Crop circles are so last century. In our lab, HeLa cell circles are all the rage:
The tissue culture incubator is currently plagued – not with aliens, but a random vibration or resonance that causes our cells to sporadically seed in perfect concentric circles. As a biologist, I haven’t the foggiest what sort of vibration or energy could cause these amazing patterns – I leave that sort of thing in the capable hands of the mysterious physicist and mathematician types who drift in and out of our lab, collaborating with my colleagues on building virtual cancers and online epithelial layers and God knows what else. (For all I know, they’re busy crafting the perfect virtual post-doc, one voxel at a time, who can pipette 24/7 without sleep, social life or access to a vending machine carbon source.)
But for us real-life biologists, such vibrations can have serious consequences. The cells within the circles are confluent, when we’d rather have them in growing exponentially without neighbors – but that’s the least of our worries. Apparently vibrations strong enough to disturb the seeding pattern can wreak havoc on the biochemical processes within. The boss even offered up a few doom-and-gloom anecdotes about the time he was a post-doc and the entire lab’s experiments went into free fall when a new lift was installed next door.
To me, all of this is just a more visible-than-usual reminder of the unavoidable fact that it is impossible for any experiment to ever be replicated with the precise conditions of the previous. If you run three identical Western blot experiments, you’ll get slightly varying band intensities. If you read about an experiment in a paper and try to reproduce it, it doesn’t always work. Apparently the most trivial-seeming things can have strong effects – such as the pH of the water mains used to feed the Milli-Q water polishing machines. Minute changes of temperature and humidity – air-co in Texas versus a sultry summer London lab – are bound to stretch the boundaries of what scientists like to call STP: standard temperature and pressure. I once had a colleague in grad school whose experiments failed miserably for years until the one fateful evening she decided to stretch her legs and took her bacterial tubes along for the ride; the corridor was several degrees cooler than the lab and the rest was history – and a mighty fine Nature paper if I recall correctly.
But I don’t see this as a bad thing. Instead, I find it infinitely reassuring. When you open up that next issue of Cell and see three back-to-back papers from three different labs all reporting the same general finding, you can be sure that the phenomenon under consideration must be remarkably robust. People often complain that science is wasteful, that having a dozen labs working on the same thing is inefficient and nonsensical. But it’s not until you see the same results half a dozen different ways that you truly know you’re onto something.
So bring on those HeLa circles. My knockdowns still worked, so I know my biology is strong enough to withstand vibrations, aliens and whatever other subtle variations the environment might throw up.
Nasty referees, however, are another matter.
Is the agar a different thickness in the peaks and troughs of cell growth?
Hi Jenny,
Your HeLa circles are very interesting! Just curious about the structures you would get in T-25 or T-75 flasks…
Been there, done that. In our case, it was a fan on the building roof (we are on the 7th of an 8 story bldg). As soon as the fan was fixed, the magic cell spreading patterns disappeared.
You know, someone looking at contact inhibition or, oh I don’t know, cancer growth for example, could find a mighty fine experimental system in your circles. *ponders*
No agar – HeLa cells grow straight on plastic. But I can imagine this could wreak havoc with soft agar assays as well.
Yes, and ironically the lab does a lot of work with micro-contact printing trying to get cells to grow in patterns just as exotic.
Do you think the periodicity of the circles is related to the frequency of the mysterious vibrations? The circles look to be about 1cm apart, and 1cm is roughly the wavelength of microwaves (as commonly used in laboratory equipment).
I’ve seen these, and like to believe (though can’t prove) that I could prevent them by being s…l…o…w….
How fast do they settle down? Can you leave them in the hood for a minute before putting the plates back in the incubator? If they touch the bottom before you put the plate in, they might be able to hold on to that spot in the plate.
Good question – I’ll have to do the experiment!
Unfortunately HeLa cells take 3-4 hours to respread – they will be round and slooshing around for at least an hour.
Bah. Melanoma cells are more grabby and sticky. Unpleasant for human bodies, but useful for plating.
Hmm, I doubt you need to posit microwaves to explain this. You’d also get a 1 cm wavelength if you had a sound (in air) of around 30 kHz, just beyond the range of normal human hearing.
