A afp concentrations were measured in the culture supernatants of huh7 and hep3b cells exposed for 18 h to the indicated concentrations of sorafenib, and a % inhibition was calculated for each cell line. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Adenoassociated virus aav protocol 9601 medical center. Splitting can be performed by centrifuging cells at 500g for 5 minutes, decanting growth medium and rinsing in sterile 1x pbs. The adaptation of cellular lineages to culture fbs or animal protein free medium can allow optimization of cell growth and expression of heterologous. To assess the utility of the rwv as a culture method for huh7 cells, huh7 cells were. 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. Prior to tumorsphere culture, breast cancer cell lines are grown as monolayers according to the suppliers recommendations on tissue culturetreated 10 cm dishes. Threedimensional huh7 cell culture system for the study of. Quantitative analysis of cell proliferation by a dye. Jcrb cell lines may be supplied in a glass ampoule or in a plastic vial glass ampoules are prescored at the neck position, and are not difficult to open. Perform all steps of protocol in a laminar flow cell culture hood using sterile techniques cell plating optimal cell densities will vary with growth characteristics that are unique to each cell type and need to be determined empirically. 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.
During the penultimate 10 min of serum starvation cells were stimulated with either insulin or. Huh7 cells should be cultured in dmem supplemented with 10% fbs. For commercially available cell lines we recommend. Aav6 vectors were generated with the icasp9 suicide gene. Cells in monolayer culture were prepared by culturing huh7 or cd44. All further steps are described in the subculture section. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. The huh7 cell line was derived from a liver tumor of a 57. I need to figure out what is the problem, either the method i stored the cells or cultured the cells is not proper. Protocol of cell growth cells grow on standard culture plates and should be incubated at 37c in the presence of 5% co 2. Simply select the cell type, nucleic acid, and culture format to receive a qiagen transfection protocol to print out or download in convenient pdf format. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant. Heidcamp, gustavus adolphus college a large collection of protocols related to cell biology work from microscopy to cell culture. Original research article highresolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources reka a.
Would anyone has experience culturing huh7 cell line please help me. Wash cells briefly with hanks solution prior trypsin treatment note. These findings show that when cultured in 3d, huh7 cells acquire a more differentiated hepatocytelike phenotype. The majority of huh7 cells show a chromosome number between 55 and 63 mode 60. Quantitative analysis of cell proliferation by a dye dilution. This unit describes the methods, equipment, supplies, and reagents used in a cell culture. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure. Cell culture protocols thermo fisher scientific hk. Development of an infectious hcv cell culture system. 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. Threedimensional huh7 cell culture system for the study. Cell culture basics handbook thermo fisher scientific.
The huh7 hepatoma cell line consists of welldifferentiated tumorigenic liver cells that are useful for carrying out transfection studies. Amaxa 4dnucleofector optimization protocol for cell lines. Original research article highresolution proteomic. All culture supplies were obtained from thermo fisher scientific, unless otherwise indicated. Hepg2 is an immortalized cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15yearold caucasian male who. Huh7 cell culture info and resources huh7 cell line. Qiagen protocols two types of protocol are provided in the transfect protocol database. Brightfield micrographs showing the structure of hepg2 cells cultured for 7 days on 22 mm diameter alvetex discs presented in the 6well plate format.
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. Product information huh7 cells cls cell lines service. Example protocol for the culture of the hepg2 cell line on alvetex 22 mm disc in 6well insert format, ams. You may be able to find more information about this cell line by contacting the japanese collection. Heidcamp, gustavus adolphus college a large collection of protocols related to cell biology work from microscopy to cell culture, histochemistry, etc. The hepg2 cell line was derived from a liver hepatocellular carcinoma of a 15yearold caucasian male atcc, atcc hb8065. Mammalian cell culture is the process of growing animal cells in vitro in a flask or dish. Cell culture guidelines the following is a general guideline for culturing of cell lines. Given the there are many formulations of dmem and different versions of serum, we would like to provide the catalog. Culture and functional characterization of human hepatoma. Alphafetoprotein is a biomarker of unfolded protein response.
