beads in the devices. Then, we investigated the effects of flow rate on inertial focusing of floating MDA-MB-231 cells. At an optimal flow condition, purification of viable cells was performed and the purity of live cells was increased significantly from 19.9% to 76.6%, with a recovery rate of 69.7%. After separation, we studied and compared the floating and adherent MDA-MB-231 cells in terms of cell proliferation, protrusive cellular structure, and the expression of cyclooxygenase (Cox-2) which is related to epithelial-mesenchymal transition (EMT) changes. Meanwhile, drug screening of both floating and adherent cancer cells was conducted using a chemotherapeutic drug, doxorubicin (Dox). The results revealed that the floating cancer cells possess 30-fold acquired chemoresistance as compared to the adherent cancer cells. Furthermore, a three-dimensional (3D) double-cellular coculture model of human mammary fibroblasts (HMF) spheroid and cancer cells using the floating liquid marble technique was developed.

Lapatinib inhibits doxorubicin induced migration of HER2-positive breast cancer cells.

Inflammatory breast cancer (IBC) is an uncommon and highly aggressive form of breast cancer. The disease is characterized by rapid progression with approximately 50% of IBC patients to have human epidermal growth factor receptor 2 (HER2) amplification. HER2-positive IBC is associated with unfavourable prognosis and increased risk of brain metastasis. Ironically, HER2-positive metastatic breast cancer is still prevalent where therapeutic targeting of HER2-receptor is well developed. In addition, the ability to accurately predict the risk of metastatic potential in these cells poses a substantial challenge. Lapatinib (Lap), a dual kinase inhibitor of HER2 and epidermal growth factor receptor is used in the treatment of advanced HER-2 positive breast cancers and is currently being evaluated in the adjuvant setting. In this study, we report the effectiveness of Lap in the suppression of low-dose response to doxorubicin (Dox) in HER2-positive SKBR3 cells. Upon treatment of SKBR3 cells with 0.1 µM of Dox, the cell viability was significantly increased as compared to the human mammary fibroblasts, and triple-negative human breast cancer MDA-MB-231 cells. Interestingly, the effect of 0.1 µM Dox revealed morphological changes consistent with a significant increase in the formation of prominent F-actin filaments and mitochondrial spread compared with the control SKBR3 cells. Furthermore, an enhanced migration was also evident in these cells. However, a combinational dose of 0.1 µM Dox + 5 µM Lap suppressed the observed phenotypic changes in the 0.1 µM Dox treated SKBR3 cells. There was a significant difference in the prominent F-actin filaments and the mitochondrial spread compared with the 0.1 µM Dox versus combination regimen of 0.1 µM Dox + 5 µM Lap. In addition, the combinational therapy showed a decrease in the percentage of wound closure when compared to the control. Hence, the combinational therapy in which Lap suppresses the low-dose effect of Dox in SKBR3 cells may provide an effective intervention strategy for reducing the risk of metastasis in HER2-positive breast cancers.

Long-Lived, Transferred Crystalline Silicon Carbide Nanomembranes for Implantable Flexible Electronics.

Implantable electronics are of great interest owing to their capability for real-time and continuous recording of cellular-electrical activity. Nevertheless, as such systems involve direct interfaces with surrounding biofluidic environments, maintaining their long-term sustainable operation, without leakage currents or corrosion, is a daunting challenge. Herein, we present a thin, flexible semiconducting material system that offers attractive attributes in this context. The material consists of crystalline cubic silicon carbide nanomembranes grown on silicon wafers, released and then physically transferred to a final device substrate (e.g., polyimide). The experimental results demonstrate that SiC nanomembranes with thicknesses of 230 nm do not experience the hydrolysis process (i.e., the etching rate is 0 nm/day at 96 °C in phosphate-buffered saline (PBS)). There is no observable water permeability for at least 60 days in PBS at 96 °C and non-Na+ ion diffusion detected at a thickness of 50 nm after being soaked in 1× PBS for 12 days. These properties enable Faradaic interfaces between active electronics and biological tissues, as well as multimodal sensing of temperature, strain, and other properties without the need for additional encapsulating layers. These findings create important opportunities for use of flexible, wide band gap materials as essential components of long-lived neurological and cardiac electrophysiological device interfaces.

Transparent crystalline cubic SiC-on-glass electrodes enable simultaneous electrochemistry and optical microscopy.

This work presents crystalline SiC-on-glass as a transparent, robust, and optically stable electrode for simultaneous electrochemical characterization and optical microscope imaging. Experimental results show a large potential window, as well as excellent stability and repeatability over multiple cyclic voltammetric scans in common redox biomarkers such as ruthenium hexaammine and methylene blue. The high optical transmittance and biocompatibility of SiC-on-glass were also observed, enabling cell culture, electrical stimulation, and high resolution fluorescence imaging. This new platform opens exciting opportunities in multi-functional biosensing-probes and observation.

