Scaleup quantities and volumes according to the number of cellswells to be transfected see table 1. A more convenient way to subculture these cells is to add a small amount of. The following protocols have been used successfully in our laboratories. Mdck nbl2 atcc ccl34 canis familiaris kidney normal. Mdck cells, an equal volume of medium was added after 48 hours and a complete medium change was performed on day 4 or 5. On day 0, dilute the matrigel matrix to 5 mgml with icecold mdck complete cell culture medium. Haredy, nobuyuki takenaka, hiroshi yamada, yoshihiro sakoda, masatoshi okamatsu, naoki yamamoto, takeshi omasa, hisao ohtake, yasuko mori. Mdck cells were nucleofected using the cell line nucleofector kit l, program a 24. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin pna. The adherent mdck cells were adapted to suspension culture by the serum reduction and serial passaging. Mdck cells are used for a wide variety of cell biology studies including cell polarity, cellcell adhesions termed adherens junctions, collective cell motility, as well as responses to growth factors. Product information mdck nbl2 cells cls cell lines service. Pdf madindarby canine kidney mdck cells can grow in synthetic medium. Serumfree suspension culture of mdck cells for production.
Id like some tips about the culture of mdck cells nbl2, so its the parental. The study protocol was approved by the ethics committee of the national institute of infectious diseases, japan. The appropriate cell lines to be used for isolation of influenza a or b viruses depend on the clinical information and the host of origin. C 0 store at 4 genjet in vitro dna transfection reagent. Heterogeneity of the mdck cell line and its applicability. Jcc 22511 from james nelsons lab, cells from lance kams lab materials. Cells are usually subcultured by prewashing with pbsedta and strong trypsin solutions. To determine the viral growth curve in transduced mdck cells and parental mdck cells, the cells were infected with pr8 virus at a moi of 0. Mammalian cell tissue culture techniques protocol abcam. Louis, mo, usa introduction vaccines developed from viruses grown in mammalian cell cultures are of major importance in both human and animal health. Mdck cells adhere very strongly to tissue culture plastics. Semiperfusion cultures of suspension mdck cells enable high cell. Correlation between ecadherin interactions, survivin. Cell culture and transfection of mdck cells mdck strain ii cells were cultured on plastic or to obtain polarized cell monolayers, on transwell polycarbonate.
Mdck crispr transfection kit for kidney cells, ccl34. Cells were nucleofected with program a24 or a024and 5 g of pmaxgfp. Protocol page 1 culture of the canine idne epithelial mdc cell line on alvetex scaffold in ell insert and ell plate formats introduction mdck is a canine kidney cell line isolated in the late 1950s from normal cocker spaniel tissue 1. Results and discussion several serumfree medium formulations, including medium k1 for mdck cells4, were examined for their ability to support mdbk and mdck cells in both static and dynamic culture systems. Useful strains, cell culture, and cell line maintenance. Mdck cells are the preferred cell line for isolation of human influenza viruses from clinical specimens. The cell viability of the parental and the transduced mdck cells expressing each shrna showed at least 90% throughout the culture period.
A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture counting cells in a hemocytometer. Grow mdck cells in tissue culture flasks t75 with conventional basal medium plus fetal bovine. Steps 1014 should be performed for each sample separately. High yield production of influenza virus in madin darby. The purpose of this paper is to provide a protocol for adapting cells to a serumfree environment. 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.
Stiffness of mdck ii cells depends on confluency and cell. Culturing three dimensional mdck cells for analyzing. Guidelines are described for management of a successful program in fish cell culture. The following protocol is given for transfecting mdck in 6well plates, refer to table 1 for optimal cell number per well per culture vessels surface area. Mdck cell morphological change on genipintreated matrigel. Three discrete cell morphotypes were identified using light microscopy. Protocol for reverse transfection of adherent and suspension cells in a 24well plate note. The invention provides a method for culturing mdck medindarby canine kidney cell proliferation recombinant h5n1 subtype avian influenza virus comprises the following steps. How to count and calculate the number of cells from a stock flask or culture dish counting cells in a countess ii. The following is a general guideline for culturing of cell lines. Proliferating cultures the cell culture flasks, 2xt25, come filled with cell culture medium.
The protocol is optimized for transfection in a 24well plate format. Singlecell clones have been established from the mdck cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Conversion of mdck cell line to suspension culture by. Mdck cells have been used to study processing of beta amyloid precursor protein and sorting of its proteolytic products.
Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Optimum transfection condition is possibly chaged by passage number and culture condition. Cell viability %pi negative is around 83% after 24h postnucleofection. Cell culture avoids an egg allergy contraindication and, as a closed system, carries a reduced risk of microbial or chemical contamination. Hi, folks, please help me figure out whats going on with my mdck cells. Cell culture basics handbook thermo fisher scientific. Pdf growth of madin darby canine kidney epithelial cell mdck. Dulbeccos modified eagle medium, low glucose, with lglutamine, with 110mgl sodium pyruvate, without sodium bicarbonate.
Plus mdck animal componentfree medium plus mdck acfm is a componentdefined cell culture medium, formulated without any human or animalderived components. Plus mdck acfm is designed to support the serumfree growth of mdck cell line of interest in the areas of virology, virus production, and biotechnology. Mdck ii madindarby canine kidney is a subclone derived from the heterogenous parent line mdck ecacc catalogue no. To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon. Cell seeding 1 rinse mdck available from atcc in 10cm dish briefly with pbs.
