Microfluidic Device Fabrication and Cell Manipulation

Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to “native” hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages. Full article

This study investigated the partitioning characteristics of red blood cells (RBCs) within capillaries, with a specific focus on ladder structures observed near the end of the capillaries. In vitro experiments were conducted using microfluidic channels with a ladder structure model comprising six bifurcating channels that exhibited an anti-parallel flow configuration. The effects of various factors, such as the parent channel width, distance between branches, and hematocrit, on RBC partitioning in bifurcating channels were evaluated. A decrease in the parent channel width resulted in an increase in the heterogeneity in the hematocrit distribution and a bias in the fractional RBC flux. Additionally, variations in the distance between branches affected the RBC distribution, with smaller distances resulting in greater heterogeneity. The bias of the RBC distribution in the microchannel cross section had a major effect on the RBC partitioning characteristics. The influence of hematocrit variations on the RBC distribution was also investigated, with lower hematocrit values leading to a more pronounced bias in the RBC distribution. Overall, this study provides valuable insights into RBC distribution characteristics in capillary networks, contributing to our understanding of the physiological mechanisms of RBC phase separation in the microcirculatory system. These findings have implications for predicting oxygen heterogeneity in tissues and could aid in the study of diseases associated with impaired microcirculation. Full article

Electrostatic traveling wave (ETW) methods have shown promising performance in dust mitigation of solar panels, particle transport and separation in in situ space resource utilization, cell manipulation, and separation in biology. The ETW field distribution is required to analyze the forces applied to particles and to evaluate ETW design parameters. This study presents the numerical results of the ETW field distribution generated by a parallel electrode array using both the charge simulation method (CSM) and the boundary element method (BEM). A low accumulated error of the CSM is achieved by properly arranging the positions and numbers of contour points and fictitious charges. The BEM can avoid the inconvenience of the charge position required in the CSM. The numerical results show extremely close agreement between the CSM and BEM. For simplification, the method of images is introduced in the implementation of the CSM and BEM. Moreover, analytical formulas are obtained for the integral of Green’s function along boundary elements. For further validation, the results are cross-checked using the finite element method (FEM). It is found that discrepancies occur at the ends of the electrode array. Finally, analyses are provided of the electric field and dielectrophoretic (DEP) components. Emphasis is given to the regions close to the electrode surfaces. These results provide guidance for the fabrication of ETW systems for various applications. Full article

The timely detection and diagnosis of diseases and accurate monitoring of specific genetic conditions require rapid and accurate separation, sorting, and direction of target cell types toward a sensor device surface. In that regard, cellular manipulation, separation, and sorting are progressively finding application potential within various bioassay applications such as medical disease diagnosis, pathogen detection, and medical testing. The aim of this paper is to present the design and development of a simple traveling wave ferro-microfluidic device and system rig purposed for the potential manipulation and magnetophoretic separation of cells in water-based ferrofluids. This paper details in full: (1) a method for tailoring cobalt ferrite nanoparticles for specific diameter size ranges (10–20 nm), (2) the development of a ferro-microfluidic device for potentially separating cells and magnetic nanoparticles, (3) the development of a water-based ferrofluid with magnetic nanoparticles and non-magnetic microparticles, and (4) the design and development of a system rig for producing the electric field within the ferro-microfluidic channel device for magnetizing and manipulating nonmagnetic particles in the ferro-microfluidic channel. The results reported in this work demonstrate a proof of concept for magnetophoretic manipulation and separation of magnetic and non-magnetic particles in a simple ferro-microfluidic device. This work is a design and proof-of-concept study. The design reported in this model is an improvement over existing magnetic excitation microfluidic system designs in that heat is efficiently removed from the circuit board to allow a range of input currents and frequencies to manipulate non-magnetic particles. Although this work did not analyze the separation of cells from magnetic particles, the results demonstrate that non-magnetic (surrogates for cellular materials) and magnetic entities can be separated and, in some cases, continuously pushed through the channel based on amperage, size, frequency, and electrode spacing. The results reported in this work establish that the developed ferro-microfluidic device may potentially be used as an effective platform for microparticle and cellular manipulation and sorting. Full article

Malignant pleural effusion is a common clinical problem, which often occurs in cases of malignant tumors, especially in lung cancer. In this paper, a pleural effusion detection system based on a microfluidic chip, combined with specific tumor biomarker, hexaminolevulinate (HAL), used to concentrate and identify tumor cells in pleural effusion was reported. The lung adenocarcinoma cell line A549 and mesothelial cell line Met-5A were cultured as the tumor cells and non-tumor cells, respectively. The optimum enrichment effect was achieved in the microfluidic chip when the flow rates of cell suspension and phosphate-buffered saline achieved 2 mL/h and 4 mL/h, respectively. At the optimal flow rate, the proportion of A549 increased from 28.04% to 70.01% due to the concentration effect of the chip, indicating that tumor cells could be enriched by a factor of 2.5 times. In addition, HAL staining results revealed that HAL can be used to identify tumor cells and non-tumor cells in chip and clinical samples. Additionally, the tumor cells obtained from the patients diagnosed with lung cancer were confirmed to be captured in the microfluidic chip, proving the validity of the microfluidic detection system. This study preliminarily demonstrates the microfluidic system is a promising method with which to assist clinical detection in pleural effusion. Full article

Mammalian blood cell separation methods contribute to improving the diagnosis and treatment of animal and human diseases. Microfluidic deterministic lateral displacement (DLD) devices can sort cells based on their particle diameter. We developed microfluidic DLD devices with poly(propylene)-based resin and used them to separate bovine and human red blood cells (RBCs) and white blood cells (WBCs) without electric devices. To determine the critical cut-off diameter (D_c) of these devices, we used immunobeads with a diameter of 1–20 μm. The D_c values of the microfluidic DLD devices for the immunobeads in the experiments were similar to the calculated D_c values (8–10 μm). Results from bovine blood cell separation experiments suggest that lymphocytes and neutrophils can be separated from diluted, whole blood. Human RBCs were occasionally observed in the left outlet where larger particles with diameters closer to the D_c value were collected. Based on the D_c values, human neutrophils were sorted to the left outlet, whereas lymphocytes were observed in both outlets. Although microfluidic channel optimization is required for the concentration of sorted cells, the microfluidic DLD device prepared with a poly(propylene)-based resin has the potential for clinical use. Full article

Live-cell microscopy is crucial for biomedical studies and clinical tests. The technique is, however, limited to few laboratories due to its high cost and bulky size of the necessary culture equipment. In this study, we propose a portable microfluidic-cell-culture system, which is merely $15 \mathrm{cm}\times 11 \mathrm{cm}\times 9 \mathrm{cm}$ in dimension, powered by a conventional alkali battery and costs less than USD 20. For long-term cell culture, a fresh culture medium exposed to 5% CO₂ is programmed to be delivered to the culture chamber at defined time intervals. The 37 $°\mathrm{C}$ culture temperature is maintained by timely electrifying the ITO glass slide underneath the culture chamber. Our results demonstrate that 3T3 fibroblasts, HepG2 cells, MB-231 cells and tumor spheroids can be well-maintained for more than 48 h on top of the microscope stage and show physical characters (e.g., morphology and mobility) and growth rate on par with the commercial stage-top incubator and the widely adopted CO₂ incubator. The proposed portable cell culture device is, therefore, suitable for simple live-cell studies in the lab and cell experiments in the field when samples cannot be shipped. Full article

To understand the influence of indigestible particles like particulate matter 2.5 (PM2.5) on macrophages, we examined the time course of the series phagocytosis of indigestible 2 μm polystyrene spheres (PS). Five kinds of antigens were used as samples for phagocytosis; Zymosan, non-coated 2 $\mathsf{\mu }$m PS, bovine serum albumin (BSA)-coated PS (BSA-PS), IgG-coated PS (IgG-PS), and IgG-BSA-coated PS (IgG/BSA-PS). To keep the surrounding concentration of antigens against single macrophages constant, antigens flowed at a continuous rate of $0.55$ μm/s within a culture dish as a free-flow measurement assay (on-chip free-flow method). The interval of series phagocytosis for IgG/BSA-PS was the shortest among five samples; it was six times faster than Zymosan in terms of engulfment frequency, and up to 50 particles were engulfed within two hours, maintaining constant intervals until reaching the maximum number. The rate of increase in the total number of phagocytozed IgG/BSA-PS over time was constant, at $1.5$ particles/min, in series phagocytosis with a 33-cell population, indicating that the phagocytosis rate constant remained constant independent of the number of phagocytoses. Reaction model fitting of the results showed that IgG/BSA-PS had the highest efficiency in terms of the phagocytosis rate constant, 2.3 × ${10}^{-2}$ particles/min, whereas those of IgG-PS, BSA-PS, PS, and Zymosan were 1.4 × ${10}^{-2}$, 1.1 × ${10}^{-2}$, 4.2 × ${10}^{-3}$, and 3.6 × ${10}^{-3}$ particles/min, respectively. One-by-one feeding of IgG/BSA-PS with optical tweezers was examined to confirm the phagocytosis intervals, and we found that the intervals remained constant until several times before the maximum number of antigens for engulfment, also indicating no change in the phagocytosis rate constant regardless of the history of former phagocytosis and phagocytosis number. Simultaneous phagocytosis of two IgG-BSA-decorated microneedle engulfments also showed that the initiation and progress of two simultaneous engulfments on the two different places on a cell were independent and had the same elongation velocity. Therefore, each phagocytosis of indigestible antigens does not affect both in series or in simultaneous subsequent phagocytosis until reaching the maximum capacity of the phagocytosis number. The results suggest (1) no change in the phagocytosis rate constant regardless of the history of phagocytosis numbers and attachment timing and positions, and (2) IgG-BSA decoration of indigestible microparticles in blood accelerates their engulfment faster, resulting in a severe shortage of macrophages within the shortest time. Full article

This study presents a novel technique for fabricating microfluidic devices with microbial transglutaminase-gelatin gels instead of polydimethylsiloxane (PDMS), in which flow culture simulates blood flow and a capillary network is incorporated for assays of vascular permeability or angiogenesis. We developed a gelatin-based device with a coverslip as the bottom, which allows the use of high-magnification lenses with short working distances, and we observed the differences in cell dynamics on gelatin, glass, and PDMS surfaces. The tubes of the gelatin microfluidic channel are designed to be difficult to pull out of the inlet hole, making sample introduction easy, and the gelatin channel can be manipulated from the cell introduction to the flow culture steps in a manner comparable to that of a typical PDMS channel. Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) were successfully co-cultured, resulting in structures that mimicked blood vessels with inner diameters ranging from 10 µm to 500 µm. Immunostaining and scanning electron microscopy results showed that the affinity of fibronectin for gelatin was stronger than that for glass or PDMS, making gelatin a suitable substrate for cell adhesion. The ability for microscopic observation at high magnification and the ease of sample introduction make this device easier to use than conventional gelatin microfluidics, and the above-mentioned small modifications in the device structure are important points that improve its convenience as a cell assay device. Full article

Alginate microcapsules are one of the attractive non-invasive platforms for handling individual cells and clusters, maintaining their isolation for further applications such as imaging cell sorter and single capsule qPCR. However, the conventional cell encapsulation techniques provide huge numbers of unnecessary empty homogeneous alginate microcapsules, which spend an excessive majority of the machine time on observations and analysis. Here, we developed a simple alginate cell encapsulation method to form content size-dependent alginate microcapsules to eliminate empty microcapsules using microcapillary centrifugation and filtration. Using this method, the formed calcium alginate microcapsules containing the HeLa cells were larger than 20m, and the other empty microcapsules were less than 3m under 4000 rpm centrifugation condition. We collected cell-containing alginate microcapsules by eliminating empty microcapsules from the microcapsule mixture with simple one-step filtration of a 20 m cell strainer. The electrical surface charge density and optical permeability of those cell-encapsulated alginate microcapsules were also evaluated. We found that the surface charge density of cell-encapsulated alginate microbeads is more than double that of cells, indicating that less voltage is required for electrical cell handling with thin alginate gel encapsulation of samples. The permeability of the alginate microcapsule was not improved by changing the reflective index of the medium buffer, such as adding alginate ester. However, the minimized thickness of the alginate gel envelope surrounding cells in the microcapsules did not degrade the detailed shapes of encapsulated cells. Those results confirmed the advantage of alginate encapsulation of cells with the centrifugation method as one of the desirable tools for imaging cell sorting applications. Full article

Drug-resistant bacterial strains seriously threaten human health. Rapid screening of antibiotics is urgently required to improve clinical treatment. Conventional methods of antimicrobial susceptibility testing rely on turbidimetry that is evident only after several days of incubation. The lengthy time of the assay can delay clinical treatment. Here, we proposed a single-cell level rapid system based on a microfluidic chip. The detection period of 30 min to 2 h was significantly shorter than the conventional turbidity-based method. To promote detection efficiency, 16 independent channels were designed, permitting the simultaneous screening of 16 drugs in the microfluidic chip. Prepositioning of drugs in the chip permitted prolonged transportation and storage. This may allow for the widespread use of the novel system, particularly in the regions where medical facilities are scarce. The growth curves were reported rapidly through a custom code in Matlab after tracking and photographing the bacteria during microscopy examination. The capability of the proposed system was validated by antimicrobial susceptibility testing trials with standard strains. The system provides a potentially useful detection tool for drug-resistant bacteria. Full article