Bewo cell culture protocol pdf

Department of pharmaceutical chemistry, school of pharmacy, the university of kansas, lawrence kansas 66047. Viral isolation protocol for cell culture introduction the optimal method for determining specific etiology of an arbovirus infection requires isolation of the virus from a specimen obtained from the patient during the acute stage of the disease and. At 24, 48, 72, and 96 h after seeding, the culture media were removed. Cells in midlog growth can generally be rendered more competent to uptake plasmid dna than can cells at stationary phase. We examined the effects of leptin and progesterone on the invasion of bewo cells, a human trophoblastic cell line, and the effect of concurrent treatment. These cell lines are more resilient when compared to cultures of primary trophoblast cells, including extended lifespans in culture, and are thus advantageous for studies encompassing more detailed investigations of trophoblast function and molecular biology. In brief, cell culture media containing dulbeccos modi. Cell biologycell culture protocols protocol online your. Some vessels were frozen immediately 0 h and others were cultured and frozen after 24, 48, 72 or 96 h umbilical.

Hplc method development for quantification of doxorubicin in. Permeabilization if the target protein is intracellular, it is very important to permeabilize the cells. Introduction cellcell interactions are key to man y biological processes. Add required amount of prewarmed cell culture media to fresh flask. Mammalian cell tissue culture techniques protocol abcam. Maintaining healthy cells is the key to experimental success and reproducible research results. Add appropriate aliquots of the cell suspension to new culture vessels. Forskolin treatment, which is known to induce differentiation and decrease cell proliferation, significantly decreased bewo cell numbers at 48 hours by %. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. Crisprcas9directed genome editing of cultured cells. Bewo induced smc apoptosis in an ex vivo vessel model.

Serumfree media for cell culture compiled by focus on alternatives this document provides an overview of the range of commercially available serumfree media for cell culture, and has been compiled by focus on alternatives foa, a group of british organisations working together to advance the replacement of animal experiments. Bewo exosomes carrying placentaassociated mirnas e. The bewo trophoblastic cell line was the first human endocrine cell type to be maintained in. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e.

Secure a 6cm plate of hek293t cells from the stock incubator. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Immunofluorescence with monoclonal antibodies demonstrates that bewo ecm contains laminin subunits beta1 and gamma1. The presence of inducible cytochrome p450 types 1a1 and 1a2. Bewo exosomal mirnas were then transferred into jurkat cells. Variations in the level of glucose contained in the culture medium exert profound changes on cell growth and glycogen content. Protocols for the revival, maintenance, passage, and growth of bewo cells for.

Trophoblast cell lines have become an invaluable tool to study various aspects of trophoblast biology. This application protocol describes how to handle icell neurons for use on. Bewo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The culture collections represent deposits of cultures from worldwide sources. Coculture of jeg3, bewo and syncbewo cell lines with adrenal h295r cell line. Cell culture guidelines the following is a general guideline for culturing of cell lines. The use of bewo cells as an in vitro model for placental.

Cell culture basics introduction to cell culture, covering topics such as laboratory setup, safety and aseptic technique including basic cell culture protocols and video training. Proteomic profiling of forskolininduced differentiated bewo. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Cnn3 regulates trophoblast invasion and is upregulated by. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Immunocytochemistry and immunofluorescence protocol. To define the signal transduction pathway utilized by heme to induce apoptosis, cell.

To confirm the microarray data nine differentially expressed transcripts representing a range of cellular function were selected for secondary analysis. In vitro models for studying trophoblast transcellular transport. Adiponectin promotes syncytialisation of bewo cell line and primary trophoblast cells. Regulation of adrp expression by longchain polyunsaturated fatty. Mouse embryonic stem cell culturing protocols 4 of 6 form 105 rev b072214 7. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to fo. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. When using accutase, cells can be plated directly into culture vessel without centrifuging to pellet cells as long as accutase is inactivated by the addition of the. They have proven to be extremely valuable for the study of the cellular, molecular, and endocrine aspects of human trophoblasts. Cannabidiol changes pgp and bcrp expression in trophoblast. A dynamic model for studies of human trophoblast function. Glucose uptake by bewo cells was carried out as described 31 with a few adaptations. The presence of inducible cytochrome p450 types 1a1 and 1a2 in the bewo cell line.

A scheme of the in vitro transfer system using bewo cells as donors and jurkat cells as recipients. After counting the cells, seed them in the desired collagen coated cell culture plates with mhp see table 3. Adiponectin promotes syncytialisation of bewo cell line and. The original bewo cell line is also available through american type culture. Importantly, bewo cell lines have fusogenic properties and, in culture with.

Remove the prewarmed mnc culture medium and laminincoated plates from the incubator during centrifugation. Detailed instructions for carrying out transplacental transport experiments with bewo cells in transwell inserts appear in the protocols that follow. The proper procedure for genome editing on tissuecultured hipscs is described in this protocol. Hplc method development for quantification of doxorubicin. Transport of large neutral amino acids into bewo cells.

