Mef not only provide a substrate for pluripotent stem cells to grow on, but also secrete critical growth factors to maintain stem cell pluripotency. Transfer cellular suspension to t75 flask or 10cm plate and add 615ml mef media. Isolation and handling of primary mouse embryonic fibroblasts mefs accompanying protocol to mouse embryonic stem es cell culture basic procedures isolation of embryonic fibroblasts embryonic stem cells are usually grown on a layer of mitotically inactivated primary mouse embryonic fibroblasts to promote growth and prevent differentiation. Nia biospecimen best practices series induced pluripotent. Immunofluorescence is a powerful tool for elucidating the complex signaling events that underlie. Induced pluripotent stem cells ipscs have the capacity to give rise to differentiated progeny representative of all three germ layers of the body. Transfer cellular suspension to t75 flask or 10cm plate and add 615ml mef.
Cell derivation cell culture cell characterization frozen, fully characterized ipscs feederdependent and feederfree antibodies and gene expression pro. Mouse es cell culture and manipulation protocols encode. Cell culture guidelines the following is a general guideline for culturing of cell lines. Isolation and handling of primary mouse embryonic fibroblasts. Preparation of mouse embryonic fibroblast cells suitable for jove. Transfer the stem cell culture medium wash to the conical tube containing the cells. Addition of medium containing serum will inhibit further trypsinization. In addition to these protocols, we highly recommend stembook, which is publishing a growing list of methods to generate induced pluripotent stem cells and differentiate them into various lineages. Mef medium should contain penicillinstreptomycin when preparing initial mefs i. Transfer the 15 ml of cell suspension to a t75 tissue culture flask. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Jul 05, 2016 to obtain cells for culture, carefully place a 5 ml pipette through the supernatant and pipette up the pellet along with 0.
Mouse embryonic fibroblast cell culture and stimulation ncbi. Although bme and mtg are extensively used as antioxidant supplements for stem cell culture and differentiation 78 9 101112, their effect on cardiac lineage specification has not been. When determining mef density, thaw mefs and plate at varying densities between 6 x to 5 x 105 cells per well. Pdf isolation and handling of mouse embryonic fibroblasts. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. First, an expanding culture is washed in pbs to remove residual. Transfer the detached cell aggregates to a 15 ml conical tube.
Approximate growth surface areas and recommended mef cell number for cell culture. Trypsinize cells and resuspend cell pellet in cold freeze medium at twice the desired final cell. Culturing bg01v human embryonic stem cells in mouse embryonic fibroblast mef conditioned media the protocol below has been used with the bg01v human embryonic stem cell line. Pipet cells up and down in the culture medium to disperse the cell clumps. Please refer to more detailed protocols in the literature before starting the experi ments. Fetuses should be counted and dissected from the uterus. Mouse embryonic fibroblast cell culture and stimulation. When the cell layer has loosened, add an equal volume of mef medium and mix by trituration to produce a singlecell suspension. Center for ips cell research and application cira institute for integrated cell material sciences icems kyoto university. Our scientists screen a large number of antibodies and recommend only those. Human embryonic stem cell culture basement membranes are continuous sheets of specialized extracellular matrix that are found at the dermalepidermal junction, at the base of all lumenlining epithelia throughout the digestive, respiratory, reproductive, and urinary tracts, and that underlie parenchyma of endocrine and exocrine glands. Passaging hues human embryonic stem celllines with.
After testing your components, thaw a fresh vial of our mef and plate them into a new flask with medium and fbs that you have tested separately and found to be free of contamination. Mef medium total volume 500 ml amountfor500ml product partnumber 440ml thermoscienti. Induced pluripotent stem cell culture protocols sigma. This protocol was adapted from previously published studies, lai et al. How to count and calculate the number of cells from a stock flask or culture dish. Preparation of mouse embryonic fibroblast cells suitable for. Is it okay if lglutamine is already added to the dmem like the media mentioned in the preceding paragraph.
