Laborless, Automated Microfluidic Tandem Cell Processor for Visualizing Intracellular Molecules of Mammalian Cells.

Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency. Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ∼30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of laborless, automated microfluidic systems that integrate cell processing and analysis operations and would pave a new path to high-throughput biological experiments.

A numbering-up strategy of hydrodynamic microfluidic filters for continuous-flow high-throughput cell sorting.

Even though a number of microfluidic systems for particle/cell sorting have been proposed, facile and versatile platforms that provide sufficient sorting throughput and good operability are still under development. Here we present a simple but effective numbering-up strategy to dramatically increase the throughput of a continuous-flow particle/cell sorting scheme based on hydrodynamic filtration (HDF). A microfluidic channel equipped with multiple branches has been employed as a unit structure for size-based filtration, which realizes precise sorting without necessitating sheath flows. According to the concept of resistive circuit models, we designed and fabricated microdevices incorporating 64 or 128 closely assembled, multiplied units with a separation size of 5.0/7.0 µm. In proof-of-concept experiments, we successfully separated single micrometer-sized model particles and directly separated blood cells (erythrocytes and leukocytes) from blood samples. Additionally, we further increased the unit numbers by laminating multiple layers at a processing speed of up to 15 mL min-1. The presented numbering-up strategy would provide a valuable insight that is universally applicable to general microfluidic particle/cell sorters and may facilitate the actual use of microfluidic systems in biological studies and clinical diagnosis.

Pharmacokinetics and postoperative analgesia of epidural tramadol: A prospective, pilot study.

BACKGROUND: Tramadol, a centrally acting analgesic drug, can be administered via multiple routes and is generally well tolerated. OBJECTIVE: This study was designed to assess the pharmacokinetics of epidural tramadol administered preoperatively in Japanese patients undergoing upper abdominal surgery. METHOD: Japanese patients who were scheduled to undergo upper abdominal surgery in The Kitasato Institute Hospital, Tokyo, Japan, were included. Patients received tramadol 2 mg/kg with 5 mL of 1% mepivacaine epidurally 10 minutes before incision. The serum concentration of tramadol was determined by high-performance liquid chromatography for 21 hours after administration. Serum concentration was determined before tramadol administration and 10, 20, 30, and 60 minutes after tramadol administration, first postoperative night, and first postoperative day. Pain score and adverse events (AEs) were assessed at 1, 3, 6, 12, 18, 24, 36, and 48 hours after surgery by patient interview. RESULTS: Eleven patients were assessed for enrollment. Seven patients (6 men, 1 woman; mean [SD] age, 61.3 [12.6] years; mean [SD] weight, 59.9 [8.9] kg) provided consent and completed the study. The mean (SD) serum Cmax of tramadol was 1385.5 (390.8) ng/mL, Tmax was 0.33 (0.22) hour, and terminal elimination half-life (t1/2ß) was 10.5 (2.3) hours. Four patients complained of nausea; however, only 1 patient was administered an antiemetic. No other AEs were reported. CONCLUSION: This pilot study found that epidural tramadol administered before incision induced a Cmax within 30 minutes of administration. The drug was detected in serum at ∼21 hours after surgery.