Label-free density difference amplification-based cell sorting

The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities.Separating specific cells from heterogeneous or homogeneous mixtures has been considered as a key step in a wide variety of applications ranging from biomedicine to energy harvesting. For example, the separation and sorting of rare circulating tumor cells (CTCs) from whole blood has gained significant importance in the potential diagnosis and treatment of metastatic cancers.^1,2 Similarly, malaria detection relies on the collection of infected red blood cells (RBCs) from whole blood.^3,4 In addition, the selective separation of lipid-rich microalgae from homogeneous mixtures of microalgae is a promising technique in biomass conversion.⁵To date, conventional cell separation can be done by labelling cells with biomolecules to induce differences in physical properties. For instance, in a fluorescence-activated cell sorter (FACS), cells to be separated are labelled with antibodies or aptamers with fluorescent molecules, and then sorted by applying an electrical potential.^6,7 Similarly, magnetic-activated cell sorter (MACS) uses magnetic.^8,9 Alternatively, label-free cell separation methods have exploited inherent differences in the physical properties (e.g., size and dielectric properties) of different kinds of cells. For example, acoustophoresis forces particles larger than a desired size to move into the center of a fluidic channel by using ultrasonic standing waves.^10–12 Inertial microfluidics takes advantage of curved fluidic channels in order to amplify the size differences between particles.^13,14 Mass-dependent separation of particles based on gravity and hydrodynamic flow was also reported.¹⁵ Particles with different dielectric properties can also be sorted by dielectrophoresis which induces the movement of polarizable particles.^16–18The disadvantage of these methods, however, is that they require external forces and labels that may cause unexpected damage to biological cells.^19–21 More importantly, most methods are limited in separating cells of the same kind or cells with similar sizes and dielectric properties.Here we designed a novel, label-free density difference amplification-based cell sorting (dDACS) that allows the separation of particles with the same size and charge by exploiting subtle differences in density without the use of external forces. Figure 1(a) illustrates the proposed microfluidic model and its underlying mechanism. The conceptual microfluidic system consists of an inlet, a separation chamber (hydraulic jump cavity), and multiple outlets. Particles entering through the inlet experience gravity (F_G), buoyancy (F_B), and drag (F_D) forces in the separation chamber. The net force acting on the particles can be described as F = F_G + F_B + F_D.(1)As particles enter the separation chamber (i.e., hydraulic jump cavity), F_D acting on the particles changes its direction along the streamline. The particles experience additional forces in the y direction due to large tangential angle (Fig. 1(b)). For lighter particles, whose densities are close to that of the surrounding water, F_D becomes comparable to F_G (i.e., in the y direction), while the net force for heavier particles is less affected by this additional contribution of F_D due to a large F_G. As a result, the height (H) and distance (D) that each particle can travel are different depending on its density. The difference in the maximum height (ΔH_max) between two particles with different density (ρ_p1 and ρ_p2) can be further approximated as ΔHmax∝(vyp0)2(vyf−vyp0), (ρp1−ρp2) ,(2)where v_yp0 and v_yf represent the velocity of particle and fluid along the y direction at the entrance of hydraulic jump cavity, respectively.Open in a separate windowFIG. 1.Schematic illustration of label-free density difference amplification-based cell sorting (dDACS), which exploits differences in the densities (ρ₁ > ρ₂) of particles with similar diameters (d) and charge. (a) The conceptual microfluidic design consists of an inlet, a separation chamber (hydraulic jump cavity), and multiple outlets. Incoming particles experience gravity (F_G), buoyancy (F_B), and drag (F_D) forces in the separation chamber, and depending on their densities, the height (H) and distance (D) that each particle is able to reach will be different, allowing the particles to be separated into multiple outlets. (b) Possible microfluidic channel configurations for density-based separation: Uniform channel height (left), gradual channel expansion (middle), and hydraulic jump cavity with sudden channel expansion (right). The height difference between particles with different densities can be amplified by the sudden channel expansion compared to the other two cases due to the relatively large tangential angle, θ of F_D. (|θ₁|≪ |θ₂|) (see Fig. S1 in the supplementary material²²).In comparison with the other two cases (Fig. 1(b) uniform channel height and gradual channel expansion), the height difference between the particles with different densities can be amplified by the sudden channel expansion in the hydraulic jump cavity due to relatively large tangential angle (see supplementary material²²). Therefore, the particles can be separated through the multiple outlets, depending on their height and distance.In order to analyze the separation behavior of particles in the chamber according to differences in their densities, H and D are systematically investigated. The numerical simulations are performed using a commercial CFD software (CFX 14.0; ANSYS 14.0; ANSYS, Inc.). Particles with the same density may have different trajectories in the separation chamber depending on their inlet positions (Fig. 2(a)). Prior to this investigation, the maximum height (H_max) and distance (D_max) for each particle are compared by examining H and D of 100 identical particles at different inlet positions since the inlet position of particles could be controlled.²⁰ Fig. 2(b) shows H_max and D_max of particles with respect to density at a fixed Reynolds number (Re = 0.1). Note that Reynolds number is defined as Re = ρ_fv_fD_h/μ, where ρ_f, v_f, D_h, μ are density of fluid, velocity of the fluid, hydraulic diameter of a channel, and dynamic viscosity of the fluid, respectively. The hydraulic diameter in the Reynolds number is determined with the inlet channel. Particle densities in the range of 1.1 to 2.0 g/cm³ are chosen with the increase of 0.1 g/cm³. These values are quite reasonable in that the densities of many microorganisms such as microalgae are typically within this range and their densities can be varied by 0.2 g/m³ depending on their cellular context.²³ The lighter particles travel with a higher H_max, and longer D_max. With the separation chamber, the height difference between particles with densities of 1.1 and 1.2 g/cm³ can be amplified by about 10 times as compared to that in a channel without the chamber, judging from the position where the 1.1 g/cm³ particle reaches its H_max.Open in a separate windowFIG. 