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Culturing Cells
! Subculture / splitting cells process of dividing cells into new medium and flask/container.
" Cells must be split to provide growing space
" Frequency of subculture depends on doubling rate
" Split too often or seed to lightly and cells may be lost
" Split too late or dense and cells will slough off, die, exhaust medium and even change gene expression
" Allowing cells to come to high confluency often results in genetic drift and a significant change in population
! Subculture involves:
" Removing medium and washing cells with isotonic solution (typically PBS)
" Dislodging cells from plastic (if monolayer)
Typically done with enzyme trypsin
" Adding complete medium back to cells
Serum includes trypsin inhibitor (a protein) to stop
digestion of cell surface proteins
" Seed new dishes with a fraction of the original cells
Observing Cells
! Check Media Color and Clarity
! Microscopic Check
" Confluence
" Morphology
" Contamination
Media Color
pH indicator in media
! Most cells grow best at pH
7.4
Yellowish medium is acidic and can indicate an overgrown culture, bacterial
contamination or too much CO2 in the incubator
! Purplish medium is alkaline and can indicate a sparse and non growing
culture or mold contamination or not enough CO2
Media Clarity
! Look for cloudiness in the media.
Could indicate contamination or a grossly overgrown
culture.
Morphology
! Clumped Cells. Cells not evenly dispersed, Peeling Cells
" Adherent monolayers lifting off surface
Maintaining Cells in Culture
" To maintain cells in culture the cells must be:
Fed supplied with fresh media
Split provided with space to grow
Frozen stored for future use
Maintaining Cells in Culture
! The faster cells grow the more often they must be fed and split.
Feeding Cells
" Removing old media and replacing with fresh media.
Splitting Cells
! When cells reach 60 80 % confluency they need to be passed or split into new culture vessels.
! Splitting Includes
" Washing cells
" Removing cells from cell surface

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" Adding Fresh media and transferring cells to new flasks
! Splitting refers to the fraction of cells of the original culture transferred into a new culture dish.
" If one half of the cells are maintained then it is a 1 to 2 (1:2) split
" If you use one-tenth of the cells (1 ml out of 10 OR ! ml of 5 ml) you have a 1:10 split.
" Do NOT use a CV=CV calculation!
" Final volume of media in the new flask does not influence the calculation.
Basic Protocols
Trypsinizing:
! Remove media from flask
! Wash with 5 mL CMF-PBS
! Remove CMF-PBS
! Add 1 mL 1X Trypsin to each flask
! Let stand 5-8 minutes
! CMF-PBS
! Calcium and Magnesium Free Phosphate Buffered Saline
Trypsin
" Protease
" Enzyme that breaks down proteins
Passing Cells
1:2 Split
! Add 9 ml growth media to flask with trypsin.
! Pipette cells up and down to mix.
! Add 5 ml of cell suspension from original flask to each of the new flasks and pipette up and down to mix cells.
1:5 Split
! Add 9 ml growth media to flask with trypsin.
! Add 3 ml of media to each of the new flasks you wish to seed.
! Add 2 ml of cell suspension from original flask to each of the new flasks and pipette up and down to mix cells.

Aseptic Technique see culturing protocol
Sub Culture Cells see culturing protocol
Key Points
! Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the
culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+
! Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage
surface receptors.
! Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.

Common Mistakes
! Letting stock cultures overgrow because you do not have time to split them
Using cultures that are obviously the wrong density for experiments
Using old media instead of preparing fresh media
Freezing and Thawing Cells
! Long term storage must be performed to provide a stock of healthy, low passaged cells.
! Cells should be frozen in log phase and placed in suspension to maintain viability.
! Store banked cells at -180
o
C (liquid nitrogen)
! Cells have a limited lifespan while frozen and partial thaw can kill the cells
! 10% glycerol or 10% dimethylsulfoxide (DMSO) acts as a cyropreservative in complete media
! Rate of freezing must be slowed to avoid ice crystals from lysing cells. 1o per min is ideal. ~4 hrs to get LN2 temp
from room temp. Use commercial freezing container or tight fitting Styrofoam holder.
! Thawing Cells should be thawed as quickly as possible without exposing cells to undue heat
" Immerse vial in 73
o
C water bath and gently shaken until thawed

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" Optionally centrifuge (400 x g for 5 min) to separate cell from DMSO
" May directly add thawed cells to T25 flask.
" DO NOT leave cells in DMSO containing solution longer than necessary. Once cells have plated, exchange
media. DMSO can have unwanted effects on cells.