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.

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.

Evidence that the PsbK polypeptide is associated with the photosystem II core antenna complex CP43.

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.