Feederdependent culture of human embryonic stem cells hescs. Add 1 ml of warm growth medium to cells and, using cell scraper, gently dislodge cells from plate. Icc and if protocol multicolor immunostaining optional step to examine the codistribution of two or more different antigens in the same sample, use a double immunofluorescence procedure. Optimized protocol for mouse embryonic fibroblasts mef. Note this protocol only gives an outline for the isolation and culture of primary mef.
Place the appropriate amount of cellmedia mixture into each well of the previously prepared mef plate to achieve the desired split ratio. Feeder dependent mef culture protocol collagenase passaging 32. Gelatin coating procedure to prepare gelatincoated tissue culture dishes, distribute a thin layer of 0. All cell culture incubations are performed in a humidified 37c, 5% co2 incubator. Human induced plutipotent stem cell ipsc handling protocols. Rock plates gently back and forth, sideways and diagonally to achieve uniform cell distribution. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Moreover the elisabased system used to evaluate cytokine. Rapid generation of mature hepatocytelike cells from. Mouse embryonic stem cell culturing protocols coriell institute. Browse our database of protocols for cell culture, nucleic acid, protein analysis, rna and dna applications, cloning, cell analysis, and drug discovery research. Breseagen protocol human embryonic stem cell protocols. In addition to these protocols, we highly recommend stembook.
Preparation of mouse embryonic fibroblast cells suitable. Stem cell culture medium kosr mef virus testing was completed for this cell line using this lot. Mouse embryonic fibroblast mef cells are required to support the growth of undifferentiated mouse or human es cells, ips cells, thus they are also called feeder cells. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. These involve growing cells on mouse embryonic fibroblast feeder. Before hepatogenic differentiation, cells were passaged using matrigelcoated feederfree culture. Add more mef culture medium to the tube containing the remaining tissue.
Wicell recommends that stem cells should be thawed and established in the. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary. Optimal culture conditions for each pluripotent stem cell line must be. This can be performed either simultaneously in a mixture or sequentially one antigen after another. Center for ips cell research and application cira institute for integrated cellmaterial sciences icems kyoto university. Passage cells by trypsinizing see support protocol, steps 1 to 4. Mouse embryonic feeder cell protocol thermo fisher scientific. Take up 12 ml stem cell culture medium in a 5 ml pipette and add it to the. Breseagen protocol human embryonic stem cell protocols this material was cultured and frozen using bresagens protocols.
Preparation, culture, and immortalization of mouse embryonic fibroblasts. The mefs isolation procedure presented here enables the establishment of a standardized culture condition for hescs and hipscs. Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem es cells. Briefly rinse the cell layer with 1xpbs scrr2201 solution to remove all traces of serum, which contain trypsin inhibitor. Mouse embryonic fibroblasts mef reagents and supplies item vendor catalog number knockout dmem, high glucose gibco 10829018. F6 es cell clones revised on 21909 cell line information jm8 derived from c57bl6n mice and the subline jm8. Brand amountfor200ml product partnumber thermoscienti. Add 20 ml mef culture medium see recipe below to inactivate the trypsin, and pipet up and down several times. Embryonic fibroblast mef cells in order to to keep them pluripotent. Feeder dependent mef culture protocol collagenase passaging. Mef feeder layer page 4 of 6 form 006 rev f061516 5. Mef cell culture instructions all media and reagents used in the culture of this product should be warmed to 37c before use.
Here, we provide a protocol to derive mefs and describe the method to inactivate the cells using mitomycin c treatment. This line of mes cells has been culture adapted to grow without mef feeders but the procedures. Numbers can vary between plastic ware from different suppliers. Mef cf1 atcc scrc1040 mus musculus the cell line was. Basic pluripotent stem cell culture protocol stembook. Preparation, culture, and immortalization of mouse. A comparative study of protocols for mouse embryonic stem. Cell culture protocols thermo fisher scientific au. There is a wide range of mef plating densities used by researchers for mouse and human pluripotent stem cell culturing. Immunocytochemistry and immunofluorescence protocol. Isolation and handling of primary mouse embryonic fibroblasts mefs accompanying protocol to mouse embryonic stem es cell culture basic procedures isolation of embryonic fibroblasts embryonic.
