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 JoVE Biology

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

1, 1, 1

1David Geffen School of Medicine, University of California, Los Angeles

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    Summary

    This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

    Date Published: 6/04/2008, Issue 16; doi: 10.3791/722

    Cite this Article

    Zhang, J., Khvorostov, I., Teitell, M. From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs. J. Vis. Exp. (16), e722, doi:10.3791/722 (2008).

    Abstract

    This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.

    Protocol

    Splitting human embryonic stem cells (hESCs) plated on mouse embryonic fibroblasts (MEFs)

    Usually a confluent hESC plate can be split 1:6 to 1:10, depending on the particular hESC line. The split plate will become confluent again 5-7 days after splitting.

    1. Two days before splitting, gelatinize plates using a 0.1% gelatin solution For a 6-well plate, add 2ml into each well and incubate plate in a 37°C, 5% CO2 tissue culture incubator overnight.
    2. Plate MEFs on gelatinized 6-well plates the day before you plan on splitting the hESCs. (NOTE: MEFs can be plated as many as 3 days earlier if needed. After ~ 3 days, MEFs become flatter and more spread-out and cannot sustain hESCs as well as fresher MEFs can.) Take a vial of g-irradiated or mitomycin C treated CF1 MEFs, containing 5-6 x 106 cells, from liquid nitrogen and thaw for 2 min in a 37°C water bath Wash cells once with warm MEF Culture Media in a 50ml falcon tube and resuspend in the final volume of warm MEF Culture Media. At 5-6 x 106 cells, this is enough to plate two or three 6-well plates.
    3. While the MEFs are being washed, take out the gelatinized plates, remove all solution from the wells and add 2.5 ml of MEF Culture Media per well.
    4. Add 0.5 ml of MEF suspension per well to achieve 3ml as a final volume in each well. Make sure MEFs are evenly dispersed and place plates back in the incubator overnight to settle.
    5. On the day of hESC splitting, prepare fresh or use < 2 week-old sterile collagenase IV solution at 1mg/ml. Remove all the media from the hESC wells you want to split, wash once with 2ml per well of warm 1×PBS, pH 7.4, and add 1ml of collagenase IV solution. Incubate at 37°C for 5-10 min. Take the 6-well hESC plate out of the incubator and add 1ml of ES media (without bFGF) to each well. Using the solution in each well, with a 1ml pipette suck up the media and blow the stem cells off of the plate. For each well this will take about 5 to 10 repetitions. Then, transfer the suspended hESCs into a 50ml falcon tube. Do this for all the wells being split and combine in one 50ml falcon tube. Pellet the hESCs at room temperature at 200g for 5 min and wash one time with ES media lacking bFGF.
    6. While the hESCs are being washed, take out the MEF plates, remove all media from the wells and wash once with sterile, warm 1×PBS, pH 7.4 then add 2.5 ml of ES media (now supplemented with 10 ng/ml bFGF) per well.
    7. Resuspend the pelleted hESCs in an appropriate volume of ES media, supplemented with 10ng/ml bFGF. (NOTE: When the hESCs are resuspended, they should be of roughly uniform colony size and shape. The resuspension volume depends on the splitting ratio.) Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated. Add 0.5 ml of hESC suspension per well to the MEF plates to achieve 3 ml as a final volume in each well. Visually check to make sure that the hESCs are distributed evenly before placing the plates back in the incubator overnight to settle. After plating, it will usually take a few days for colonies to take on their characteristic shape and border appearance.

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    Discussion

    This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. In the last step before plating, when the hESCs are resuspended, they should be of roughly uniform colony size and shape. Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated.Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state during culture.

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    Disclosures

    Acknowledgements

    Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.

