The emergence of microfluidic devices and computational fluid dynamics (CFD) has propelled the need for next-generation biomimetic cell culture platforms that are flexible for monitoring and regulation. Therefore, this study evaluated a CFD application in an in silico-designed and spheroid-based flow integration 3D cell culture chip (SFI chip) to illustrate cell culture, drug screening, cytokine delivery, and differentiation of cells in a platform that partially recapitulates the natural environment. Our results show that a flow rate of 0.05 mL h-1 or less induced no physical stress in the SFI chip (15 mm), and uniform cell spheroids (approximately 200 μm) were formed across the platform. The cultured cells were tested in several experimental contexts (co-culture, drug screening, cytokine delivery, and differentiation), demonstrating the usefulness of computational simulation in expediting discovery and simple and effective means to scale the production of standardized cell spheroids cultured under dynamic and natural conditions. Advanced cell culture technologies can be used to accelerate research and discovery and the preclinical and clinical development of cell and cell-free therapies for urgent medical needs.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623–630

Epidermal electronics are extensively explored as an important platform for future biomedical engineering. Epidermal devices are typically fabricated using high‐cost methods employing complex vacuum microfabrication processes, limiting their widespread potential in wearable electronics. Here, a low‐cost, solution‐based approach using electroconductive reduced graphene oxide (RGO) sheets on elastic and porous poly(dimethylsiloxane) (PDMS) thin films for multifunctional, high‐performance, graphene‐based epidermal bioelectrodes and strain sensors is presented. These devices are fabricated employing simple coatings and direct patterning without using any complicated microfabrication processes. The graphene bioelectrodes show a superior stretchability (up to 150% strain), with mechanical durability up to 5000 cycles of stretching and releasing, and low sheet resistance (1.5 kΩ per square), and the graphene strain sensors exhibit a high sensitivity (a gauge factor of 7 to 173) with a wide sensing range (up to 40% strain). Fully functional applications of dry bioelectrodes in monitoring human electrophysiological signals (i.e., electrocardiogram, electroencephalography, and electromyogram) and highly sensitive strain sensors for precise detection of large‐scale human motions are demonstrated. It is believed that our unique processing capability and multifunctional device platform based on RGO/porous PDMS will pave the way for low‐cost processing and integration of 2D materials for future wearable electronic skin.

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell–cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

This paper provides a method for microscale fiber spinning and the in situ construction of a 3D fibrous scaffold on a single microfluidic platform. This platform was also used to fabricate a variety of fibrous scaffolds with diverse compositions without the use of complicated devices. We explored the potential utility of the fibrous scaffolds for tissue engineering applications by constructing a fibrous scaffold encapsulating primary hepatocytes. The cells in scaffold were cultured over seven days and maintained higher viability comparing with 3D alginate non-fibrous block. The main advantage of this platform is that the fibrous structure used to form a scaffold can be generated without damaging the mechanically weak alginate fibers or encapsulated cells because all procedures are performed in a single platform without the intervention of the operator. In addition, the proposed fibrous scaffold permitted high diffusion capability of molecules, which enabled better viability of encapsulated cells than non-fibrous scaffold even in massive cell culture.

ABSTRACTLeftover uncured polydimethylsiloxane (PDMS) polymer is generally discarded. In an effort to minimize this waste, we have developed a method for cryo‐preserving PDMS prepolymer involving a simple storage technique for the preservation and repeated use over 1 month. Aliquots of the uncured PDMS prepolymer were stored at −20 or −80°C, then conveniently and easily thawed at body temperature (37°C). Proposed cryo‐preservation was successfully evaluated using diverse biological and physical tests. This method of cryo‐preserving PDMS reduces both the material waste and labor of soft lithography process and may enable soft lithography to be environmentally friendly relative to previous methods. The method may be popularly accepted for application to a variety of common microdevice fabrication procedures. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40378.

The encapsulation of living cells in a variety of soft polymers or hydrogels is important, particularly, for the rehabilitation of functional tissues capable of repairing or replacing damaged organs. Cellular encapsulation segregates cells from the surrounding tissue to protect the implanted cell from the recipient's immune system after transplantation. Diverse hydrogel membranes have been popularly used as encapsulating materials and permit the diffusion of gas, nutrients, wastes and therapeutic products smoothly. This review describes a variety of methods that have been developed to achieve cellular encapsulation using microscale platform. Microtechnologies have been adopted to precisely control the encapsulated cell number, size and shape of a cell-laden polymer structure. We provide a brief overview of recent microtechnology-based cell encapsulation methods, with a detailed description of the relevant processes. Finally, we discuss the current challenges and future directions likely to be taken by cell microencapsulation approaches toward tissue engineering and cell therapy applications.

