The caco2 cell line is a continuous line of heterogeneous human epithelial colorectal. Jan 22, 20 caco2 cells from american type culture collection atcc grown in basic culture medium in tissue culture flasksdiscs. Modified eagles minimum essential medium mem earles balanced salt solution bss 2 mm lglutamine 0. Using our low density ld protocol, caco2 cells retained a higher proliferation potential resulting in a cell population, which, reaching confluence, was able to differentiate almost synchronously, forming a. The sixwell culture plates with culture medium are incubated at 37 c 5% co2 with medium change every second day. Caco2 cells, a human colonic epithelial cell line atcc, rockville, md, were maintained routinely in 100. Cell growing density affects the structural and functional. Surachai unchern laboratory practice on caco2 cell culture. Protocol for growing and staining cells for immunofluorescence.
Effects of butyrate on intestinal barrier function in a. Cytokine responses of intestinal epitheliallike caco2 cells. Caco2 permeability assay caco 2 permeability goal to measure directional caco2 permeability of test compounds in cultured caco2 monolayer. Using our low density ld protocol, caco 2 cells retained a higher proliferation potential resulting in a cell population, which, reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a higher cell growing density natoli et al. Feed cells by changing media approximately once every two days. This chapter will however, focus on describing how to handle and cultivate the original caco2 cell line as. Individual permeation of digoxin, quinidine and verapamil across caco2 cell monolayers at 37c 102 6. Caco2 permeability assay, in vitro drug metabolism bioduro. Caco2 cells harvested after overnight culture with and without f. The addition of antibiotics to media during transfection may result in cell death, and has not. Caco2 cells were seeded onto polyethylene membranes in 96well falcon insert systems at 2 x 10 5 cellscm 2 until to 2128 days for confluent cell monolayer formation. Caco 2 studies are incapable of simulating the changes of intestinal ph system since it is performed at constant ph conditions apical ph of 6. Cell culture caco2 cells were culture in mem supplemented with 20% vv fetal bovine serum, 1% vv nonessential aminoacids and 1% vv glutamax at 37.
Caco2 cell line, with or without collagen i alvetex scaffold. Any ph modification in design of caco 2 cell protocol may affect its cytoarchitecture and underestimate the permeability characteristics. Gene expression,transient transfection recommended transfection reagents plasmid dna transfection. Caco 2 cells in this image had been kept in culture for 21 days, allowing them to grow confluent and epithelialize. Cytokine responses of intestinal epitheliallike caco2. I read that pbmcs will produce low readings using mtt assays. Good caco2 cell culture practices request pdf researchgate. Caco2 cells atcc, htb37 were routinely maintained in t75 flasks. The number of viable cells in each suspension was estimated by the pourplate count method, using potato. Do i have to trypsinize the cells, count cells, and seed on 96 well plate. To shorten and simplify the caco2 cell culture method, rapid protocols have been developed lentz. Elucidation of the impact of cell culture conditions of. However, it is widely reported in literature that culture. Vigorously resuspend caco 2 cell pellet with 4610 ml media for 1.
Nutritional biotechnology department, national agrifood biotechnology institute, mohali, india source. Phase contrast micrograph of caco2 cells grown in conventional 2d culture plates. Protocol example protocol for the culture of the caco2. Take out required amount of cell suspension from the flask using pipette and place into new flask. The human epithelial cell line caco2 has been widely used as a model of the intestinal epithelial barrier. Application of caco2 cell line in herbdrug interaction. Elucidation of the impact of cell culture conditions of caco. Using our low density ld protocol, caco2 cells retained a higher proliferation potential resulting in a cell population, which, reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a higher cell growing density natoli et al. Cell culture growth conditions,protein expression,rnai,stable cell transfection,transfection.
Culture surface area per well suggested working volume per well suggested cell seeding densitywell cat. Caco2 studies are incapable of simulating the changes of intestinal ph system since it is performed at constant ph conditions apical ph of 6. Use of caco2 cell monolayers to study drug absorption and. Protocol example protocol for the culture of the caco2 cell. Serumreduced and serumfree media for differentiation of caco2. Detailed protocols for handling the caco2 cells in the laboratory are provided. The experiment must include both the genetically modified caco 2 knockout cells and wild type caco 2 cells. Caco2 engineered control cells mtoxccp96 technical. Example protocol for the culture of the caco2 cell line on. Pdf the caco2 cell line as a model of the intestinal barrier.
Background information on ncipbcfhtb37 caco 2 cell line designations. Caco2 cell line, with or without collagen i alvetex scaffold plates and inserts page 2 of 7 methods preparation for 3d cell culture on alvetex scaffold 1. Protocol for polarizing caco2 cells in tissue culture inserts. Discussion on the variability problem of caco2 cell permeability is ongoing since there is not a standard protocol for cell culture, and attempts are being continued for the standardization of.
For transport studies, caco2 cells are seeded on polycarbonate membranes inside transwell cell culture chambers. Add required amount of prewarmed cell culture media to fresh flask. Propagation of caco2 2 4 add 8mls of accutase and return to incubator for 1015 minutes. Caco2 permeability background information cyprotexs caco2 permeability assay uses an established method that measures the rate of flux of a compound across polarised caco2 cell monolayers and. Example protocol for the culture of the caco2 cell line. Pdf caco2 cell permeability assays to measure drug. Following an overnight attachment period 24 h after seeding, the cell medium is replaced with fresh medium in both. This protocol is designed to assess drug transporter functionality in caco 2 cells. When polarized and confluent, the cell layer forms a continuous barrier between the upper and lower compartments apicalmucosal and basolateralserosal. The caco 2 cell culture protocol here described has been recently optimized in our laboratory, producing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of the intestinal enterocytes.
