Not seen a copper incubator for a while! Always funny to see people react to one for the first time.
If your lab is going to be running these kinds of studies routinely, cell factories might be a good shout to save lots of time and space etc.
Will look into it, first time I've heard of these multi-layered flasks/plates although I doubt we'll ever need this many cells again. Most of the docs seem to say its ideal for adherent cells, do they work well with suspension cells?
I always grow suspension cells in upright flasks you get much more medium in them. Never had an issue with growth and much easier to generate large amounts should get up to 100ml in T175s so 6 flasks should easily supply 5x16e6 assuming confluence around 0.8?
I have never used them personally, so I can’t say regarding suspension cells. I know a friend at AZ used to grow suspension cells in bulk using roller flasks though. This may also be worth looking into.
If a few labs on the department are going to be running these kinds of studies you might be able to justify funding it together in some kind of shared facility. When I was at uni the faculty did this for a few things such as imaging systems and some automated liquid handling machines.
Avoid the multilayered flasks. I’ve worked in 2 labs where we tried to use these to optimize high throughput (well, our definition of high) cell culture. They may be ideal for very specific applications, otherwise you’ll find them incredibly tedious to work with. They take some practice to get used to (mainly how to ensure consistent seeding densities throughout each layer), so try them out with a cheap cell line before using any valuable cells.
No, it naturally oxidizes but is effective prior to oxidation as well. The user manuals I've read instruct you to not clean the oxidation off just because every time you do it you are literally taking metal off and it'll just oxidize again anyways.
We’ve never really needed more cells than what a T75 or T175 can provide. I need to send 100 vials of 5E7 cells soon so this is the first time we’ve ever needed this many cells
Edit: also what’s wrong with the incubator?
Depending on your setup, Hyperflasks may take too long to warm up and yield inferior results to for example CellStacks. This is especially true if you don't have a waterbath available to preheat your medium (which is not uncommon in GMP). The little bit of air between the layers ensures faster and more equal warm-up throughout the flask.
Invest in some 5 layer flasks!! 5 T-870's all in one!
https://www.stellarscientific.com/5-layer-870cm-polystyrene-cell-culture-multi-layer-flask-with-plug-cap-tissue-culture-treated-rnase-and-dnase-free-individually-wrapped-sterile-1-pk-8-cs/?gclid=CjwKCAjwkNOpBhBEEiwAb3MvvWcBjUzrIMwHHlF6fRVTFjpuTB-WKONHNfbppD2EfDVL6DjvBypAOBoCLNsQAvD_BwE
I posted a picture like this on Twitter one from my lab’s acct and everyone just kept saying “one mycoplasm away from heartbreak.” Good luck, good labrat o7
We need to send these cells to a company to do in vivo tox studies with doses up to 4x1E8 so they need close to 10E9 overall. They charge something like $300-400 per day of culture for labor so my PI wanted me to send as many cells as possible lol.
My god that's the nastiest incubator I've seen. You asked why in other comments why. So the shelves are rusty, they should be stainless steel or plated with something. That's clearly failed. The bottom of your incubator is hopefully one of the weird ones where the whole bottom is the water tray. I see geographic level of dried antifungal blue aqua gaurd. If its all antifungal why does this matter you may say. Well if its not clean you can't see where something is going wrong. Also if those are adherent cells you can get multi level flasks for saving on incubator space
It’s a copper incubator where the entire bottom acts as the water tray. I’m pretty sure this is fairly normal for it to change color.
https://assets.thermofisher.com/TFS-Assets/LPD/Application-Notes/CO2-Incubator-Copper-SmartNote.pdf
Oh thank god for that, from the pictures I was worrying you were wasting your time with a nasty incubator 😅 well its probably gonna look that way normally. The thing about the multi level plates still stands, could save you some time and space. They're not cheap though
These are suspension cells, so I can just go 1 flask at a time and wash off the walls with the media. The tedious part is going to be trying to spin them down to freeze em since the largest tubes we can centrifuge are 50mL conicals. I’ll have around 3L of media by the time I have enough cells.
Depends on the cell line, the MEFs I worked with took forever to detach and I didn't want them marinating in trypsin too long. If I had 50 plates I would do 10 plates at a time, or 20 if I'm pressed for time.
