Tissue culture can sometimes seem like a black art. Too careful—your cells go down. Not careful enough—your cells go down. A butterfly flutters its wings in the middle of the Atlantic Ocean—your cells go down. It’s annoying, it’s frustrating, and there are times (and I’m speaking from personal experience here) that you’ll end up chucking everything in the bin and walking out, and the cause of death is not due to contamination. The cause is the diva behavior that THP-1 cells seem to have in bucket loads.
I have committed many crimes when culturing these cells, however, I have paid my penance. In doing so, I have picked up a few tips and tricks to successfully culture, freeze down and bring back these cells.
My Diva’s Won’t Come Back from Frozen for Less Than 20% FBS
Going through my PhD, I encountered many a sales rep who tried to sell me the best and brightest reagent for tissue culture. They promised it would make that aspect of my research a breeze. When they asked what cells I was working with, I would respond with “DIVA cells.” That name seemed to fit their unpredictable behavior, particularly when bringing them back from frozen.
Regardless of what cells you are working with, speed is vitally important when freezing them. This is particularly true for THP-1 cells.
To Successfully Freeze THP-1 Cells, Follow These Tips:
Thaw frozen vials in a 37°C water bath, but DO NOT LET THE WATER COME IN CONTACT WITH THE LID. Thaw the contents until there’s a small lump of ice in the bottom—trust me, it may seem counter-intuitive, but it works.
Transfer the cells to a flask containing warmed media, then transfer some of this back into the vial to get as many cells as you can.
Put the flask in the incubator either at an angle or upright. I usually put the cells in a T25 flask with 5 mL of media standing upright. The flasks are small and take up even less space this way. As these cells are suspension cells, you want to keep them in close contact to one another.
Also, add extra FBS to the flask to bring the final concentration of FBS up to 20%. This cell line in particular doesn’t deal well under stress. The additional FBS gives that extra TLC that the cells need.
It’s also worth noting that these cells take between a week to ten days to come back and proliferate well. Check them every few days but don’t attempt to split them. If they require media, add media to the flask. But keep some of the original media as this contains growth factors which will help the cells proliferate.
As tempting as it may be to start working with the cells as soon as they proliferate, resist the temptation. A rule of thumb I use is to wait until there are enough cells to maintain 2 T75 flasks before I start working with them.
When you begin working with them, don’t split the flask every day—the cells need time to replenish. Working with the cells, a good routine is to split/seed cells Monday, Wednesday and Friday. Again, you’re limiting stress placed on the cells while maintaining growth factors in the media, both of which will aid their proliferation.
Splitting Your THP-1 Cells Does Not Mean You Have to Spin out Your Media
When THP-1s start to proliferate, they excrete growth factors that aid their growth. And, while it seems counterintuitive, spinning THP-1 cells out to split them causes stress. Stress to them, because I am subjecting them to centrifugal force; and stress to myself, when I see the pitiful pellet at the bottom of a falcon.
About 4 days after they come back from frozen, instead of replacing the media, add fresh media. If you need to split your cells, remember these are suspension cells—remove X amount from your flask and replace what you’ve removed with fresh media. It also means that you’re in and out of tissue culture in about 5 minutes.
Trypan Blue Is Your Friend
In tissue culture, I am known as having green fingers. No matter the cell, I can get it to grow. I’m pretty sure this is a direct result of having to battle with THP-1 cells. Because of this, I have a knack for looking down the microscope and knowing which cells would come back and which wouldn’t. However, it took many months before I developed this ‘skill,’ especially as a newbie.
Therefore, if I suspect my cells are dead, before calling time of death, I confirm it with trypan blue. I used it routinely for tissue culture during my PhD, in which I focused on living cells. However, it’s equally beneficial when trying to figure out if your cells are in fact dead. Furthermore, it is also useful in determining if you have contamination—trypan blue is excluded from living cells. Bacteria are also living entities…sigh.
Freezing THP-1 Cells
Be Prepared. Be Quick. Be Gentle.
Similar to bringing THP-1 cells back from frozen both preparation and speed are important when freezing cells. To work efficiently:
Have your cryovials labelled and ready to go and your freezing medium prepped (personally—I’m a fan of 10% DMSO and 90% FBS).
When you add the freezing medium, do so gently, especially the first few drops. It takes a minute or two longer, but it’s worth it. Make sure you use enough freezing medium for your cells or the cells will not survive the tundra.
Once the cells are suspended, seal the tube, invert a few times, and then divide the cells into the freezing vials.
Transfer the vials to your equipment for freezing.
Do Not Leave Your THP-1 Cells in the -80°C Freezer for More Than 24 Hours
THP-1 cells do not like being in the -80°C freezer! I cannot stress this point enough. If you transfer the cells to liquid nitrogen after 24 hours, then you are as good as guaranteed that your cells will come back. Any longer, well…may the force be with you. You will have limited success if, having left your cells in the -80oC, you decide to transfer your cells to liquid nitrogen.
If you have, however, forgotten to move your cells to liquid nitrogen, fear not. THP-1’s will come back from -80°C. Thaw your cells using the tips above from -80oC but this time, freeze the cells down using the correct procedure. Transfer them to liquid nitrogen after 24 hours this time!
Have Your Own Stash…of Everything
I have witnessed contamination of a lot of things—trypan blue, DMSO, FBS, you name it, and I’ve seen it. That’s why it is imperative that you maintain your own stash of cells and reagents. Aliquot everything that comes in to the lab, from pen/strep to DMSO. If you get contamination, then there isn’t much of a loss if you have to chuck everything. Also, if others get contamination, you know your stuff is safe because you aren’t sharing. But don’t dip into anyone else’s stash without permission!
And if you do experience contamination—do not panic!
The most important thing to do is to dispose of your cells immediately. You won’t have to worry, because you know you have frozen some down successfully. Then, find the source of your problem – pipette boys are a common source, but don’t forget about your media. If there’s any doubt, chuck it out.
Tissue culture with THP-1 cells isn’t all bad, as it’s where all the experiments begin. It can be long, it can be monotonous and it can be time consuming—so have fun in there, otherwise you’ll begin to resent it. I have been known to sing in there (sadly no one would bring me through to the audition stages had it been an X Factor audition), while we as a group have had 10 second dance parties (a la Grey’s Anatomy). It helped get rid of bad juju as well as entertain us during centrifugation steps. It passes the time, but it also puts out good energy.
So, if it all seems to be going down the river, sing a song, have a dance, and leave it for the day. Because you can start afresh tomorrow.
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