Editorial for the Special Issue on Microfluidic Device Fabrication and Cell Manipulation.

Microfluidic devices have been utilized for separation sciences, environmental sciences, food processing, drug delivery, bioimaging, diagnostics, and cell cultures [...].

Blood Cell Separation Using Polypropylene-Based Microfluidic Devices Based on Deterministic Lateral Displacement.

Mammalian blood cell separation methods contribute to improving the diagnosis and treatment of animal and human diseases. Microfluidic deterministic lateral displacement (DLD) devices can sort cells based on their particle diameter. We developed microfluidic DLD devices with poly(propylene)-based resin and used them to separate bovine and human red blood cells (RBCs) and white blood cells (WBCs) without electric devices. To determine the critical cut-off diameter (Dc) of these devices, we used immunobeads with a diameter of 1-20 µm. The Dc values of the microfluidic DLD devices for the immunobeads in the experiments were similar to the calculated Dc values (8-10 µm). Results from bovine blood cell separation experiments suggest that lymphocytes and neutrophils can be separated from diluted, whole blood. Human RBCs were occasionally observed in the left outlet where larger particles with diameters closer to the Dc value were collected. Based on the Dc values, human neutrophils were sorted to the left outlet, whereas lymphocytes were observed in both outlets. Although microfluidic channel optimization is required for the concentration of sorted cells, the microfluidic DLD device prepared with a poly(propylene)-based resin has the potential for clinical use.

Point-of-care semen analysis of patients with infertility via smartphone and colorimetric paper-based diagnostic device.

Male infertility affects millions of males worldwide and is rising in prevalence due to social and environmental conditions. However, men often feel too embarrassed to receive a semen analysis in the hospital due to social stigmas. To overcome this problem, we developed a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test strip to distinguish semen samples with low total motile sperm concentration from those with normal motile sperm concentration. This is a point-of-care colorimetric semen analytical method with a one-step, inexpensive, equipment-free evaluation process, and adequate accuracy validated in a 42-sample clinical trial. In this study, results were evaluated visually and with a smartphone application. Using visual observation methods, the area under the curve (AUC) was 0.71 (95% of confidence interval = 0.55-0.86; p = 0.021), sensitivity was 41%, specificity was 95%, positive predictive value was 90%, negative predictive value (NPV) was 59.4%, and accuracy was 67%. Using a smartphone recording and analytical system, AUC was 0.766 (95% of confidence interval = 0.612-0.92; p = 0.003), sensitivity was 96%, specificity was 65%, PPV was 75%, NPV was 92.9%, and accuracy was 80.9%. This work demonstrated a screening tool that could elevate semen analysis to the level of routine healthcare and provide for private, in-home self-assessment.

Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm.

Porcine sperm motility was assessed via resazurin reduction color change in sperm cells using a novel paper-based assay of our own design. We applied mixtures of resazurin solution and porcine semen onto hydrophilic test circles on our paper-based device and investigated the resulting reduction reaction expressed as red and blue color intensity (RBCI). We quantified this reaction using a blue/pink color ratio from our 8 × 3 = 24 bit RGB color image. To examine enzymatic reactivity in sperm cells, we used two inhibitors: 3-Nitropropanoic acid (3-NPA) and 3-Bromopyruvic acid (3-BP). 3-NPA inhibits the citric acid cycle and electron transfer reaction in mitochondria, but did not strongly reduce sperm motility in our tests. 3-BP decreases reactivity of both mitochondrial electron transfer and glycolytic enzymes in cytosol, which significantly lowers porcine sperm motility. RBCIs of 3-NPA- and 3-BP-treated samples were significantly lower compared to our untreated control (p < 0.025). Based on these results, we feel that resazurin can be used to estimate the amount of reductants with and without inhibitor treatment. For continued research assessing the molecular mechanisms of resazurin reduction in porcine sperm, a combination assay using two or more redox indicators (e.g., resazurin and Thiazolyl Blue Tetrazolium Bromide (MTT)) embedded into our paper-based device could further our understanding of sperm cell bioenergetics.

Spiral Trajectory Modulation of Rheotaxic Motile Human Sperm in Cylindrical Microfluidic Channels of Different Inner Diameters.

