Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Differentiated heparg cells cryopreserved description and user guide for thawing, culture and use catalog number. Development of stable cell lines in 28 days that overexpress a protein of interest or shrna construct are vital research tools, especially for the production of recombinant antibodies. A robust cell culture system supporting the complete life. Mcf7, human hepatocarcinoma hepg2 and african green monkey kidney cell lines vero, respectively, were incubated with different doses to 1. Total protein extraction from cells cultured in alvetex in.
Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food. This technology provides a significant increase in delivery efficiency of the difficulttotransfect cell line hep g2. C 0 genmute sirna transfection reagent for hepg2 cell. Warm emem media with 10% dialyzed fetal bovine serum prepared according to. Protocol example protocol or the culture o the hepg2 cell. Hepg2 labeling and cell growth protocol for iroa phenotypic. Culturing these cell lines in human serum hs has been reported to improve functionality. Proliferation of hepg2 cells after kaempferol treatment andor mir21 mimic transfection were measured using 5bromo2. Protocol page 2 example protocol or the culture o the hepg2 cell line on alvetex sca. A highly efficient method of transfecting hep g2 cells. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. This method can be applied to the use of alvetex scaffold in 12well insert format, avp005.
Hepg2 cells have been demonstrated to be neomycin g418 resistant 400 gml. The protocal is from thermo fisher scientific important guidelines for transfection 1. Example protocol for the culture of the hepg2 cell. This protocol shows optimum transfection condition using hilymax in hepg2 cells. Od450 values were measured at 24 h, 48 h, 72 h and 96h.
Ive been trying to culture these hepg2 cells for a while now, and i get hit with the same problem each time. I too have been working on hepg2 cells in the same media composition. Growth medium for hepg2 dmem 10% fbs penstrep 1x procedure a. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Prepare a singlecell suspension of cells in media using trypsin to remove cells from 2d plasticware. Example protocol for the culture of the hepg2 cell line on. This differentiation makes them very helpful in the study of intracellular transport of molecules between the different liver cell types, dysfunction of which can. Culture of hepg2 liver cells on three dimensional polystyrene. No significant signs of cellular toxicity were observed. Expand populations of hepg2 cells in emembased media supplemented as described above as monolayer cultures in conventional 2d plasticware according to standard procedures. After 24 hours of incubation, cell viability was determined by the mtt assay.
It is critical to ensure dissociation has occurred to yield a single cell suspension, when passaging and plating the cells. Hepg2 sable cell line hepg2 liver hepatocellular carcinoma. Characterization of insulinlike growth factor ii receptors in human small cell lung cancer cell lines. Applic on n hepg2 labeling and cell growth protocol for. Example data was obtained using this protocol to extract protein from hepg2. I was wondering as hepg2 is the cancer cell line fcs would be the better option to choose over fbs in terms of avoiding contamination.
Materials and methods cell culture hepg2 cell line was. Example protocol or the culture o the hepg2 cell line on alvetex sca. The cell number and concentration determined can vary significantly depending on what method is used for counting. Try when possible oil red coloration on cell layer to check the. Protocol for hela cell culture and plasmid transfection av. When the cells reach approximately 90% of confluency, they. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3d.
Cell culture guidelines the following is a general guideline for culturing of cell lines. This study used the cell culturing procedure described by the global bioresource center atcc with the following modifications. Cell culture techniques are ubiquitous in areas of developmental biology, drug discovery, regenerative medicine and protein production. Hepg2 was derived from a liver hepatocellular carcinoma of a 15 year old caucasian male. Phase contrast micrographs of hepg2 cells grown in conventional 2d culture plates. Celltiterglo luminescent cell viability assay technical. Phase contrast micrographs of hepg2 cells grown in. Development of hepg2 stable cell line services are provided by altogen labs cro.
Kaempferol inhibits proliferation, migration, and invasion. Published protocols differ in cell numbers, culture conditions and durations, and droplet volumes. The utility of hepg2 cells to identify direct mitochondrial. Complete culture medium with serum and antibiotics is freshly added to each well 60 minutes before transfection. Transfection protocol transfection components component cat. In vitro cellular models of human hepatic fatty acid. Hpr116 background heparg cells have the unique properties of maintaining significant levels of hepatic cell functions, of being cyp450 inducible and supporting the complete replicative cycle of hbv. Having a single cell suspension is also very important to ensure efficient transfection. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7. In this chapter, freezing, thawing, and subculturing procedures for hepg2 cells are described. Since the introduction of cell culture techniques, cells have been cultured in twodimensions, attached to tissue culture plasticware or ecm attachment proteins. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Culture and functional characterization of human hepatoma.
Hep g2 is an immortal cell line which was derived from the liver tissue of a 15yearold american adolescent boy of european ancestry with a welldifferentiated hepatocellular carcinoma. A cell culture, therefore, refers to cultures derived from dispersed cells taken from the original tissue, from a primary culture, or from a cell line or cell strain. Hepg2 cell line alvetex scaffold plates and inserts page 2 of 5 methods preparation for 3d cell culture on alvetex scaffold 1. Transfection protocol lipofectamine 3000 transfection reagent. It is critical to ensure dissociation has occurred to yield a single cell suspension when passaging and plating the cells. The base medium for this cell line is atccformulated. It is critical to ensure dissociation has occurred to yield a. Mammalian cell tissue culture techniques protocol cryopreservation is a method whereby cells are frozen, maintaining their viability, until they are defrosted months or years later. Hepg2 cells are nontumorigenic cells with high proliferation rates and an epitheliallike morphology that perform many differentiated hepatic functions. The hepg2 cell line is commonly used in drug metabolism and hepatoxicity studies. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Purchase hepg2 cells hb8065 as a frozen cell culture from atcc manassas, va. Aspirate and add fresh culture medium every 23 days. Cgms was spherical, unlike that of cells cultured under traditional two.
Having a singlecell suspension is also very important to ensure efficient transfection. Hepg2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. Characterization of primary human hepatocytes, hepg2 cells. Applic on n hepg2 labeling and cell growth protocol for iroa.
Dmem and rpmi1640 are also alternatives that work well. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Cell culture basics handbook thermo fisher scientific. This method can be applied to the use of alvetex scaffold in 24 well. Investigation of testosterone, androstenone, and estradiol.
H e p g 2 labe l ing and cell growth col hepg2 labeling and cell growth protocol for iroa phenotypic metabolic profiling candice z. In our lab, we culture hepg2 cells in dulbeccos minimal essential medium dmem supplemented with 5% fetal bovine serum, penicillin 100 unitsml, streptomycin 100. It is critical to ensure dissociation has occurred to yield a singlecell suspension when passaging and plating the cells. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic. Hepg2 cell line alvetex scaffold plates and inserts. Gibcodmem with glutamaxsupplement 10566016 10% gibcofbs a3160401 proper culture techniques and procedures are an essential part of ensuring successful transfection. Human hepg2 hepatic carcinoma cells were purchased from the american type culture collection atcc, manassas, vc, usa. Remove frozen cells from dry ice packaging and store cell line under. The specific inhibition of hepg2 cells proliferation by. So, if you need liver cells, hep2 are a bad choice. Hepg2 cell culturing protocol hepg2 complete medium. 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. Cultures of primary hepatocytes and hepatoma cell line hepg2 are frequently used in in vitro models for human biotransformation studies.
Hep2 is a cell line derived from hela they were are contamination in an originally hepatic cell line see atcc for details and so are a cervix carcinoma cell line, most useful, because they have a rather big nucleus. When cultured properly, they differentiate, forming apical and basolateral cell surfaces, resembling in vivo liver structures. In our hands hepg2 cells grow very slow and with big vaculoes in dmem high. To obtain healthy hepg2 cells, the cells must be grown on a culture dish pretreated with collagen type i. The comet assay applied to hepg2 liver spheroids sciencedirect. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. When using the other vessel, refer to table 2 and adjust the amounts of cells, medium, dna and hilymax in proportion to the relative surface area. Cells were fixed, embedded in paraffin wax, sectioned 10 m and.