Stretching cells - An approach for early cancer diagnosis.

Cells express multiple biophysical cues during migration, differentiation, and transformation. Probing and quantifying these biophysical cues could serve as a diagnostic tool for differentiating healthy with neoplastic cells. These biophysical cues may be utilized for diagnostic screening in cancer, as the tumor cells interact with the surrounding extracellular matrix (ECM). Stress and strain induced by the cancer cells and applied to the cancer cells have effects in cancer progression due to its influence in cell migration. It was reported that the introduction of compressive forces on cancerous cells triggers them to undergo apoptosis. In this report, we evaluated the effects of stretching forces on cancer cells by morphological analyses. We observed that cancer cells decrease their roundness (as determined by perimeter: area); increase their length and form filopodia in the initial stretching cycle. However, due to the increasing rigidity of the cells, they undergo apoptosis in later stretching cycles. These morphological changes were unique to breast cancer (MDA-MB-231) cells compared to the non-cancerous control. Elucidating and quantifying these morphological changes is potentially an early cancer diagnostic tool that may predict the propensity of the cancerous cells undergoing a metastatic transformation.

Cryoprotectant-Free Freezing of Cells Using Liquid Marbles Filled with Hydrogel.

Cryopreservation without cryoprotectant remains a significant challenge for the re-establishment of cell culture after freeze-thaw. Thus, finding an alternative and a simple cryopreservation method is necessary. Liquid marble (LM)-based digital microfluidics is a promising approach for cryoprotectant-free cryopreservation. However, the use of this platform to efficiently preserve samples with low cell density and well-controlled serum concentrations has not been investigated. We addressed this issue by embedding an agarose-containing fetal bovine serum (FBS) inside the LM. A low density of 500 cells/µL of murine 3T3 cells was selected for evaluating the postcryogenic survivability. The effects on the post-thaw cell viability of the concentration of agarose, the amount of FBS inside the agarose, and the volume of the LM were investigated systematically. This paper also presents an analysis on the changes in shape and crack size of post-thawed agarose. The results revealed that the embedded agarose gel serves as a controlled release mechanism of FBS and significantly improves cell viability. Post-thaw recovery sustains major cellular features, such as viability, cell adhesion, and morphology. The platform technology reported here opens up new possibilities to cryopreserve rare biological samples without the toxicity risk of cryoprotectants.

Pneumatically actuated cell-stretching array platform for engineering cell patterns in vitro.

Cellular response to mechanical stimuli is a well-known phenomenon known as mechanotransduction. It is widely accepted that mechanotransduction plays an important role in cell alignment which is critical for cell homeostasis. Although many approaches have been developed in recent years to study the effect of external mechanical stimuli on cell behaviour, most of them have not explored the ability of mechanical stimuli to engineer cell alignment to obtain patterned cell cultures. This paper introduces a simple, yet effective pneumatically actuated 4 × 2 cell stretching array for concurrently inducing a range of cyclic normal strains onto cell cultures to achieve predefined cell alignment. We utilised a ring-shaped normal strain pattern to demonstrate the growth of in vitro patterned cell cultures with predefined circumferential cellular alignment. Furthermore, to ensure the compatibility of the developed cell stretching platform with general tools and existing protocols, the dimensions of the developed cell-stretching platform follow the standard F-bottom 96-well plate. In this study, we report the principle design, simulation and characterisation of the cell-stretching platform with preliminary observations using fibroblast cells. Our experimental results of cytoskeleton reorganisation such as perpendicular cellular alignment of the cells to the direction of normal strain are consistent with those reported in the literature. After two hours of stretching, the circumferential alignment of fibroblast cells confirms the capability of the developed system to achieve patterned cell culture. The cell-stretching platform reported is potentially a useful tool for drug screening in 2D mechanobiology experiments, tissue engineering and regenerative medicine.

Superior Robust Ultrathin Single-Crystalline Silicon Carbide Membrane as a Versatile Platform for Biological Applications.

Micromachined membranes are promising platforms for cell culture thanks to their miniaturization and integration capabilities. Possessing chemical inertness, biocompatibility, and integration, silicon carbide (SiC) membranes have attracted great interest toward biological applications. In this paper, we present the batch fabrication, mechanical characterizations, and cell culture demonstration of robust ultrathin epitaxial deposited SiC membranes. The as-fabricated ultrathin SiC membranes, with an ultrahigh aspect ratio (length/thickness) of up to 20 000, possess high a fracture strength up to 2.95 GPa and deformation up to 50 µm. A high optical transmittance of above 80% at visible wavelengths was obtained for 50 nm membranes. The as-fabricated membranes were experimentally demonstrated as an excellent substrate platform for bio-MEMS/NEMS cell culture with the cell viability rate of more than 92% after 72 h. The ultrathin SiC membrane is promising for in vitro observations/imaging of bio-objects with an extremely short optical access.

