Pumps for microfluidic cell culture.

In comparison to traditional in vitro cell culture in Petri dishes or well plates, cell culture in microfluidic-based devices enables better control over chemical and physical environments, higher levels of experimental automation, and a reduction in experimental materials. Over the past decade, the advantages associated with cell culturing in microfluidic-based platforms have garnered significant interest and have led to a plethora of studies for high throughput cell assays, organs-on-a-chip applications, temporal signaling studies, and cell sorting. A clear concern for performing cell culture in microfluidic-based devices is deciding on a technique to deliver and pump media to cells that are encased in a microfluidic device. In this review, we summarize recent advances in pumping techniques for microfluidic cell culture and discuss their advantages and possible drawbacks. The ultimate goal of our review is to distill the large body of information available related to pumps for microfluidic cell culture in an effort to assist current and potential users of microfluidic-based devices for advanced in vitro cellular studies.

Productive chemical interaction between a bacterial microcolony couple is enhanced by periodic relocation.

This paper describes a system to study how small physical perturbations can affect bacterial community behavior in unexpected ways through modulation of diffusion and convective transport of chemical communication molecules and resources. A culture environment that mimics the chemically open characteristic of natural bacterial habitats but with user-defined spatiotemporal control of bacteria microcolonies is realized through use of an aqueous two phase system (ATPS). The ATPS is formulated with nontoxic dextran (DEX) and poly(ethylene glycol) (PEG) dissolved in cell culture media. DEX-phase droplets formed within a bulk PEG-phase stably confine the bacteria within it while small molecules diffuse relatively freely. Bacteria-containing DEX droplets can also be magnetically relocated, without loss of its bacterial content, when DEX-conjugated magnetic particles are included. We found that decreasing the distance between quorum-sensing (QS)-coupled microcolonies increased green fluorescent protein (GFP) expression due to increased inter-colony chemical communication but with upper limits. Periodic relocation of the chemical signal receiver colony, however, increased GFP expression beyond these typical bounds predicted by quorum sensing concepts alone by maintaining inter-colony chemical communication while also relieving the colony of short-range resource depletion effects. Computer simulations suggest that such increased productive output in response to periodic nonlethal physical perturbations is a common feature of chemically activated interactive cell systems where there is also a short-range inhibitory effect. In addition to providing insights on the effect of bacteria relocation, the magnetic ATPS droplet manipulation capability should be broadly useful for bioanalyses applications where selective partitioning at the microscale in fully aqueous conditions is needed.

Anti-Melanogenic Effects of Fractioned Cynanchum atratum by Regulation of cAMP/MITF Pathway in a UVB-Stimulated Mice Model.

Based on traditional pharmacological applications and partial in vitro data, Cynanchum atratum (CA) is proposed to act on skin whitening. However, its functional evaluation and underlying mechanisms have yet to be identified. This study aimed to examine the anti-melanogenesis activity of CA fraction B (CAFB) on UVB-induced skin hyperpigmentation. Forty C57BL/6j mice were exposed to UVB (100 mJ/cm2, five times/week) for eight weeks. After irradiation, CAFB was applied to the left ear once a day for 8 weeks (the right ear served as an internal control). The results showed that CAFB significantly reduced melanin production in the ear skin, as indicated by the gray value and Mexameter melanin index. In addition, CAFB treatment notably decreased melanin production in α-MSH-stimulated B16F10 melanocytes, along with a significant reduction in tyrosinase activity. Cellular cAMP (cyclic adenosine monophosphate), MITF (microphthalmia-associated transcription factor), and tyrosinase-related protein 1 (TRP1) were also noticeably downregulated by CAFB. In conclusion, CAFB is a promising ingredient for treating skin disorders caused by the overproduction of melanin and its underlying mechanisms involving the modulation of tyrosinase, mainly mediated by the regulation of the cAMP cascade and MITF pathway.

