I am relatively new to cell culturing and I am currently working with several lines of cancer cells, some of them being HeLa, A549, HCT116, etc. I have some very basic doubts regarding the culturing techniques and I'd really appreciate some insights on the same:
1. Some cells seem to get quite confluent within a day post splitting. Even though I plate them at low density, I end up having to split them every day or the other. I try not to seed lower than 2 x 10^5 cells while passaging into a T-25 flask. I understand that cells need some time to settle, so is it okay to split them everyday given that they are at high confluency right before splitting?
2. If by any chance I end up seeding more cells than intended, is it okay to change the media the next day to get rid of the floating cells to avoid crowding? This way I think I will be able to reduce the frequency of passing cells throughout the week. But I'm not sure if that's suggested as I might end up loosing more cells than estimated.
3. What if while working in the hood, the pipette tip or anything else falls into the flask during pipetting? Is it okay to remove the tip with a sterilized item like tweezers and continue working with the flask? Or is it suggested to start from scratch?
4. How to maintain cells over weekends or long holidays (by long I don't mean longer than a week) without having them get confluent? I understand that there is a trade-off between seeding at low density and the flask becoming over confluent. As far as I am aware, we cannot seed at a density less than 20%. My question is, what if you seed at that density and the cells still get really confluent over the weekend?
5. A lot of times I have noticed the cells growing at high confluency in the center of the flask or in some other patch of the flask but the other surrounding areas are relatively less confluent. I thought that maybe I am not mixing it right post seeding. But I follow the suggested movements and view the flask under the microscope before placing it in the incubator. They look evenly distributed at that time, but once they attach to the bottom, they get very...centralized?
I hope someone can take the time out to read this and respond. It would be a great help so Thank You in advance! :)
Dear Srutartha,
1. proliferation of some cell lines are very high, definitely it is true for cancer cell lines such as 4T1,... , so you can passage them every day And if you would not like to do it, seed less number at first. After a while by working you will understand cell lines difference in growth and proliferation time.
2. After a while cells will attached to plate so it is no problem to change medium after a day, but not to reduce cell numbers. In this condition i prefer to passage it to more dishes/flasks.
3. For this part if the hood and everything is sterilized as well i think it is no barrier to remove it, but i do not suggest!
4. I never propose it to leave your cells for more than two days, it depends on your cell lines but at least you should check them every other day.
Good wishes in your research!
1. If you commonly seed into flasks, a T-25 can hold about 2 million cells at ~75 confluent. You never want your cells to get over confluent (above 85%). I would seed HCT116 in 6-well dish at about 100,000 (or less) cells/mL. 6-well plate holds 3mL, so I would have a max of about 300,000 cells if I needed them within 56 hrs (treatment).
2. Better to seed less than more. And with cells, once you seed them, they start in a lag phase before growing. And every cell type has a different doubling rate so you want to seed cells so you can change media every 3-5 days (if not actively using them but just keeping them alive).
3. When working in cell culture, aseptic techniques are key. Treat everything single use until you build confidence. My view point is contamination is very simple to introduce in cell culture, so if a scenario like what you explained happens, start over.
4. Maintaining cell lines is all about learning proper seeding densities and timing. As I mentioned before every cell line has a different doubling time. So once you figure out your proper seeding density to have to change media every 3-5 days then its all about timing on your side, to make sure you dont have to feed cells on the weekends.
5. If you get crowding in the center of your dish, rock it gently horizontally and vertically long the axis of the flask. If you do a circular motion you can get them to deposit into the center.
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