Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

High efficiency rare sperm separation from biopsy samples in an inertial focusing device.

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.

An automated instrument for intrauterine insemination sperm preparation.

Sperm preparation is critical to achieving a successful intrauterine insemination and requires the processing of a semen sample to remove white blood cells, wash away seminal plasma, and reduce sample volume. We present an automated instrument capable of performing a sperm preparation starting with a diluted semen sample. We compare our device against a density gradient centrifugation by processing 0.5 mL portions of patient samples through each treatment. In 5 min of operating time, the instrument recovers an average of 86% of all sperm and 82% of progressively motile sperm from the original sample while removing white blood cells, replacing the seminal plasma, and reducing the volume of the sample to the clinically required level. In 25 min of operating time, density gradient centrifugation recovers an average of 33% of all sperm and 41% of progressively motile sperm. The automated instrument could improve access to IUI as a treatment option by allowing satellite doctor's offices to offer intrauterine insemination as an option for patients without the clinical support required by existing methods.

Social Cost of Blindness Due to AMD and Diabetic Retinopathy in the United States in 2020.

BACKGROUND AND OBJECTIVE: To estimate the social cost of blindness due to wet age-related macular degeneration (wAMD), diabetic macular edema (DME), and proliferative diabetic retinopathy (PDR) in the United States in 2020. PATIENTS AND METHODS: Excess costs that occur because of blindness were estimated as the difference in costs in blind versus non-blind individuals. Per-patient costs were aggregated using the number of cases of blindness due to wAMD, DME, and PDR projected in 2020. RESULTS: Associated annual excess direct costs, indirect costs, and quality-adjusted life year loss per blind individual were $4,944, $54,614, and 0.214, respectively. Combining estimates with 246,423 projected cases of blindness due to wAMD, DME, and PDR translated to total societal costs of $20 billion in 2020, estimated to triple by 2050. CONCLUSION: Excess social costs associated with blindness in individuals with wAMD, DME, and PDR are substantial, with more than half of the burden attributed to indirect costs. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:S6-S14.].

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

The energy pump and the origin of the non-equilibrium flux of the dynamical systems and the networks.

The global stability of dynamical systems and networks is still challenging to study. We developed a landscape and flux framework to explore the global stability. The potential landscape is directly linked to the steady state probability distribution of the non-equilibrium dynamical systems which can be used to study the global stability. The steady state probability flux together with the landscape gradient determines the dynamics of the system. The non-zero probability flux implies the breaking down of the detailed balance which is a quantitative signature of the systems being in non-equilibrium states. We investigated the dynamics of several systems from monostability to limit cycle and explored the microscopic origin of the probability flux. We discovered that the origin of the probability flux is due to the non-equilibrium conditions on the concentrations resulting energy input acting like non-equilibrium pump or battery to the system. Another interesting behavior we uncovered is that the probabilistic flux is closely related to the steady state deterministic chemical flux. For the monostable model of the kinetic cycle, the analytical expression of the probabilistic flux is directly related to the deterministic flux, and the later is directly generated by the chemical potential difference from the adenosine triphosphate (ATP) hydrolysis. For the limit cycle of the reversible Schnakenberg model, we also show that the probabilistic flux is correlated to the chemical driving force, as well as the deterministic effective flux. Furthermore, we study the phase coherence of the stochastic oscillation against the energy pump, and argue that larger non-equilibrium pump results faster flux and higher coherence. This leads to higher robustness of the biological oscillations. We also uncovered how fluctuations influence the coherence of the oscillations in two steps: (1) The mild fluctuations influence the coherence of the system mainly through the probability flux while maintaining the regular landscape topography. (2) The larger fluctuations lead to flat landscape and the complete loss of the stability of the whole system.