Quantitative inference of cellular parameters from microfluidic cell culture systems

Microfluidic cell culture systems offer a convenient way to measure cell biophysical parameters in conditions close to the physiological environment. We demonstrate the application of a mathematical model describing the spatial distribution of nutrient and growth factor concentrations in inferring cellular oxygen uptake rates from experimental measurements. We use experimental measurements of oxygen concentrations in a poly(dimethylsiloxane) (PDMS) microreactor culturing human hepatocellular liver carcinoma cells (HepG2) to infer quantitative information on cellular oxygen uptake rates. We use a novel microchannel design to avoid the parameter correlation problem associated with simultaneous cellular uptake and diffusion of oxygen through the PDMS surface. We find that the cellular uptake of oxygen is dependent on the cell density and can be modeled using a logistic term in the Michaelis–Menten equation. Our results are significant not only for the development of novel assays to quantitatively infer cell response to stimuli, but also for the development, design, and optimization of novel in vitro systems for drug discovery and tissue engineering. Biotechnol. Bioeng. 2009;103: 966–974. © 2009 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63052/1/22334_ftp.pd

Microenvironmental Control in Microfluidic Bioreactors for Long Term Culture of Bone Marrow Cells.

The goal of this research is to create in vitro microenvironments for long term culture of hematopoeitic stem cell (HSC) in microfluidic bioreactors. In vivo, HSCs reside in the bone marrow osteoblastic and vascular niches in adult mammals. Defining features of their in vivo niche include: small number of HSCs, heterogeneous population of bone marrow cells that support HSCs, and low oxygen tension. We engineer niche elements in microfluidic bioreactors by modulating oxygen tension, optimizing attachment and growth of HSC-supporting bone marrow stromal cells, and culturing small numbers of HSCs in their physiologically relevant ratios between HSCs and supporting cells. By using a combination of a mathematical model and quantitative experiments, we have created a design tool to manipulate and control oxygen tension for cell culture inside the poly(dimethyl siloxane) (PDMS) microbioreactors. Dissolved oxygen concentrations in the microbioreactor are quantified in real time using fluorescence lifetime imaging of an oxygen sensitive dye. Experimental results are consistent with the mathematical model and give insight into operating conditions required for a desired oxygen tension in cell culture regions of the microbioreactor. We used microfluidic perfusion systems to develop nanocoatings made from electrostatic self assembly of PDDA (poly(diallyldimethyl ammonium chloride)), clay, type IV collagen and fibronectin to optimize attachment of primary murine bone marrow cells (support cells for HSCs) onto PDMS bioreactors. PDDA-topped coatings were found to be cytotoxic, while coatings with two or more bilayers of proteins collagen and fibronectin were found to optimize spreading, proliferation, and viability as compared to other surfaces. On-chip erythropoiesis was achieved with a 3-D co-culture of HSCs with supporting cells in PDMS bioreactors. In addition, an optimal ratio of support cells to HSCs was found to maximize self renewal potential of HSCs in vitro. By the combination of hypoxia (which simulates in vivo bone marrow oxygen tension), biofunctional surfaces, and 3-D co-cultures, we are moving towards a ‘microfluidic HSC niche’, in which hypothesis-driven studies about crosstalk between HSCs and stromal cells can be carried out.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60852/1/mehtagee_1.pd