Human embryonic stem cell culture basement membranes are continuous sheets of specialized extracellular matrix that are found at the dermalepidermal junction, at the base of all. 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. You need to use gelatincoated tissue culture dishes in order to facilitate and increase es cell adhesion. Mef medium should contain penicillinstreptomycin when preparing. Mef cell isolation, culture, freezing, thawing and mitomycin c treatment himmelbauer lab, maxplanckinstitute for molecular genetics procedures for isolation of mef cells, culturing, freezing, thawing and mitomycin c treatment.
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. The embryos should be weighed andor measured to ensure that they are at the predicted gestational stage. Perform all activities under aseptic culture conditions. Place the appropriate amount of cell media mixture into each well of the previously prepared mef plate to achieve the desired split ratio. In this communication we describe a protocol for the isolation of mefs from day. Culturing jm8 and jm8 university of california, davis. Mouse es cell culture r1 mouse es cell line from p33 onwards was cultured following the standard protocol. Es cells were first derived in 1981 from a mouse embryo nature 292. Transfer the board or pad with the mouse to the tissue culture hood.
Genetically manipulated transgenic and genetargeted mouse models are indispensible tools used in defining the role of genes in development and organ function. Culture of mouse embryonic fibroblast mef cells represents a powerful system to. Human embryonic stem cells hescs are generally maintained on a layer of inactive murine embryonic fibroblast mef feeder cells. Every other day, aseptically remove spent medium from mef culture and replace with an equivalent volume of fresh mef medium. Take up the medium and transfer it into each subsequent well to collect cells. Add 1 ml of warm growth medium to cells and, using cell scraper, gently. Aliquot 20 ml per 50 ml conical centrifuge tube and freeze. Suggested concentration antibiotic mouse es cells human es cells neomycin 200 gml 50150 gml puromycin 1 gml 0.
Maintenance of mouse embryonic stem cells in culture. At cell signaling technology cst, our goal is to provide highly specific antibodies that yield strong, specific signal with minimal background. This protocol was based on the method of todaro and green 1963 and. Examine the culture medium and cells for any contamination. After testing your components, thaw a fresh vial of our mef and plate them into a new flask with medium and fbs that you have tested separately and found to be free of. Successful hpsc culture requires the recreation of the in vivo stem cell microenvironment, or niche, which includes growth factors, celltocell interactions and cell to matrix adhesions. When the cell layer has loosened, add an equal volume of mef medium and mix by trituration to produce a single cell suspension. Pdf primary mouse embryonic fibroblasts mefs are the most commonly used feeder.
Preparation, culture, and immortalization of mouse embryonic. Mouse embryonic fibroblasts mefs have been used as feeder cell layers for the culture of embryonic stem cells escs since the first mouse escs were derived. Set up timed pregnancies, fetuses should be harvested between day 12. Related protocols sc protocol 00002 mouse embryonic feeder cell protocol. Embryonic stem es cell culturemouse embryonic fibroblast. Please note that the use of other psc lines may require modifications of this protocol. Mouse embryo fibroblast mef feeder cell preparation. Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal hues human embryonic stem cell culture. Moreover the elisabased system used to evaluate cytokine production by feeder cells is a useful indicator of the quality of the mef derived conditioned media. Cells should be passaged when the culture reaches 95% confluence. Wicell recommends that stem cells should be thawed and established in the conditions in which they were initially frozen prior to transfer to alternate culture platforms. Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts mefs, preparation of conditioned medium. Nyscf sf ips trainingmanualv3nodate new york stem cell. Mef plating density there is a wide range of mef plating densities used by researchers for mouse and human pluripotent stem cell culturing.
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