Culture of the canine kidney epithelial mdck cell line on. In the steady state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments and in response to external. To tranfect mdck cells in 24well plate, follow optimum condition for transfection and transfection procedure. The suitability of mdcksiat1 cells, engineered to express increased levels of. Crisprcas9 transfection protocol for 24 well plate. Corning matrigel basement membrane matrix for 3d culture. The adherent mdck cells were adapted to suspension culture by the serum reduction. Additionally, prepare the virus growth medium for virus propagation as noted on the product sheet.
Cell culture guidelines the following is a general guideline for culturing of cell lines. Results transfection of mdck cells with human siat7e and its effects on cellcell adhesion and cell spreading anchoragedependent mdck cells exhibited changes in cellcell adhesion and cell spreading behavior following the incorporation of the human siat7e gene as shown in fig. A reprocell brand culture of the canine kidney epithelial mdck cell line on alvetex scaffold in well insert and well plate formats introduction mdck is a canine kidney cell line isolated in the late 1950s from normal cocker spaniel tissue1. The confocal microscopy images show the localization of cell junction protein e. Isolation of influenza viruses in mdck 33016pf cells and. This protocol will describe plating of mdck cells in collagen i matrix, which is a major component of the extracellular matrix. Protocol culture of the canine idne epithelial mdc cell. Over the first adaptation period, the content of xeno medium was increased by 10 % steps.
Madindarby canine kidney mdck cells are a model mammalian cell line used in biomedical research. Mdcksiat1 cells show improved isolation rates for recent. This protocol does not replace optimization experiments. All further steps are described in the subculture section. In addition, the performance of vero, mdbk, and mdck cell growth on the nunclon delta surface will be compared to. Adapting vero, mdbk, and mdck cells to a serumfree. 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. Serumfree systems for mdbk and mdck epithelial cells. Here, we present protocols for the culture of mdck cells on both 6 well insert and. It is one of few cell culture models that is suited for 3d cell culture and multicellular. Viral growth medium is usually supplemented with a lower. Comparison of suspension mdck cells, adherent mdck cells.
Growth of mdck cells growth of mdck cells objective due to their adhesive properties, both intercellularly as well as to surfaces, mdck cells possess several challenges when they are to be used in large scale cell culture. Threedimensional morphogenesis of mdck cells induced by. The mdck cell line can be easily adapted to excell mdck using a direct adaptation method or a sequential. Thaw corning matrigel basement membrane matrix overnight by submerging the vial in ice in a 4c refrigerator before use.
We have found that modification on protocols may be required to obtain optimum results with plus mdck acfm. The mdck cells can either be on tissue culture plastic, or, for higher efficiency, on permeable cell culture inserts of 25mm diameter and 3. This protocol shows optimum transfection condition using hilymax in mdck cells. Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. An mdck cell culturederived formalininactivated influenza virus wholevirion vaccine from an influenza virus library confers crossprotective immunity by intranasal administration in mice ahmad m. Using the conventional, recommended reference viruses, these vaccines still originate from eggderived. Detach the mdck cells with trypsinedta and stop the. In advance, prepare the cell growth medium for growing the host cell line. Values are suitable for easy for transfect cell lines. Several companies are currently developing cell culturebased influenza vaccines and the first of those vaccines, produced in mdck and vero cells, have been licensed and distributed as interpandemic trivalent and pandemic h 1 n 1 vaccines. If transfection efficiency is low by followed this protocol, refer to. Culture mdck cells to around 70% confluence in complete growth medium. Clinical efficacy of cell culturederived and eggderived.
The adherent mdck cells ccl34, atcc were cultivated on cytodex tm 3 microcarriers 3 gl, ge healthcare in dmem gibco supplemented with 10% vv fetal bovine serum fbs, gibco or a proprietary serumfree medium developed by the authors mdcksfm1. Supplemental assay method for titration of canine adenovirus in canine kidney cell culture uncontrolled copy 2. Cell culture protocols thermo fisher scientific jp. The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. General details of cell culturing and subculturing. Finally, mdck cells adapted to rapid growth in suspension media do not require microcarriers, enzyme treatment for subculture, or protein supplements to promote adhesion to a substrate 31. This application note describes several alternatives to reduce the difficulties usually encountered in the culture of these. To induce 3d culture formation of mdck cells, extracellular matrix components need to be provided.
Mdck and tssrc mdck cell phenotypes and the expression of survivin in various culture environments. Intracellular traffic of fcrn in epithelial mdck cells. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Just mix nucleic acids with the reagent to form complexes and. Becton dickinson labware, lincoln park, nj, which fit into 35mm wells. This method can be applied to the use of alvetex scaffold in 12well. The optimal transfection conditions for mdck cells are given in the standard protocol described below. How to count and calculate the number of cells using an automated cell counter. Mdck is good morphology condtion in dmem when passaged in our lab shows slightly different and bigger in dmem prepared by us. This strain is thought to be derived from the distal tubule or collecting duct of the nephron. Once matrigel matrix is thawed, swirl vial to ensure the material is dispersed. The adherent mdck cells ccl34, atcc were cultivated on cytodex tm 3 microcarriers 3 gl, ge healthcare in dmem gibco supplemented with 10% vv fetal bovine serum fbs, gibco or a proprietary serumfree medium developed by the authors mdck sfm1.
161 1286 434 805 149 552 263 745 512 675 1243 114 1415 236 164 244 1301 972 987 75 988 467 244 859 1476 275 1412 1191 750 779 749 696 1301 281 650