A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture counting cells in a hemocytometer. Huh7 is an immortal cell line composed of epitheliallike, tumorigenic cells. Culture huh7 cells to around 70% confluence in complete growth medium. Hepg2 stable cell line generation and rnai applications sirna transfection. Subculture and maintenance 1 maintain culture at a cell density between 1x105 and 3x106 cellsml. We therefore recommend that you familiarize yourself with your cell line of interest, and closely follow the instructions provided with each product you are. When cultured properly, they differentiate, forming apical and basolateral cell. How to count and calculate the number of cells from a stock flask or culture dish. Lipofectamine 3000 5179% transfection efficiency and 4 fold protein expression improvement over lipofectamine 2000. Tumorsphere culture of breast cancer cell lines mdamb231, mcf7, skbr3, au565, bt474, sum149.
These cells are adherent to the surface of flasks or plates and typically grow as 2d monolayers. Protocols for jfh1 in vitro transcription and hcv rna. Mammalian cell tissue culture techniques protocol abcam. Cells were fixed, embedded in paraffin wax, sectioned 10 m and. Cells should be passaged using the following conditions. Most cell lines will grow on culture flasks without the need for special matrixes etc. The huh7 cell line was derived from a liver tumor of a 57yearold japanese male patient in 1982. The in vitro infectivity of the cell culture generated jfh1 virus was monitored by inoculation of naive huh7 cells with the culture medium of jfh1 rnatransfected cells. Cell replication and division are normally very tightly. The huh7 cell line is a hepatocytederived carcinoma cell line that was originally taken from a liver tumor in a 57yearold japanese male jcrb cell bank, jcrb0403. Incubation of naive huh7 cells with conditioned medium. Phase contrast micrographs of hepg2 cells grown in. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment.
Hepg2 cells are useful as a model system in vitro for human hepatic cancer cells. Hepg2 liver hepatocellular carcinoma cell culture and. However, some cells, particularly primary cells, will require growth on special. Despite the increasing demand for knowledge on viral replication and pathogenesis, detailed. Secure a 6cm plate of hek293t cells from the stock incubator. 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. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. You may be able to find more information about this cell line by contacting the japanese collection of research bioresources jcrb. Hcv rna titer in the inoculated cells was inhibited more than 1 log, indicating that infection by secreted viral particles is at least partially.
Alphafetoprotein is a biomarker of unfolded protein. Please acknowledge cellbank australia, the jcrb cell bank, and the originator of the cell line, in all publications and patent applications that make reference to the cell line. Assay for phenotype or target gene expression 48 72. Add prepared transfection complexes from step 3 or 4 7.
See table 2 for cell line specific cell density recommendations in 96well format. Sub culture and maintenance 1 maintain culture at a cell density between 1x105 and 3x106 cellsml. To investigate whether the antiproliferative effect of ailanthone on huh7 cells was triggered by cell cycle arrest, we measured the cell cycle phase ratio by flow cytometry with propidium iodide pi staining. Importantly, we show that these 3d cultures are highly permissive for hcv infection, thus providing an opportunity to study hcv entry and the effects of hcv infection on host cell function in a more physiologically relevant cell culture system. Adaptation to serumfree culture of hek 293t and huh7. Huh7 is a type of immortal cell line, as hepatoma cells, they exhibit an epithelial cell morphology. Cell culture and infection system for hepatitis c virus. Mebiolgel, a thermoreversible polymer as a scaffold for. Despite the increasing demand for knowledge on viral replication and pathogenesis, detailed examinations of the viral life cycle have been hampered by the lack of efficient viral culture systems, owing in part to its narrow host range. Primary hepatic cancer or liver cancer is the fifthmostcommon global malignancy and the thirdmostcommon cause of cancerrelated death 1. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Considering all these reasons, it is essential to develop a simple cell adaptation process for the culture of cells in serumfree, animal componentfree, or proteinfree medium.
We selected the 120 split from a few days previous. Huh7 transfection reagent liver cancer cells altogen. Crisprcas9 transfection protocol for 24 well plate. Renew medium 23 times a week, or as needed according to media ph, and maintain cell confluency between 3090%. Tumorsphere culture of human breast cancer cell lines. Ailanthone induces cell cycle arrest by regulating cell cycle regulatory proteins. Ailanthone inhibits huh7 cancer cell growth via cell cycle. Characterization of vexosomes pelleted from aav vectorproducing aav293 cell culture media a transmission electron microscopy the representative image shows the presence of vexosomes of varying size. Cancer is a broad term to describe over 200 diseases that are caused by cells proliferating in an outofcontrol manner. Doing so indicates to funding bodies the importance of cellbank australia and jcrb cell bank to the australian and international research communities, and signifies the importance of the. The following is a general guideline for culturing of cell lines.
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. A afp concentrations were measured in the culture supernatants of huh7 and hep3b cells exposed for 18 h to the indicated concentrations of sorafenib, and a % inhibition was. Renew medium 23 times a week, or as needed according to media ph, and maintain cell confluency between 30. Dulbeccos modified eagles medium with 10% heatinactivated fetal bovine serum. We isolated fulllength hcv clone, jfh1strain, from a. Dec 21, 2006 the in vitro infectivity of the cell culture generated jfh1 virus was monitored by inoculation of naive huh7 cells with the culture medium of jfh1 rnatransfected cells. Cell culture protocols thermo fisher scientific jp. Brightfield micrographs showing the structure of hepg2. Hepg2 liver hepatocellular cells description, culturing medium conditions, transfection efficiency and protocols, and optimized transfection kits.
Cell culture growth conditions,stable cell transfection,transient transfection recommended transfection reagents plasmid dna transfection and cotransfection. Amaxa 4dnucleofector optimization protocol for cell. A novel three dimensional 3d culture system purely synthesised from copolymer which is free from biological contamination for huh7 cell cultivation and hepatitis c virus. This protocol describes the use of mission trc shrna lentiviral particles and provides a system for longterm silencing and phenotypic observation. Dharmafect transfection reagentssirna transfection protocol. In vitro cellular models of human hepatic fatty acid. Hepatitis c virus hcv infection causes chronic liver diseases and is a health problem worldwide. The majority of huh7 cells show a chromosome number between 55 and 63 mode 60 and are highly heterogeneous. Transfection of a differentiated human hepatoma cell line huh7. Considering all these reasons, it is essential to develop a simple cell adaptation process for the culture of cells in serumfree, animal componentfree, or proteinfree medium conditions 1, 2.
Methods in molecular biology methods and protocols, vol 1250. Prior to tumorsphere culture, breast cancer cell lines are grown as. Perform all steps of protocol in a laminar flow cell culture hood using sterile techniques cell plating optimal cell densities will vary with growth characteristics that are unique to each cell. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. Doxorubicin blocks proliferation of cancer cells through. General details of cell culturing and subculturing. Huh7 crispr transfection kit for liver cancer cells. Protocol for hela cell culture and plasmid transfection av. Dharmafect transfection reagentssirna transfection.
Cell culture plates of your choice for detaching adherent cells. If you already have preformed sgrna complexes, proceed to step 4, if you have crrna and. In this study, we differentiated the human hepatoma cell line huh7. Cd44 standard isoform is involved in maintenance of cancer. Characterization of vexosomes pelleted from aav vectorproducing aav293 cellculture media a transmission. Note that cell culture conditions vary for each cell type. Human serum leads to differentiation of human hepatoma. Proliferating cultures the cell culture flasks, 2xt25, come filled with cell culture medium.
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