Our current theory is that it’s the fan inside the incubator. I say “our”, but really I don’t know why we all instinctively feel this is true. Perhaps I should try popping some corn in the agar microwave to test this…hang on, I’m getting my blog posts muddled up.
I thinking the same myself. (Not the popcorn, the fan!)
Not that I know anything about this…! 🙂 (I’m strictly a theoretical computational biologist, not an experimental one.)
Is the fan slightly out of balance?
One more: there must a practical use for concentric circles of HeLa cells. surely? 😉
Oh for an edit option: my last comment should start: “I *was* thinking …” (My pathetic excuse is it’s midnight on Friday here.)
ooh, that’s a good one. Let’s assume you’ve got a 10 cm fan, doing 1,000 rpm (computer fans do about 2,000 rpm), so that’s about 30,000 centimetres per minute, or 5 m s-1. That would mean it’d be vibrating at about 500 Hz if the wavelength is 1 cm. So you should hear a low hum.
I think it’s time to call a halt to all this speculation and call Mulder and Scully.
I’ve seen a documentary on those, it’s two old blokes with sticks and ropes!
Oh you said cell circles, my mistake!
You need to be a bit careful there. The minute you say something about cells being “sensitive to vibrations”, a whole horde of homeopaths, electrosensitivity types and other alternative loons will descend on your lab claiming you have validated their belief system.
Come to think of it, most of those folk live by Fox Mulder’s other slogan:
I like the idea of ‘alternative loons’. I’m sure I used to have a pair back in the ’70s.
Ah, a man with numbers. Scary 🙂
If you had two incubators of the same make, you could try the “standard” hack of swopping parts, trying the same tests on both, etc…
My labmates complained that this exact thing happened a bit over a year ago (before I had joined the lab) when the second wing of our institute was under construction. Apparently the vibrations from the construction caused similar “crop circles” in tissue culture. After the construction finished up, the circles disappeared.
Hopefully the answer will be found for your circles, and readily fixed…
With this supernatural events stuff, all I can say is – circles, schmircles.
In order to break reaaally big, Jenny’s cell cultures will have to come up with something more like this.
Hm… do you have a nitrogen (or air) table handy? Sticking the incubator on it *might* isolate it from outside vibrations… if your circles disappear, it’s something in the building. If not, it might be in the incubator itself (fan, motor, whatever)… or something in the building being transmitted down, say, the power cord (unlikely as power cords are flexible, but you never know).
Since I am a genomics guy, I suggest an enormously expensive, multi-center collaborative project involving hundreds and hundreds of incubators. That should sort it out.
Would only work if it was building structure vibrating… (I blame the buses) not if it was pulsatile airflow…
Physiologists who own anti-vibration tables with patch-clamp rigs (or other electrophys) on them will know whereof I speak…
That makes me think that maybe the site for the UK Centre for Medical Research and Innovation at St Pancras isn’t particularly well chosen. There are St Pancras International and King’s Cross train stations very close by, and the Circle, Hammersmith and City, Metropolitan, Northern, Piccadilly and Victoria underground lines passing by. Even here at Nature Towers, which is a little way away next to Regent Canal, I get my LCD screen wobbling from, I assume, passing trains. Oh and there is a cement factory nearby, with heavy lorries moving around the entire area. If even a roof fan can induce strange behaviour in cell cultures, I doubt whatever precautions the architects took will prevent unusual results from appearing.
Clearly in the case of Jenny’s HeLa cells the Circle Line must be a prime suspect.
@stephenemoss – Argh.
Also, I used to work in a lab on the 11th floor of SickKids hospital in Toronto. On the roof of the tower was (and still is) the heliport. Not only did the building shake like crazy when the chopper landed, if the wind was in the right direction we’d get a strong smell of aircraft fuel fumes coming in to the lab. Probably also not terribly good for cells in culture.
Gimbals. Lots of gimbals. Problem solved.*
*Possibly not.
Ah, a gimbal* each way.
*bob.
So… has The Truth been found?
(Or has the search been called off for lack of evidence?)
I second the argh!
The Truth has not been found but I can report that an engineer was sent out and he determined that there are no moving parts within the incubator that could cause the problem. And then he whipped out the most amazing, high-tech piece of vibration-sensing equipment I’ve ever seen – a bottle of water – and set it on top of the incubator, the fridge and other surfaces in the TC room.
Dear reader, the whole damned room is shaking. But only in the morning.
The search continues…
Awesome..so has the search ended or are you after what makes the whole damned room shake in the morning?
Perhaps you should test other rooms with the sophisticated vibration-detector and see if the TC room is shaking alone (is that possible at all?) or with other rooms and to what extent.
And are you going to plate cells in the morning versus evening and see how that affects cell circles? Or what times of plating in the morning are critical for well-formed cell-circles?
Ooh! the possibilities….
Hi Jenny, It might be nitric oxide due to vibrations of the fluid in the petri dish. The wavelength looks like capillary waves.
http://en.wikipedia.org/wiki/Capillary_waves
These are standing waves, so the point at the center has to be a stationary node which looks to be low density. The high density regions must be the non-stationary nodes, where there is fluid movement. This movement is radial and leads to the media flowing across the surfaces of the attached cells. This generates shear which activates various things and stimulates growth. NO is one thing that can be generated by shear and which then stimulates growth, but there are other shear stress mediated growth pathways too. They would change the properties of the cells by a lot.
You could add a surfactant which changes the surface tension of the media, but your cells might not like that. You could drive the vibrations at different frequencies so the nodes move and you get more of an average. You could use smaller area growth surfaces, the whole surface acts like an antenna so the amplitude of the growth differences would be less. You could interrupt the surface with a mesh or grid of some type, square mesh at 2 meshes per inch breaks the surface up into ½ inch squares. Those are square so they don’t have the same circular resonance and they are small so the amplitude can’t get so high. The amplitude probably goes as the number of wavelengths in the surface. Putting something off center at the middle so the center node is blocked might help a lot. The resonant frequency and the wavelength won’t change, but if it is no longer centered at the center of the dish, but moves around some, the patterns won’t be stationary.
One solution might be to add an orbital component to the movement of the incubator and use deeper media (if you want suspension grown cells). If you want confluent cells I think the mesh approach would be best, but that would leave a mesh shaped gaps when you pull it out.
You could also lay a piece of hydrophobic porous PTFE over the surface. This makes the surface not move which then prevents any shear but still allows gas exchange. This is the best solution, but it means you need a lot of porous PTFE (you could use PTFE pipe tape cut into a circle). But then with no shear you don’t have any of the shear mediated biophysical effects.
You could use a hydrophobic mesh that is not wet by the media. That sinks in the fluid and generates surface tension discontinuities that distort the capillary waves preventing a standing wave from occurring.
During my PhD, a Drosophila lab moved to our institute from the US. Their flies started dying almost immediately, and no-one could understand why there were doing so poorly in their new environment… until some bright spark noticed that the lab’s air flowed from an intake vent located in the wall above the sheltered corner of the building where all the smokers liked to gather. You couldn’t smell the smoke in the lab, but the flies were obviously extremely sensitive to it. The smokers were made to go and smoke somewhere else, and the flies did just fine from that point on.
Damnit. Imagine a closed html tag in there, please.
Fixed it. 😉
Did any of the flies get hooked? Start hanging out underneath the bleachers and not coming home before curfew?
I think ideally I just need a non-shaking incubator. We are thinking of putting it on a special platform. I’ll keep everyone posted!
That may not work. It is most likely a resonance so the degree of isolation needed is very high. If you just put a small obstruction in the middle of a dish but off center, that may do it.
Another thing to try might be a different diameter dish.
Just because you can’t see visible patterns, may not mean that differences in cell growth and expression are not present.
I am not sure if it is the incubator. I saw perfect concentric circles in my HeLa culture plate too. But there were other people growing Hela in the same incubator. And their cells didn’t show the concentric rings.
Hy Jenny, do you solved the mistery? I think the solution is in the dish. I also see the circles in other cell types
I am a molecular biology graduate student. I noticed my HeLa cells were also growing in concentric circles. Interestingly, I have a batch of Hek cells which are growing normally. The standard joke in the lab, is that its a message from “above!”. I found your forum and It was helpful.
Herman
I thought it was related to the phases of the moon or a message from above. I asked for an explanation to the BD and they answered to me that they knew the problem and so they gave me some free dishes.