Total protein extraction from cells cultured in alvetex in. In this chapter, freezing, thawing, and subculturing procedures for hepg2 cells are described. Hbv particles obtained from the serum of a chronic hepatitis b surface antigen subtype ad carrier were used to inoculate hepg2 cells. Growth medium for hepg2 dmem 10% fbs penstrep 1x procedure a. Cultures of primary hepatocytes and hepatoma cell line hepg2 are frequently used in in vitro models for human biotransformation studies. Having a single cell suspension is also very important to ensure efficient transfection. It is critical to ensure dissociation has occurred to yield a single cell suspension when passaging and plating the cells.
Example data was obtained using this protocol to extract protein from hepg2. 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. There is no evidence of a hepatitis b virus genome in this cell line. Assay apoptosis in magnetically bioprinted hepg2 spheroids. To apply this protocol to the established cell lines, hepg2 cells were treated with dmso or left untreated, and incubated with hbvpositive human serum. American type culture collection and kac respectively. C3a hepg2c3a, derivative of hep g2 atcc hb8065 atcc. A robust cell culture system supporting the complete life. A robust cell culture system supporting the complete life cycle of hepatitis b virus eleftherios michailidis w, jonathan pabon w, kuanhui xiang, paul park w, vyas ramanan y, hansheinrich homann w, william m. Huh6 and huh4 both also produce alphafetoprotein and albumin in cell culture 12, but unlike the huh7, do not allow replication of hcv genomic replicon and, as stated earlier, cannot be propagated in purely chemically defined media, thus limiting their use 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.
Reproducing hepatic tumor gene and protein expression in in vitro scaffolds as for 3d cell culture in general, it is also true for tumor cells that 3d conditions create a spatial microenvironment that is stimulated by concentration gradients of nutrients and gases. Hepg2 cells were routinely maintained in t75 flasks. Herein, a galactoseappended naphthalimide galnpa with paminophenylether as a new receptor and galactose moiety as hepatoma targeting unit was synthesized and employed to detect endogenous hocl in living hepg2 cells. Ive been trying to culture these hepg2 cells for a while now, and i get hit with the same problem each time. In our lab, we culture hepg2 cells in dulbeccos minimal essential medium dmem supplemented with 5% fetal bovine serum, penicillin 100 unitsml, streptomycin 100. If you want hepg2 cells healthy and behaving like hepatocytes, feed them the medium in attached file. Rna was extracted for the expression of the indicated isgs by rtqpcr. The utility of hepg2 cells to identify direct mitochondrial dysfunction in the absence of cell death. Thaw cryopreserved cell line using an oring in warm water bath for 12 min. Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 70% confluency at the time of transfection. The impact of the four test bps on hepg2 cell viability was assessed using the mts assay zhang et al. Rational design of a hepatomaspecific fluorescent probe. The human hepatoma hepg2 cell line and c2c12 myoblasts were obtained from the chinese academy of sciences shanghai, china.
Also, at every passage, squeeze them through a 21g needle three to four times. Im gonna start to work with hepg2 cells and i would to know what are best culture conditions for these cells. 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. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen freezer storage until ready to culture. Mangiferin improved palmitateinducedinsulin resistance. In addition to the hepatocellular carcinoma or hepatoma cell line based on the original publication pubmed. It is critical to ensure dissociation has occurred to yield a single cell suspension, when passaging and plating the cells. Hep, g2, hepg2 human caucasian hepatocyte carcinoma the hep g2 cell line has been isolated from a liver biopsy of a male caucasian aged 15 years, with a well differentiated hepatocellular carcinoma. Published protocols differ in cell numbers, culture conditions and durations, and droplet volumes. Size of u266 peripheral blood, multiple myeloma cell. Maintaining healthy cells is the key to experimental success and reproducible research results. Cells growing in the 384 well plates as described were treated for 4 days with concentrations of camptothecin ranging from 0. The protocal is from thermo fisher scientific important guidelines for transfection 1.