2.Microfluidic particle separation with respect to Reynolds number (Re). (a) Trajectories in the separation chamber of a hundred particles with the same density starting from inlet positions chosen arbitrarily in order to investigate the effect of the inlet positions on the maxima of the height (H_max) and distance (D_max) prior to further simulation. (b) Representative trajectories of particles having different densities from 1.1 to 2.0 g/cm³. (c) The maximum height (H_max) of each particle with respect to Re. (d) Representative maximum distance (D_max) of each particle at Re = 0.1. (Left) Streamline of fluid and representative trajectories of particles with densities of 1.1 and 2.0 g/cm³ in the separation chamber at Re = 0.1 (right).In Fig. 2(c), the values for H_max of particles with respect to Reynolds number (Re) are presented. Since in our study, the maximum height (H_max) and distance (D_max) for each particle were compared by examining H and D of 100 identical particles that are randomly distributed in the channel (throughout all figures), there is little variation in H_max and D_max between each simulation. However, the standard deviation between each simulation is quite small and can be negligible. The H_max values particles at Re = 0.5 with densities of 1.1 g/cm³ and 1.2 g/cm³ are 2.21 × 10³μm and 2.17 × 10³μm, respectively. The difference between H_max of different particles, ΔH_max, increases with decreasing Re. For example, ΔH_max between particles with densities of 1.1 and 2.0 g/cm³ becomes 0.26 × 10³μm at Re = 1.0, but increases to 1.38 × 10³μm as Re decreases to 0.1. As Re increases (velocity of fluid increases), the relative velocity in the y direction between the fluid and the particle increases resulting in increasing of F_D in the y direction since the velocity of particle in the y direction is very small at the entrance of the separation chamber. Thus, contribution of F_D becomes comparable to the net force in the y direction. As a result, most of the particles even in the case of heavier ones travel quite similarly with the streamline, and ΔH_max subsequently decreases. On the other hand, as Re decreases, the contribution of F_G becomes dominant due to the decrease of F_D in the y direction. Consequently, the particles start to cross downwards streamlines as the density of the particles increases and H_max gradually decreases. In addition, irrespective of their densities, ΔH_max of the particles increases with decreasing Re.Fig. 2(d) shows D_max with respect to the density of the particles (left). Different densities of particles show different trajectories due to the relative contribution of F_D to the net force in the y direction depending on the particle density (right). At Re = 0.1, D_max of particles with densities of 1.1 cm³ and 1.2 g/cm³ are 2.91 × 10⁴μm and 1.43 × 10⁴μm, respectively. As the density of a particle increases, its D_max dramatically decreases. The difference in D_max between particles with densities of 1.1 and 1.2 g/cm³ is 1.48 × 10⁴μm, and 0.0037 × 10⁴μm for particles with densities of 1.9 and 2.0 g/cm³. The effect of F_D is stronger compared to that of F_G on lighter particles. Thus, lighter particles travel quite similarly with the streamline and finally have a large D_max. On the other hand, heavier particles where effect of F_G is stronger compared to that of F_D cross downwards streamlines and finally have a small D_max.Next, in order to investigate the separation behavior of particles with respect to the geometry of the microfluidic device, the effect of the ratio of the height of the separation chamber (h_c) to the inlet (h_i) on H_max is investigated as shown in Fig. ​Fig.3.3. Interestingly, H_max of particles with density of 1.1 g/cm³ increases from 1.93 × 10³μm to 6.48 × 10³μm while that of particles with density of 1.9 g/cm³ slightly changes from 0.70 × 10³μm to 0.73 × 10³μm as h_c/h_i increases from 5 to 20.Open in a separate windowFIG. 3.Microfluidic particle separation with respect to the ratio of the height of the inlet (h_i) to the separation chamber (h_c).This result can be attributed to two effects: (1) the change in the streamline and (2) the relative contribution of drag force to the net force depending on the density. With increasing h_c/h_i, dramatic increase in H_max for lighter particles is because the streamline for the lighter ones experiences more vertical displacement in the separation chamber and the contribution of F_D to the net force acting on the lighter one is more significant (see Fig. S2 in the supplementary material²²).Based on this approach, we propose a microfluidic device for the selective separation of the lightest particle. Fig. 4(a) shows one unit (with three outlets) of the proposed microfluidic device that can be connected in series. The ratio of channel heights (h_c/h_i) is set to 20, and the particle densities are in the range of 1.1 ∼ 1.5 g/m³. Fig. 4(b) shows the representative separation behavior of the particles. A portion of the lightest particles (1.1 g/cm³) is selectively separated into the upper and middle outlets, while remaining light particles together with four other heavier particles with densities in the range of 1.2 to 1.5 g/cm³ leave through the lowest outlet. With a single operation of this unit, 40% of the lightest particles are recovered. In addition, the yield increases with increasing number of cycles (Fig. 4(c)).Open in a separate windowFIG. 4.(a) One unit of the proposed microfluidic device for the selective separation of the lightest particle based on the simulation results. Particles are separated into two outlets based on differences in both the height and distance travelled stemming from differences in density. (b) Representative separation behavior of particles observed in the device. (c) The yield of the lightest particle (1.1 g/cm³) with the proposed microfluidic device according to the number of cycles (i.e., this unit is assumed to be connected in series).In summary, we have demonstrated a label-free microfluidic system for the separation of particles according to subtle differences in their densities without external forces. Our microfluidic design consists simply of an inlet, a separation chamber, and multiple outlets. When entering the separation chamber, the particles experience an additional drag force in the y direction, amplifying the difference in both the height and the distance that the particles with different densities can travel within the chamber. At a fixed Reynolds number, with increasing particle density, H_max decreases monotonously, and D_max decreases dramatically. On the other hand, as Reynolds number increases, the difference between the heights of particles with different densities is attenuated. In addition, the simulation reveals that increasing the ratio of the channel heights increases the difference between the heights of particles only when their densities are close to that of the surrounding water. Based on this approach, a microfluidic device for the separation of the lightest particles has been proposed. We expect that our density-based separation design can be beneficial to the selective separation of specific microorganisms such as lipid-rich microalgae for energy harvesting application.     

Beyond Interaction: The relational construct of 'Transactional Presence'

This paper presents the novel construct of 'Transactional Presence' in consideration of a distance student's perceptions of psychological presence on the part of teachers, peers, and institutions. Relevant distance education literature is analyzed through the lens of interpersonal relationships, and concepts such as telepresence and social presence are drawn from media and communication studies. When the two streams of literature reviewed are merged, it is conjectured that the perception of presence concerned must be influenced by distance students' idiosyncratic needs for connection with learning resources and sources of support. This notion of presence, designated 'Transactional Presence', is defined as the degree to which a distance student perceives the availability of, and connectedness with, other parties involved in a given distance education setting. The construct's potential significance is explored in view of enriching distance education theory and research.     

Density-dependent separation of encapsulated cells in a microfluidic channel by using a standing surface acoustic wave

This study presents a method for density-based separation of monodisperse encapsulated cells using a standing surface acoustic wave (SSAW) in a microchannel. Even though monodisperse polymer beads can be generated by the state-of-the-art technology in microfluidics, the quantity of encapsulated cells cannot be controlled precisely. In the present study, mono-disperse alginate beads in a laminar flow can be separated based on their density using acoustophoresis. A mixture of beads of equal sizes but dissimilar densities was hydrodynamically focused at the entrance and then actively driven toward the sidewalls by a SSAW. The lateral displacement of a bead is proportional to the density of the bead, i.e., the number of encapsulated cells in an alginate bead. Under optimized conditions, the recovery rate of a target bead group (large-cell-quantity alginate beads) reached up to 97% at a rate of 2300 beads per minute. A cell viability test also confirmed that the encapsulated cells were hardly damaged by the acoustic force. Moreover, cell-encapsulating beads that were cultured for 1 day were separated in a similar manner. In conclusion, this study demonstrated that a SSAW can successfully separate monodisperse particles by their density. With the present technique for separating cell-encapsulating beads, the current cell engineering technology can be significantly advanced.     

