Bewo cell culture protocol pdf

Protocols for the revival, maintenance, passage, and growth of bewo cells for. Pdf coculture of jeg3, bewo and syncbewo cell lines. Cells in midlog growth can generally be rendered more competent to uptake plasmid dna than can cells at stationary phase. Culture and transfection of human choriocarcinoma cells. Grow cells on glass coverslips or prepare cytospin or smear preparation. The bewo trophoblastic cell line was the first human endocrine cell type to be maintained in. Unidirectional influx of lphenylalanine lphe and lleucine lleu across the microvillous border of the cells was studied using a rapid pairedtracer dilution technique. Midlog cells are used in the classic transformation protocol described in laboratory 10. Coculture of jeg3, bewo and syncbewo cell lines with adrenal h295r cell line. 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. Maintaining healthy cells is the key to experimental success and reproducible research results. For commercially available cell lines we recommend. The roller tube method of culture 3 was used with constant. The recommended seeding density for each lot is stated on the.

The use of bewo cells as an in vitro model for placental. Bacteria can be isolated from nearly any surface including inanimate objects and human skin. The presence of inducible cytochrome p450 types 1a1 and 1a2. However, a considerable amount of cell death occurred after forskolin or hcg treatment as compared to the control sirnatransfected cells. Trophoblast cell fusion and differentiation are mediated by.

A threedimensional culture system recapitulates placental. A glucose concentration of approximately 300340 mg percent has been found to be optimal for the cells. Hplc method development for quantification of doxorubicin. Methods and protocols methods in molecular biology pdf. The effect of rottlerin on parasite growth was evaluated in bewo cells infected for 3 hours. Immunofluorescence with monoclonal antibodies demonstrates that bewo ecm contains laminin subunits beta1 and gamma1. 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 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. Add required amount of prewarmed cell culture media to fresh flask. The use of bewo cells as an in vitro model for placental iron.

To define the signal transduction pathway utilized by heme to induce apoptosis, cell. 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. 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. Cnn3 regulates trophoblast invasion and is upregulated by hypoxia in bewo cells sarah appel1, janina ankerne1, jan appel1, andre oberthuer2, peter mallmann3,jo. 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. Cnn3 regulates trophoblast invasion and is upregulated by.

In vitro models for studying trophoblast transcellular. 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. When subculturing cells 6075% confluency, discard old media from flask. The very first method, and one of the most important, used to study. Bewo cells stimulate smooth muscle cell apoptosis and elastin. Bewo cells are a placental cell line that has been widely used as an in vitro model for the placenta. Bewo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into columns and superfused. Rottlerinmediated inhibition of toxoplasma gondii growth in. Importantly, bewo cell lines have fusogenic properties and, in culture with. 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.

The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to fo. A human hormonesynthesizing trophoblastic cell system has. Coculture of jeg3, bewo and syncbewo cell lines with. To subculture the cells they need to be brought into suspension. 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. Regulation of adrp expression by longchain polyunsaturated fatty. 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. Hememediated apoptosis and fusion damage in bewo trophoblast. Mouse embryonic stem cell culturing protocols 4 of 6 form 105 rev b072214 7. Fusion of the trophoblastderived choriocarcinoma cell line bewo can be triggered by forskolin. Variations in the level of glucose contained in the culture medium exert profound changes on cell growth and glycogen content. Wolf, university of maryland, baltimore county general guidelines to cell and tissue culture techniques, including. These results suggest that the nr4a subfamily of nuclear orphan receptors has a ro le in trophoblastic cell differentiation. Regulates trophoblast differentiation in the bewo cell.

Resuspend the cell pellet in r10 and count viable cells using a hemocytometer and trypan blue. They have proven to be extremely valuable for the study of the cellular, molecular, and endocrine aspects of human trophoblasts. Pdf adiponectin promotes syncytialisation of bewo cell line. Dislodge cells from the flask substrate with a cell scraper.

Part c provides a protocol for preparing a midlog culture of e. Crisprcas9directed genome editing of cultured cells. 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. This study investigated the roles of progesterone and leptin in placenta invasion, which is closely related to pregnancy prognosis.

Rottlerinmediated inhibition of toxoplasma gondii growth. Cell culture on microelectrode arrays axion biosystems. All further steps are described in the subculture section. 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. Oct 29, 2010 bewo cell culture the human choriocarcinoma cell line bewo was obtained from american type collection of cell manassas, va, usa. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Outlined procedures include cell revival, maintenance of the bewo cells in culture, freezing cells, plate preparation, seeding the cells, and transport protocols.

Fundamental techniques in cell culture sigmaaldrich. Some vessels were frozen immediately 0 h and others were cultured and frozen after 24, 48, 72 or 96 h umbilical. Research paper progesterone inhibits leptininduced. Permeabilization if the target protein is intracellular, it is very important to permeabilize the cells. Note that cell culture conditions vary for each cell type. Pdf coculture of jeg3, bewo and syncbewo cell lines with. The presence of inducible cytochrome p450 types 1a1 and. The cells secrete placental hormones including chorionic gonadotrophin hcg, polypeptide hormones, lactogen, estrogenic and progestational steroids, estrone, estradiol, estriol and progesterone. Hplc method development for quantification of doxorubicin in. Cell culture plates of your choice for detaching adherent cells.

All culture incubations should be performed in a humidi. Superfused microcarrier cultures of bewo choriocarcinoma cells. Product information bewo cells cls cell lines service gmbh. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Cells were maintained at 37c under 5% co 2, 95% air atmosphere in phenolred free dmemf12 medium with 15% fcs, streptomycin 10. Introduction cellcell interactions are key to man y biological processes. A dynamic model for studies of human trophoblast function. Exosomes were collected from culture supernatants of bewo cells. Bewo induced smc apoptosis in an ex vivo vessel model. The new eplate insert enables investigation of spe cific cellcell interactions in real time, while maintaining the cells in separate com partments. The protocol begins with an overnight suspension culture. 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. 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.