Cell culture basics handbook thermo fisher scientific. Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. Pdf plant cell culture protocols methods in molecular biology full online. Pdf coculture of jeg3, bewo and syncbewo cell lines with. A human hormonesynthesizing trophoblastic cell system has been established in vitro and. The protocol begins with an overnight suspension culture. Culture and transfection of human choriocarcinoma cells. The roller tube method of culture 3 was used with constant. A human hormonesynthesizing trophoblastic cell system has. Cell culture on microelectrode arrays axion biosystems. The human trophoblast cell line, bewo displays morphological and biochemical markers common to a cytotrophoblast, and elevation of intracellular camp concentrations by addition of forskolin, resulting in morphological and biochemical changes of cytotrophoblastlike bewo cells into syncytiotrophoblastlike cells wice et al.

Relevance of the nr4a subfamily of nuclear orphan receptors. Fusion of the trophoblastderived choriocarcinoma cell line bewo can be triggered by forskolin. Protocol human ips cell culture under feederfree conditions. Bewo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Pdf plant cell culture protocols methods in molecular. Placental cytotrophoblast cells were isolated by the method of kliman 30.

Exosomes were collected from culture supernatants of bewo cells. Human choriocarcinoma the first human, trophoblastic endocrine cell type to be maintained in continuous culture. Unidirectional influx of lphenylalanine lphe and lleucine lleu across the microvillous border of the cells was studied using a rapid pairedtracer dilution technique. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. The cell line was submitted to the american type culture collection in the 187th passage. Cells that are difficult to detach may be placed at 37c to facilitate dispersal. However, a considerable amount of cell death occurred after forskolin or hcg treatment as compared to the control sirnatransfected cells. Pdf coculture of jeg3, bewo and syncbewo cell lines.

The alamarblue stock solution or cell counting kit8 cck8 reagent in amounts equal to 10% of the incubation volume was added, according to the manufacturers protocol. To help illustrate differences in the bewo and jeg3 cell lines, a small number of differentially expressed transcripts that are involved in various biological functions were selected from the complete lists and are shown in table 2, table 3. The use of bewo cells as an in vitro model for placental iron. Co culture of jeg3, bewo and syncbewo cell lines with adrenal h295r cell line. Grow cells on glass coverslips or prepare cytospin or smear preparation. Once matrigel matrix is thawed, swirl vial to ensure the material is dispersed. The mean cell viability of bewo and njg cells declined to about 58 and 63% compared to controls after 72 h of culture in the presence of antifas mab ch11 while the fassensitive jurkat cells showed viability of only 10%. Superfused microcarrier cultures of bewo choriocarcinoma cells. A glucose concentration of approximately 300340 mg percent has been found to be optimal for the cells. Oct 29, 2010 bewo cell culture the human choriocarcinoma cell line bewo was obtained from american type collection of cell manassas, va, usa. Wash cells briefly with hanks solution prior trypsin treatment note. Resistance to fasmediated cell death in bewo and njg. Bewo atcc ccl98 homo sapiens placenta choriocarcinoma.

To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell health. Outlined procedures include cell revival, maintenance of the bewo cells in culture, freezing cells, plate preparation, seeding the cells, and transport protocols. Rottlerinmediated inhibition of toxoplasma gondii growth. Centrifuge conical tube containing cells at 1100 rpm for 2 minutes at room temperature. Visit our website for a detailed protocol on using a hemocytometer to obtain a viable cell count 6. Corning matrigel basement membrane matrix for 3d culture. The new eplate insert enables investigation of spe cific cellcell interactions in real time, while maintaining the cells in separate com partments. Bewo cells stimulate smooth muscle cell apoptosis and. Trophoblast cell fusion and differentiation are mediated by. Live cell imaging of in vitro human trophoblast syncytialization.

Wolf, university of maryland, baltimore county general guidelines to cell and tissue culture techniques, including. The presence of inducible cytochrome p450 types 1a1 and. Methods and protocols methods in molecular biology pdf. This chapter describes basic methods for culture and maintenance of the jeg3, jar, and bewo human choriocarcinoma cell lines as well as an effective paradigm for introducing dna into the cells. The effect of rottlerin on parasite growth was evaluated in bewo cells infected for 3 hours. Fusion of the trophoblastderived choriocarcinoma cell line bewo can be. Cell culture medium for the analytical method validation of dxr was prepared as per the protocol in which bewo b30 cells are typically grown 7. These results suggest that the nr4a subfamily of nuclear orphan receptors has a ro le in trophoblastic cell differentiation. Lower pr expression noted in co culture of syncbewo cell could be explained by lower p4 secretion corresponding with high levels of e2 secretion and er expression. Trophoblast cell fusion and differentiation are mediated. To subculture the cells they need to be brought into suspension.