Liquid Marble as Bioreactor for Engineering Three-Dimensional Toroid Tissues.

Liquid marble is a liquid droplet coated with hydrophobic powder that can be used as a bioreactor. This paper reports the three-dimensional self-assembly and culture of a cell toroid in a slow-releasing, non-adhesive and evaporation-reducing bioreactor platform based on a liquid marble. The bioreactor is constructed by embedding a hydrogel sphere containing growth factor into a liquid marble filled with a suspension of dissociated cells. The hydrogel maintains the water content and concurrently acts as a slow-release carrier. The concentration gradient of growth factor induces cell migration and assembly into toroidal aggregates. An optimum cell concentration resulted in the toroidal (doughnut-like) tissue after 12 hours. The harvested cell toroids showed rapid closure of the inner opening when treated with the growth factor. We also present a geometric growth model to describe the shape of the toroidal tissue over time. In analogy to the classical two-dimensional scratch assay, we propose that the cell toroids reported here open up new possibilities to screen drugs affecting cell migration in three dimensions.

Single-Crystalline 3C-SiC anodically Bonded onto Glass: An Excellent Platform for High-Temperature Electronics and Bioapplications.

Single-crystal cubic silicon carbide has attracted great attention for MEMS and electronic devices. However, current leakage at the SiC/Si junction at high temperatures and visible-light absorption of the Si substrate are main obstacles hindering the use of the platform in a broad range of applications. To solve these bottlenecks, we present a new platform of single crystal SiC on an electrically insulating and transparent substrate using an anodic bonding process. The SiC thin film was prepared on a 150 mm Si with a surface roughness of 7 nm using LPCVD. The SiC/Si wafer was bonded to a glass substrate and then the Si layer was completely removed through wafer polishing and wet etching. The bonded SiC/glass samples show a sharp bonding interface of less than 15 nm characterized using deep profile X-ray photoelectron spectroscopy, a strong bonding strength of approximately 20 MPa measured from the pulling test, and relatively high optical transparency in the visible range. The transferred SiC film also exhibited good conductivity and a relatively high temperature coefficient of resistance varying from -12 000 to -20 000 ppm/K, which is desirable for thermal sensors. The biocompatibility of SiC/glass was also confirmed through mouse 3T3 fibroblasts cell-culturing experiments. Taking advantage of the superior electrical properties and biocompatibility of SiC, the developed SiC-on-glass platform offers unprecedented potentials for high-temperature electronics as well as bioapplications.

Liquid marbles as bioreactors for the study of three-dimensional cell interactions.

Liquid marble as a bioreactor platform for cell-based studies has received significant attention, especially for developing 3D cell-based assays. This platform is particularly suitable for 3D in-vitro modeling of cell-cell interactions. For the first time, we demonstrated the interaction of olfactory ensheathing cells (OECs) with nerve debris and meningeal fibroblast using liquid marbles. As the transplantation of OECs can be used for repairing nerve injury, degenerating cell debris within the transplantation site can adversely affect the survival of transplanted OECs. In this paper, we used liquid marbles to mimic the hostile 3D environment to analyze the functional behavior of the cells and to form the basis for cell-based therapy. We show that OECs interact with debris and enhanced cellular aggregation to form a larger 3D spheroidal tissue. However, these spheroids indicated limitation in biological functions such as the inability of cells within the spheroids to migrate out and adherence to neighboring tissue by fusion. The coalescence of two liquid marbles allows for analyzing the interaction between two distinct cell types and their respective environment. We created a microenvironment consisting of 3D fibroblast spheroids and nerve debris and let it interact with OECs. We found that OECs initiate adherence with nerve debris in this 3D environment. The results suggest that liquid marbles are ideal for developing bioassays that could substantially contribute to therapeutic applications. Especially, insights for improving the survival and adherence of transplanted cells.

An Electromagnetically Actuated Double-Sided Cell-Stretching Device for Mechanobiology Research.

Cellular response to mechanical stimuli is an integral part of cell homeostasis. The interaction of the extracellular matrix with the mechanical stress plays an important role in cytoskeleton organisation and cell alignment. Insights from the response can be utilised to develop cell culture methods that achieve predefined cell patterns, which are critical for tissue remodelling and cell therapy. We report the working principle, design, simulation, and characterisation of a novel electromagnetic cell stretching platform based on the double-sided axial stretching approach. The device is capable of introducing a cyclic and static strain pattern on a cell culture. The platform was tested with fibroblasts. The experimental results are consistent with the previously reported cytoskeleton reorganisation and cell reorientation induced by strain. Our observations suggest that the cell orientation is highly influenced by external mechanical cues. Cells reorganise their cytoskeletons to avoid external strain and to maintain intact extracellular matrix arrangements.

