passaging 293T cells - (Sep/23/2005 )

We just got some 293T cells and are having trouble passaging them. The seem to not plate very evenly. Some areas of the plate are 100% confluent while other areas are 10-20% confluent. I would say the problem has gotten worse and worse the more we try different things. We typically passage cells using 0.05% Trypsin 0.53 mM EDTA by adding 1 ml to a 10mm plate and removing it an adding a fresh 1 ml of trypsin. Wait with gentle rocking to see when the cells come off the plate. Then we add fresh DMEM/10%FBS/pen/Strep and pipet the cells up and down to disperse them. Transfer the appropriate amount of cells the new dish which already contains the fresh media and move the dish up and down then left and right to get the cells evenly spread across the plate. They appear to be evenly spread out before they sit down on the plate but the next day they are not.

i usually split 293T 1:10 every 4th day. add 1ml trypsin, just like you, then tap the plate for all the cells to come off the bottom. then add 10 ml of medium, pipet a lot - no clumps or other stuff, and then take 1 ml and spread drop by drop all over the surface of the medium on fresh plate. then i shake the plate in all directions like million times then carefully put them in the incubator.

so, nothing different then you do.
if you have problem splitting then this way, try like this: trypsinize, add 10ml of medium, and then , when splitting 1:10, add this 10ml to 90ml of fresh medium in the sterile bottle or something. there you can shake and shake, and in principle when you add 10ml on the plate you dont have to shake too much - they shuld be spreaded.
good luck.

hi
well i've got these problems too. And i've solved them by 2 ways. I dulite trypsin 1:3 and let it more time in order to get all cells detached. I pippet up and down cells 3/4 times and it enhances cell detachment. and when platting them i swirl gently the plate 3times "N/S" and 3 times too "E/W". That wqas the main point. Second possibility was that the desk which i put my plates on was not total horizontal. Check it and you can adjust by a pipette tip.

Last suggestion from Fred is the same as mine. Our shelves weren't horizontal. So how nice I had dispersed them before... After overnight incubation it looked terrible. Simple but definetly something you want to check!

Well, i usually incubate the cells after adding the trypsin for about 3-4 min before adding the media. After transfering, you can try to pipette the media and the cells up and down a few times so that the cells are evenly spread out. You can also try to shake the dish up and down and left and right after you have placed them into the incubator.

cricket,

When you look at the dispersment of the cells, are there higher concentrations in one area of the plate, then a cresent shaped area with less cells near one of the walls of the plate? Then a higher density near the wall? If this is the case, this means there is a leveling problem. There could also be a vibration you are not aware of. Equipment near an incubator, especially equiment that turns on and off, can affect the attachment of cells. You can get all sorts of strange attachment problems with vibrations.

If the highest concentration is near the center of the plate and the lower concentration is near the walls, there is a swirling effect when you set the plates in the incubator, this causes the cells to all deposit in the center. When I plate cells in a round plate, I add the cells and media, rock the dish to coat the entire bottom with media, then walk to the incubator and just before I set the dish on the shelf I tip the dishso the media all slides to one side (without going over the edge of course) and tap the dish on the side a few times gently. Then when you set the dish back to level, the cells all sweep across the dish with no swirling motion. They should be evenly dispersed.

Also sometimes certain epithelial cell lines can clump with excess pipetting and swirling.

Hope this helps,
Beverly