Basic Principles of Flow Cytometer Operation: The Make your Own Flow Cytometer.

Flow cytometry is a critical technology for biomedical analysis and is an essential component of almost any study of the immune system. Widespread usage and increasing instrument complexity have, however, led to increasing neglect of education in their basic operating principles, a common situation with many technologies. This chapter describes the basics of flow cytometer operation using the Make Your Own Flow Cytometer ( https://www.cytometryworks.com ), a working cytometer than can be assembled by students into a functional instrument. This project and others like it is seeing widespread usage in biomedical education and can serve as models for like-minded investigators who wish to build their own systems. They also provide a good mechanism to introduce the key operational principles of flow cytometry as illustrated here.

Continuous Flow Separation of Red Blood Cells and Platelets in a Y-Microfluidic Channel Device with Saw-Tooth Profile Electrodes via Low Voltage Dielectrophoresis.

Cell counting and sorting is a vital step in the purification process within the area of biomedical research. It has been widely reported and accepted that the use of hydrodynamic focusing in conjunction with the application of a dielectrophoretic (DEP) force allows efficient separation of biological entities such as platelets from red blood cell (RBC) samples due to their size difference. This paper presents computational results of a multiphysics simulation modelling study on evaluating continuous separation of RBCs and platelets in a microfluidic device design with saw-tooth profile electrodes via DEP. The theoretical cell particle trajectory, particle cell counting, and particle separation distance study results reported in this work were predicted using COMSOL v6.0 Multiphysics simulation software. To validate the numerical model used in this work for the reported device design, we first developed a simple y-channel microfluidic device with square "in fluid" electrodes similar to the design reported previously in other works. We then compared the obtained simulation results for the simple y-channel device with the square in fluid electrodes to the reported experimental work done on this simple design which resulted in 98% agreement. The design reported in this work is an improvement over existing designs in that it can perform rapid separation of RBCs (estimated 99% purification) and platelets in a total time of 6-7 s at a minimum voltage setting of 1 V and at a minimum frequency of 1 Hz. The threshold for efficient separation of cells ends at 1000 kHz for a 1 V setting. The saw-tooth electrode profile appears to be an improvement over existing designs in that the sharp corners reduced the required horizontal distance needed for separation to occur and contributed to a non-uniform DEP electric field. The results of this simulation study further suggest that this DEP separation technique may potentially be applied to improve the efficiency of separation processes of biological sample scenarios and simultaneously increase the accuracy of diagnostic processes via cell counting and sorting.

A low-cost flow cell for flow cytometry.

Flow cytometry is an essential analytical technique used in biomedical diagnostics to measure properties of cells, micro-organisms, and particles. Laser light is scattered from particles focused in a flow cell and collected by light sensors, where the intensity of the scattered light is a function of the scattering angle, the refractive index of the particle and surrounding medium, the wavelength of light, and the size and the shape of the particle. One of the critical parts of the cytometer is the flow cell where the particle stream is constrained into a tight region within 10-30 µm using hydrodynamic focusing. The conventional flow cells use thick quartz flow cells, which are expensive and therefore not suitable for instruments targeted for resource-constrained settings. We demonstrate a compact, economical, bio-compatible flow cell assembly design that incorporates inexpensive and easily available capillaries attached to sturdy polymer fixtures in a simple manner that performs the focusing of a sample stream of particles. The flow cell has been tested by studying the relation between sample core diameter, and sample and sheath flow rates. Small-angle scattering (forward scatter) and wide-angle scattering (side scatter) have been captured for the enumeration and characterization of particles. We show excellent agreement between the size distribution obtained via direct imaging and that obtained from light scattering. The flow cell was also used to successfully size white blood cells in human blood samples.

One-step Production of Sterically Stabilized Anionic Nanoliposome Using Microfluidic Device.

Anionic liposomes (AL) are very attractive for nanomedicine and some formulations have already been launched for clinical development. Despite the excellent potential, their application presents two major challenges: laborious production methods and rapid degradation and elimination from blood by the immune system. In this work, we optimized the production of AL and its stealth form (SAL) using a onestep microfluidic process. We obtained unilamellar and near-monodisperse (< 10%) AL composed by the commercial composition (DMPC:DMPG) with mean size small as 53.7 nm, which is optimized for application in drug delivery. We also obtained SAL with similar characteristics using the microfluidic technique, overcoming the limitation of conventional methods where SAL presents high polydispersity (> 30%). This study demonstrates the great potential of the microfluidic technique for one-step production of stealth anionic nanoliposomes with controlled sizes and reproducible characteristics.

Tunable hydrodynamic focusing with dual-neodymium magnet-based microfluidic separation device.

Microfluidic separation technologies are the focus of various biological applications, such as disease diagnostics, single-cell analysis, and therapeutics. Different methods and devices were proposed in the micro-separation field, focusing on minimizing the chemical deformation and physical damage to the particles throughout the separation process; however, it is still a challenge. This paper proposes a hydrodynamic focusing-based microfluidic separation device equipped with a dual-neodymium magnet for positive magnetophoretic microparticles and cell separation. Hydrodynamic focusing is used to help to sort the particles and minimize the damage to the microparticles through the proposed different inlet flow rates between the two focusing channels. The dual magnets help to separate the particles in two stages. The system's novelty is integrating the hydrodynamic focusing with the dual magnetics system, where the hydrodynamic focusing is with variable inlet flow rates. The performance of the proposed microfluidic particle separator is numerically assessed under various operating parameters, including the concentration of the particle in the injected solution and flow rate ratios of high to the low focusing flows on the efficiency of the separation. Following the proposed separation method, it was possible to separate the 16 and 10 [Formula: see text] microparticles with the first-round efficiency of 21% with a quality of 92%, respectively. The developed particle separation system can significantly broaden its applications in a variety of biomedical research studies.

Performance Comparison of Flow-Through Optofluidic Biosensor Designs.

Optofluidic flow-through biosensors are being developed for single particle detection, particularly as a tool for pathogen diagnosis. The sensitivity of the biosensor chip depends on design parameters, illumination format (side vs. top), and flow configuration (parabolic, two- and three-dimensional hydrodynamic focused (2DHF and 3DHF)). We study the signal differences between various combinations of these design aspects. Our model is validated against a sample of physical devices. We find that side-illumination with 3DHF produces the strongest and consistent signal, but parabolic flow devices process a sample volume more quickly. Practical matters of optical alignment are also discussed, which may affect design choice.

A microfluidic cytometer for white blood cell analysis.

Despite the wide use of cytometry for white blood cell classification, the performance of traditional cytometers in point-of-care testing remains to be improved. Microfluidic techniques have been shown with considerable potentials in the development of portable devices. Here we present a prototype of microfluidic cytometer which integrates a three-dimensional hydrodynamic focusing system and an on-chip optical system to count and classify white blood cells. By adjusting the flow speed of sheath flow and sample flow, the blood cells can be horizontally and vertically focused in the center of microchannel. Optical fibers and on-chip microlens are embedded for the excitation and detection of single-cell. The microfluidic chip was validated by classifying white blood cells from clinical blood samples.

Particle separation in microfluidics using different modal ultrasonic standing waves.

Microfluidic technology has great advantages in the precise manipulation of micro and nano particles, and the separation of micro and nano particles based on ultrasonic standing waves has attracted much attention for its high efficiency and simplicity of structure. This paper proposes a device that uses three modes of ultrasonic standing waves to continuously separate particles with positive acoustic contrast factor in microfluidics. Three modes of acoustic standing waves are used simultaneously in different parts of the microchannel. According to the different acoustic radiation force received by the particles, the particles are finally separated to the pressure node lines on both sides and the center of the microchannel. In this separation method, initial hydrodynamic focusing and satisfying various equilibrium constraints during the separation process are the key. Through numerical simulation, the resonance frequency of the interdigital transducer, the distribution of sound pressure in the liquid, and the relationship between the interdigital electrode voltage and the output sound pressure are obtained. Finally, the entire separation process in the microchannel was simulated, and the separation of the two particles was successfully achieved. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.

Optical Investigation of Individual Red Blood Cells for Determining Cell Count and Cellular Hemoglobin Concentration in a Microfluidic Channel.

We demonstrate a blood analysis routine by observing red blood cells through light and digital holographic microscopy in a microfluidic channel. With this setup a determination of red blood cell (RBC) concentration, the mean corpuscular volume (MCV), and corpuscular hemoglobin concentration mean (CHCM) is feasible. Cell count variations in between measurements differed by 2.47% with a deviation of -0.26×106 µL to the reference value obtained from the Siemens ADVIA 2120i. Measured MCV values varied by 2.25% and CHCM values by 3.78% compared to the reference ADVIA measurement. Our results suggest that the combination of optical analysis with microfluidics handling provides a promising new approach to red blood cell counts.

Detecting cells rotations for increasing the robustness of cell sizing by impedance measurements, with or without machine learning.

The Coulter principle is a widespread technique for sizing red blood cells (RBCs) in hematological analyzers. It is based on the monitoring of the electrical perturbations generated by cells passing through a micro-orifice, in which a concentrated electrical field is imposed by two electrodes. However, artifacts associated with near-wall passages in the sensing region are known to skew the statistics for RBCs sizing. This study presents numerical results that emphasize the link between the cell flow-induced rotation in the detection area and the error in its measured volume. Based on these observations, two methods are developed to identify and reject pulses impaired by cell rotation. In the first strategy, the filtering is allowed by a metric computed directly from the waveform. In the second, a numerical database is employed to train a neural network capable of detecting if the cell has experienced a rotation, given its electrical pulse. Detecting and rejecting rotation-associated pulses are shown to provide results comparable to hydrodynamical focusing, which enforces cells to flow in the center of the orifice, the gold standard implementation of the Coulter principle.

3D-Printed Microfluidic Nanoelectrospray Ionization Source Based on Hydrodynamic Focusing.

Nanoelectrospray ionization (nESI) mass spectrometry (MS) is an ideal detection method for microfluidic chips, and its performances depend on nESI emitters. However, the fabrication of monolithic nESI emitters in chips was difficult. Herein, we propose a three-dimensional (3D) printing method to develop a microfluidic nanoelectrospray ionization source (NIS), composed of a nESI emitter and other components. Firstly, the NIS was compatible with a 50 - 500 nL min-1 nanoflows by imposing 3D hydrodynamic focusing to compensate for the total flow rate, achieving a 7.2% best relative standard deviation in the total ion current (TIC) profiles. Additionally, it was applied to probe thirteen organic chemicals, insulin, and lysozyme with adequate signal-to-noise ratios and an accuracy of m/z between 9.02 × 10-1 and 1.48 × 103 ppm. Finally, the NIS achieved comparable limits of detection compared with its commercial counterpart. Considering the standardized preparation of NIS, it would be a potential option to develop 3D-printed customized Chip-MS platforms.

3D Hydrodynamic Focusing in Microscale Optofluidic Channels Formed with a Single Sacrificial Layer.

Optofluidic devices are capable of detecting single molecules, but greater sensitivity and specificity is desired through hydrodynamic focusing (HDF). Three-dimensional (3D) hydrodynamic focusing was implemented in 10-µm scale microchannel cross-sections made with a single sacrificial layer. HDF is achieved using buffer fluid to sheath the sample fluid, requiring four fluid ports to operate by pressure driven flow. A low-pressure chamber, or pit, formed by etching into a substrate, enables volumetric flow ratio-induced focusing at a low flow velocity. The single layer design simplifies surface micromachining and improves device yield by 1.56 times over previous work. The focusing design was integrated with optical waveguides and used in order to analyze fluorescent signals from beads in fluid flow. The implementation of the focusing scheme was found to narrow the distribution of bead velocity and fluorescent signal, giving rise to 33% more consistent signal. Reservoir effects were observed at low operational vacuum pressures and a balance between optofluidic signal variance and intensity was achieved. The implementation of the design in optofluidic sensors will enable higher detection sensitivity and sample specificity.

Facile Fabrication of Microfluidic Chips for 3D Hydrodynamic Focusing and Wet Spinning of Polymeric Fibers.

Microfluidic wet spinning has gained increasing interest in recent years as an alternative to conventional wet spinning by offering higher control in fiber morphology and a gateway for the development of multi-material fibers. Conventionally, microfluidic chips used to create such fibers are fabricated by soft lithography, a method that requires both time and investment in necessary cleanroom facilities. Recently, additive manufacturing techniques were investigated for rapid and cost-efficient prototyping. However, these microfluidic devices are not yet matching the resolutions and tolerances offered by soft lithography. Herein, we report a facile and rapid method using selected arrays of hypodermic needles as templates within a silicone elastomer matrix. The produced microfluidic spinnerets display co-axially aligned circular channels. By simulation and flow experiments, we prove that these devices can maintain laminar flow conditions and achieve precise 3D hydrodynamic focusing. The devices were tested with a commercial polyurethane formulation to demonstrate that fibers with desired morphologies can be produced by varying the degree of hydrodynamic focusing. Thanks to the adaptability of this concept to different microfluidic spinneret designs-as well as to its transparency, ease of fabrication, and cost-efficient procedure-this device sets the ground for transferring microfluidic wet spinning towards industrial textile settings.

On the competition between mixing rate and uniformity in a coaxial hydrodynamic focusing mixer.

Fast microfluidic mixers for use with line-of-sight integrating detection schemes pose unique challenges. Such detectors typically cannot discriminate signal from slow moving (e.g. near internal walls) and fast-moving portions of the fluid stream. This convolves reaction rate dynamics with fluid flow residence time dynamics. Further, the small cross sections of typical three-dimensional hydrodynamic focusing devices lead to lower detection signals. The current study focuses on achieving both small time scales of mixing and homogenous residence times. This is achieved by injecting sample through a center capillary and hydrodynamically focusing using a sheath flow within a tapered second capillary. The current design also features a third, larger coaxial capillary. The mixed stream flows into the large cross-section of this third capillary to decelerate and expand the stream by up to 14-fold to improve line-of-sight signal strength of reaction products. Hydrodynamic focusing, mixing, and expansion are studied using analytical and numerical models and also studied experimentally using a fluorescein-iodide quenching reaction. The experimentally validated models are used to explore trade-offs between mixing rate and uniformity. For the first time, this work presents detailed analysis of the Lagrangian time history of species transport during mixing inside coaxial capillaries to measure mixing nonuniformity. The mixing region enables order 100 µs mixing times and residence time widths of the same order (140 µs).

Efficient separation of tumor cells from untreated whole blood using a novel multistage hydrodynamic focusing microfluidics.

Significant progress on circulating tumor cells (CTCs) has profound impact for noninvasive tumor profiling including early diagnosis, treatment monitoring, and metastasis recognition. Therefore, CTCs based liquid biopsy technology is taking a rapid growth in the field of precision oncology. The label-free approaches relied on microfluidic chip stand out from a crowd of methods that suffer from time consuming, extensive blood samples, lost target cells and labor-intensive operation. In this paper, a label-free separation microfluidic device was developed using multistage channel, which took full advantage of inertial lift force. Our strategy demonstrated CTCs were efficiently isolated from untreated human blood samples including antibody conjugation and erythrocyte lysis. This device was applied for isolating human brain malignant glioma cells that were spiked in human peripheral blood samples. The experimental condition was optimized and exhibited an average separation efficiency of ≥ 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is >95%, which was better than other one-step CTCs separation methods. Furthermore, the CTCs were successfully separated from untreated clinical blood sample of cancer patient on the proposed microfluidic device. The entire experimental procedures are extremely low-cost and easy manipulation. It is believed that the proposed multistage microfluidic chip can become a promising tool for CTCs separation and early diagnosis of cancer.

Photo-Cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels: Spherical Microparticles to Bow Tie-Shaped Microfibers.

Bow tie-shaped fibers and spherical microparticles with controlled dimensions and shapes were fabricated with poly(ethylene glycol) diacrylate hydrogel utilizing hydrodynamic shear principles and a photopolymerization strategy under a microfluidic regime. Decreasing the flow rate ratio between the core and sheath fluids from 25 (50:2) to 1.25 (100:80) resulted in increasing the particles size and reducing the production rate by 357 and 86%, respectively. The width of the fibers increased by a factor of 1.4 when the flow rate ratio was reduced from 2.5 to 1 due to the decrease of the shear force at the fluid/fluid interface. The stress at break and Young's modulus of the fibers were enhanced by 32 and 63%, respectively, when the sheath-to-core flow rate ratio decreased from 100:40 to 100:80. The fiber fabrication was simulated using the finite element method, and the numerical and experimental results were in agreement. Adult hippocampal stem/progenitor cells and bone-marrow-derived multipotent mesenchymal stromal cells were seeded onto the fibrous scaffolds in vitro, and cellular adhesion, proliferation, and differentiation were investigated. Microgrooves on the fibers' surface were shown to positively affect cell adhesion when compared to flat fibers and planar controls.

Enhanced Detection of Single Viruses On-Chip via Hydrodynamic Focusing.

Planar optofluidics provide a powerful tool for facilitating chip-scale light-matter interactions. Silicon-based liquid core waveguides have been shown to offer single molecule sensitivity for efficient detection of bioparticles. Recently, a PDMS based planar optofluidic platform was introduced that opens the way to rapid development and prototyping of unique structures, taking advantage of the positive attributes of silicon dioxide-based optofluidics and PDMS based microfluidics. Here, hydrodynamic focusing is integrated into a PDMS based optofluidic chip to enhance the detection of single H1N1 viruses on-chip. Chip-plane focusing is provided by a system of microfluidic channels to force the particles towards a region of high optical collection efficiency. Focusing is demonstrated and enhanced detection is quantified using fluorescent polystyrene beads where the coefficient of variation is found to decrease by a factor of 4 with the addition of hydrodynamic focusing. The mean signal amplitude of fluorescently tagged single H1N1 viruses is found to increase with the addition of focusing by a factor of 1.64.

3D hydrodynamic focusing in microscale channels formed with two photoresist layers.

3D hydrodynamic focusing was implemented with channel cross-section dimensions smaller than 10 µm. Microchannels were formed using sacrificial etching of two photoresist layers on a silicon wafer. The photoresist forms a plus-shaped prismatic focusing fluid junction which was coated with plasma-enhanced chemical-vapor-deposited oxide. Buffer fluid carried to the focusing junction envelopes an intersecting sample fluid, resulting in 3D focusing of the sample stream. The design requires four fluid ports and operates across a wide range of fluid velocities through pressure-driven flow. The focusing design was integrated with optical waveguides to interrogate fluorescing particles and confirm 3D focusing. Particle diffusion away from a focused stream was characterized.

Flow Cytometry Instrumentation - An Overview.

The term flow cytometry, used since the seventies, describes a technology employed mainly in biology and medicine to measure and classify suspended particles, e.g., cells or microspheres. Measurable cell parameters include: geometric properties, such as cell size (diameter, surface area, volume); physiological properties (membrane potential, integrity, vitality); and quantities of DNA, RNA, cytokines, surface antigens, nuclear antigens, enzymes, and proteins. © 2018 by John Wiley & Sons, Inc.

Cardiac Stem Cell Patch Integrated with Microengineered Blood Vessels Promotes Cardiomyocyte Proliferation and Neovascularization after Acute Myocardial Infarction.

Cardiac stem cell (CSC) therapy has shown preclinical and clinical evidence for ischemic heart repair but is limited by low cellular engraftment and survival after transplantation. Previous versions of the cardiac patch strategy improve stem cell engraftment and encourage repair of cardiac tissue. However, cardiac patches that can enhance cardiomyogenesis and angiogenesis at the injured site remain elusive. Therapies that target cardiomyocyte proliferation and new blood vessel formation hold great potential for the protection against acute myocardial infarction (MI). Here, we report a new strategy for creating a vascularized cardiac patch in a facile and modular fashion by leveraging microfluidic hydrodynamic focusing to construct the biomimetic microvessels (BMVs) that include human umbilical vein endothelial cells (HUVECs) lining the luminal surface and then encapsulating the BMVs in a fibrin gel spiked with human CSCs. We show that the endothelialized BMVs mimicked the natural architecture and function of capillaries and that the resultant vascularized cardiac patch (BMV-CSC patch) exhibited equivalent release of paracrine factors compared to those of coculture of genuine human CSCs and HUVECs after 7 days of in vitro culture. In a rat model of acute MI, the BMV-CSC patch therapy induced profound mitotic activities of cardiomyocytes in the peri-infarct region 4 weeks post-treatment. A significant increase in myocardial capillary density was noted in the infarcted hearts that received BMV-CSC patch treatment compared to the infarcted hearts treated with conventional CSC patches. The striking therapeutic benefits and the fast and facile fabrication of the BMV-CSC patch make it promising for practical applications. Our findings suggest that the BMV-CSC patch strategy may open up new possibilities for the treatment of ischemic heart injury.