We investigated the relationship between human sperm rheotaxis and motile sperm trajectories by using poly-(dimethylsiloxane) (PDMS)-based cylindrical microfluidic channels with inner diameters of 100 µm, 50 µm, and 70 µm, which corresponded to the inner diameter of the human isthmus, the length of a sperm and a diameter intermediate between the two, respectively. We counted the number of rheotaxic sperm and sperm with spiral motion. We also analyzed motile sperm trajectories. As the cylindrical channel diameter was decreased, the percentage of sperm cells exhibiting rheotaxis, the percentage of sperm cells exhibiting spiral motion, the frequency-to-diameter ratio of the sperm cells' spiral trajectories, and the surface area of the microfluidic channel increased, while the flagellar motion at the channel wall decreased. The percentage of sperm exhibiting a spiral trajectory and the frequency-to-diameter ratio of the sperm cells' spiral trajectories were thus affected by the channel diameter. Our findings suggest that the oviduct structure affects the swimming properties of sperm cells, guiding them from the uterus to the ampulla for egg fertilization. These results could contribute to the development of motile sperm-sorting microfluidic devices for assisted reproductive technologies.

Surface modification effects on the tensile properties of functionalised graphene oxide epoxy films.

Graphene oxide (GO) is a candidate for nanofillers to improve the mechanical and thermal stability of nanocomposites. In order to determine the molecular interaction to improve the mechanical properties of GO-epoxy resin composites, we investigated the relationship between GO oxidation properties and the tensile strength of the epoxy resin. With respect to GO preparation, graphite was oxidised by the Brodie or Hummers method, and the oxidised GO was reduced or chloride substituted. The X-ray photoelectron spectroscopy (XPS) spectral patterns indicate that a shorter Brodie oxidation method GO (B-GO) is associated with a higher proportion of hydroxyl groups. The oxidised GO materials, with the exception of the sample produced by the 54 h Brodie oxidation method, improved the tensile strength of the composites while the epoxy resin with reduced or chlorinated GO did not increase the tensile strength of the film. Based on XPS and elemental analyses, the improvement in the tensile strength is due to the presence of O atom based functional groups, such as hydroxyl groups, on the GO surface. The interaction between the epoxy resin and O atom based functional groups on the GO contributes to improving the tensile strength of the composites.

Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices.

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

Development of Microfluidic Systems for Fabricating Cellular Multilayers.

We designed a microfluidic system comprising microfluidic channels, pumps, and valves to enable the fabrication of cellular multilayers in order to reduce labor inputs for coating extracellular matrices onto adhesive cells (e.g., centrifugation). Our system was used to fabricate nanometer-sized, layer-by-layer films of the extracellular matrices on a monolayer of C2C12 myoblasts. The use of this microfluidic system allowed the fabrication of cellular multilayers in designed microfluidic channels and on commercial culture dishes. The thickness of the fabricated multilayer using this microfluidic system was higher than that of the multilayer that was formed by centrifugation. Because cellular multilayer fabrication is less laborious and the mechanical force to the cell is reduced, this novel system can be applied to tissue modeling for cell biology studies, pharmaceutical assays, and quantitative analyses of mechanical or chemical stimuli applied to multilayers.

Numerical calculations for diffusion effects in the well-of-the-well culture system for mammalian embryos.

Recent studies suggest that the microenvironment and embryo density used during embryo culture considerably affect development to the blastocyst stage. High embryo density allows for autocrine secretions to diffuse to neighbouring embryos during group culture, with a positive effect on further development. A variation of group culture is the well-of-the-well (WOW) culture system, allowing for individual identification of embryos cultured in small holes in a microdroplet. Bovine blastocyst development is higher in the WOW culture system than in conventional group culture. To compare the concentration of chemical factors between conventional and WOW culture, a model was constructed to calculate the concentration of secreted factors based on Fick's second law of diffusion using spreadsheet software. Furthermore, model was used to determine the concentration of growth factors and waste materials adjacent to the embryo periphery. The results of these calculations suggest that the highest difference in the concentration of secreted small molecules and macromolecules was at the most two- to threefold, with the concentrations reduced more and diffusion kinetics facilitated to a greater extent in the WOW culture system. The average ratio of the concentration of secreted macromolecules (10nm diameter) around the embryos was also compared between systems with well widths of 0.1 and 0.3mm. The concentration of secreted materials surrounding embryos increased in a narrow tapered well. The findings suggest that the WOW culture system is better than conventional group culture because of the increased final concentration of autocrine factors and higher diffusion kinetics of waste materials.

Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems.

We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida.

Detection of Micrococcus luteus biofilm formation in microfluidic environments by pH measurement using an ion-sensitive field-effect transistor.

Biofilm formation in microfluidic channels is difficult to detect because sampling volumes are too small for conventional turbidity measurements. To detect biofilm formation, we used an ion-sensitive field-effect transistor (ISFET) measurement system to measure pH changes in small volumes of bacterial suspension. Cells of Micrococcus luteus (M. luteus) were cultured in polystyrene (PS) microtubes and polymethylmethacrylate (PMMA)-based microfluidic channels laminated with polyvinylidene chloride. In microtubes, concentrations of bacteria and pH in the suspension were analyzed by measuring turbidity and using an ISFET sensor, respectively. In microfluidic channels containing 20 µL of bacterial suspension, we measured pH changes using the ISFET sensor and monitored biofilm formation using a microscope. We detected acidification and alkalinization phases of M. luteus from the ISFET sensor signals in both microtubes and microfluidic channels. In the alkalinization phase, after 2 day culture, dense biofilm formation was observed at the bottom of the microfluidic channels. In this study, we used an ISFET sensor to detect biofilm formation in clinical and industrial microfluidic environments by detecting alkalinization of the culture medium. 

