Microelectromechanical systems Coulter counter for cell monitoring and counting

This note describes the design, fabrication, and testing of a novel microelectromechanical systems Coulter counter. The Coulter counter will be used to detect and monitor impedance changes of cells as a function of time in response to different experimental extracellular environments. The device consists of SU-8 (negative photoresist ) microchannels, vertical electroplated electrodes, polydimethylsiloxane cover, and is divided into a passive mixing region, a focusing region using negative dielectrophoretic forces, and a measuring region defined by multiple electroplated electrode pairs. The devices were tested using both microbeads in saline water and fibroblast cells in phosphate buffered saline solution. The results show that the proposed microsystem is capable of monitoring impedance of cells at different positions along the Coulter microchannel

Osmotic characteristics and fertility of murine spermatozoa collected in different solutions

Osmotic stress is an important factor that can result in cell damage during cryopreservation. Before ejaculation or collection for cryopreservation, murine spermatozoa are stored in epididymal fluid, a physiologically hyperosmotic environment (w415 mmol/kg). The objectives of this study were to determine the osmotic tolerance limits of sperm motion parameters of ICR and C57BL/6 mouse spermatozoa collected in isosmotic (290 mmol/kg) and hyperosmotic (415 mmol/kg) media, and the effect of the osmolality of sperm collection media on sperm fertility after cryopreservation. Our results indicate that murine spermatozoa collected in media with different osmolalities (290 and 415 mmol/kg Dulbecco’s phosphate buffered saline (DPBS)) appeared to have different osmotic tolerances for the maintenance of sperm motility and other motion parameters in both mouse strains. The hypo- and hyperosmotic treatments decreased motility and affected other motion parameters of spermatozoa collected in 290 mmol/kg DPBS. The extent of the change of motion parameters after treatments corresponded with the levels of osmotic stress. However, for spermatozoa collected in 415 mmol/kg DPBS, exposure to 290 mmol/kg DPBS tended to increase sperm motility and the quality of their motion parameters. The osmolality of sperm collection medium can affect murine sperm fertility. Spermatozoa collected in 415 mmol/kg medium showed higher fertility compared with spermatozoa collected in 290 mmol/kg as assessed by IVF. Results characterizing murine sperm osmotic tolerance collected in media with different osmolalities from different strains and the effect of collection media osmolality on sperm fertility after cryopreservation will be useful in designing cryopreservation protocols

Existence, functional impairment, and lung repair potential of endothelial colony-forming cells in oxygen-induced arrested alveolar growth

BACKGROUND: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth. METHODS AND RESULTS: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension. CONCLUSIONS: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage

Reversible Disassembly of the Actin Cytoskeleton Improves the Survival Rate and Developmental Competence of Cryopreserved Mouse Oocytes

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method

Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes

Background: Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the oocyte oolemma permeability to water and diverse cryoprotectants. Methods: We prospectively investigated volume changes over time at different temperatures (30°C, 22°C and 8°C) of human metaphase II (MII) oocytes (obtained in stimulated ICSI cycles and matured in vitro from the germinal vesicle stage) when exposed to changes in external osmolality. We also investigated human in vitro matured (IVM) oocytes membrane permeability characteristics at 22°C to 1,2-propanediol (PG) and dimethylsulphoxide (DMSO) and at 30°C, 22°C and 8°C to ethylene glycol (EG), and calculated corresponding oocyte oolemma permeability coefficients (Lp and Pcpa). Furthermore, we investigated the osmotic tolerance limits of IVM oocytes exposed to changes in external osmolality as assessed by their developmental competence during the course of 72h after ICSI. Results: The results of our studies describe human oocyte membrane permeability coefficients for EG at 30°C (2.85 ± 0.15 × 10-3cm/min), 22°C (1.17 ± 0.60 × 10-3cm/min) and 8°C (0.37 ± 0.15 × 10-3cm/min). Furthermore, at 22°C the EG oolemma permeability coefficient was lower than that of PG and DMSO (1.17 ± 0.60 × 10-3cm/min versus 2.15 ± 0.70 × 10-3 and 1.56 ± 0.38 × 10-3cm/ min, respectively). Our results also indicate, that human IVM MII oocytes tolerated exposure to solutions in the range of 39-2264 mOsmol/ kg H2O as assessed by the oocytes' developmental competence after exposure. Conclusions: The results of the present study may contribute to a better understanding of the biology and cryobiology of human oocytes, and to the design of better and more robust cryopreservation (freezing or vitrification) protocols. © The Author 2007.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A magnetic bead inside of a mouse oocyte.

<p>A photomicrograph of a CD1 mouse oocyte having been injected with a 5 µm superparamagnetic bead, and subsequently embedded in fibrin gel, is shown.</p

Oocyte survival after cryopreservation.

<p>(A) The percentage of mouse oocytes that survived slow-cooling with (left) and without (right) LATA pretreatment. LATA pretreatment increased the cryosurvival rate by 26.2% (<i>p</i><0.05). (B) The percentage of blastocysts developed from 2-cell embryos with (left) and without (right) LATA pretreatment. LATA pretreatment increased developmental competence by 81% (<i>p</i><0.05). Error bars represent the SEM.</p

The viscoelastic model for the ooplasmic environment.

<p>The ooplasmic environment is modeled as a Voight body (<i>k</i>,<i>µ</i>) in series with a dashpot (<i>µ</i>₁). The ideal trajectory of the bead under constant force (creep, <i>F</i>≠0) and during relaxation (<i>F</i> = 0). <i>F</i> = magnetic force; <i>k</i> = elastic coefficient; <i>µ</i> and <i>µ</i>₁ = friction coefficients.</p

The oocyte viscoelastic parameters.

<p>Viscoelastic parameters before freezing without (Fresh, n  =  29 cells) or with LATA treatment (Fresh-LATA, n  =  28 cells) and after freezing without (Thawed, n  =  16 cells) or with (Thawed LATA, n  =  19 cells) LATA pretreatment. (A) Relaxation time. (B) Friction coefficient <i>µ</i>₁. (C) Elastic coefficient <i>k</i>. (D) Friction coefficient <i>µ</i>. All viscoelastic parameters show significant statistical difference (see <i>p</i> values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002787#pone-0002787-t001" target="_blank">Table 1</a>) between the Fresh and Thawed groups and most show statistical difference between Fresh and Fresh LATA groups. There is no significant statistical difference between the Fresh and the Thawed LATA groups. Bars represent geometric mean±geometric SE (panel A) and mean±SEM (panels B, C and D).</p