Single-cell mechanical analysis and tension quantification via electrodeformation relaxation.

The mechanical behavior and cortical tension of single cells are analyzed using electrodeformation relaxation. Four types of cells, namely, MCF-10A, MCF-7, MDA-MB-231, and GBM, are studied, with pulse durations ranging from 0.01 to 10 s. Mechanical response in the long-pulse regime is characterized by a power-law behavior, consistent with soft glassy rheology resulting from unbinding events within the cortex network. In the subsecond short-pulse regime, a single timescale well describes the process and indicates the naive tensioned (prestressed) state of the cortex with minimal force-induced alteration. A mathematical model is employed and the simple ellipsoidal geometry allows for use of an analytical solution to extract the cortical tension. At the shortest pulse of 0.01 s, tensions for all four cell types are on the order of 10^{-2} N/m.

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency.

Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m2). The energy of the pulse application primarily dictated cell viability, with Mg2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg2+ also hinders eTE compared to K+-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

Coherent Timescales and Mechanical Structure of Multicellular Aggregates.

Multicellular aggregates are an excellent model system to explore the role of tissue biomechanics in specifying multicellular reorganization during embryonic developments and malignant invasion. Tissue-like spheroids, when subjected to a compressive force, are known to exhibit liquid-like behaviors at long timescales (hours), largely because of cell rearrangements that serve to effectively dissipate the applied stress. At short timescales (seconds to minutes), before cell rearrangement, the mechanical behavior is strikingly different. The current work uses shape relaxation to investigate the structural characteristics of aggregates and discovers two coherent timescales: one on the order of seconds, the other tens of seconds. These timescales are universal, conserved across a variety of tested species, and persist despite great differences in other properties such as tissue surface tension and adhesion. A precise mathematical theory is used to correlate the timescales with mechanical properties and reveals that aggregates have a relatively strong envelope and an unusually "soft" interior (weak bulk elastic modulus). This characteristic is peculiar, considering that both layers consist of identical units (cells), but is consistent with the fact that this structure can engender both structural integrity and the flexibility required for remodeling. In addition, tissue surface tension, elastic modulus, and viscosity are proportional to each other. Considering that these tissue-level properties intrinsically derive from cellular-level properties, the proportionalities imply precise coregulation of the latter and in particular of the tension on the cell-medium and cell-cell interfaces.

The Effect of Adding Subarachnoid Epinephrine to Hyperbaric Bupivacaine and Morphine for Repeat Cesarean Delivery: A Double-Blind Prospective Randomized Control Trial.

BACKGROUND: Spinal anesthesia has become the most common type of anesthetic for cesarean delivery. The major limitation to spinal anesthesia is that the duration of the anesthetic may not be adequate in the event of a prolonged surgery. Some practitioners add epinephrine to hyperbaric bupivacaine to increase the duration, although its effect has not been fully studied. We therefore aimed to evaluate whether adding epinephrine to the spinal medication prolongs the duration of action of the resultant block in women presenting for repeat cesarean delivery. METHODS: Sixty-eight patients were randomized to receive no epinephrine (NE group), epinephrine 100 µg (low-dose [LD] group), or epinephrine 200 µg (high-dose [HD] group) with a standardized spinal mixture (1.5 mL 0.75% hyperbaric bupivacaine with 0.25 mg morphine). Sixty-five patients were included for primary analysis. Our primary outcome was time to intraoperative activation of the epidural catheter or postoperative regression of sensory blockade to T-10 dermatome level as measured by pinprick sensation; motor recovery was a secondary outcome, and graded via a Modified Bromage scale. RESULTS: Block onset time, vital sign changes, and the incidence of hypotension; nausea, and vomiting were similar among groups. Median difference in time to T-10 regression was greatest in the HD group compared to the NE group (median difference [min] [95% confidence interval]: 40 [15-60]; P = .007), followed by the HD group to the LD group (30 [15-45]; P = .007). Comparisons of LD to NE were not significant, but trended to an increase in T-10 regression time (10 [-15 to 30]; P = .76). Median difference in time to knee extension (Bromage 3) was also greatest in the HD group when compared to both the LD and NE group (median difference [min] [95% confidence interval]: 30 [0-60]; P = .034, 60 [0-93]; P = .007). Median difference time to knee extension (min) between the LD and NE group was also significant (37.5 [15-60]; P = .001]. Pain scores during the procedure were higher in the NE group (median [interquartile range] HD: 0 [0-0], LD: 0 [0-0], NE: 0 [0-3]; P = .02) during uterine closure and were otherwise not significantly different from the other groups. CONCLUSIONS: In this single center, prospective, double-blind, randomized control trial, the addition of epinephrine 200 µg to hyperbaric bupivacaine and preservative-free morphine for repeat cesarean delivery prolonged the duration of the sensory blockade. Motor blockade was similarly prolonged and block quality may have been enhanced.

Inhibition of glioblastoma dispersal by the MEK inhibitor PD0325901.