The medium was subpackaged into 200 ml culture flasks. Hepg2 cells were maintained in dmem dulbeccos modified eagles medium supplemented with 10% fbs, 100 unitsml penicillin and 100. Warm emem media with 10% dialyzed fetal bovine serum prepared according to manufacturers recommendations in warm water bath for 2030 min. Yost1,3 1 department of chemistry, university of florida, gainesville, fl. Phase contrast micrographs of hepg2 cells grown in. The cells were fixed and stained, and images acquired using the opera system. However, the cell selective fluorescent probe still remains a difficult task among current reports. An in vitro system for production of hepatitis b virus hbv was established by infection of human hepatoma hepg2 cells. Keywords multimode microplate reader, plate reader, 3d spheroids, hepg2 cells, imaging cytometer, imaging cytometry, spectramax i3x, spectramax minimax, cell culture, apoptosis, homogeneous. The cells collected from the culture flask were seeded in the 96well plates corning. Brightfield image of hepg2 cells in culture, prior to transfection. Cstimulated lx2 culture sn on isgs expression in hepg2 cells.
Pdf sodium valproate induces mitochondrial respiration. Author links open overlay panel laleh kamalian a 1 amy e. Learn how hepg2 cells can be bioprinted using a magnetic 3d cell culture system and imaged on the spectramax i3x plate reader with minimax 300 imager. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Hepg2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. Human hepatoma cell lines, hepg2 and heparg cells, were obtained from a. Hepg2 cells as an in vitro model for evaluation of. They should be limited to 20 passages otherwise you get very weird cellular behaviors foci, slow. To quickly thaw the cells, the hepg2 cells were removed. Cell culture basics handbook thermo fisher scientific.
Materials and methods cell culture hepg2 cell line was purchased fromamerican type culture collection manassas, va in july 2009, and maintainedas subconfluent monolayers in dmem invitrogen with 10% fetal bovine serum hyclone, logan. Culture sn was collected for treatment of hepg2 cells 20% vv for 12 h. Phase contrast micrographs of hepg2 cells grown in conventional 2d culture. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food. Although various in vitro assays have been developed to evaluate the cytochrome p450 cypinducing potential of drug candidates, there is a continuing need for the development of a reliable model in drug discovery. Culture and functional characterization of human hepatoma. Original article construction of human hepg2 cells. The cells secrete a variety of major plasma proteins e. Recombinant protein therapeutics from cho cells 20 years and counting karthik p. Tlr3 activation of hepatic stellate cell line suppresses.
Ii for transfecting hepg2 cells was formulated for tanasfection of hepg2 cells. Recombinant protein therapeutics from cho cells 20 years. Hep g2 is an immortal cell line which was derived from the liver tissue of a 15yearold african american adolescent boy with a welldifferentiated hepatocellular carcinoma. Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog numbers of. Molecular cell article pgc1a, a key modulator of p53, promotes cell survival upon metabolic stress nirmalya sen,1 yatendra kumar satija,1 and sanjeev das1, 1molecular oncology laboratory, national institute of immunology, aruna asaf ali marg, new delhi 110067, india correspondence. Transfecting plasmid dna into hepg2 cells using lipofectamine 3000 reagent. Size of hepg2 liver carcinoma cell human homo sapiens. Materials and methods cell culture hepg2 cell line was purchased fromamerican type culture collection manassas, va in july 2009, and maintainedas subconfluent monolayers in dmem invitrogen with 10% fetal bovine serum hyclone, logan, ut and 100 unitsml penicillinplus 100. Propagation of hepg2 2 5 immediately remove cells and pellet at 500 xg for 5 minutes 4oc. In vitro infection of human hepatoma hepg2 cells with. We found that the composition of the cell culture media greatly a ected cell growth and sensitivity to selenium cytotoxicity. Hepg2 cells atcc were thawed at passage 37 and cultured in two separate batches with. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection.
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. High throughput cytotoxicity testing with hepg2 cells. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in nude mice. Nuclear staining of hepg2 cells in 3d cell culture with draq5. Hepg2 cells are nontumorigenic cells with high proliferation rates and an epitheliallike morphology that perform many differentiated hepatic functions. Hepg2cyp cell panel new tools to assist in identifying human routes of metabolism, cyp phenotyping and drugdrug interactions hepg2 is one of the most commonly used cell systems for in vitro screening, but hepg2 cells have low expression and activity of cyp enzymes which leads to missed or underestimated cytotoxicity of test compounds andor metabolites we have assembled a panel of. Risk of vpainduced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with polg1 gene mutations.
972 724 843 1086 1242 1189 1518 294 923 75 466 1121 869 373 978 1416 16 562 203 103 466 952 1236 1055 1411 1352 496