Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

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.

In silico design and fabrication of an SFI chip-based microspheroid culture system.

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.

A Cell Programmable Assay (CPA) chip.

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.

High-throughput microfluidics: improved sample treatment and washing over standard wells.

Fluid flow in microchannels is used to treat or wash samples and can be incorporated into high-throughput applications such as drug screening, which currently use standard microtiter wells for performing assays. This paper provides theoretical and experimental data comparing microchannels and standard wells on the metrics of sample washing and experimental error in treatment concentrations. It is shown numerically and experimentally that microchannel concentration can be approximated with an inverse linear relationship to input volume. The experimentally supported mathematical approximation and error propagation methods are used to compare the accuracy and precision of treatments in microchannels vs. standard wells. Mathematical results suggest microchannels can provide 10 or more times the treatment precision of standard wells for volume ratios typical of high-throughput screening. Passive-pumping and diffusion are utilized to improve microchannel accuracy and precision even further in a treat-wait-treat method. The advantages of microchannels outlined here can have large-scale effects on cost and accuracy in screening applications.

Cell encapsulation via microtechnologies.

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.

Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

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.

Passive micromixer using by convection and surface tension effects with air-liquid interface.

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.

Large-scale, ultrapliable, and free-standing nanomembranes.

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.

Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects.

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.

3D co-culturing model of primary pancreatic islets and hepatocytes in hybrid spheroid to overcome pancreatic cell shortage.

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.

Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel.

Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of alpha-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis.