JoVE Biology

Trypsinizing and Subculturing Mammalian Cells

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1Molecular Pathology Laboratory Network, Inc

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    As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

    Date Published: 6/12/2008, Issue 16; doi: 10.3791/755

    Cite this Article

    Ricardo, R., Phelan, K. Trypsinizing and Subculturing Mammalian Cells. J. Vis. Exp. (16), e755, doi:10.3791/755 (2008).


    As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.


    The complete text protocol for this experimental approach is available in Current Protocols in Cell Biology.

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    The authors have nothing to disclose.



    the video isnot playing

    Posted by: AnonymousJuly 6, 2008, 10:23 PM

    how to prepare cell growth curve of  cancer cell line

    Posted by: AnonymousSeptember 11, 2008, 3:10 AM

    can i know the passaging protocol for MDA MB ²31 Breast cancer cells.

    Posted by: AnonymousSeptember 18, 2008, 12:10 PM

    sir can u tell me that how to transfer cell ( frozen into liq. nitrogen) into media and that will be the mother culture so in that also we have to do passaging , means we have to add HBSS and Trypsin and EDTA. and how to count cells. thank you waiting for your reply.... sam A Masih

    Posted by: AnonymousSeptember 24, 2008, 12:24 PM

    Hi, For subculturing suspension cells, what is the normal routine? is it better if changed everyday or only after the media contents turns trubid and yellowish colour? Regards Kamal

    Posted by: AnonymousOctober 22, 2008, 7:45 PM

    Hello kamal bro..
    paras sir le bhannu bhayeko ta tyahi ho kya re...

    Posted by: AnonymousJuly 28, 2009, 7:18 AM

    Thank you

    Posted by: AnonymousMarch 24, 2009, 4:09 PM

    Could you elaborate on how to culture cancer cells? The precautions to be taken and when to subculture them. Thank you

    Posted by: Preethi S.April 2, 2009, 12:37 PM

    I can see the video

    Posted by: AnonymousJune 22, 2009, 12:25 PM

    We were having a technical issue when you posted your comment. Please let us know at if you are unable to view this video.

    Posted by: AnonymousJune 23, 2009, 7:52 PM

    every time I trypsinize my BBe cells - they clump together (even I don't shake the flask at all..)
    how can I prevent it ? because the clumps are super strong and I can not brake them down again.. I do something wrong?
    I wash ²x with PBS (37C) and 5-15min trypsinize the cells and add RPMI 1640 media carefully to it and mix gently..

    can you help me?

    thank you

    Posted by: Alexandra K.April 12, 2010, 5:19 PM

    thank u

    Posted by: AnonymousOctober 21, 2010, 6:32 AM

    How long dŒs it take for an MCF-7 cell lines grow confluent? What's the best media for culturing this cell?

    Posted by: AnonymousSeptember 22, 2011, 11:44 AM

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