A STUDY TO EVALUATE THE EFFECT OF ADDING CLONIDINE TO ROPIVACAINE FOR AXILLARY PLEXUS BLOCKADE

Aims and objectives- The present study was undertaken to evaluate the effect of adding Clonidine to Ropivacaine for axillary plexus blockade. Material and methods- A total of 60 adult patients having physical status grade I or II according to American Society of Anaesthesiologists ( ASA ) undergoing hand or forearm surgery under axillary plexus blockade using nerve stimulator were included in the study. Patients were randomly allocated to one of the two groups. Each group consisted of 30 patients. Group 1 patients received 35 ml of Ropivacaine 0.5 % + 1 ml of normal saline.Group 2 patients received 35 ml of Ropivacaine + 1 ml of clonidine ( 150 µg). Sensory block, motor block and sedation were assessed every 5 minutes for 30 minutes. Postoperatively assessment was done every 15 minutes till complete regression of sensory and motor block. Results- Mean sensory onset time in patients of group 1 was 26.48 ± 7.88 min and in patients of group 2 was 26.55 ± 8.06 min, which was insignificant statistically. Patients of group 1 had a mean motor onset time 35.51 ± 10.4 min and patients of group 2 had a mean motor onset time 37.06 ± 14.19min, the difference being statistically comparable. Mean duration of sensory block in patients of group 1 was 422 ± 163.10 min and in patients of group 2 was 438 ± 133.93 min, which was statistically comparable. Patients belonging to group 1 had a mean duration of motor block 404 ± 160.60 min and patients belonging to group 2 had a mean duration of motor block 388 ± 151.63 min, which was statistically comparable. Conclusion- Addition of Clonidine ( 150 µg) is of no benefit in the onset and duration of axillary plexus block

Ethnobotanical Survey of Wild Edible Plants of Leh District, Ladakh

Ethnobotanical exploration was undertaken to document the Wild edible plants (WEPs) utilised by the inhabitants of the Leh district. Traditionally exploited WEPs are an indispensable part of every household culinary. This place is known for its unique culture and cuisine which diversifies it from the rest of India. Pre-structured questionnaires, guided field visits, field examinations, and group discussions were conducted to gather ethnobotanical data. Detailed inquiries were made on the plant’s part used, time of collection and method of food preparation. In the present study, 40 wild consumable plants were documented, these belong to 18 families and spreads to 31 genera; out of which four are new records as wild food plants. They are Cotoneaster integerrimus, Dracocephalum heterophyllum, Astragalus frigidus, and Turritis glabra. Diverse use of wild leafy plants was observed and some of the most famous dishes are ‘Shangsho tsodma’ and ‘Kabra tsodma’ vegetables. The utilisation of wild plants helps enrich diet diversity and enhances the availability of green vegetables hence broadening food choices. Our study also reveals that the gathering of wild plants is confined to village people, shepherds, and farmers, and a majority of this knowledge exists in the memory of the elderly and these wild resources are under threat due to various anthropogenic activities. Therefore, the present finding highlights the value of these plants along with maintaining regional traditional knowledge and preserving the old ethnic traditional way of living and eating. Further, this information will provide baseline data to upcoming researchers dealing with nutrition and nutraceutical aspects. In addition, these wild plants are nutritionally rich and their consumption should be encouraged

Phytochemical Screening, In Vitro Antidiabetic and Antioxidant Activity of Rabdosia rugosa (Wall. ex Benth.) H. Hara Extract from Kinnaur District, Himachal Pradesh

Rabdosia rugosa (Wall. ex Benth.) H. Hara. (Syn. Plectranthusrugosus Wall.) belongs to the Lamiaceae family and is used by local communities of the Kinnaur district to cure diabetes. The selection of the plant material is based upon data that aerial parts of the plant (leaves along with flowers) are used in folklore to manage the postprandial hike in blood sugar levels. Therefore, in the present investigation, antiradical and antidiabetic activities of the ethanolic extract of the plant were checked. Preliminary tests for phytochemicals, total phenols, and flavenoids were also determined. Our results manifested that R. rugosa (Rabdosia rugosa) extract exhibited superior antioxidant activity (p˂0.05) by DPPH and H2O2 assays with IC50 = 9.62 ± 2.70 μg/ml and IC50 = 28.22 ± 2.94 μg/ml. This plant also exhibited preeminent repression action against the enzyme, i.e., alpha-amylase and alpha-glucosidase (p˂0.05) with IC50 = 10.49 ± 6.17 μg/ml (Porcine α – amylase), IC50 = 1.87 ± 1.79 μg/ml (Yeast α - glucosidase), ( IC50 = 16.89 ± 0. 06 μg/ml(Intestinal rat α - glucosidase) Hence, we concluded that present plant possessed excellent antiradical and antidiabetic activity. The antidiabetic activity was estimated higher than the standard Acarbose. As per our information, this may be the first time to report on the extract of R. rugosa for its antioxidant and antidiabetic capacity. The current findings divulge that this plant can further be studied to verify its therapeutic activities

