Some Thoughts On Creating Stable Cell-Lines

Creating stable cell-lines can be a straightforward or tricky process. In this post I will share some techniques or quirks, which I found worked when creating stable cell-lines.

* Use cells with low passage number – Thaw out your cell stocks^1,2 and start with “new” cells with low passage number.

* Culture cells in antibiotic-free media – By cutting out penicillin/streptomycin, I found increases in my transfection efficiencies. It also avoids any possibility of the penicillin/streptomycin interfering with the selection antibiotic during the selection process.

* Make the selection media fresh – Have aliquots of your selection antibiotic at a higher concentration and dilute it into fresh media each time you need to do a media change.

* Change the selection media daily – After transfection, change the selection media daily up until you reach the limiting dilution stage. It may seem excessive or wasteful but I have found that by having freshly made selection media and a daily media change ensures that the untransfected cells are effectively killed off.

* Have high concentrations of your antibiotics aliquoted – Freeze-thaws and heat-cool cycles can affect the efficacy of some antibiotics so to avoid any issues calculate the approximate amount that you will need for selection and aliquot small amounts into separate tubes.

Cell Culture and Antibiotics

There are arguments for and against the use of antibiotics (e.g. penicillin/streptomycin) in cell culture. On the one hand, it is a useful prophylactic to prevent cultures from contamination by providing a layer of protection. However, arguments have been made against its use with claims that constant usage allows for unintentional selection of antibiotic resistant bacteria, researchers become reliant on it and becoming lax in applying aseptic techniques and good cell culture practice.

I am inclined towards the latter view of avoiding the use of antibiotics, but mainly because antibiotics can interfere with particular experiments, namely transfections. However, I do find that it is better to use aseptic techniques because it is better to know quickly that your cells are contaminated rather than have low level contamination hang around which is not visible. The danger of low level contamination is that it will affect and alter your cells and interfere with your experiments.

In regards to students or researchers learning cell culture for the first time, it is probably better for them to see how lapse in aseptic practice can easily lead to contamination. People learn from their mistakes and will only improve with practice.

Cell Freezing Protocol

To continue the cell culture theme of the previous two posts^1,2, I thought I would share my protocol for freezing down cell culture stocks.

Lets assume that I have HeLa cells.

Defrosting Cells

If you receive a cryovial of cells shipped in dry ice, either store the vial of cells in liquid nitrogen or defrost the contents. To defrost, immerse the entire vial in a 37 degree waterbath. When you see that the contents of the vial has defrosted to a partial liquid state but still has a frozen block inside, spray and wipe down the vial with 70% ethanol, open the vial in a laminar flow hood and empty the contents into a sterile tube containing warm (heated to 37 degrees) cell culture media. Spin down to pellet the cells. Discard the supernatant and resuspend the cells in 5ml of fresh cell culture media. Transfer the contents into a T25 flask for culturing and expansion.

 

Cell Expansion

With a T25 flask of cells, grow it to full confluence and transfer (by trypsinising or whatever protocol you use to detach/passage your cells) all the cells to a T75 flask. From here, grow the T75 flask to full confluence and split the cells into two T175 flasks. Again, grow the cells to confluence and expand them into a further two T175 flasks. When the cells in all 4 flasks are at full confluence and ready for passaging, use your standard protocol for cell detachment, but take an aliquot to count on a haemocytometer. Centrifuge the tube to pellet the cells as you carry out the cell counting. Calculate how many cells you have in your cell pellet. You will need ~1x10⁷ cells/ml for the Master cell stocks, thus for 2-3 vials, you will need at least 3.5x10⁷ cells (the extra cells you will need for further culturing and expansion).

Freezing

Prepare your freezing media. I would recommend using freezing media made up of 90% FBS + 10% DMSO for the Master and Submaster cell stocks; and 10% DMSO + cell culture media containing 10% FBS for the Working cell stocks. Make sure your DMSO is cell culture grade and sterile. Your FBS should be sterile. If you are using a new bottle of FBS, it should be sterile but if there is any uncertainty just filter it with a 0.2um pore filter.