This article describes a passive micromixer that utilizes an air-liquid interface and surface tension effects to enhance fluid mixing via convection and Marangoni effects. Performance of the microfluidic component is tested within a passive-pumping-based device that consists of three microchannels connected in succession using passive micro-mixers. Mixing was quantified at 5 key points along the length of the device using microscope images of patterned streams of Alexa 488 fluorescent-dyed water and pure DI water flowing through the device. The passive micro-mixer mixed fluid 15–20 times more effectively than diffusion between laminar flow streams alone and is a novel micro-mixer embodiment that provides an additional strategy for removing external components from microscale devices for simpler, autonomous operation.

We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.

Here, a spheroidal 3D co-culture model of primary (rat) pancreatic islets and hepatocytes with uniform size and shape was developed using hemispheric concave microwell arrays. We conducted morphological and functional analyses of hybrid spheroids versus mono-cultures of islets or hepatocytes (controls). For the establishment of a 3D hybrid model, a broad range of cell ratios - 1:1, 1:3, 1:5, 1:7, 3:1, 5:1 and 7:1 mixture - of hepatocytes and pancreatic islets were used. As control, each hepatocyte and pancreatic islet were mono-cultured forming 3D spheroids. The transient morphology of spheroid formation in 9 culture models was observed using optical microscopy. Cell viability under these culture environments was assessed, and the morphologies of the outer and inner porous cell-spheroid structures were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and imaging of stained spheroid sections. The pancreatic islet-specific function of hybrid spheroids was evaluated by measuring insulin secretion and in vivo test by xenotransplantation of encapsulated spheroids in microfibers with a consistent maintenance of normal blood glucose levels over 4 weeks, while liver-specific functions were measured in terms of albumin secretion, urea secretion and cytochrome P450 activity. These diverse observations and evaluations validated the positive and bidirectional effects of co-cultured 3D spheroids. The proposed 3D co-culture model demonstrated that both cells appeared to support each other's functions strongly in spheroids, even though smaller proportions of each cell type was evaluated compared to mono-culture models, suggesting that the proposed model could help overcome the problem of cell shortages in clinical applications. (C) 2013 Elsevier Ltd. All rights reserved.

The creation and characterization of large-area ultrathin highly pliable free-standing PDMS membranes and their application to the study of cellular epithelia is described. The ultra-thin membranes permitted the straight forward calculation of cell monolayer moduli, derived from measured stress-strain curves. These measurements allowed the unprecedented detection of cellular-level injury in the epithelia caused by the rupture of cell-cell tight junctions in response to stretching.

This article describes a novel method for the detection of amyloid-${\beta}$($A{\beta}$) peptide that utilizes a photo-sensitive field-effect transistor (p-FET). According to a recent study, $A{\beta}$ protein has been known to play a central role in the pathogenesis of Alzheimer's disease (AD). Accordingly, we investigated the variation of photo current generated from p-FET with and without intracellular magnetic beads conjugated with $A{\beta}$ peptides, which are placed on the p-FET sensing areas. The decrease of photo current was observed due to the presence of the magnetic beads on the channel region. Moreover, a similar characteristic was shown when the Raw 264 cells take in magnetic beads treated with $A{\beta}$ peptide. This means that it is possible to simply detect a certain protein using magnetic beads and a p-FET device. Therefore, in this paper, we suggest that our method could detect tiny amounts of $A{\beta}$ for early diagnosis of AD using the p-FET devices.

This paper describes an integrated lysis chip that utilize syringe pump for the detection of the Beta-amyloid protein in cells or tissues. An integrated lysis chip that has many advantages such as easy control, simply and novel design, cost and time effective. The integrated lysis chip that was fabricated in poly (dimethylsiloxane) (PDMS) form masters prepared by soft lithography using SU-8 3035 photoresist. The integrated lysis chip has two main components: the lysis channel with blades containing an input port and connects port to filter system channel, and the filter system channel which has an output port and reservoir chamber. The filter system channel was constructed using multi layers of commercial filters. The integrated lysis chip is used here to lysis, concentration and precipitation. The chip was successfully evaluated using NIH3T3 cells. The specific procedures of this tool are as follows; (1) the preparation of cells and Aβ (beta-amyloid)1-42 mixture, (2) the drop of cells mixture were achieved using micro-pipette in input port, (3) To delivery of mixture cells, the output port was created by negative pressure through a syringe pump (or by hand). The liquid through filter system channel is characterized by Immunocytochemistry. In addition, we demonstrate how its filter system channel how its using Raman spectroscopy active site was developed to observe Aβ and other materials. The integrated lysis chip can be readily modified to apply to a wide variety of common cell lysis and protein experiment procedure.