The difference in growth rates between animal and bacterial cells is such that a lowlevel contamination in an animal cell culture can quickly lead to bacterial overgrowth. Caco 2 rcb0988 cells18,19 purchased from the cell bank at riken bioresource. Briefly, caco2 cells, ecacc catalogue number 09042001. Lipofectamine 3000 5179% transfection efficiency and 2 fold protein expression improvement over lipofectamine 2000.
Discard the cell culture medium by inverting the slide and gently tapping it on a paper towel to remove the. Do anyone have any protocol for assays with caco 2. The experiment must include both the genetically modified caco2 knockout cells and wild type caco2 cells. Gene expression,transient transfection recommended transfection reagents plasmid dna transfection and cotransfection. The confluent monolayers are used between the 21th and 25th day after seeding. Caco2 wild type cell line mtoxp24 technical bulletin.
Due to this, a variety of cloned caco 2 cell lines has been established, and described in the literature. I will be growing the cells for 1 week and stimulating the cells for 2 days in 24well plates. In vitro intestinal coculture cell model to evaluate. The effects of serum fetal bovine serum, fbs in the culture medium and of the period of.
Thawing, propagating and cryopreserving of ncipbcfhtb37 caco 2 page 3 of 25 protocol for thawing, propagating and cryopreservingof ncipbcfhtb37 caco 2 atcc htb37 colorectal adenocarcinoma 1. The caco2 cell culture model is used to determine the absorption potentials of drug candidates and the transport and metabolism mechanisms of drugs and dietary chemicals. Caco2 cells in this image had been kept in culture for 21 days, allowing them to grow confluent and epithelialize. Determination of drug absorption parameters in caco2 cell. Due to this, a variety of cloned caco2 cell lines has been established, and described in the literature. Cells were seeded on to millipore millicell cell culture inserts 0. Cyprotexs caco 2 permeability assay uses an established method that measures the rate of flux of a compound across polarised caco 2 cell monolayers and from which the data generated can be used to predict in vivo absorption of drugs. Vigorously resuspend caco2 cell pellet with 4610 ml media for 1. Transepithelial electrical resistance teer measurements of. Please provide an overview of cyprotexs caco2 permeability assay. The caco2 cells are cultured to confluency, trypsinized and seeded onto a filter transwell insert at a density of 32,000 cellswell in dmem cell culture medium. This chapter will however, focus on describing how to handle and cultivate the original caco 2 cell line as obtained from cell culture collections like american type culture collection and the european collection of cell cultures.
Eagles minimum essential medium mem 450 ml bottle with. This method can be applied to the use of alvetex scaffold in 24 well insert. Reproducibility problems often reported in literature have been generally ascribed to different culturerelated conditions, such as the type of animal serum used, the supplements added to the culture. The effects of serum fetal bovine serum, fbs in the culture medium and of the period of cell culture on the barrier function and transporter activities of the monolayers were examined. Normally cultures kept at 37 c in a 5% co 2 95% air atmosphere.
Caco2 monolayer formation in transwells was assessed. This protocol differs from standard protocols mainly because caco2 cells are subcultured when they reach just 50% of confluence, instead of 80%, retaining a. The caco2 cell line is derived from a human colon carcinoma. The compound of interest, suspended in a physiological fluid, typically pbs, fig. In order to investigate the role of caco2 culture protocol might. Effects of butyrate on intestinal barrier function in a caco. Setup instruments tissue culture co2 incubator with humidity control liquid handler orbital shaker evom epithelial voltohmmeter fitted with planar. Cac, caco, caco2, caco 2 human caucasian colon adenocarcinoma generated by xstandard version 2.
This protocol is designed to assess drug transporter functionality in caco2 cells. The number of viable cells in each suspension was estimated by the pourplate count method, using potato dextrose agar eiken chemical, tokyo, japan. Surachai unchern laboratory practice on caco 2 cell culture. Although colonic in origin, in culture the caco2 cell differentiates and closely resembles mature intestinal absorptive cells.
Propagation of caco 2 2 4 add 8mls of accutase and return to incubator for 1015 minutes. To shorten and simplify the caco2 cell culture method, rapid protocols have. Discussion on the variability problem of caco 2 cell permeability is ongoing since there is not a standard protocol for cell culture, and attempts are being continued for the standardization of. Caco2 permeability background information cyprotexs caco2 permeability assay uses an established method that measures the rate of flux of a compound across polarised caco2 cell monolayers and from which the data generated can be used to predict in vivo absorption of drugs. Protocol page 1 example protocol for the culture of the caco2 cell line on alvetex scaffold in ell insert and ell plate formats it or without a collagen i gel layer introduction caco2 is a human. Caco2 cells were grown in tissue culture flasks, trypsinized by rinsing cells with phosphatebuffered saline and then treating with trypsinedta for 510 minutes at 37 c with 5% co 2 until. Cells were harvested with trypsinedta every 34 days and subcultured at 12 x 103 cellscm2.
Transport is measured in both directions apicaltobasal and basaltoapical across the cell monolayer, enabling an efflux ratio to be determined. Drug permeation of binary combinations of compounds across caco2 cell monolayers at 37c 103 6. In regards to fe, early studies of caco 2 cell monolayers docu. Pdf the human intestinal caco2 cell line has been extensively used over the last twenty years as a model of the. The caco 2 cell line is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, developed by the sloankettering institute for cancer research through research conducted by dr. Caco2 cells are widely used as an in vitro model for predicting human drug. The food and drug administration fda recognized the model system as useful in classifying a compounds absorption characteristics in the biopharmaceutics classification. Pdf caco2 cell permeability assays to measure drug absorption. Caco 2 permeability assay to investigate intestinal permeability. Cells are grown in a humidified atmosphere of 5% co 2 at 37 c.