BACKGROUND: Dispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization - processes crucial to cell adhesion, substrate attachment, and cell motility - the aim of this study was to determine whether inhibiting this pathway could also impede dispersal. METHODS: A variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods. RESULTS: We show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures. CONCLUSIONS: Inhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.

Development and validation of a microfluidic immunoassay capable of multiplexing parallel samples in microliter volumes.

Immunoassays are widely utilized due to their ability to quantify a vast assortment of biomolecules relevant to biological research and clinical diagnostics. Recently, immunoassay capabilities have been improved by the development of multiplex assays that simultaneously measure multiple analytes in a single sample. However, these assays are hindered by high costs of reagents and relatively large sample requirements. For example, in vitro screening systems currently dedicate individual wells to each time point of interest and this limitation is amplified in screening studies when the investigation of many experimental conditions is necessary; resulting in large volumes for analysis, a correspondingly high cost and a limited temporal experimental design. Microfluidics based immunoassays have been developed in order to overcome these drawbacks. Together, previous studies have demonstrated on-chip assays with either a large dynamic range, high performance sensitivity, and/or the ability to process samples in parallel on a single chip. In this report, we develop a multiplex immunoassay possessing all of these parallel characteristics using commercially available reagents, which allows the analytes of interest to be easily changed. The device presented can measure 6 proteins in 32 samples simultaneously using only 4.2 µL of sample volume. High quality standard curves are generated for all 6 analytes included in the analysis, and spiked samples are quantified throughout the working range of the assay. In addition, we demonstrate a strong correlation (R(2) = 0.8999) between in vitro supernatant measurements using our device and those obtained from a bench-top multiplex immunoassay. Finally, we describe cytokine secretion in an in vitro inflammatory hippocampus culture system, establishing proof-of-concept of the ability to use this platform as an in vitro screening tool. The low-volume, multiplexing abilities of the microdevice described in this report could be broadly applied to numerous situations where sample volumes and costs are limiting.

Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery.

This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient's inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice. Additionally, the cytokine concentration increased significantly compared to baseline values over the circulation time for all cytokines analyzed. The high plasma protein recovery (over 80%), no indication of hemolysis and low level of biofouling on the membrane surface during the experimental period (over 4 h) were all indications of effective and reliable device performance for future clinical applications. The simple and robust design and operation of these devices allow operation over a wide range of experimental flow conditions and blood hematocrit levels to allow surgeons and clinicians autonomous usage in a clinical environment to better understand the mechanisms of injury resulting from cardiac surgery, and allow early interventions in patients with excessive postoperative complications to improve surgical outcomes. Ultimately, monolithic integration of this microfiltration device with a continuous microimmunoassay would create an integrated microanalysis system for tracking inflammation biomarkers concentrations in patients for point-of-care diagnostics, reducing blood analysis times, costs and volume of blood samples required for repeated assays.

Translational research in pediatric extracorporeal life support systems and cardiopulmonary bypass procedures: 2011 update.

Over the past 6 years at Penn State Hershey, we have established the pediatric cardiovascular research center with a multidisciplinary research team with the goal to improve the outcomes for children undergoing cardiac surgery with cardiopulmonary bypass (CPB) and extracorporeal life support (ECLS). Due to the variety of commercially available pediatric CPB and ECLS devices, both in vitro and in vivo translational research have been conducted to achieve the optimal choice for our patients. By now, every component being used in our clinical settings in Penn State Hershey has been selected based on the results of our translational research. The objective of this review is to summarize our translational research in Penn State Hershey Pediatric Cardiovascular Research Center and to share the latest results with all the interested centers.

Differential immune activation during simulated cardiopulmonary bypass procedure using freshly drawn and week-old blood­a pilot study.

The goal of this study is to assess whether the use of different aged blood, used during the simulated PennState Hershey Pediatric cardiopulmonary bypass (CPB)model affects immune activation. In order to study and compare the cytokine release involved in the humoral immune response during simulated CPB, both freshly drawn whole blood used less than 1 h after donation (n = 2)and reconstituted whole blood (1 week old) (n = 3) were circulated in a simulated CPB circuit under identical perfusion conditions. Discrete samples were collected in both experiments and analyzed for the proinflammatory cytokines concentrations of tumor necrosis factor-alpha, interleukin(IL)-6, and IL-8 using immunofluorocytometry as an indicator of immune activation. The results indicated that the cytokine concentrations of freshly drawn blood increased significantly compared with the reconstituted blood over the CPB circulation time. The fresh blood activation was two to three orders of magnitude larger than the week-old blood for all cytokines analyzed. These results suggest that the use of freshly drawn blood is required to evaluate immune responses to the extracorporeal circulation.

Microfluidic devices for continuous blood plasma separation and analysis during pediatric cardiopulmonary bypass procedures.

