3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay.

BACKGROUND: Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. RESULTS: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 µm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. CONCLUSIONS: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids.

Organ-on-a-Chip for Studying Gut-Brain Interaction Mediated by Extracellular Vesicles in the Gut Microenvironment.

Extracellular vesicles (EVs) are a group of membrane vesicles that play important roles in cell-to-cell and interspecies/interkingdom communications by modulating the pathophysiological conditions of recipient cells. Recent evidence has implied their potential roles in the gut-brain axis (GBA), which is a complex bidirectional communication system between the gut environment and brain pathophysiology. Despite the evidence, the roles of EVs in the gut microenvironment in the GBA are less highlighted. Moreover, there are critical challenges in the current GBA models and analyzing techniques for EVs, which may hinder the research. Currently, advances in organ-on-a-chip (OOC) technologies have provided a promising solution. Here, we review the potential effects of EVs occurring in the gut environment on brain physiology and behavior and discuss how to apply OOCs to research the GBA mediated by EVs in the gut microenvironment.

3D-Printed Modular Microfluidic Device Enabling Preconcentrating Bacteria and Purifying Bacterial DNA in Blood for Improving the Sensitivity of Molecular Diagnostics.

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3DpmµFD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3DpmµFD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

Cell Seeding Technology for Microarray-Based Quantitative Human Primary Skeletal Muscle Cell Analysis.

Pipetting techniques play a crucial role in obtaining reproducible and reliable results, especially when seeding cells on small target areas, such as on microarrays, biochips or microfabricated cell culture systems. For very rare cells, such as human primary skeletal muscle cells (skMCs), manual (freehand) cell seeding techniques invariably result in nonuniform cell spreading and heterogeneous cell densities, giving rise to undesirable variations in myogenesis and differentiation. To prevent such technique-dependent variation, we have designed and fabricated a simple, low-cost pipet guidance device (PGD), and holder that works with hand-held pipettes. This work validates the accuracy and reproducibility of the PGD platform and compares its effectiveness with manual and robotic seeding techniques. The PGD system ensures reproducibility of cell seeding, comparable to that of more expensive robotic dispensing systems, resulting in a high degree of cell uniformity and homogeneous cell densities, while also enabling cell community studies. As compared to freehand pipetting, PGD-assisted seeding of C2C12 mouse myoblasts showed 5.3 times more myotube formation and likewise myotubes derived from PGD-seeded human primary skMCs were 3.6 times thicker and 2.2 times longer. These results show that this novel, yet simple PGD-assisted pipetting technique provides precise cell seeding on small targets, ensuring reproducible and reliable high-throughput cell assays.

A Microbore Tubing Based Spiral for Multistep Cell Fractionation.

Cells were separated with the aid of a multistep spiral fractionation device, utilizing hydrodynamic forces in a spiral tubing. The spiral was fabricated using "off-the-shelf" microbore tubing, allowing for cheap and fast prototyping to achieve optimal cell separation. As a first step, a model system with 20 and 40 µm beads was used to demonstrate the effectiveness of the multistep separation device. With an initial purity of 5%, a separation purity of 83% was achieved after a two-step separation with the addition of 0.1% polyethylene glycol (PEG)-8000. Next, doxorubicin-resistant polyploid giant breast cancer cells (MDA-MB-231) were separated from doxorubicin-sensitive monoploid small breast cancer cells in the same fashion as the beads, resulting in a purity of around 40%, while maintaining a cell viability of more than 90%. Combined with basic cell analytical methods, the hydrodynamic separation principle of the device could be envisaged to be useful for a variety of cell fractionation needs in cell biology and in clinical applications.

Solenoid Driven Pressure Valve System: Toward Versatile Fluidic Control in Paper Microfluidics.

As paper-based diagnostics has become predominantly driven by more advanced microfluidic technology, many of the research efforts are still focused on developing reliable and versatile fluidic control devices, apart from improving sensitivity and reproducibility. In this work, we introduce a novel and robust paper fluidic control system enabling versatile fluidic control. The system comprises a linear push-pull solenoid and an Arduino Uno microcontroller. The precisely controlled pressure exerted on the paper stops the flow. We first determined the stroke distance of the solenoid to obtain a constant pressure while examining the fluidic time delay as a function of the pressure. Results showed that strips of grade 1 chromatography paper had superior reproducibility in fluid transport. Next, we characterized the reproducibility of the fluidic velocity which depends on the type and grade of paper used. As such, we were able to control the flow velocity on the paper and also achieve a complete stop of flow with a pressure over 2.0 MPa. Notably, after the actuation of the pressure driven valve (PDV), the previously pressed area regained its original flow properties. This means that, even on a previously pressed area, multiple valve operations can be successfully conducted. To the best of our knowledge, this is the first demonstration of an active and repetitive valve operation in paper microfluidics. As a proof of concept, we have chosen to perform a multistep detection system in the form of an enzyme-linked immunosorbent assay with mouse IgG as the target analyte.

The nanoscale architecture of force-bearing focal adhesions.

The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100-280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion.

A thin-walled polydimethylsiloxane bioreactor for high-density hepatocyte sandwich culture.

In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24-well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long-term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days.

3D printed fluidic swab for COVID-19 testing with improved diagnostic yield and user comfort.

The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 µL of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

A multishear microfluidic device for quantitative analysis of calcium dynamics in osteoblasts.

Microfluidics is a convenient platform to study the influences of fluid shear stress on calcium dynamics. Fluidic shear stress has been proven to affect bone cell functions and remodelling. We have developed a microfluidic system which can generate four shear flows in one device as a means to study cytosolic calcium concentration ([Ca(2+)](c)) dynamics of osteoblasts. Four shear forces were achieved by having four cell culture chambers with different widths while resistance correction channels compensated for the overall resistance to allow equal flow distribution towards the chambers. Computational simulation of the local shear stress distribution highlighted the preferred section in the cell chamber to measure the calcium dynamics. Osteoblasts showed an [Ca(2+)](c) increment proportional to the intensity of the shear stress from 0.03 to 0.30 Pa. A delay in response was observed with an activation threshold between 0.03 and 0.06 Pa. With computational modelling, our microfluidic device can offer controllable multishear stresses and perform quantitative comparisons of shear stress-induced intensity change of calcium in osteoblasts.

Cells in microfluidics.

Microfluidic devices offer a realistic environment for cell cultures as it is related to scales found in biological systems. The aim is to create more in vivo like systems, in comparison to 2D plate cultures. Creating 3D cell culture constructs increase the cell's functionality. By controlling the microenvironment (e.g., cell matrix, flow rate, temperature) cell functionality can be increased even more. As microfluidic devices allow for precise control of the microenvironment, they are a paramount tool to study stem cells and their differentiation caused by external factors. We will give an overview of the use of microfluidic devices for some biological problems, and especially as a cell culture platforms. We focus on 3D cell cultures and stem cells and their microenvironment.

Systematic Review: Microfluidics and Plasmodium.

Malaria affects 228 million people worldwide each year, causing severe disease and worsening the conditions of already vulnerable populations. In this review, we explore how malaria has been detected in the past and how it can be detected in the future. Our primary focus is on finding new directions for low-cost diagnostic methods that unspecialized personnel can apply in situ. Through this review, we show that microfluidic devices can help pre-concentrate samples of blood infected with malaria to facilitate the diagnosis. Importantly, these devices can be made cheaply and be readily deployed in remote locations.

Integrated Microfluidic Preconcentration and Nucleic Amplification System for Detection of Influenza A Virus H1N1 in Saliva.

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (µFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the µFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

Towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments.

We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-microFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, the multi-channel 3D-microFCCS was designed for culturing different 3D cellular aggregates simultaneously to mimic multiple organs in the body. Four human cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-beta1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-beta1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-microFCCS could be potentially used to supplement or even replace animal models in drug screening.

Effective mixing in a microfluidic chip using magnetic particles.

We present a novel active mixing method in a microfluidic chip, where the controlled stirring of magnetic particles is used to achieve an effective mixing of fluids. To perform mixing, the ferromagnetic particles were embedded and manipulated under the influence of a rotating magnetic field. By aligning the magnetic beads along the magnetic field lines, rod-like structures are formed, functioning as small stir bars. Under higher flow conditions the particles did not form the typical rod structure but rather formed aggregates, which were even more beneficial for mixing. Our system reached a 96% mixing efficiency in a relatively short distance (800 microm) at a flow rate of 1.2-4.8 mm/s. These results demonstrate that our mixing method is useful for microfluidic devices with low aspect ratios and molecules with large molecular weights.

A microfluidic 3D hepatocyte chip for drug toxicity testing.

We have developed a microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity. The 3D HepaTox Chip is based on multiplexed microfluidic channels where a 3D microenvironment is engineered in each channel to maintain the hepatocytes' synthetic and metabolic functions. The multiplexed channels allow for simultaneous administration of multiple drug doses to functional primary hepatocytes while an incorporated concentration gradient generator enables the in vitro dose-dependent drug responses to predict in vivo hepatotoxicity. The IC(50) values of 5 model drugs derived from the dose-dependent on-chip testing correlate well with the reported in vivo LD(50) values. The 3D HepaTox Chip can be integrated with on-chip sensors and actuators as the next generation cell-based on-chip drug testing platform.

Editorial for the Special Issue on Microfluidics for Cell and Other Organisms.

It is my great pleasure to present to you this first volume of 13 papers on the subject of Microfluidics for Cells and other Organisms [...].

Microslit on a chip: A simplified filter to capture circulating tumor cells enlarged with microbeads.

Microchips are widely used to separate circulating tumor cells (CTCs) from whole blood by virtues of sophisticated manipulation for microparticles. Here, we present a chip with an 8 µm high and 27.9 mm wide slit to capture cancer cells bound to 3 µm beads. Apart from a higher purity and recovery rate, the slit design allows for simplified fabrication, easy cell imaging, less clogging, lower chamber pressure and, therefore, higher throughput. The beads were conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) to selectively bind to breast cancer cells (MCF-7) used to spike the whole blood. The diameter of the cell-bead construct was in average 23.1 µm, making them separable from other cells in the blood. As a result, the cancer cells were separated from 5 mL of whole blood with a purity of 52.0% and a recovery rate of 91.1%, and also we confirmed that the device can be applicable to clinical samples of human breast cancer patients. The simple design with microslit, by eliminating any high-aspect ratio features, is expected to reduce possible defects on the chip and, therefore, more suitable for mass production without false separation outputs.

A gel-free 3D microfluidic cell culture system.

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture.

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.