Numerical Simulation of the Behavior of Toroidal and Spheroidal Multicellular Aggregates in Microfluidic Devices with Microwell and U-Shaped Barrier.

A microfluidic system provides an excellent platform for cellular studies. Most importantly, a three-dimensional (3D) cell culture model reconstructs more accurately the in vivo microenvironment of tissue. Accordingly, microfluidic 3D cell culture devices could be ideal candidates for in vitro cell culture platforms. In this paper, two types of 3D cellular aggregates, i.e., toroid and spheroid, are numerically studied. The studies are carried out for microfluidic systems containing U-shaped barrier as well as microwell structure. For the first time, we obtain oxygen and glucose concentration distributions inside a toroid aggregate as well as the shear stress on its surface and compare its performance with a spheroid aggregate of the same volume. In particular, we obtain the oxygen concentration distributions in three areas, namely, oxygen-permeable layer, multicellular aggregates and culture medium. Further, glucose concentration distributions in two regions of multicellular aggregates and culture medium are investigated. The results show that the levels of oxygen and glucose in the system containing U-shaped barriers are far more than those in the system containing microwells. Therefore, to achieve high levels of oxygen and nutrients, a system with U-shaped barriers is more suited than the conventional traps, but the choice between toroid and spheroid depends on their volume and orientation. The results indicate that higher oxygen and glucose concentrations can be achieved in spheroid with a small volume as well as in horizontal toroid with a large volume. The vertical toroid has the highest levels of oxygen and glucose concentration while the surface shear stress on its surface is also maximum. These findings can be used as guidelines for designing an optimum 3D microfluidic bioreactor based on the desired levels of oxygen, glucose and shear stress distributions.

A lab-on-a-chip device for investigating the fusion process of olfactory ensheathing cell spheroids.

Understanding the process of fusion of olfactory ensheathing cell spheroids will lead to improvement of cell transplantation therapies to repair spinal cord injuries. The successful fusion of transplanted spheroids will enable alternative transplantation strategies to be developed for in vivo applications. This paper describes the use of a microfluidic device to trap and fuse olfactory ensheathing cell spheroids. The velocity, the pressure distribution in the device were simulated numerically to predict the trapping location. The simulation predicted the optimum flow rates for trapping the spheroids in the later experiments. Simulated particle trajectories were verified experimentally with tracing of fluorescent micro particles. The fusion process of the spheroids was investigated over a period of 48 hours. The microfluidic platform presented here can be used for testing potential drugs that can promote the fusion process and improve the transplantation therapy.

Digital microfluidics with a magnetically actuated floating liquid marble.

Controlled actuation of a floating liquid marble, a liquid droplet coated with hydrophobic particles floating on another liquid surface, is a potential digital microfluidics platform for the transport of aqueous solution with minimal volume loss. This paper reports our recent investigation on the magnetic actuation of floating liquid marbles filled with magnetic particles. The magnetic force and frictional force acting on the floating liquid marble determine the horizontal movement of the marble. We varied the magnetic flux density, flux density gradient, concentration of magnetic particles and speed of the marble to elucidate the relationship between the acting forces. We subsequently determined the suitable operating conditions for the actuation and derived the scaling laws for the actuation parameters.

An electromagnetic cell-stretching device for mechanotransduction studies of olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are primary candidates for cell transplantation therapy to repair spinal cord injury (SCI). However, the post transplantation survival of these cells remains a major hurdle for a success using this therapy. Mechanical stimuli may contribute to the maintenance of these cells and thus, mechanotransduction studies of OECs may serve as a key benefit to identify strategies for improvement in cell transplantation. We developed an electromagnetic cell stretching device based on a single sided uniaxial stretching approach to apply tensile strain to OECs in culture. This paper reports the design, simulation and characterisation of the stretching device with preliminary experimental observations of OECs in vitro. The strain field of the deformable membrane was investigated both experimentally and numerically. Heterogeneity of the device provided an ideal platform for establishing strain requirement for the OEC culture. The cell stretching system developed may serve as a tool in exploring the mechanobiology of OECs for future SCI transplantation research.

Floating mechanism of a small liquid marble.

Flotation of small solid objects and liquid droplets on water is critical to natural and industrial activities. This paper reports the floating mechanism of liquid marbles, or liquid droplets coated with hydrophobic microparticles. We used X-ray computed tomography (XCT) to acquire cross-sectional images of the floating liquid marble and interface between the different phases. We then analysed the shape of the liquid marble and the angles at the three-phase contact line (TPCL). We found that the small floating liquid marbles follow the mechanism governing the flotation of solid objects in terms of surface tension forces. However, the contact angles formed and deformation of the liquid marble resemble that of a sessile liquid droplet on a thin, elastic solid. For small liquid marbles, the contact angle varies with volume due to the deformability of the interface.