As an extension of previous work, a microfluidic device, which can separate blood plasma in a continuous, real-time fashion from a whole blood, is successfully integrated with a mock cardiopulmonary bypass circuit. The functionality of the device is demonstrated with the use of freshly harvested bovine blood. The plasma selectivities were 100% and 99.4% and the plasma separation volume percents were 18.7% and 24.5% for 26% and 37% inlet hematocrit levels, respectively. As an advanced stage of this research, a microfluidic device, which can measure the concentration of clinically relevant blood plasma protein in a continuous fashion, is being developed on the basis of fluid handling circuits coupled to fluorescent cytometric bead assays. The functionality of the device is demonstrated with the use of a biotinylated FITC solution and a streptavidin-coated, 8-mum-diameter bead. The binding event between biotinylated FITC and the streptavidin bead is continuously detected within a detection window at the outlet of the device. For a known concentration (1 microg/ml) of biotinylated FITC solution, the measured fluorescent intensity is fairly constant and shows a stable gaussian distribution of the bead fluorescence intensity. It is expected that the proposed device can be used for continuous measurement of clinically relevant proteins during cardiac surgery with the cardiopulmonary bypass procedure.

Design of a side-view particle imaging velocimetry flow system for cell-substrate adhesion studies.

Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate tumor cell and leukocyte arrest on the endothelium and subsequent sustained adhesion. However, further investigation has been limited by the lack of experimental models that allow quantitative measurement of the hydrodynamic environment over adherent cells. The purpose of this study was to develop a system capable of acquiring quantitative flow profiles over adherent cells. By combining the techniques of side-view imaging and particle image velocimetry (PIV), an in vitro model was constructed that is capable of obtaining quantitative flow data over cells adhering to the endothelium. The velocity over an adherent leukocyte was measured and the shear rate was calculated under low and high upstream wall shear. The microcapillary channel was modeled using computational fluid dynamics (CFD) and the calculated velocity profiles over cells under the low and high shear rates were compared to experimental results. The drag force applied to each cell by the fluid was then computed. This system provides a means for future study of the forces underlying adhesion by permitting characterization of the local hydrodynamic conditions over adherent cells.

Blood plasma separation in microfluidic channels using flow rate control.

Several studies have clearly shown that cardiac surgery induces systemic inflammatory responses, particularly when cardiopulmonary bypass (CPB) is used. CPB induces complex inflammatory responses. Considerable evidence suggests that systemic inflammation causes many postoperative complications. Currently, there is no effective method to prevent this systemic inflammatory response syndrome in patients undergoing CPB. The ability to clinically intervene in inflammation, or even study the inflammatory response to CPB, is limited by the lack of timely measurements of inflammatory responses. In this study, a microfluidic device for continuous, real-time blood plasma separation, which may be integrated with downstream plasma analysis device, is introduced. This device is designed to have a whole blood inlet, a purified plasma outlet, and a concentrated blood cell outlet. The device is designed to separate plasma with up to 45% hematocrit of the inlet blood and is analyzed using computational fluid dynamics simulation. The simulation results show that 27% and 25% of plasma can be collected from the total inlet blood volume for 45% and 39% hematocrit, respectively. The device's functionality was demonstrated using defibrinated sheep blood (hematocrit=39%). During the experiment, all the blood cells traveled through the device toward the concentrated blood outlet while only the plasma flowed towards the plasma outlet without any clogging or lysis of cells. Because of its simple structure and control mechanism, this microdevice is expected to be used for highly efficient, realtime, continuous cell-free plasma separation.

Subarachnoid small-dose bupivacaine versus lidocaine for cervical cerclage.

UNLABELLED: Cervical cerclage is often performed as an outpatient procedure under subarachnoid anesthesia. Lidocaine was historically the drug of choice for short procedures but has fallen out of favor because of concerns of transient neurologic symptoms (TNS). We performed this study to determine whether small-dose bupivacaine is an acceptable alternative to lidocaine for cervical cerclage. We randomized 59 women to receive either subarachnoid isobaric lidocaine 30 mg or hyperbaric bupivacaine 5.25 mg. Fentanyl 20 micro g was added to both local anesthetics, and the total volume was diluted to 3 mL with 0.9% saline. Onset and highest dermatomal level of sensory block; quality of anesthesia; hypotension; and times until T12 regression, return of lower extremity motor function, ambulation, and micturition were recorded. Symptoms of TNS were evaluated by telephone interview 24 h after surgery. We did not find any significant difference in onset or recovery times between the groups, with the exception of a longer duration until return of lower extremity motor strength in the lidocaine group. Symptoms consistent with TNS that resolved spontaneously within 48 h were reported by two women in the lidocaine group but by none in the bupivacaine group. We conclude that subarachnoid bupivacaine offers a satisfactory alternative to subarachnoid lidocaine for cervical cerclage. IMPLICATIONS: We found that small-dose subarachnoid bupivacaine (5.25 mg) with fentanyl 20 micro g provides reliable anesthesia for cervical cerclage and exhibits a pharmacodynamic profile similar to that of small-dose lidocaine.