As cellular models for in vitro liver cancer and toxicity studies, hepg2 and hep3b are the two most frequently used liver cancer cell lines. Heparg human hepatic progenitor cells were obtained from life technology carlsbad, ca, usa. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7 cell lines study of marine yeast p. Once complete media has been formulated, it should be stored at 28oc. Atcc stands ready to support our customers needs during the coronavirus pandemic.
Assuming that the same cell system is used, that the virus forms plaques on those cells, and that no procedures are added which would inhibit plaque formation, 1 ml of virus stock would be expected to have about half of the number of plaque forming units pfus as tcid50. Proliferating cultures the cell culture flasks, 2xt25, come filled with cell culture medium. While we are not currently experiencing delays due to this pandemic, we expect that we could see them as the situation evolves. 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. Phase contrast micrographs of hepg2 cells grown in.
This step is the most critical to ensure single cells for accurate counting and plating. Choose from among the worlds largest collection of bacteria, viruses, yeast, fungi, protozoa, nucleic acids, and molecular tools. In vitro cytotoxicity mtt assay in vero, hepg2 and mcf 7. An important issue in toxicity studies is the development of pertinent new in vitro tests that will be able to provide an alternative to in vivo testing methods. Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog numbers of. C3a hepg2c3a, derivative of hep g2 atcc hb8065 atcc.
Cells were cultured in dmem supplemented with 100 mll fbs, 100,000 ul penicillin, and 100 mgl streptomycin. Comparative chemosensitivity of vx2 and hcc cell lines to drugs. The growth inhibitory effect of conjugated linoleic acid. Cell culture the hepg2 human hepatoma cell line was obtained from the american type culture collection manassas, va, usa. Atcc was entrusted with its first cell line in 1962 and has consistently attained the highest standards and used the most reliable procedures to verify every cell line since. All further steps are described in the subculture section. I have cultured hepg2 cells successfully in eagles minimum essential media which contains 1gl dglucose and 2mm lglutamine, and i added. Hepatocellular carcinoma hcc is the third prevalent cause of cancer death worldwide. In our hands hepg2 cells grow very slow and with big vaculoes in dmem high glucose and tend to make foci. Cultured cells are dramatically affected by the microenvironment in which they are grown. Cell culture basics handbook includes 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. Therefore, hepg2 cells were also considered as a suitable tool for the. Activity in the endothelial cell culture medium may represent residual factorvafromthe 10% humanserum used tablei. This has been corrected in both the pdf and html versions of the article. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in nude mice. The protocal is from thermo fisher scientific important guidelines for transfection 1.
Cultures of primary hepatocytes and hepatoma cell line hepg2 are frequently used in in vitro models for human biotransformation studies. Our first job is to listen to and observe what our customers need, and meet those needs with quality products and services. Thank you for your understanding, patience and flexibility as atcc does everything it can to help reduce the impact of the coronavirus pandemic to our valued customers. Bioedip was developed as a userfriendly tool to quantify confluence levels in cell culture images. Cell culture basics handbook thermo fisher scientific. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Hepg2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. Both hepg2 and huh7 cells were grown in media containing 11 mmoll glucose and either 2% hs or 10% fetal bovine serum. Collect the entire medium in 2x 50 ml centrifuge tubes. Avoid extended exposure of the medium to room or higher temperatures. The hepg2 cell line is commonly used in drug metabolism and hepatoxicity studies. Kathiresan2 1department of zoology, annamalai university, annamalai nagar, chidambaram608002, tamilnadu, india.
In this chapter, freezing, thawing, and subculturing procedures for hepg2 cells are described. Hep g2 or hepg2 is a human liver cancer cell line 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. Behavior of hepg2c3a cell cultures in a microfluidic. Applic on n hepg2 labeling and cell growth protocol for. Current developments in the fields of tissue engineering and microtechnology make it possible to propose the use of microfluidic bioreactors as a tool for enhanced in vitro investigations. 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. Yost1,3 1 department of chemistry, university of florida, gainesville, fl. The aim of this study was to compare fa metabolism between hepg2 and huh7 cells in response to culturing in different sera. Example data was obtained using this protocol to extract protein from hepg2.