    Materials

    Name Type Company Catalog Number Comments
    Knockout Serum Replacer (KSR) Reagent GIBCO, by Life Technologies 10828-028
    DMEM/F12 Reagent GIBCO, by Life Technologies 11330-057
    Non-essential Amino Acids Reagent GIBCO, by Life Technologies 11140-050
    GlutaMax Reagent GIBCO, by Life Technologies 35050-061
    DMEM Reagent GIBCO, by Life Technologies 11995-065
    FBS Reagent Clontech Laboratories 631107
    L-glutamine Reagent GIBCO, by Life Technologies 25030-081
    BME Reagent Fisher Scientific BP176-100
    bFGF Reagent R&D Systems 233-FB-025
    Collagenase IV Reagent GIBCO, by Life Technologies 17104-019
    Dispase Reagent Stem Cell Technologies 17105-041
    Penicillin / Streptomycin Reagent GIBCO, by Life Technologies 15140-122
    Gelatin Reagent Chemicon International ES-006-B
    Matrigel Reagent BD Biosciences 354277
    Oct-4 antibody Reagent Santa Cruz Biotechnology, Inc. SC-9081
    anti-h/mSSEA-4 Phyc–rythrin Conjugated Mouse IgG3 Reagent R&D Systems FAB1435P
    FITCI-conjugated antirabbit IgG Reagent Jackson ImmunoResearch 715-095-152

    References

    1. James A. Thomson, Joseph Itskovitz-Eldor, Sander S. Shapiro, Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall, and Jeffrey M. Jones Embryonic Stem Cell Lines Derived from Human Blastocysts Science 282, 1145-1147 (1998).

    2. Chunhui Xu, Margaret S. Inokuma, Jerrod Denham, Kathaleen Golds, Pratima Kundu, Joseph D. Gold & Melissa K. Carpenter Feeder-free growth of undifferentiated human embryonic stem cells Nature Biotechnology 19, 971-974 (2001).

    Comments

    5 Comments

    Hi, may I ask why you are using 10ng/micro lt bFGF. would you please provide the refrence (s) for that? Thank you very much Shima
    Reply

    Posted by: Shima B.April 7, 2009, 11:56 PM

    Hi Shima, Different protocols are varying in the amount of bFGF.  The range is between 4 ng/ml (Thompson lab recommendation http://ink.primate.wisc.edu/~thomson/protocol.html) and ²0 ng/ml (Stem Cell Research Center Rutgers University http://cord.rutgers.edu/stemcellcourse/documents/Laboratory_Manual_091508_optimized.pdf) We are following up the core facility recommendation and probably use a bit higher concentration of bFGF than it is critically requaired. At least it saves us from variation in the quality of bFGF between different manufacturers. There is nothing wrong if you try less bFGF. Just track down your cells behavior. Ivan
    Reply

    Posted by: AnonymousApril 8, 2009, 2:46 PM

    Hi,
    Just wanted to ask about some of the small es colonies in the video. The cells in that small colony appeared elongated and not the usual compact ES cells. Did the cells change as the colony grew and how was the trasncriptome (PCR) or the proteome
    Reply

    Posted by: prasad84 p.February 11, 2010, 12:18 PM

    Hi,
    Just wanted to ask about some of the small es colonies in the video. The cells in that small colony appeared elongated and not the usual compact ES cells. Did the cell morphology change as the colony grew and how was the trasncriptome (PCR) or the proteome (Immunofluorescence). And for using CF 1 conditioned media, how long can one store the CM and retain its ability to support undifferentiated growth?
    Reply

    Posted by: prasad84 p.February 11, 2010, 12:21 PM

    hESCs prefer to stay in clumps, and single cells will not survive in normal culture of hESCs. Therefore, the small colonies and big colonies might have some different characteristics as well. We do see sometimes the small colonies contain branched cells, especially at the border of the colonies. And this phenomenon is more significant in the small colonies cultured in the chemically defined media, such as Stempro. However, the small colonies will be either eliminated , or turning into more compact and smooth-border big colonies as cells grow.


    "how was the trasncriptome (PCR) or the proteome (Immunofluorescence)"
    I don't think this is related to what our paper shows.

    For the conditioned media, we can freeze it at -²0 for a couple of months.
    Reply

    Posted by: AnonymousFebruary 11, 2010, 2:27 PM

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