Thermal processing as a means to prepare durable, submicron thickness ionomer films for study by transmission infrared spectroscopy.

A high temperature solution processing method was adapted to prepare durable, freestanding, submicrometer thickness films for transmission infrared spectroscopy studies of ionomer membrane. The materials retain structural integrity following cleaning and ion-exchange steps in boiling solutions, similar to a commercial fuel cell membrane. Unlike commercial membrane, which typically has thicknesses of >25 µm, the structural properties of the submicrometer thickness materials can be probed in mid-infrared spectral measurements with the use of transmission sampling. Relative to the infrared attenuated total reflection (ATR) technique, transmission measurements can sample ionomer membrane materials more uniformly and suffer less distortion from optical effects. Spectra are reported for thermally processed Nafion and related perfluoroalkyl ionomer materials containing phosphonate and phosphinate moieties substituted for the sulfonate end group on the side chain. Band assignments for complex or unexpected features are aided by density functional theory (DFT) calculations.

Aqueous two-phase system-derived biofilms for bacterial interaction studies.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a ß-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Electroosmotic pumps for microflow analysis.

With rapid development in microflow analysis, electroosmotic pumps are receiving increasing attention. Compared to other micropumps, electroosmotic pumps have several unique features. For example, they are bi-directional, can generate constant and pulse-free flows with flow rates well suited to microanalytical systems, and can be readily integrated with lab-on-chip devices. The magnitude and the direction of flow of an electroosmotic pump can be changed instantly. In addition, electroosmotic pumps have no moving parts. In this article, we discuss common features, introduce fabrication technologies and highlight applications of electroosmotic pumps.

Infrared spectroscopy of bis[(perfluoroalkyl)sulfonyl] imide ionomer membrane materials.

Structural properties of the proton-exchanged forms of bis[(perfluoroalkyl)sulfonyl] imide (PFSI) ionomer materials were investigated. The hydration and dehydration of samples prepared as thin films and freestanding membrane were probed by applying transmission infrared spectroscopy. Spectral bands were assigned and effects of water incorporation into membrane pores and channels were understood by drawing upon results from related measurements performed on the structurally similar, perfluorosulfonic acid ionomer, Nafion. Both PFSI and Nafion membrane materials display a prominent infrared absorbance band near 1060 cm(-1) that arises from a vibrational mode of the ionizable group present on the side chains that extend from the poly(tetrafluoroethylene) backbone on the polymers. The mode can be traced to symmetric stretching of the -SO(3)(-) (sulfonate) group in Nafion and to antisymmetric S-N-S stretching within the sulfonyl imide end group (-SO(2)(N(-))SO(2)CF(3)) in the PFSI materials. For Nafion samples, the position and width of the band near 1060 cm(-1) are strongly sensitive to membrane hydration, whereas the band position and shape change only slightly during hydration and dehydration of PFSI materials. The possibility for greater charge delocalization over the sulfonyl imide moiety and shielding of hydrophilic species by the terminal -CF(3) group are suggested to explain the differences. These effects also likely influence the stretching modes of the side chain C-O-C groups. A pair of bands, sensitive to hydration and traceable to different C-O-C groups in a side chain, is present in the 970-990 cm(-1) region of Nafion. However, the two features are not well resolved and are less sensitive to hydration in spectra of PFSI samples. The most intense ionomer spectral bands arise from modes involving C-F stretching motion and appear between 1150 and 1250 cm(-1). Toward the high energy side of the envelope, there is substantial overlap with features of sulfonate group antisymmetric SO stretching modes in Nafion, but SO stretching modes of the sulfonyl imide moiety are higher in energy and better resolved in spectra of the PFSIs. During water uptake from a dry state into PFSI materials, a progression of features characteristic of solvated H(3)O(+) species appears across the water O-H stretching (2800-3800 cm(-1)) and H-O-H bending (1500-2000 cm(-1)) regions, similar to responses observed for water inside proton-exchanged Nafion.

Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field.

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a approximately 50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.

CGplus, a standardized herbal composition ameliorates non-alcoholic steatohepatitis in a tunicamycin-induced mouse model.

BACKGROUND: The prevalence of Non-alcoholic fatty liver disease (NAFLD) including non-alcoholic steatohepatitis (NASH) has increased by 15-39% worldwide, but no pharmaceutical therapeutics exists. HYPOTHESIS/PURPOSE: This study investigated anti-hepatosteatotic effect of CGplus (a standardized herbal composition of Artemisia iwayomogi, Amomum xanthioides, and Salvia miltiorrhiza) and its underlying mechanisms in a tunicamycin-induced NASH model. METHODS: C57/BL6J male mice were orally administrated CGplus (50, 100, or 200 mg/kg), dimethyl dimethoxy biphenyl dicarboxylate (DDB, 50 mg/kg) or distilled water daily for 5 days. 18 h after a single injection of tunicamycin (ip, 2 mg/kg), the parameters for hepatic steatosis and inflammation were measured. RESULTS: Pretreatment with CGplus significantly attenuated the accumulation of triglycerides and total cholesterol as well as lipid peroxidation, evidenced by quantitative and histopathological analyses in liver tissues. The elevations of serum aspartate transaminase, alanine transaminase and lactate dehydrogenase were significantly ameliorated by CGplus. Also, it normalized the altered activities of pro- (TNF-α, IL-1ß and IL-6), anti-inflammatory (IL-10) cytokines and lipid metabolism-related molecules in protein and gene expression analyses. CONCLUSION: Our data present experimental evidence for the potential of CGplus as an herbal therapeutic against NAFLD and NASH. Its underlying mechanisms may involve the modulations of pro- and anti-inflammatory cytokines, but further study is required especially for the actions of CGplus on lipid metabolisms.

Demonstration of transgressive overyielding of algal mixed cultures in microdroplets.

Algae are ubiquitous in natural ecosystems and have been studied extensively for biofuel production due to their unique metabolic capabilities. Most studies to date have approached biofuel optimization through synthetic biology and process engineering with few industrial scale projects considering algal community interactions. Such interactions can potentially lead to increased productivity and reduced community invasability, both important characteristics for scalable algal biofuel production. It is estimated that over a million species of algae exist such that elucidating the interactions that might be beneficial for biofuel production remains extremely resource and time intensive. Here we describe a strategy for rapid, high-throughput screening of algal community combinations using a microfluidic platform to generate millions of parallel, nanoliter-scale algal mixed cultures for estimation of biomass accumulation. Model communities were first studied in a bench scale flask experiment and then examined using microfluidic droplets. These experiments showed consistent results for both positively interacting algal bicultures that increase biomass when together, and negatively interacting bicultures that decrease biomass. Specifically, these included enhanced performance of two bicultures, Ankistrodesmus falcatus and Chlorella sorokiniana, Chlorella sorokiniana and Selenastrum minutum, and reduced performance of a biculture consisting of Selenastrum capricornutum and Scenedesmus ecornis. While the ultimate techno-economic feasibility of algal bioproducts hinges on an amalgamation of scientific fields, rapid screening of algal communities will prove imperative for efficiently discovering community interactions.

Antiemetic and Myeloprotective Effects of Rhus verniciflua Stoke in a Cisplatin-Induced Rat Model.