Pdf monocultures of different placental cells are used for many physiological and. Forskolin treatment, which is known to induce differentiation and decrease cell proliferation, significantly decreased bewo cell numbers at 48 hours by %. The culture collections represent deposits of cultures from worldwide sources. Proteomic profiling of forskolininduced differentiated bewo. Resistance to fasmediated cell death in bewo and njg.

Wash cells briefly with hanks solution prior trypsin treatment note. Microarray analysis of bewo and jeg3 trophoblast cell. On day 0, dilute the matrigel matrix to 5 mgml with icecold mdck complete cell culture medium. After counting the cells, seed them in the desired collagen coated cell culture plates with mhp see table 3. 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.

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. Bewo cells stimulate smooth muscle cell apoptosis and. Oct 31, 2016 heme induces apoptosis in bewo cells through activation of stat3caspase3parp and p73 signaling pathways. In vitro models for studying trophoblast transcellular transport. Trophoblast cell fusion and differentiation are mediated. Glucose uptake by bewo cells was carried out as described 31 with a few adaptations. Bewo cells are regularly used as a cell culture model to mimic in vivo syncytialisation of placental. Remove the prewarmed mnc culture medium and laminincoated plates from the incubator during centrifugation.

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. 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. Fusion of the trophoblastderived choriocarcinoma cell line bewo can be. Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. Amaxa 4dnucleofector optimization protocol for cell lines. Adiponectin promotes syncytialisation of bewo cell line and primary trophoblast cells. 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.

This application protocol describes how to handle icell neurons for use on. Cell biologycell culture protocols protocol online your. Cells that are difficult to detach may be placed at 37c to facilitate dispersal. 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. 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. Adiponectin promotes syncytialisation of bewo cell line and. Cell culture applications resources including application notes and protocols to create an ideal environment for growing cells, right from the start. Quantifying the syncytialisation of human placental. 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. Centrifuge conical tube containing cells at 1100 rpm for 2 minutes at room temperature. Corning matrigel basement membrane matrix for 3d culture. The presence of inducible cytochrome p450 types 1a1 and 1a2 in the bewo cell line.

Detailed instructions for carrying out transplacental transport experiments with bewo cells in transwell inserts appear in the protocols that follow. At this point the cell lines should be subcultured or passaged in order to prevent the culture dying. At 24, 48, 72, and 96 h after seeding, the culture media were removed. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. Protocol for hela cell culture and plasmid transfection av.

The cell line was submitted to the american type culture collection in the 187th passage. Pdf plant cell culture protocols methods in molecular. Add appropriate aliquots of the cell suspension to new culture vessels. Cannabidiol changes pgp and bcrp expression in trophoblast. 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. The proper procedure for genome editing on tissuecultured hipscs is described in this protocol. Co culture of jeg3, bewo and syncbewo cell lines with adrenal h295r cell line. Cell culture basics handbook thermo fisher scientific. Department of pharmaceutical chemistry, school of pharmacy, the university of kansas, lawrence kansas 66047. Human choriocarcinoma the first human, trophoblastic endocrine cell type to be maintained in continuous culture. Visit our website for a detailed protocol on using a hemocytometer to obtain a viable cell count 6. Culture of hek 293293t cells charles yin last updated.

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. Mammalian cell tissue culture techniques protocol abcam. Cell seeding density of 5 104 cellswell in 24well plates was determined after testing various densities for sensitivity to glucose uptake data not shown. 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. Placental cytotrophoblast cells were isolated by the method of kliman 30. The original bewo cell line is also available through american type culture. 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 human choriocarcinoma cell line bewo was obtained from american. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. In brief, cell culture media containing dulbeccos modi. Live cell imaging of in vitro human trophoblast syncytialization. Once matrigel matrix is thawed, swirl vial to ensure the material is dispersed. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work.

A scheme of the in vitro transfer system using bewo cells as donors and jurkat cells as recipients. Secure a 6cm plate of hek293t cells from the stock incubator. Bewo exosomes carrying placentaassociated mirnas e. It was initiated from a malignant gestational choriocarcinoma of the foetal placenta. Cell culture guidelines the following is a general guideline for culturing of cell lines. Pdf plant cell culture protocols methods in molecular biology full online. Cryopreserved human ipscderived neurons axion biosystems. Progesterone inhibits leptininduced invasiveness of bewo cells. Transport of large neutral amino acids into bewo cells.

To confirm the microarray data nine differentially expressed transcripts representing a range of cellular function were selected for secondary analysis. Bewo exosomal mirnas were then transferred into jurkat cells. 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%. Human exosomal placentaassociated mir517a3p modulates. Immunocytochemistry and immunofluorescence protocol. Protocol human ips cell culture under feederfree conditions. Trophoblast cell line an overview sciencedirect topics. Coculture of jeg3, bewo and syncbewo cell lines with adrenal. A human hormonesynthesizing trophoblastic cell system has been established in vitro and. Effect of human telomerase reverse transcriptase transfection. Stain the cells with trypan blue and count the number of living cells with the countess. Differential effects of inducers of syncytialization and. Optimization of bewo model to investigate placental responses.

Trophoblast cell lines have become an invaluable tool to study various aspects of trophoblast biology. We selected the 120 split from a few days previous. Bewo atcc ccl98 homo sapiens placenta choriocarcinoma. Relevance of the nr4a subfamily of nuclear orphan receptors. Cell culture protocols thermo fisher scientific kr. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price.