Exercise-Mobilized Platelet-Rich Plasma: Short-Term Exercise Increases Stem Cell and Platelet Concentrations in Platelet-Rich Plasma.

PURPOSE: To evaluate the effects of vigorous short-term exercise on the platelet and other cellular components of 2 point-of-care blood-processing devices: a buffy coat-based platelet-rich plasma (PRP) product and a plasma-based PRP product. METHODS: Twenty healthy subjects (aged 21-45 years) participated in a 20-minute vigorous exercise regimen on an upright stationary bike at 70% to 85% of maximum target heart rate. Pre- and post-exercise blood was processed in either a plasma-based or automated buffy coat-based PRP system. Complete blood counts were used to compare the cellular components in whole blood and the PRP products. RESULTS: Exercise significantly increased the concentrations of platelets by over 20% in whole blood (P < .001) and in both PRP products (P = .002 and P = .018). Both devices performed consistently with pre- and post-exercise blood. Buffy coat-based PRP prepared after exercise was also significantly larger in volume and had a significantly higher concentration of mobilized hematopoietic stem cells (hematopoietic progenitor cells [HPCs], from 1.7/µL to 2.7/µL, P = .043). The concentrations of all white blood cell types were increased, which could be differentially collected in the devices studied. CONCLUSIONS: Exercise can be used to consistently alter the composition of PRP. Twenty minutes of vigorous exercise can increase platelet concentrations in plasma-based and buffy coat-based PRP products and can increase HPC concentrations and volume in buffy coat-based PRP. CLINICAL RELEVANCE: This study shows a nonpharmacologic method to increase platelet and HPC harvests from peripheral blood. This is important because it highlights a method for altering biological therapies with limited comorbidity.

Assessment of Sub-Micron Particles by Exploiting Charge Differences with Dielectrophoresis.

The analysis, separation, and enrichment of submicron particles are critical steps in many applications, ranging from bio-sensing to disease diagnostics. Microfluidic electrokinetic techniques, such as dielectrophoresis (DEP) have proved to be excellent platforms for assessment of submicron particles. DEP is the motion of polarizable particles under the presence of a non-uniform electric field. In this work, the polarization and dielectrophoretic behavior of polystyrene particles with diameters ranging for 100 nm to 1 µm were studied employing microchannels for insulator based DEP (iDEP) and low frequency (<1000 Hz) AC and DC electric potentials. In particular, the effects of particle surface charge, in terms of magnitude and type of functionalization, were examined. It was found that the magnitude of particle surface charge has a significant impact on the polarization and dielectrophoretic response of the particles, allowing for successful particle assessment. Traditionally, charge differences are exploited employing electrophoretic techniques and particle separation is achieved by differential migration. The present study demonstrates that differences in the particle's surface charge can also be exploited by means of iDEP; and that distinct types of nanoparticles can be identified by their polarization and dielectrophoretic behavior. These findings open the possibility for iDEP to be employed as a technique for the analysis of submicron biological particles, where subtle differences in surface charge could allow for rapid particle identification and separation.

Dielectrophoretic manipulation of particle mixtures employing asymmetric insulating posts.

A novel scheme for particle separation with insulator-based dielectrophoresis (iDEP) was developed. This technique offers the capability for an inverted order in particle elution, where larger particles leave the system before smaller particles. Asymmetrically shaped insulating posts, coupled with direct current (DC) biased low-frequency alternating current (AC) electric potentials, were used to successfully separate a mixture of 500 nm and 1 µm polystyrene particles (size difference of 0.5 µm in diameter). In this separation, the 1 µm particles were eluted first, demonstrating the discriminatory potential of this methodology. To extend this technique to biological samples, a mixture containing Saccharomyces cerevisiae cells (6.3 µm) and 2 µm polystyrene particles was also separated, with the cells being eluted first. The asymmetric posts featured a shorter sharp half and a longer blunt half; this produced an asymmetry in the forces exerted on the particles. The negative DC offset produced a net displacement of the smaller particles toward the upstream direction, while the post asymmetry produced a net displacement of the larger particles toward the downstream direction. This new iDEP approach provides a setup where larger particles are quickly concentrated at the outlet of the post array and can be released first when in a mixture with smaller particles. This new scheme offers an extra set of parameters (alternating current amplitude, DC offset, post asymmetry, and shape) that can be manipulated to obtain a desired separation. This asymmetric post iDEP technique has potential for separations where it is important to quickly elute and enrich larger and more fragile cells in biological samples.

Isolation and enrichment of low abundant particles with insulator-based dielectrophoresis.

Isolation and enrichment of low-abundant particles are essential steps in many bio-analytical and clinical applications. In this work, the capability of an insulator-based dielectrophoresis (iDEP) device for the detection and stable capture of low abundant polystyrene particles and yeast cells was evaluated. Binary and tertiary mixtures of particles and cells were tested, where the low-abundant particles had concentration ratios on the order of 1:10 000 000 compared to the other particles present in the mixture. The results demonstrated successful and stable capture and enrichment of rare particles and cells (trapping efficiencies over 99%), where particles remained trapped in a stable manner for up to 4 min. A device with four reservoirs was employed for the separation and enrichment of rare particles, where the particles of interest were first selectively concentrated and then effectively directed to a side port for future collection and analysis. The present study demonstrates that simple iDEP devices have appropriate screening capacity and can be used for handling samples containing rare particles; achieving both enrichment and isolation of low-abundant particles and cells.

Assessment of cell viability after manipulation with insulator-based dielectrophoresis.

The effects of insulator-based DEP (iDEP) manipulation on cell viability were investigated by varying operating conditions and the shape of the insulating structures. Experiments were conducted with Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cells by varying the applied potential (300-1000 V), exposure time (1-4 min), and composition of the suspending medium (0-10% glucose); using devices made from polydimethylsiloxane. Cell viability was quantified employing Trypan blue staining protocols. The results illustrated a strong decrease in cell survival at higher applied electric potentials and exposure times; and an increase in cell viability obtained by increasing suspending medium osmolality. The composition and structure of the cell wall also played a major role on cell survival, where prokaryotic Gram-positive B. subtilis was the most resilient cell strain, while eukaryotic S. cerevisiae had the lowest survival rate. Due to the popularity of iDEP in applications with biological cells, characterizing how iDEP operating conditions affect cell viability is essential.

Effect of insulating posts geometry on particle manipulation in insulator based dielectrophoretic devices.

In this study, the effect of the geometry of insulating posts on microparticle trapping in insulator based dielectrophoresis (iDEP) was analyzed. The motivation for this research was to study how to improve particle trapping and enrichment by modifying the shape of insulating posts used in iDEP microdevices, while keeping post spacing constant. Mixtures of inert polystyrene particles were employed for demonstrating the effects of insulator shape on particle capture and enrichment. A series of experiments were carried out using an array of devices with different insulating post shapes. All the different post shapes employed had a width of 200 µm and were arranged in a square array of 250 µm center-to-center, thus, the spacing between posts was 50 µm in all cases. Mathematical modeling with COMSOL Multiphysics was employed to assess the magnitude of electric field gradients achieved with each one of the geometries tested. The results showed that the electric potential required to obtain effective particle trapping and enrichment can be significantly reduced by modifying the geometry of the insulating posts, without having to modify the separation distance between posts, thus, preserving the porosity of the microchannels. The separation of a mixture of 1-µm and 2-µm diameter particles was achieved in the form of dielectropherograms employing two different insulating post geometries (circle and diamond). Concentrated particles were released as peaks from the insulating post arrays where higher peak resolution separation was obtained with the sharper diamond geometry. Concentration enrichment above one order or magnitude was obtained for both particle types in both dielectropherograms. The results demonstrate that more efficient iDEP separations can be achieved at lower applied electric potentials by carefully selecting the geometry of the insulating structures.