Rhus verniciflua Stoke has been commonly used in traditional medicine to treat gastrointestinal (GI) dysfunction diseases. In order to investigate pharmacological properties of Rhus verniciflua Stoke water extract (RVX) on cisplatin-induced amnesia, RVX (0, 25, 50, or 100 mg/kg) was orally administrated for five consecutive days after a single intraperitoneal injection of cisplatin (6 mg/kg) to SD rat. Cisplatin injection significantly increased the kaolin intake (emesis) but reduced the normal diet intake (anorexia) whereas the RVX treatment significantly improved these abnormal diet behaviors at both the acute and delayed phase. The serotonin concentration and the related gene expressions (5-HT3 receptors and SERT) in small intestine tissue were abnormally altered by cisplatin injection, which were significantly attenuated by the RVX treatment. Histological findings of gastrointestinal tracts, as well as the proteins level of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß), revealed the beneficial effect of RVX on cisplatin-induced gastrointestinal inflammation. In addition, RVX significantly improved cisplatin-induced myelosuppression, as evidenced by the observation of leukopenia and by histological examinations in bone marrow. Our findings collectively indicated Rhus verniciflua Stoke improved the resistance of rats to chemotherapy-related adverse effects in the gastrointestinal track and bone marrow.

A quantitative model to evaluate the ion-enrichment and ion-depletion effect at microchannel-nanochannel junctions.

In a nanometer-scale fluidic channel (nanochannel), coions are depleted while counterions are concentrated due to the electric double layer (EDL) overlap. When an electric field is applied across the nanochannel, ions are enriched at one end and depleted at the other end of the nanochannel. This phenomenon is termed the ion-enrichment and ion-epletion (IEID) effect. In this paper, we develop a theoretical model to evaluate this effect. The model takes into accounts not only the biased electrophoretic migrations but also the net charge transportation caused by electroosmotic flow. In addition, we consider the conductance change inside the nanochannel in assessing the electric field strength across it. We employ our recently developed protocol to measure these values. We establish a protocol to measure/quantitate the IEID effect. Finally, we compare the calculated results with the experimentally measured data and show good agreements between them.

Electroosmosis-based nanopipettor.

Decreasing the volume of reagent solutions consumed in each assay is an effective means to reduce the overall cost in high-throughput analysis laboratories. Recently, increasing attention has been paid to investigate the behavior of individual cells. If one wishes to transfer solution to or from a single cell, a picoliter pipettor is needed since the entire cell volume is commonly less than 1 nL. While pressure ejection and iontophoresis have been used to deliver picoliter volumes of solutions, these techniques cannot yield routine pipettors which perform both solution "picking up" and "dispensing" functions. The state-of-the-art pipettors can handle liquids down to approximately 100 nL, although the pipetting accuracy and precision deteriorate considerably from microliters to nanoliters. If one wishes to pipet reagents of less than 100 nL, new pipettors need to be developed. Electroosmosis has been utilized to pump solutions at flow rates of nanoliters to approximately picoliters per second, which is ideal for nanopipettors. The issue is how to arrange fluidic/electrical connections so that pipetting functions can be performed conveniently. In this paper, we present the results of our initial attempt to develop an electroosmosis-based nanopipettor. The first version of this pipettor consists of a microfabricated electroosmotic (EO) flow pump, a polyacrylamide grounding interface, and a nanoliter-to-picoliter pipet tip. The detailed configuration and fabrication process of the pipettor are discussed. An excellent feature of an EO-driven pipettor is that it has no moving parts. Good reproducibilities (RSD = 6% at 140 pL, 2% at 950 pL, and 2% at 13 nL) and accuracies (9% at 0.13 nL, 4% at 1.0 nL, and 3% at 10 nL) of this pipettor have been demonstrated to aliquot/transport nanoliter-to-picoliter solutions.

Determination of trace isoflavone phytoestrogens in biological materials by capillary electrochromatography.

Capillary electrochromatography using a specialty monolithic matrix was utilized in developing a rapid and highly efficient separation of isoflavones in biological materials. Without a preconcentration technique, it is relatively easy to reach ppm-ppb concentrations of these compounds in soy-based foods and verify them structurally using a photodiode array detector. With on-column preconcentration, we were able to measure low-ppb levels in human serum. Using blood samples from human volunteers, whose diet was supplemented by a soy-based product, the method has been validated for high-throughput screening of isoflavones in clinical studies.