Do Teachers’ Instructional Practices Moderate Equity in Mathematical and Scientific Literacy?: an Investigation of the PISA 2012 and 2015

Many efforts have been made to reach educational equity, especially to reduce mathematics and science achievement gaps by students’ socioeconomic status. Across countries, educators strive to reform traditional teacher-centered instructional approaches to more student-centered/inquiry-based instruction to improve equity in education. In this context, this study examines whether relationships between socioeconomic status and scientific or mathematical literacy are moderated by student-centered instruction. Ten countries covering a wide range of achievement levels as well as equity in education are selected for an international comparison. A linear regression analysis is applied to student achievement, equity, and frequency of student-centered instruction data from the PISA 2012 and PISA 2015. We find mixed results: As student-centered instruction is offered more frequently, the gap in mathematical and scientific literacy between low and high socioeconomic status is generally narrowed or maintained. In most countries, students’ mathematical and scientific literacy scores are expected to decrease across all socioeconomic status as student-centered instruction is given more frequently. The findings necessitate further scrutiny of how teachers implement student-centered instruction in various educational systems. This further research need to consider the complexity of implementation related to sociological and pedagogical aspects.     

Challenges and strategies of building and sustaining inter-organizational partnerships in sport for development and peace

While sport management scholars have explored inter-organizational partnerships and their associated challenges, they have devoted less attention to inter-organizational partnership development and sustainability in sport for development and peace (SDP), particularly across a wide range of organizations with varied missions and foci. Hence, the purpose of this qualitative study was to examine challenges faced by SDP organizations when forming and sustaining inter-organizational partnerships across contexts and partnership types, and to uncover strategies they have employed to overcome these challenges. Common challenges encountered across 29 SDP organizations included competition for resources, skepticism of sport as a development tool, unequal power relations, misaligned goals and mission drift, and implementation issues. Strategies included focusing on building relationships and networks, demonstrating benefits to partner, starting small then diversifying, keeping focused on mission and goals, involving partner, and treating the partnership as a business relationship. Theoretical extensions and practical implications are discussed, along with directions for future research.     

How does gender relate to social skills? Exploring differences in social skills mindsets,academics, and behaviors among high‐school freshmen students

Boys struggle academically and behaviorally more than girls and are more likely to have difficulty with social skills. It seems likely that boys and girls do not perceive social skills in the same light. Past research has not investigated this or its relationship to academic and behavioral performance. Using data from a cohort of 9th‐grade students (n = 323) in one high school in central Illinois, this study involves interaction analyses of student mindsets about their social skills and gender. Findings indicated that females who perceive social skills as more important had higher grade point averages (GPAs), higher attendance rates, and fewer disciplinary problems than their male counterparts. Conversely, females who perceive social skills as of lesser importance have lower GPAs, poorer attendance, and more disciplinary referrals than their male counterparts. Findings highlight pertinent gender differences in the relation between social skills mindsets and outcomes among high‐school freshmen students.     

A critique of Korean National Information Strategy: Case of national information infrastructures

In 2004, the Korean government launched the IT839 project with the objective of converting Korea into a ubiquitous information society. This study investigates the role of the Korean government in the development of the national information infrastructure and the realization of IT839 vision. This paper reviews the historic and individual data related to the infrastructure project, draws on the social construction of technology theory as a framework for interpreting such data, traces the transformations and translation of this data in the public, political, and social discourse, and discusses the next generation of information infrastructure. Findings imply that despite optimistic prospects and proactive drive, uncertainty still remains with respect to where IT839 will evolve and how it will impact the new information milieu.     

Attending to Diversity Representation Among Council for Accreditation of Counseling and Related Educational Programs (CACREP) Master’s Programs: A Pilot Study

Extant research has neglected to explore the diversity representation of counselor education programs. This pilot study sought to examine the extent to which CACREP-accredited master’s-level programs attend to the representation of people of color, individuals with dis/abilities, lesbian, gay, bisexual (LGB), and transgender persons within admissions, enrollment, and graduation data. The findings indicated little consistency in the maintenance of students’ demographic characteristics among participating programs. The authors contend that counseling programs need to critically analyze their policies and procedures to ensure a responsive milieu for students from diverse backgrounds. Implications for the counseling field are presented.