Preparation and Characterization of Celecoxib Entrapped Guar Gum Nanoparticles Targeted for Oral Drug Delivery against Colon Cancer: An In-Vitro Study

The present study is an attempt to synthesize nanosized guar gum carriers encapsulating celecoxib as the chemopreventive agent for experimental colorectal cancer (CRC).  Guar gum nanoparticles without celecoxib (eGGNPs) and celecoxib loaded guar gum nanoparticles (cGGNPs) were prepared by oil-in-water emulsification and in situ polymer crosslinking method. Electron microscopy, zeta potential and fourier transform infrared spectra analysis was used to affirm the size, stability and morphology of the nanoparticles. In-vitro drug release was investigated using dialysis method. Further, the effect of nanoparticles (eGGNPs & cGGNPs) was evaluated on Caco 2 colon cancer cell lines. Spherical guar gum nanoparticles were obtained in the size range of 200±6nm with zeta potential of -32.1mV indicating good stability of the GGNPs with drug loading of 30±3.2%, and drug release following zero order kinetics. The eGGNPs had no effect on Caco2 cell viability whereas the cGGNPs showed time and concentration dependent growth inhibition of Caco 2 cells. These findings suggest the successful preparation of chemopreventive nanoparticles that can be targeted as the prophylactic agent for experimental colorectal cancer. Keywords: nanoparticles, guar gum, celecoxib, colorectal cancer, release kinetics, cytotoxicit

Acute myocardial infarction in labor: a rare case report

Myocardial infarction (MI) presenting first time in labor is rare with incidence of 3 case per 1,00,000 and its management during labor is rarely seen in literature. In this case, a 29-year-old gravida 2 para 1 with previous caesarean section 1 year back with history of postpartum eclampsia presented with acute breathlessness and lower abdominal pain. She was diagnosed with acute MI. Patient had emergency caesarean section due to scar tenderness. Post operatively patient was managed in ICU with multi-disciplinary team of critical care, cardiologist and obstetrician.

In Vivo Antidiabetic Activity and GCMS Analysis of Ethanolic Extracts of Rabdosia Rugosa (Wal ex Benth) H Hara

The current study examined the effects of an ethanolic extract of R. rugosa (Rabdosia rugosa (Wall. ex Benth.) H. Hara) on alloxan-induced diabetes rats, detailing its hypoglycemic potential and using gas chromatography-mass spectrometry to analyze its phytoconstituents. The FTIR spectrum verified the presence of various functional groups in the active ingredients. This research opted to use an extract from fresh leaves and flowers. Chichiri is the local name for the shrub R. rugosa, which belongs to the sage family (Labiatae) and is used as traditional medicine. To determine the hypoglycemic activity of plant extracts the rats were randomly assigned to one of four treatment groups for two weeks of experiments: a normal control group that received no treatment, a diabetic control group that received only alloxan (150 mg/kg BW), a drug control group that received glibenclamide (5mg/kg BW), and a treatment group that received R. rugosa extract (50 mg/kg BW). Our results demonstrated that the extract and medication group saw statistically significant improvement (p≤0.001) in body weight, blood glucose levels, lipid profile, liver and renal parameters. The GCMS analysis showed that numerous active phytoconstituents were present. Phenols, alkanes, alcohols, and other compounds were detected in the FTIR spectrum. After examining the data, we determined that the leaves and inflorescences of R. rugosa have hypoglycemic potential. Continued study of the naturally separated chemicals can aid in the creation of organic medications for diabetic treatment

GCMS Analysis In Vitro Antioxidant and Antidiabetic Activity of Oxyria digyna (L.) Hill