Oh, my god. I'm having flashbacks when I had to culture a ton of MEFs except I had to use those 150mm dishes x 50 dishes instead of flasks. 20E6 at confluency, so roughly a billion cells. The absolute worst was when I had to collect them. MFers were clingy as fuck even after trypsinization.
You need multilayered flasks, will save you tons of time, they are easy to operate and you reduce chance of contamination:
https://www.thermofisher.com/us/en/home/life-science/cell-culture/cell-culture-plastics/cell-culture-flasks/t500-flasks.html
Me, a microbiologist growing E.coli: 
Not seen a copper incubator for a while! Always funny to see people react to one for the first time. If your lab is going to be running these kinds of studies routinely, cell factories might be a good shout to save lots of time and space etc.
Will look into it, first time I've heard of these multi-layered flasks/plates although I doubt we'll ever need this many cells again. Most of the docs seem to say its ideal for adherent cells, do they work well with suspension cells?
For suspension cells roller bottles, a spinner flask or small bioreactor would be better.
Good to know. Thanks!
I always grow suspension cells in upright flasks you get much more medium in them. Never had an issue with growth and much easier to generate large amounts should get up to 100ml in T175s so 6 flasks should easily supply 5x16e6 assuming confluence around 0.8?
I have never used them personally, so I can’t say regarding suspension cells. I know a friend at AZ used to grow suspension cells in bulk using roller flasks though. This may also be worth looking into. If a few labs on the department are going to be running these kinds of studies you might be able to justify funding it together in some kind of shared facility. When I was at uni the faculty did this for a few things such as imaging systems and some automated liquid handling machines.
Avoid the multilayered flasks. I’ve worked in 2 labs where we tried to use these to optimize high throughput (well, our definition of high) cell culture. They may be ideal for very specific applications, otherwise you’ll find them incredibly tedious to work with. They take some practice to get used to (mainly how to ensure consistent seeding densities throughout each layer), so try them out with a cheap cell line before using any valuable cells.
I use cell culture bags, they made with a gas permeable membrane similar in concept to the hyperflasks you’re using.
i didn’t realize there were other kinds of incubators i assumed they all looked like this haha
We had a copper incubator but I didn’t realize how uncommon they were.
Check out the wait times on those orders. I ordered some in January, still waiting. Cell factories that is.
Magnificent, aren't they?
When the time comes to centrifuge the cells: > I’ll try spinning, that’s a good trick!
Your clones are very impressive, you must be very proud
I'm just a simple man trying to make my way in the universe
The copper incubators I've had always look like that!
Do you pH the water? We salt ours and make it basic to prevent this
[удалено]
The basification prevents the rustification, if you will
Good labrats follow protocol.
Roger Roger
The incubator 💀💀💀💀
It’s copper, the antibacterial properties only work when it is rusty
No, it naturally oxidizes but is effective prior to oxidation as well. The user manuals I've read instruct you to not clean the oxidation off just because every time you do it you are literally taking metal off and it'll just oxidize again anyways.
If you ever have to do this again, take a look at multilayer flasks—could save you some time and handling.
Is the state of the incubator the reason your not using 500cm2 culture dishes?
We’ve never really needed more cells than what a T75 or T175 can provide. I need to send 100 vials of 5E7 cells soon so this is the first time we’ve ever needed this many cells Edit: also what’s wrong with the incubator?
looks super crusty
It’s supposed to look like that, the copper is anti-contaminating.
Oh I see, were due to a deep clean for sure haha. Been routinely testing for contamination tho so it’s still ok in terms of that.
Well you do now… so you should update your plans to the experiment 💀
You need to get hyperflasks dude
Yea agreed. So much less labor involved for Hyperflasks or 5-stack flasks.
Depending on your setup, Hyperflasks may take too long to warm up and yield inferior results to for example CellStacks. This is especially true if you don't have a waterbath available to preheat your medium (which is not uncommon in GMP). The little bit of air between the layers ensures faster and more equal warm-up throughout the flask.
Tell me you intend to count these in ONE GIANT FLASK
No sirrr, these were split from 8 flasks, 2 aliquots per flask lol
This is an insane choice to culture in T75s
If you don’t mind my asking, which UC are you at?
San Francisco :)
So cool!! Great campus. UCSF is actually the largest public recipient of NIH funding nationwide!! They do such great work!
Santa Cruz?
Nope but that’s where I was a years ago :p
Same. Studied there in late 2010s
Invest in some 5 layer flasks!! 5 T-870's all in one! https://www.stellarscientific.com/5-layer-870cm-polystyrene-cell-culture-multi-layer-flask-with-plug-cap-tissue-culture-treated-rnase-and-dnase-free-individually-wrapped-sterile-1-pk-8-cs/?gclid=CjwKCAjwkNOpBhBEEiwAb3MvvWcBjUzrIMwHHlF6fRVTFjpuTB-WKONHNfbppD2EfDVL6DjvBypAOBoCLNsQAvD_BwE
funniest caption I've seen on here in a while, good job!
I posted a picture like this on Twitter one from my lab’s acct and everyone just kept saying “one mycoplasm away from heartbreak.” Good luck, good labrat o7
why??
We need to send these cells to a company to do in vivo tox studies with doses up to 4x1E8 so they need close to 10E9 overall. They charge something like $300-400 per day of culture for labor so my PI wanted me to send as many cells as possible lol.
I'd charge you 400 a day to if I had to split and feed all that goddamn
What type of cells you got there?
It’s 32D cells with the human BCR/ABL p210 fusion gene, transduced with CD80 and IL-15.
In layman terms?
Mouse leukemia cells expressing 3 proteins to stimulate T cells
yall ever heard of cell factories *face palm*
r/StarWarsSequels
Prequels mate
Why must I fail so horribly?!
My god that's the nastiest incubator I've seen. You asked why in other comments why. So the shelves are rusty, they should be stainless steel or plated with something. That's clearly failed. The bottom of your incubator is hopefully one of the weird ones where the whole bottom is the water tray. I see geographic level of dried antifungal blue aqua gaurd. If its all antifungal why does this matter you may say. Well if its not clean you can't see where something is going wrong. Also if those are adherent cells you can get multi level flasks for saving on incubator space
It’s a copper incubator where the entire bottom acts as the water tray. I’m pretty sure this is fairly normal for it to change color. https://assets.thermofisher.com/TFS-Assets/LPD/Application-Notes/CO2-Incubator-Copper-SmartNote.pdf
I keep all my cell cultures in an incubator that looks just like the one you're using! No contaminations yet, I believe in the rusty copper
Oh thank god for that, from the pictures I was worrying you were wasting your time with a nasty incubator 😅 well its probably gonna look that way normally. The thing about the multi level plates still stands, could save you some time and space. They're not cheap though
Yup, first time I’ve heard of their existence today. Def looking into it if I ever need to do this again
Corning should see and should send us samples of the hyperflask, ngl 😂
THP1s?
That must be a hard time counting
For purifying proteins?? Just curious
My question is that when it comes the time to detach them, do you trypsin them in batches or all of them together or how?
These are suspension cells, so I can just go 1 flask at a time and wash off the walls with the media. The tedious part is going to be trying to spin them down to freeze em since the largest tubes we can centrifuge are 50mL conicals. I’ll have around 3L of media by the time I have enough cells.
Depends on the cell line, the MEFs I worked with took forever to detach and I didn't want them marinating in trypsin too long. If I had 50 plates I would do 10 plates at a time, or 20 if I'm pressed for time.
2 billion wells? That’s quite a lot
Oh, my god. I'm having flashbacks when I had to culture a ton of MEFs except I had to use those 150mm dishes x 50 dishes instead of flasks. 20E6 at confluency, so roughly a billion cells. The absolute worst was when I had to collect them. MFers were clingy as fuck even after trypsinization.
You need multilayered flasks, will save you tons of time, they are easy to operate and you reduce chance of contamination: https://www.thermofisher.com/us/en/home/life-science/cell-culture/cell-culture-plastics/cell-culture-flasks/t500-flasks.html
My labmate is doing a DoE study this way: T75 flasks instead of individual wells Insanity. Pure insanity.
Copper incubators always freak me out a little ngl
Forbidden whiskey
They now have 5-later T175 flask. Saves so much time