Transiently transfected cells are typically harvested 2496 hours posttransfection and are often used for studying the effects of short. Hepg2 cell line was purchased from american type culture collection manassas, va. The presence of socalled vacuoles are often seen in hepg2 cells but they could also be the result of fatty acids accumulation. The inhibition of cell proliferation serially became more marked figure 1a. Transfecting plasmid dna into hepg2 cells using lipofectamine.
Cells that are difficult to detach may be placed at 37c to facilitate dispersal. Example protocol for the culture of the hepg2 cell line on alvetex 22 mm disc in 6well insert format, ams. Hepg2 liver hepatocellular carcinoma cell culture and. The aim of this study was to compare fa metabolism between hepg2 and huh7 cells in response to culturing in different. Hepg2 liver hepatocellular cells description, culturing medium conditions, transfection efficiency and protocols, and optimized transfection kits. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen freezer storage until ready to culture. To tranfect hepg2 cells in 24well plate, follow optimum condition for transfection and transfection procedure. 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. Microscope growth medium for hepg2 dmem 10% fbs penstrep 1x procedure a. In vitro treatment of hepg2 cells with saturated fatty. Hepg2 is an immortalized cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15yearold caucasian male who had a welldifferentiated hepatocellular carcinoma, which is the fifth mostcommon cancer worldwide. The cell culture must have 90% viability and be 60% confluent on the day of transfection.
Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog numbers of the stuff that we are using. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. The implementation of the developed protocol in two independent laboratories, using hepg2 cells from different sources and with different cell culture media, confirmed a high reproducibility, in contrast to a study by hurrell et al. Hepg2 cells are useful as a model system in vitro for human hepatic cancer cells. Remove frozen cells from dry ice packaging and store cell line under liquid n 2 c until use. The morphology of human hepatoma cell lines hepg2 cells cultured on the 3d. Eagles minimum essential medium emem supplemented with 10% fbs. Transfex transfection of plasmid dna into hepg2 cells. Plates were reincubated and maintained by complete media exchange after every 23 days. Nov 30, 2017 establishing an hbv infection system in hepg2 ntcp cells.
Prepare transfection complexes by mixing 40 l of serum. Lipofectamine 3000 transfection reagent hepg2 liver. Transfection efficiency of transfex reagent on hepg2. Weve used rpmi 1640 with 10% fbs and 2mm lglu for the cell culture. P urchase hepg2 cells b8065 as afroz en cll lt re m atcc m nassas, v. Here we demonstrate that using gp3k in a rapid, sameday transfection protocol with hep g2 cells will result in a 3fold improvement in transfection efficiency over the. I guess hepg2 is a cancer cell line so it could be ok to culture in lower serum medium.
For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Add appropriate aliquots of the cell suspension to new culture vessels. Cells are cryopreserved to minimize genetic change and avoid loss through contamination. Alvetex scaffold 12well plates were prepared for seeding with 70% ethanol 2 ml per well and media washes twice with 3 ml of media each.
I trypsinise a 10 cm culture dish with 1 ml of trypsin for maximum 3 minutes when they are 70% confluent. Brightfield image of hepg2 cells in culture, prior to transfection. Images show cells at low left and high right confluency. Prepare a single cell suspension of cells in media using trypsin to remove cells from 2d plasticware. Transfection protocol lipofectamine 3000 transfection. Usa based on an established method that utilizes bromocresol green which. Some of the extracts induced cell cytotoxicity in a. Propagation of hepg2 2 5 immediately remove cells and pellet at 500 xg for 5 minutes 4oc. Yost1,3 1 department of chemistry, university of florida, gainesville, fl.
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