A tilting embryo culture system increases the number of high-grade human blastocysts with high implantation competence.

Human embryos normally experience mechanical stimuli during development in vivo. To apply appropriate stimuli to embryos, this study group developed a tilting embryo culture system (TECS) and investigated whether it could improve the grade of fresh human embryos compared with a control static culture system. A total of 450 retrieved oocytes from 32 IVF or intracytoplasmic sperm injection cycles of 32 women were cultured for 5-6 days. Oocytes were divided randomly into TECS and control groups and then were inseminated in vitro. All embryos were evaluated at days 3 and 5 using standard grading criteria for embryo quality. The rates of fertilization per mature oocyte and high-grade cleavage-stage embryo formation in the TECS group were similar to those in the control group. The rates of blastocyst formation and of blastocysts graded 3BB or higher at day 5 were significantly higher in the TECS group than those in the control group: 45.3% (67/148) versus 32.1% (51/159) (P=0.018) and 29.1% (43/148) versus 17.6% (28/159) (P=0.018), respectively. The TECS group produced more high-grade blastocysts than the control group. Embryo movement or mechanical stimulation during embryo culture may be beneficial for human embryonic development.

A microfluidic device to reduce treatment time of intracytoplasmic sperm injection.

OBJECTIVE: To develop a microfluidic device that can reduce the intracytoplasmic sperm injection (ICSI) treatment time by increasing sperm concentration. DESIGN: We compared the ICSI treatment time required for porcine sperm using a method employing the microfluidic device and one using the conventional microdroplet method. SETTINGS: Academic research laboratories at Okayama University. ANIMAL(S): Reproductive cells of porcine sperm, oocytes, and embryos. INTERVENTION(S): Cell manipulations, ICSI, and embryo culture. MAIN OUTCOME MEASURE(S): Average ICSI treatment time and sperm concentration. RESULT(S): The average ICSI treatment time (mean ± SEM) using the method with the microfluidic device for poor-quality semen (sperm concentration, 2.0 × 10(4) cells/mL) was significantly shorter than the treatment time using the conventional microdroplet method (265 ± 15 seconds [n = 43] vs. 347 ± 19 seconds [n = 50]). When diluted semen with a sperm concentration of 2.0 × 10(5) cells/mL was used, no significant difference was observed between the two methods (n = 50 and n = 48). CONCLUSION(S): The microfluidic device can reduce the time required for ICSI treatment that is used to increase sperm concentration in poor-quality semen samples. The results suggest that this device may be clinically useful for ICSI treatment in human assisted reproductive technology.

Scanning and non-scanning surface plasmon microscopy to observe cell adhesion sites.

We observe adhesion sites of a cell on a substrate with high resolution. Since this observation requires interfacial measurements between the cell and the substrate, we employ scanning localized surface plasmon microscopy. We experimentally show that focal adhesion sites of a mouse muscle cell can be observed without fluorescent labeling. We also show that a non-scanning surface plasmon microscope combined with the scanning localized surface plasmon microscope contributes to observing an entire cell adhesion site and identify regions of interest.

Screening of sperm velocity by fluid mechanical characteristics of a cyclo-olefin polymer microfluidic sperm-sorting device.

The microfluidic sperm-sorting (MFSS) device is a promising advancement for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection. However, these disposable devices were not clinically suitable for assisted reproduction, so a cyclo-olefin polymer MFSS (COP-MFSS) device was developed. By micromachining, two microfluidic channels with different heights and widths (chip A: 0.3 × 0.5 mm; chip B: 0.1 × 0.6 mm) were prepared. Sorted sperm concentrations were similar in both microfluidic channels. Linear-velocity distribution using the microfluidic channel of chip B was higher than that of chip A. Using confocal fluorescence microscopy, it was found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than in the middle because of the low fluid velocity. These results experimentally demonstrated that the width of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity.

Hydrophobic silicone elastomer chamber for recording trajectories of motile porcine sperms without adsorption.

Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P<0.05) and ALHD (P<0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels.

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration.

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

In-vitro culture with a tilting device in chemically defined media during meiotic maturation and early development improves the quality of blastocysts derived from in-vitro matured and fertilized porcine oocytes.

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.

Improved development of mouse and human embryos using a tilting embryo culture system.

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P<0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P<0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube.