this video will show you how to passage pscswith edta in essential 8™ medium on vitronectin coated plates. the following protocol uses a 6-well plate.if you’re using another type of cell culture vessel, please refer to table 2 on the writtenprotocol for volumes there are three major differences that you’llobserve with cells cultured in essential 8™ medium on vitronectin compared to other feeder-freesystems such as mtesr and stempro hesc sfm: • cells are typically passaged ~24 hourssooner than they would be with other feeder-free media.• passaging should take place when cells are at ~85% confluency. if cells are passagedwhen they are more than 85% confluent, the
health of the cells and final cell yield maybe compromised. • cells must be passaged in edta. collagenaseand dispase are not recommended. cells will reach optimal confluency typicallyevery four to five days. you should also split cultures or passagecells when psc colonies become too dense or too large or show increased differentiation; the split ratio can vary, although it’sgenerally between 1:2 and 1:4 for early passages and between 1:3 and 1:12 for established cultures.occasionally, cells will grow at a different rate and the split ratio will need to be adjusted. a good rule is to observe the last split ratioand adjust the ratio according to the appearance
of the psc colonies. if the cells look healthy,and the colonies have enough space, split your cultures using the same ratio. if thecolonies are overly dense and crowding, increase the ratio; if they are sparse, decrease theratio. newly derived psc lines may contain a fairamount of differentiation through passage 4. it’s not necessary to remove differentiatedmaterial prior to passaging. by propagating/splitting the cells, the overall health of the cultureshould improve throughout the early passages. prepare 0.5 mm edta by combining 50 âµl ofultrapureâ„¢ 0.5 m edta, ph 8.0 with 50 ml of dpbs without calcium and magnesium. filter,sterilize the solution. and store at room temperature.
pre-warm complete essential 8â„¢ medium andvtn-n-coated culture vessels to room temperature. aspirate the spent medium from the vesselcontaining pscs and rinse the vessel twice with dpbs without calcium and magnesium. add 0.5 mm edta in dpbs to the vessel containingpscs. swirl the vessel to coat the entire cell surface. incubate the vessel at room temperature for5 to 8 minutes or at 37 degrees celsius for 4 to 5 minutes. when the cells start to separateand round up, and the colonies appear to have holes in them when viewed under a microscope,they are ready to be removed from the vessel. note: in larger vessels or with certain celllines, this may take longer than 5 minutes.
aspirate the edta solution, and add pre-warmedessential 8â„¢ medium to the vessel. remove the cells from the well(s) by gentlysquirting medium and pipetting the colonies up. avoid creating bubbles. try to work with no more than 1 to 3 wellsat a time, and work quickly to remove cells after adding essential 8â„¢ medium to thewell(s), which quickly neutralizes the initial effect of the edta. some lines re-adhere veryrapidly after medium addition, and must be removed 1 well at a time. others are slowerto re-attach, and may be removed 3 wells at a time. collect cells in a 15ml conical tube. theremay be obvious patches of cells that were
not dislodged and left behind. don’t scrapethe cells from the dish in an attempt to recover them. add an appropriate volume of pre-warmed essential8™ medium to each well of a vitronectin coated 6-well plate so that each well contains2 ml of medium after the cell suspension has been added. move the vessel in several quick figure eightmotions to disperse the cells across the surface of the vessels. place the vessel gently intothe 37 degrees celsius, 5% co2 incubator and incubate the cells overnight. feed the psc cells with essential 8™ mediumbeginning the second day after splitting and
replace the spent medium daily. it is normal to see cell debris and smallcolonies after passage.
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