Oxyria digyna (mountain sorrel) is an edible and medicinal herb with a wide array of ethnopharmacological uses. A literature review revealed that this species is less explored for its pharmacological efficacy. Methanolic leaf extract of O. digyna was screened for antioxidant potential utilizing “2,2-diphenyl-1-picrylhydrazyl” DPPH and “Hydrogen peroxide” H202 assay, in addition, the antidiabetic potential was evaluated using enzyme alpha-amylase. Further, the bioactive compounds were analyzed through GC/MS and FTIR techniques. The antioxidant results demonstrated that the extract showed strong reducing potential for both DPPH and Hydrogen peroxide assay, plant extract revealed scavenging activity with an IC50 value of 42.55±0.7311 µg/ml for DPPH and an IC50 value of 51.77±1.855 µg/ml for H2O2. Furthermore, O. digyna showed a moderate inhibitory outcome towards alpha-amylase with an IC50 =131.02±1.90 µg/ml while the standard showed an IC50 = of 22.05±3.9 µg/ml. The extract exhibited an enormous amount of total flavonoid and phenolic. Moreover, the FTIR spectrum showed the presence of alcohol, alkanes, alkyne, aldehyde, etc. and the GC/MS study reveals the presence of sixty compounds. The most prevalent one is 9,12,15- Octadecatrienoic acid (Z, Z, Z) 38.33 %. According to our knowledge, this study is the first to validate its antidiabetic potential and identification of numerous phytoconstituents through GC/MS and identification of several functional groups employing FTIR analysis. The above finding suggests that O. digyna possess a high amount of phenols, and flavonoids, showing significant antioxidant properties, which makes it a promising source of natural antioxidant, also it can be used in food industries and for future drug synthesis. Further, the extract showed potential alpha-amylase inhibition but the potential was less, further, the active biochemical constituent can be isolated and utilized in therapeutic applications

Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments

Abstract Background Innate immune cells such as macrophages are abundantly present within malignant ascites, where they share the microenvironment with ovarian cancer stem cells (CSC). Methods To mimic this malignant ascites microenvironment, we created a hanging-drop hetero-spheroid model to bring CSCs and macrophages in close association. Within these hetero-spheroids, CD68+ macrophages (derived from U937 or peripheral blood monocytes) make up ~ 20% of the population, while the rest are ovarian cancer cells and ovarian cancer stem cells (derived from the high grade serous ovarian cancer cell line, OVCAR3). Results Our results indicate that CSCs drive the upregulation of M2 macrophage marker CD206 within hetero-spheroids, compared to bulk ovarian cancer cells, implying an inherently more immuno-suppressive program. Moreover, an increased maintenance of elevated aldehyde dehydrogenase (ALDH) activity is noted within hetero-spheroids that include pre-polarized CD206+ M2 macrophages, implying a reciprocal interaction that drives pro-tumoral activation as well as CSC self-renewal. Consistent with enriched CSCs, we also observe increased levels of pro-tumoral IL-10 and IL-6 cytokines in the CSC/M2-macrophage hetero-spheroids. CSC/M2-macrophage hetero-spheroids are also less sensitive to the chemotherapeutic agent carboplatin and are subsequently more invasive in transwell assays. Using inhibitors of WNT secretion in both CSCs and macrophages, we found that CSC-derived WNT ligands drove CD206+ M2 macrophage activation, and that, conversely, macrophage-derived WNT ligands enriched ALDH+ cells within the CSC compartment of hetero-spheroids. Upon examination of specific WNT ligand expression within the monocyte-derived macrophage system, we observed a significant elevation in gene expression for WNT5B. In CSCs co-cultured with macrophages within hetero-spheroids, increases in several WNT ligands were observed, and this increase was significantly inhibited when WNT5B was knocked down in macrophages. Conclusions Our data implies that macrophage- initiated WNT signaling could play a significant role in the maintenance of stemness, and the resulting phenotypes of chemoresistance and invasiveness. Our results indicate paracrine WNT activation during CSC/M2 macrophages interaction constitutes a positive feedback loop that likely contributes to the more aggressive phenotype, which makes the WNT pathway a potential target to reduce the CSC and M2 macrophage compartments in the tumor microenvironment.https://deepblue.lib.umich.edu/bitstream/2027.42/152174/1/40425_2019_Article_666.pd