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APPENDIX
Troubleshooting
The table below lists some potential problems and solutions for culturing MSCs.
Problem
Low cell recovery
rate
Slow cell growth
Cause
Solution
Thestorageconditiondoes
notmeettherequirements
Purchaseareplacementandstoreinliquid
nitrogenforlongͲtermpreservation.
Thawingofthecellstakestoo
long
Thawcellsfornomorethan3minutes.
Cellsareincompletely
recoveredafterthawing
Afteraspiratingoffmedium,washthetube
withculturemediumtwiceandtransferallof
thecellstothedish.
Cellsarehandledroughly
Careshouldbetakentoavoidintroducing
bubblesduringpipetting.Alsoavoidvortexing
andhighͲspeedcentrifugation
MediumisnotpreͲwarmed
Warmmediumto37°Cbeforerecovery.
Mycoplasmacontamination
Discardthecellsinquestionanddisinfectthe
laboratoryenvironmentbeforerecoveringthe
nextbatchofcells.
Overdigestion
WashthecellswithPBS2Ͳ3timestoremove
serumpriortotrypsinization(serumwillinhibit
thefunctionoftrypsin).
Controlthe digestiontime.
Cell aging
Platingdensityistoolow
Increasetheplatingdensity.
Inappropriateserumand
medium
UseCyagentailorͲmadeculturemedia. If
otherserumandmediaproductsareused,
pleaseperformvalidationtoensure
compatibility.
Deadcellsarenotremoved
promptly
Changethemediumnextdayafterrecovery
toensureremovalofalldeadcells.
CellContamination
Discardthecellsinquestionanddisinfectthe
laboratoryenvironmentbeforerecoveringthe
nextbatchofcells.
Platingdensityistoolow
Somestemcellscansecretefactorsto
supportcellgrowth.Therefore,acertain
degreeofplatingdensitymustbemaintained;
otherwise,itwillleadtocellproliferation
slowdownandcellaging.
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