Generation of cytochrome p450 3a4overexpressing hepg2 cell. Hepg2 cells grow slow initially and tend to grow in clusters rather than as an intact monolayer. The homogenates contain phase i enzymes that convert chemicals to. Distinctive pharmacological differences between liver. Growth medium for hepg2 dmem 10% fbs penstrep 1x procedure a. Receipt of frozen cells and starting cell culture 1 immediately place frozen cells in liquid nitrogen freezer storage until ready to culture. Resuspend the cells carefully in 10ml fresh cell culture medium and transfer them into one t25 cell culture flask. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food. The atcc cell biology collection is one of the largest bioresources in the world, and offers a complex array of human, animal, insect, fish and stem cell lines from which to choose. Thank you for your understanding, patience and flexibility as atcc does everything it can to help reduce the impact of. Use of huh6 and other humanderived hepatoma lines for the. Hepg2 human hepatocellular carcinoma expansion media is designed to be used with hepg2 human hepatocellular carcinoma cells, which are available separately. However, we did detect losses of the chromosome 4q3 region, which have been.
Cell culture hepg2 cells were purchased from atcc manassas, va, cat. Can anyone tell me the difference between hepg2 and hep3b. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating. The human liver cancer cell line hepg2, the human lung cancer cell line a549, and the human oral squamous carcinoma cell line sas were purchased from the riken bioresource center tsukuba, ibaraki, japan. Apolipoproteinb100 apob100 is the essential protein for the assembly and secretion of very low density lipoproteins vldl from liver. Investigating effect of rapamycin and metformin on angiogenesis in. Culture and functional characterization of human hepatoma. The cells express 3hydroxy3 methylglutarylcoa reductase and hepatic triglyceride lipase activities. Biosynthesis coagulation factor human carcinoma cell line. Culture of hepg2 liver cells on three dimensional polystyrene. What is the most efficient method to culture hepg2 cells.
Protective effects of an ethanol extract of angelica. Because of their similarities they are often treated as the same in experimental studies. Medium should be equilibrated in a water bath set at 37oc before adding media to any cell culture. Interestingly, the atcc american type culture collection catalog indicates that hepg2 carries a chromosome 1 rearrangement, as reported in 1980 3, which was not detected by a cgh, probably due to its balanced nature. The parental cells were stably transfected at passage 48 with a human cholesterol 7 alphahydroxylase cyp7 minigeneluciferase construct. Hepg2 cells human hepatocellular carcinoma cells liver tumor were cultured in 75cm 2 cell culture flasks to confluence and harvested using a solution containing 0. Microbiology collections atcc has been the premier source for microbial reference strains since 1925, placing the highest standards on producing quality materials you can depend on for reproducible results. For experiments, cells were grown in 96well plates, and inspected under an inverted system microscope, car.
Hepg2 cells were routinely maintained in t75 flasks. The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, hepg2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells. After 3 days, insulin and insulinlike growth factor1 signaling were measured. The human liver cancer cell line, hepg2 atcc hb8065, manassas, usa was cultured in rpmi1640 medium thermo fisher scientific. Atcc converting tcid50 to plaque forming units pfu124. Established from the tumor tissue of an 8yearold black boy species. The utility of hepg2 cells to identify direct mitochondrial dysfunction in the absence of cell death. Huh6 and huh4 both also produce alphafetoprotein and albumin in cell culture 12, but unlike the huh7, do not allow replica. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Author links open overlay panel laleh kamalian a 1 amy e. When used as directed, this media will support growth and expansion of. Prepared by heena shah 23 august 2016 page 1 of 36 m205.
Hepg2 human hepatocellular carcinoma expansion media hepg2. Hb8065 and cultured according to the suppliers instructions. Despite recent advances in diagnosis and treatment, hcc is frequently diagnosed at an advanced stage and has a poor prognosis. Volume contraction occurs on conversion of monomer to polymer, which. Propagation of hepg2 2 5 immediately remove cells and pellet at 500 xg for 5 minutes 4oc. Hepg2 cells are nontumorigenic cells with high proliferation rates and an epitheliallike morphology that perform many differentiated hepatic functions. Total protein extraction from cells cultured in alvetex in. I have followed all the steps shown in the atcc manual for the hepg2 cell line. I have used hepg2 in my study about erythropoietin but i would like to know if i might expect any differences on hep3b cell culture.
Based on the widely used hepatocellular carcinoma cell line hepg2 and a. In this study, we have investigated whether hepg2 liver cells grown in three dimensional 3d cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional 2d flat plastic surfaces. The low level ofactivity in the endothelial cell culture mediumwasnotduetothepresence ofan inhibitor, as shown by the mixing experiments in table i. However, there are many differences that have been largely oversighted or ignored between them. We also working on cell culture since 3 years experienced various problems on protocol of different cell lines. In vitro cellular models of human hepatic fatty acid.