For commercially available cell lines we recommend. Bewo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into columns and superfused. Part c provides a protocol for preparing a midlog culture of e. We selected the 120 split from a few days previous. Bewo cells stimulate smooth muscle cell apoptosis and elastin. Oct 31, 2016 heme induces apoptosis in bewo cells through activation of stat3caspase3parp and p73 signaling pathways. We have generated lines of bewo cells that constitutively and stably express either histone h2b tagged with the green fluorescent protein gfp, or the mitochondrial targeting sequence of subunit viii of cytochrome c oxidase fused with a red fluorescent protein. While every effort is made to ensure details distributed by culture collections are accurate, culture collections cannot be held responsible for any inaccuracies in the data supplied. Spiral arteries or umbilical arteries were denuded of endothelium, perfused with control culture medium, bewo cells or bewo conditioned medium 50% vv and tied off. Midlog cells are used in the classic transformation protocol described in laboratory 10. The very first method, and one of the most important, used to study. Protocol for hela cell culture and plasmid transfection av.

Optimization of bewo model to investigate placental responses. Bewo and jeg3 choriocarcinoma cells were purchased from the european collection of cell cultures ecacc, salisbury, uk and were maintained in 75 cm 2 falcon culture flasks bd biosciences, erembodegem, belgium under standard culture conditions of 5% co 2 in air containing 20% o 2 at 37c with medium renewal every 23 days. Some vessels were frozen immediately 0 h and others were cultured and frozen after. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. Cells were maintained at 37c under 5% co 2, 95% air atmosphere in phenolred free dmemf12 medium with 15% fcs, streptomycin 10. Cell culture protocols thermo fisher scientific kr. The effect of rottlerin on parasite growth was evaluated in bewo cells infected for 3 hours and then treated with the compound for 24 and 48 hours. Jun 01, 1998 bewo is a choriocarcinoma cell line that generates an extracellular matrix ecm rich in laminin and is a useful model for human trophoblast. Note that cell culture conditions vary for each cell type.

Stain the cells with trypan blue and count the number of living cells with the countess. Bewo cells are regularly used as a cell culture model to mimic in vivo syncytialisation of placental. A threedimensional culture system recapitulates placental. Further, the expression patterns of dysf in bewo cells, both before and after cell fusion, recapitulate the in vivo expression in the ctb and stb respectively, reiterating the usefulness of bewo culture model as a surrogate system for studying trophoblast differentiation. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. On day 0, dilute the matrigel matrix to 5 mgml with icecold mdck complete cell culture medium. Dislodge cells from the flask substrate with a cell scraper. Microarray analysis of bewo and jeg3 trophoblast cell. Amaxa 4dnucleofector optimization protocol for cell lines. The cells secrete placental hormones including chorionic gonadotrophin hcg, polypeptide hormones, lactogen, estrogenic and progestational steroids, estrone, estradiol, estriol and progesterone. Pdf monocultures of different placental cells are used for many physiological and. Quantifying the syncytialisation of human placental. Coculture of jeg3, bewo and syncbewo cell lines with. Regulates trophoblast differentiation in the bewo cell.

Coculture of jeg3, bewo and syncbewo cell lines with adrenal. When subculturing cells 6075% confluency, discard old media from flask. In vitro models for studying trophoblast transcellular. Effect of human telomerase reverse transcriptase transfection.

Human exosomal placentaassociated mir517a3p modulates. The profile of pr protein expression, in co culture of jeg3 and bewo, increased over time of cell culture, while there was no change in syncbewo cells. The rwvbioreactor is a rotating cylindrical bioreactor th at attaches to slowturning lateral vessels stlvs, which are completely filled with cell culture medium and contain cells attached to porous, extracellular matrixcoated dextran beads or other scaffolds 9. Trophoblast cell line an overview sciencedirect topics. The effect of htert transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cellcell fusion was delayed and differentiation did not occur in hterttransfected cells.

Progesterone inhibits leptininduced invasiveness of bewo cells. Pdf adiponectin promotes syncytialisation of bewo cell line. Resuspend the cell pellet in r10 and count viable cells using a hemocytometer and trypan blue. Cell culture plates of your choice for detaching adherent cells. Rottlerinmediated inhibition of toxoplasma gondii growth in. Product information bewo cells cls cell lines service gmbh. All culture incubations should be performed in a humidi. Bacteria can be isolated from nearly any surface including inanimate objects and human skin. Hememediated apoptosis and fusion damage in bewo trophoblast. Differential effects of inducers of syncytialization and. Cell seeding density of 5 104 cellswell in 24well plates was determined after testing various densities for sensitivity to glucose uptake data not shown. Culture of hek 293293t cells charles yin last updated. All further steps are described in the subculture section. The recommended seeding density for each lot is stated on the.

It was initiated from a malignant gestational choriocarcinoma of the foetal placenta. Cell culture applications resources including application notes and protocols to create an ideal environment for growing cells, right from the start. Cnn3 regulates trophoblast invasion and is upregulated by hypoxia in bewo cells sarah appel1, janina ankerne1, jan appel1, andre oberthuer2, peter mallmann3,jo. This study investigated the roles of progesterone and leptin in placenta invasion, which is closely related to pregnancy prognosis. The human choriocarcinoma cell line bewo was obtained from american. Research paper progesterone inhibits leptininduced. Fundamental techniques in cell culture sigmaaldrich.