Deposition chamber technology as building blocks for a standardized brain-on-chip framework.

The in vitro modeling of human brain connectomes is key to exploring the structure-function relationship of the central nervous system. Elucidating this intricate relationship will allow better studying of the pathological mechanisms of neurodegeneration and hence result in improved drug screenings for complex neurological disorders, such as Alzheimer's and Parkinson diseases. However, currently used in vitro modeling technologies lack the potential to mimic physiologically relevant neural structures. Herein, we present an innovative microfluidic design that overcomes one of the current limitations of in vitro brain models: their inability to recapitulate the heterogeneity of brain regions in terms of cellular density and number. This device allows the controlled and uniform deposition of any cellular population within unique plating chambers of variable size and shape. Through the fine tuning of the hydrodynamic resistance and cell deposition rate, the number of neurons seeded in each plating chamber can be tailored from a thousand up to a million. By applying our design to so-called neurofluidic devices, we offer novel neuro-engineered microfluidic platforms that can be strategically used as organ-on-a-chip platforms for neuroscience research. These advances provide essential enhancements to in vitro platforms in the quest to provide structural architectures that support models for investigating human neurodegenerative diseases.

Rapid mask prototyping for microfluidics.

With the rise of microfluidics for the past decade, there has come an ever more pressing need for a low-cost and rapid prototyping technology, especially for research and education purposes. In this article, we report a rapid prototyping process of chromed masks for various microfluidic applications. The process takes place out of a clean room, uses a commercially available video-projector, and can be completed in less than half an hour. We quantify the ranges of fields of view and of resolutions accessible through this video-projection system and report the fabrication of critical microfluidic components (junctions, straight channels, and curved channels). To exemplify the process, three common devices are produced using this method: a droplet generation device, a gradient generation device, and a neuro-engineering oriented device. The neuro-engineering oriented device is a compartmentalized microfluidic chip, and therefore, required the production and the precise alignment of two different masks.

Dielectrophoretic properties of engineered protein patterned colloidal particles.

This work determines the dielectrophoretic response of surface modified polystyrene and silica colloidal particles by experimentally measuring their Clausius-Mossotti factors. Commercial charged particles, fabricated ones coated with fibronectin, and Janus particles that have been grafted with fibronectin on one side only were investigated. We show that the dielectrophoretic response of such particles can be controlled by the modification of the chemistry or the anisotropy of their surface. Moreover, by modelling the polarizabilities of those particles, the dielectric parameters of the particles and the grafted layer of protein can be measured.

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat.

In the initial stage of ascidian fertilization sequential sperm-egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66 kDa, 120 kDa and 140 kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120 kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata.

Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity.

Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.

Isolation, characterization, and localization of a sperm-bound N-acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata.

N-Acetylglucosaminidase (GlcNAc'ase), which possesses by far the highest activity of all Phallusia mammillata sperm glycosidases, was isolated and purified using DEAE-cellulose, phenyl-Sepharose, and concanavalin A affinity chromatography. The molecular size of the native enzyme estimated by G-200 gel permeation was 158 kDa. On SDS-PAGE, the denatured enzyme migrated as a single band with a Mr of 78 kDa. This indicates that under nondenaturing conditions the GlcNAc'ase prevails as a dimer. The molecular activity of the enzyme was determined to be 3.7 x 10(5) U/mumole, the Km for p-NP-GlcNAc was 0.65 mM, and the Ki for GlcNAc was 5.5 mM. It has been suggested that gamete binding in ascidians might be mediated by an enzyme-substrate complex established between a sperm glycosidase and corresponding glycosides on the vitelline coat. Thus, the GlcNAc'ase should be present as an exoenzyme at the proper place on the sperm surface membrane, i.e., on the sperm tip and possibly over the mitochondrial region. We localized the enzyme with fluorescence and electron microscopy using the neoglycoprotein BSA-p-aminophenyl-N-acetyl-beta-D-glucosaminide (BSA-GlcNAc) or concanavalin A coupled either to fluorochromes or gold particles. Labeling of unreacted and activated sperm revealed three distinct binding sites, namely at the sperm tip, over the mitochondrion, and at the head-tail junction. In reacted sperm strong labeling was observed over the translocated mitochondrion as well as at the sperm tip. An intensive binding was observed along the rim which borders the cap-like structure at the sperm tip. The distribution of the enzyme reflected by these binding patterns accounts well for the suggested function. Using N-acetylglucosaminono-1,5-lactone oxime, a novel, highly specific inhibitor of GlcNAc'ase, we were able to show that this enzyme is indispensable for fertilization of intact eggs, but not of eggs deprived of their vitelline coat. These observations are discussed in terms of functional relationships which may exist between this enzyme, sperm binding, gamete recognition, and penetration of the vitelline coat.

A single layer of microtubules is part of a complex cytoskeleton in mature nematocytes of hydra.

The architecture of microtubules in mature nematocytes (stinging cells) of Hydra attenuata was investigated in detail by an indirect immunofluorescence study and by scanning and transmission electron microscopy in order to comprehend the function of the cytoskeleton in this extremely complex cell type. Microtubules were detected in all types of nematocytes in the tentacles and were found to be arranged in parallel arrays forming a highly organized basket-like structure around the nematocysts.

Oogenesis in Hydra carnea: A new model based on light and electron microscopic analyses of oocyte and nurse cell differentiation.

Oogenesis in Hydra carnea starts with an accumulation of a great number of I-cells in the interstitial spaces of the ectoderm of the body column. One centrally located I-cell becomes the future oocyte, the others differentiate into nurse cells. Presumptive oocyte and nurse cells are not easily distinguishable at that time. The earliest stage of an oocyte we could identify on ultrastructural criteria was in prophase of its first meiotic division. Only at this stage autosynthesis of nutritive substances predominates, the following rapid increase of the oocyte volume relies on the successive adoption of cytoplasmic fragments from nurse cells. Extending fingerlike processes between the epitheliomuscular cells, the oocyte then starts to phagocytose apoptotic nurse cells. Nurse cell differentiation is indicated by the appearance of lipid vesicles in I-cells. As differentiation proceeds glycogen, rEr and Golgi complexes appear and the cells increase due to a continuous production and accumulation of lipid, glycogen and yolk-like electron dense material. Then the loss of cytoplasmic fragments and degenerative changes typical of apoptosis, a morphologically defined form of cell death, converts the nurse cells into apoptotic bodies. The bulk of nurse cells becomes phagocytosed by the oocyte at late stages of their transformation into apoptotic bodies. At the end of oogenesis which in Hydra carnea takes about 4 days, the egg consists for the largest part of apoptotic nurse cells which persist in the developing embryo until hatching.