Have your sterile cryovials ready and labeled. After your cells have pelleted, in a laminar flow hood, pour off the supernatant and resuspend your cell pellet in 5mls of freezing media. Aliquot 1ml per cryovial, cap your vials, and place them into a freezing container (e.g. Mr. Frosty or a DIY chamber). Store the freezing container of cells in -80 degrees for 24 hours.

With the cells that are left over, dilute them in serum-free media and spin down. Pour off the supernatant to remove the freezing media and resuspend the pellet. Transfer everything into one T175 flask. Repeat the expansion process to freeze down your Submaster cell stocks (@ 5x10⁶ cells/ml; ~8 vials) and then again for your Working cellstocks (@ 1x10⁶ cells/ml; as many vials as you need).

After freezing the cells in the freezing container at -80 degrees for 24 hours, transfer the cells to liquid nitrogen for long-term storage. If you need to transport the freezing container from one location to another, use dry ice to keep the contents frozen.

Note

There may be times when a collaborator will give you a T25 flask of cells with media filling up the entire flask. If this is the case, spray and wipe down the flask and put it in the incubator for a couple of hours so that the cells can settle and acclimatize (cells tend to shrivel when cooled).

In a laminar flow hood, open up the flask and pipette out the excess media and filter it into a sterile tube or bottle for later use. Leave enough media in the flask for the cells to continue to grow. 

Cell Culture Freezing Media

Following on from the last post in regards to freezing cell culture stocks, I thought it would interest you in knowing the type of freezing media I generally use for each stock type. For my Master and Submaster cell stocks, I generally use a freezing media composition of 10% DMSO + 90% FBS. In contrast, for the Working cell stocks, I would use 10% DMSO + cell culture media containing 10% FBS.

High Percentage Serum

As you may know, DMSO is typically used because it has low toxicity to mammalian cells and importantly, prevents the formation of disruptive ice crystals during the freezing process. I opt for the higher serum percentage for the Master and Submaster cell stocks because it contains less water compared to cell culture media + serum. As such, the high percentage serum in combination with DMSO makes for a “gentle” freezing media capable of preserving cell viability or enhancing recovery after thawing. This is particularly important if the cell stocks are going to stay stored in liquid nitrogen for very long periods (i.e. years).

Lower Percentage Serum

Regarding the Working cells, I generally opt for a lower percentage serum for freezing because the Working cell stocks are not meant for long-term storage as compared to the Master cells and because they have a higher turnover in terms of usage. There is nothing wrong with using 10% serum; cell viability can be just as good as if you had frozen your cells using 90% serum. The main difference I have noticed comes from long-term storage (e.g. 2+ years).

Another reason to use a lower percentage serum for the Working cells is cost. A high number of Working cell stocks are generally frozen down so if you are using 90% serum, it will get expensive.

But despite what I have said above, if you are using a cell type that is just bad to freeze-thaw (i.e. not very good at recovering quickly), use the higher percentage serum to freeze down your Working cell stocks.

Ways To Ensure Experimental Consistency #1

When reproducing experiments, it is important to keep experimental variation and human error to a minimum.

Cell-line Stocks

One way to ensure consistency is to keep track of the passage numbers of your cell-lines. When you order in new cells or are given cells from a collaborator, make a note of how many passages they have already been through.  Once this is done, proceed to create your own stocks. I have typically used the organization as set out below.

The Master cells are cells that you expand upon receipt. Freeze a high concentration (e.g. ~1x10⁷ cells/ml) of these down as your Master cell stocks and save an aliquot to continue growing for the Submaster cell stocks. For the Submaster stocks, you can freeze at a lower cell concentration (e.g. ~5x10⁶ cells/ml). After freezing down aliquots of your Submaster cell stocks, once again, save an aliquot for further expansion for your Working cell stocks. The Working cells can be frozen anywhere between 1-5x10⁶ cells/ml.

It is intended that the Working cells be used for x number of experiments before a new vial is thawed. It is only once you exhaust the Working stocks that you thaw out one vial of your Submaster cell stocks for expansion to create a new Working cell stock. From this you can see that it is only once your Submaster stocks are exhausted do you then thaw out one of your Master cells vials to repeat the entire process.

Keep a log book of your cell stocks and make sure that the numbers are regularly updated.