I’ve seeded 6-well, 12-well, and 100mm dishes and continually find a heterogeneous distribution of my microvascular endothelial cells (primary cell line), regardless of seed density or technique used to deposit them in the plate. For example, I’ve tried creating a “stock” solution of DMEM, 10% FBS + cells and used both plastic serological pipets and micropipetters to deposit the correct amounts into each well or dish (to the center of the well, on the side, and in multiple locations have been tried). I’ve also tried adding media first, then adding cells dropwise around the well. All techniques continually, but not always, yield overconfluent centers and subconfluent edges. There is a graduate student in my lab who has experienced the same issue; however, there is also a lab tech with a great deal more experience than either of us who does not (although on rare occasions she may get overconfluent edges and subconfluent centers). Any suggestions as to what could be causing this?
I’ve made a list of possible explanations but don’t know if any of these could explain my results:
1. Shaking the dish immediately after seeding causes the cells to aggregate in the center
2. Incubator shelves are not level
3. I’m using Corning Cell-bind 6-well and 12-well plates. Is there an issue with uneven distribution of whatever treatment the wells are given (I believe they treat the polymer instead of coating it with something)? Is this plate incompatible with 10% FBS or higher? …however, a different company manufactures my 100mm dishes and this problem seems to arise there as well, so I tend to discount this.
4. Improper splitting, seeding technique causes this problem (such as depositing cell stock on the edges of the well instead of at the center or all around) – probably most likely issue
5. The filter I’m using to remove cell clumps before seeding does not remove all clumps; clumps tend to aggregate in the center of the well, growing somewhat faster than single cells on the periphery
6. Passage number issue: cells have adapted to the plastic and bind in weird ways (all of my experiments have been conducted using passage 9 and above – usually between 13 and 20)
7. Latent contamination of the cell line depletes something in the media, causing weird growth pattern
8. Use of plastic pipettes instead of glass serological pipets
9. Size of the pipette opening (1ml micropipetter or 5ml serological) causes this issue
10. At some step in the past, the cell line became too acidic, causing this weird growth pattern down the line
11. Initial seed density is too high for wells (300K for 6-well, for example). I tend to discount this.
12. Something I’m doing causes a physical deformation of the wells – extremely unlikely, I think.
Most probably the shaking. On a circular well this will cause the cells to aggregate on the center, much like sugar in the bottom of a cup of tea after you mix it circularly. Less likely, but also possible, the incubator (or the surface where the incubator is on) is vibrating slightly, causing the same effect above (I already had concentric circles on a well because of this). To solve this I would check that there is no vibration, then shake the plate North-South of East-West a few times before putting it on the incubator. But don't fret too much about it, this still happens with me sometimes, even after 10 years working with cells.
When I was first learning to plate cells I was told that the natural motion you have when you walk (i.e from the hood to the incubator) can cause a subtle circular motion that will cause the cells to gather primarily in the center of the dish. Or if the way you shake the dish is to swirl it.
A simple way to counteract it is after you first plate the cells is to gently shake/move the dish first side to side, then shake it perpendicular to the motion you just did by moving it up and down (i.e. Towards you than away from you) a few times, while you do this you should always keep it parallel to the surface of the hood or table (5 or 6 times each way) it best if you just slide it on the table to make sure you aren't rocking it. Then walk to the hood and parallel to the shelf repeat the same motion again a few times each direction before you set it down.
Most probably the shaking. On a circular well this will cause the cells to aggregate on the center, much like sugar in the bottom of a cup of tea after you mix it circularly. Less likely, but also possible, the incubator (or the surface where the incubator is on) is vibrating slightly, causing the same effect above (I already had concentric circles on a well because of this). To solve this I would check that there is no vibration, then shake the plate North-South of East-West a few times before putting it on the incubator. But don't fret too much about it, this still happens with me sometimes, even after 10 years working with cells.
Its most likely the pattern of shaking the plate to distribute cells evenly. Kari Herrington and Luiza Mendonca has given excellent descriptions of how to go about it. It has always worked for me for every cell type I have worked with.
Hi,
I totally agree with the previous comments. We too had a similar problem with larger cell culture dishes, where cell density was alternating higher and lower in a pattern of concentric rings. This was due to the vibrations of the incubator's cooling compressor. Haven't encountered this since we used another model. Also, excessive orbital swirling before cells are attached to the plates (a few hours) will lead to accumulation of the cells in center of the plates.
Hi, I too agree with the others comments. In my experience, using cellular lines, not primary cultures, the abnormal growing cells is also due to the loss of the adherents features and changes in the extracellular matrix. After these, can occur others alterations to conduce to deteriorate the culture characteristic.
After adding the cell suspension in the circular plate with additional media already there, I mix them with a pipette again rather than doing circular shaking and that gives me a uniform cell density.
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