How to culture cancer cells the right way?

09 September 2019 3 6K Report

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! :)

Jordan R. Yaron

A T25 can generally hold about 2 million cells, depending on the type of cell. 2e5 would then be, roughly a 1:10 split of a confluent flask. This is fine, but you shouldn't be worried about splitting even lower. 1:20 or even 1:40 is sometimes necessary if you will be away from your cultures for too long. This is all dependent on the cell type, some are more sensitive than others. HeLa, for example, is happy to be split very, very low and will recover just fine. Test empirically.

Avoid changing the medium every day. The medium is basically essential components for the cells, but the conditioned medium is where the cells will truly be happy. The nice thing for us, as researchers, is that the cells will do the conditioning themselves. But they put all that work into it, don't take it away right away! I would try changing medium maximally every other day if you really need it. 2-3 times per week is ideal.

A pipette tip is supposed to be sterile if you're using it for cell culture. I would leave it. Trying to retrieve is just adding additional opportunities for exposure to your cells. Just let the cells be, they won't be bothered by the tip. When you passage the cells the next time, just be careful not to drop another tip in.

Depending on the cell type you should be fine to seed the cells way lower than 20% for generalized maintenance. However, try to keep the cells in their happiest, logarithmic phase for a few passages prior to experiments for data collection. If you are going to be gone longer than a week I would either (1) have a lab member take care of your cells or (2) toss them and thaw a new vial when you return.

It is possible you have excess vibrations in your incubator that are bringing your cells to the middle. It is also possible you are looking at them too much. Cells are "shy," don't bother them more than you absolutely need to. The more you perturb the cells (in and out of the incubator, in and out of light exposure, etc), the more you bother them. Leave them be unless you absolutely must do something with them (culture, experiments, etc).

Mohammad Taleb

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!

Shawn Krueger

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.

How to culture cancer cells the right way?

No title

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

Issues with Permeabilization during Flow Cytometry?

Which cell line should we use to perform biological dosimetry experiments?

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

What could be some reasons why unstimulated monocytes are getting activated?

Is there a deep reason for using the squared modulus of the wave function as the probability density of the existence of the particle?