Microfluidic Approaches for Affinity-Based Exosome Separation.

As a subspecies of extracellular vesicles (EVs), exosomes have provided promising results in diagnostic and theranostic applications in recent years. The nanometer-sized exosomes can be extracted by liquid biopsy from almost all body fluids, making them especially suitable for mainly non-invasive point-of-care (POC) applications. To achieve this, exosomes must first be separated from the respective biofluid. Impurities with similar properties, heterogeneity of exosome characteristics, and time-related biofouling complicate the separation. This practical review presents the state-of-the-art methods available for the separation of exosomes. Furthermore, it is shown how new separation methods can be developed. A particular focus lies on the fabrication and design of microfluidic devices using highly selective affinity separation. Due to their compactness, quick analysis time and portable form factor, these microfluidic devices are particularly suitable to deliver fast and reliable results for POC applications. For these devices, new manufacturing methods (e.g., laminating, replica molding and 3D printing) that use low-cost materials and do not require clean rooms are presented. Additionally, special flow routes and patterns that increase contact surfaces, as well as residence time, and thus improve affinity purification are displayed. Finally, various analyses are shown that can be used to evaluate the separation results of a newly developed device. Overall, this review paper provides a toolbox for developing new microfluidic affinity devices for exosome separation.

Simultaneous Determination of Human Erythrocyte Deformability and Adhesion Energy: A Novel Approach Using a Microfluidic Chamber and the "Glass Effect".

The simultaneous determination of adhesion and deformability parameters of erythrocytes was carried out through a microfluidic device, which uses an inverted optical microscope with new image acquisition and analysis technologies. Also, an update of the models describing erythrocyte adhesion and deformation was proposed. Measurements were carried out with red blood cells suspended in saline solution with human serum albumin at different concentrations. Erythrocytes adhered to a glass surface were subjected to different low shear stress (from 0.04 to 0.25 Pa), causing cellular deformation and dissociation. The maximum value obtained of the erythrocyte deformability index was 0.3, and that of the adhesion energy per unit area was 1.1 × 10-6 Pa m, both according to previous works. The obtained images of RBCs adhered to glass reveal that the adhesion is stronger in a single point of the cell, suggesting a ligand migration that concentrates the adhesion in a "spike-like tip" in the cell. Moreover, adhesion energy results indicate that the energy required to separate erythrocytes in media with a lower albumin concentration is greater. Both results could be explained by the mobility of membrane receptors.

A treatment approach for couples with disrupted sperm DNA integrity and recurrent ART failure.

OBJECTIVE: To test a novel method to select spermatozoa with high chromatin integrity. DESIGN: Specimens with high sperm chromatin fragmentation (SCF) were selected by density gradient selection (DGS) and microfluidic sperm sorting (MSS). SETTING: Academic medical center. PATIENT(S): Ejaculates from consenting men were processed by DGS/MSS. Couples underwent ICSI cycles with spermatozoa processed by DGS/MSS. Clinical outcomes were evaluated after embryo transfer. INTERVENTION(S): SCF was measured by TUNEL. ICSI with spermatozoa selected by DGS and MSS was performed. MAIN OUTCOME MEASURE(S): Fertilization, embryo implantation, and pregnancy outcomes were compared between DGS and MSS. RESULT(S): A total of 23 men had an average SCF of 20.7 ± 10%. After DGS and MSS, the SCF was 12.5 ± 5% and 1.8 ± 1%, respectively. In couples who underwent ICSI, the average SCF was 28.8 ± 9%, which fell to 21.0 ± 9% after DGS and 1.3 ± 0.7% after MSS. Four couples underwent 11 ICSI cycles with DGS and achieved one (25%) pregnancy that resulted in pregnancy loss. In four subsequent ICSI cycles with MSS, an ongoing clinical pregnancy rate of 50% was achieved. Five additional couples underwent 12 cycles of ICSI with DGS. After preimplantation genetic testing for aneuploidy, 30.3% of the embryos were euploid. One pregnancy was achieved, resulting in pregnancy loss. With MSS, 31.5% of the embryos were euploid and 4 couples obtained a pregnancy. Finally, sixteen couples underwent 20 ICSI cycles solely with MSS at our center. Of these couples, 8 had failed 13 ICSI cycles with DGS elsewhere. These couples achieved an overall implantation of 34.5% (10/29) and a pregnancy rate of 58.8% (10/17). CONCLUSION(S): Microfluidic selection yielded spermatozoa with optimal genomic integrity and improved chances of obtaining a euploid conceptus.

Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices.

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Here, we present our protocol for culture of primary neurons in microfluidic devices and their chronic treatment with the Parkinson's disease (PD) relevant toxicant rotenone. In addition, we present a method for reuse of devices for culture. This culture methodology presents advantages for evaluating early pathogenic cellular and molecular changes in neurons in a compartment-specific manner.

Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs).

The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) are believed to be involved in many disorders of the brain such as Alzheimer's disease (AD), schizophrenia, sleep disorders and drug abuse ( Mobley et al., 1986 ). Primary cultures of BFCNs will provide an important tool for studying the mechanism of these diseases. This protocol provides a detailed description of experimental procedures in establishing in vitro primary culture of rat embryonic BFCNs.

Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.

Molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. Disruption in the structure and function of NMJs is a hallmark of various neurodegenerative processes during both development and pathological events. Still due to the complexity of this process, it is very difficult to elucidate the cellular mechanisms underlying it, generating a keen interest for developing better tools for investigating it. Here we describe a simplified method to study mechanisms of NMJs formation, maintenance and disruption. A spinal cord explant from mice expressing the Hb9::GFP motoneuron marker is plated on one side of a compartmental chamber, and myotubes derived from muscle satellite progenitor cells are plated on the other. The GFP labeled motoneurons extend their axons via microgrooves in the chamber to innervate the muscle cells and to form functional in-vitro NMJs. Next we provide procedures to measure axon growth and to reliably quantify NMJ activity using imaging of both muscle contractions and fast intracellular calcium changes. This platform allows precise control, monitoring and manipulation of subcellular microenvironments. Specifically, it enables to distinguish local from retrograde signaling mechanisms and allows restricted experimental intervention in local compartments along the muscle-neuron route.

A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.

Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells.

UNLABELLED: Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP. DATABASE: The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/webMathematica/UItester.jsp?modelName=gustavsson5. [Database section added 14 May 2014 after original online publication].

Investigation of nerve injury through microfluidic devices.

Traumatic injuries, both in the central nervous system (CNS) and peripheral nervous system (PNS), can potentially lead to irreversible damage resulting in permanent loss of function. Investigating the complex dynamics involved in these processes may elucidate the biological mechanisms of both nerve degeneration and regeneration, and may potentially lead to the development of new therapies for recovery. A scientific overview on the biological foundations of nerve injury is presented. Differences between nerve regeneration in the central and PNS are discussed. Advances in microtechnology over the past several years have led to the development of invaluable tools that now facilitate investigation of neurobiology at the cellular scale. Microfluidic devices are explored as a means to study nerve injury at the necessary simplification of the cellular level, including those devices aimed at both chemical and physical injury, as well as those that recreate the post-injury environment.