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Millipore Technical Publications - Frequently Asked Questions


Why are my mouse embryonic stem cells differentiating and what preventative measures do you recommend?


Causes of DifferentiationRecommendations
Incorrect concentration of mLIF ESGRO Supplement (mLIF) should be used at the recommended concentration of 1000 units/mL in ES cell media.
Expiry date of ESGRO Supplement Always check the date of each batch prior to use. Medium should be less than 4 weeks old as glutamine by-products could be toxic to ES cells. ES cells should be fed with fresh media every 2–3 days or when media discolors.
SerumNew batches of Fetal Bovine Serum should be tested for the effect of inducing differentiation. ES cell medium usually requires a serum concentration between 10% and 20%. We recommend the use of EmbryoMax ES qualified serums.
Lack of passagingES cells should be passaged every 2–3 days as frequent passaging removes differentiated cells. Only undifferentiated ES cells will survive frequent passaging. Refer to the attached figures for illustrations of ES cell confluency and differentiated ES cells.
DisinfectantsDisinfectants such as Roccal or Lysol should be avoided in the Tissue Culture Room and incubator where ES cells are cultured. Some disinfectants have been suspected to cause differentiation by use in water baths and aerosols created by spray wiping. The use of 70% ethanol to clean tissue culture surfaces is recommended.
Feeder cellsIf differentiation cannot be controlled using ESGRO Supplement with gelatinized plates, it may be necessary to culture ES cells on a feeder layer. ESGRO Supplement should be added at a concentration of 1000 units/mL to assist in maintaining undifferentiated ES Cells.
Incubator settings Ensure that the incubator readings are correct at 37°C and 5% CO2.
Rate of growth of ES cellsSlow growing ES cells will be most likely to undergo differentiation. Increase serum concentration if cells are not growing quickly enough.
Concentration of ES cellsLow confluency of ES cells can result in differentiation. ES cells should be plated at a minimum density of
1x106 cells / 100 mm dish. Refer to the attached figures for illustrations of ES cell confluency.
GelatinIt is preferable to use cell culture grade gelatin at all times (even if using feeder layers) as gelatin minimizes surface differences on the tissue culture plates. EmbryoMax ES Cell qualified 0.1% gelatin solution is recommended
Low level of contaminationRegular use of Pen/Strep in media can often mask a low level contamination with agents such as mycoplasma. It is recommended to routinely test your ES cell lines for mycoplasma contamination on a periodic basis.
MediaMedia should be less than 4 weeks old as glutamine by-products are toxic to ES cells. ES cells should be fed with fresh media every 2-3 days or when media turns yellow.

 
Optimal confluence of ES cells (10x).Over confluent ES cells (10x).


ES cells at a density ready for passage (10x).ES cells plated at low density (10x). Cells require
another passage to increase individual colony numbers.


ES cells plated at too low density with fibroblasts
(10x). Cells require another passage to increase
individual colony numbers. Note the darkened areas
in the center of each colony where ES cells are dying.
Highly differentiated ES cells (25x). Note the loss of
discreet ES cell colony border, the formation of
cobblestone-like cells and ES cells differentiating
into fibroblasts that extend outwards from the colony.
It is possible to attempt to rescue this colony by passing
several times, however it is not usually recommended.
Highly differentiated ES cells - plated at too
low density (10x). Note that these cells are beyond
rescue by passaging.

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