This paper reports a technique for quantification of beta-amyloid protein by the use of photo sensitive thin film transistor (p-TFT). In recent studies, a beta-amyloid protein has strongly related a central role of the pathogenesis of Alzheimer’s disease (AD) and AD-related olfactory sensory loss. In the p-TFT, we observed the difference of the photo-current according to the presence of the particles with beta-amyloid, RAW 264.7 cells, and olfactory receptor neuron cells. The p-TFT shows the drain current reduction of about 50μA is caused by the RAW 264.7 cells with magnetic beads and beta-amyloid. Therefore, we suggest a new method of detecting small quantity of A-beta for early diagnosis of AD by using photo-sensitive thin film transistor (p-TFT) devices.

It is well known that Alzheimer's disease (AD) is pathologically defined by the presence of amyloid-beta plaques and neurofibrillary tangles within the brain by advanced medical imaging technique such as computed tomography or magnetic resonance imaging. Currently, in AD-related olfactory sensory loss studies, early olfactory perceptual loss is likely contributed by nonfibrillar, versus fibrillar, amyloid-beta related mechanism in the olfactory system and nonfibrillar amyloid beta deposition is observed within the olfactory bulb. Therefore, the results of the olfactory dysfunction studies represents that if the amount of nano-sized amyloid beta proteins (nano-Abs) is quantified in in vivo olfactory system, in vivo early diagnosis of AD is possible. In this paper, we have measured the amount of nano-Abs by the use of olfactory cells and nano particles (100nm bead) in a micro-fluidic channel. For sample preparation, nano-particles are sequentially conjugated with linkers (biotin, straptavidin and so on) and the particles are absorbed (or adhere) into in vivo olfactory system. The amount of the conjugated nano-Abs is measured by evanescent field-nano particle coupling effect where we use micro-fluidic channels. As results, in the in vivo olfactory system, the coupled optical power with single nano-particle is around 1nW. The results of quantification with amyloid-related nano-particles in in vivo olfactory system will be briefly introduced.

This article describes two kinds of "Cell Programmable Assay" (CPA) chips that utilize passive pumping for the culture and autonomous staining of cells to simply common protocols. One is a single timer channel CPA (sCPA) chip that has one timer channel and one main channel containing a cell culture chamber. The sCPA is used to culture and stain cells using Hoechst nuclear staining dye (a 2 step staining process). The other is a dual timer channel CPA (dCPA) chip that has two timer channels and one main channel with a chamber for cell culture. The dCPA is used here to culture, fix, permeablize, and stain cells using DAPI. The additional timer channel of the dCPA chip allows for automation of 3 steps. The CPA chips were successfully evaluated using HEK 293 cells. In addition, we provide a simplified equation for tuning or redesigning CPA chips to meet the needs of a variety of protocols that may require different timings. The equation is easy to use as it only depends upon the dimensions of microchannel and the volume of the reagent drops. The sCPA and dCPA chips can be readily modified to apply to a wide variety of common cell culture methods and procedures.

Urinary hippuric acid (HA), of molecular weight 180 Da, is one of the major metabolites in toluene-exposed humans and is a major biological indicator. Simple and ubiquitous monitoring of exposure to toluene is very important in occupational health care, and a microfluidic chip-based electrochemical immunoassay for rapid and quantitative detection of HA in human urine is proposed in this paper. The system employs a conjugate of ferrocene (Fc) and hippuric acid (HA). The competition between hippuric acid (HA) and the ferrocene-hippuric acid complex (Fc-Lys-HA) to bind with a HA antibody coated onto polybeads generated electrical signals proportional to the HA concentration in the range of 0-40 mg mL(-1). All the complicated HA detection processes were integrated on the single microfluidic platform. The quantitative advantages of our HA detection chip are as follows: (1) the total chip size was reduced to 3.0 x 2.0 x 0.5 cm and is small enough to be portable, (2) the assay time took 1 min, and is shorter than that of conventional electrochemical HA immunoassay systems (about 20 min) and (3) 40 microL of the sample solution was enough to detect HA in the range of 0-40 mg mL(-1), which is enough range to be used for the point-of-care system. In addition, we suggest the improved chip-based HA assay method by the combination of electrochemical and enzymatic amplification processes for the detection of greater electrical signals. The sensitivity of the combined method was increased about three times compared to that of the non-enzymatic process.