11 replies to this topic

[lydialing]

Dear all,

I got a problem in making primary-culture cells well distributed when seeding!
I’m doing rat cardiomyocytes culture.
The tissues has been subjected to collagenase for half an hour of digestion, and filtered by a cell strainer (100um) before seeding. But I get cells very close to each other, or tangled. (I intended to attach a photo(133Kb) to show you what it looks like, but the system said I'm not permitted to upload this kind of file and I don't know why.)

I want them to be single cells with clear border for I’ll have to count the cell surface area, but the cells distributed as patches, making it hard to outline each cell.
Any ideas to improve the condition? Thank you very much in advance.

[bob1]

Primary cultures usually grow best in small patches or clumps as the cells like to have company.  This is because the cells need items that the other cells secrete to stay happy.  It may well be that these cells naturally grow in this conformation, or that you have started forming colonies (if you have left the cells for a day or two before looking at them after seeding).

The file attachment problem may be because you only have a limited number of posts.  I think you need to have 5 before being able to attach files - this is to prevent spam and viruses being spread by the site.

The site also doesn't support particular file types, I think JPG, PNG and BMP are all OK.

[steinpdh]

Actually this is just a matter of two things:

1. After plating the cells - I recommend 10.000 cells per cm^2 - you need to shake/rock the dish in a star-like pattern. Rock it back and forth in at least 3 different directions. This is most conveniently done when they are already placed in the incubator. Then let rest for at least 1-3 hours before you change the medium the first time.

2. The dishes need to be coated in order for the rat cardiomyocytes to attach to the surface where they drop. The "tissure culture treated" surfaces from the manufacturers are not sufficient for these cells. We use laminin as it works best by far. Otherwise the cells will slowly float to the center of the dish over time.

In case you still find aggregates keep in mind that this might also be due to incomplete digestion of the ECM.

[bob1]

steinpdh, on 06 August 2012 - 12:35 AM, said:

1. After plating the cells - I recommend 10.000 cells per cm^2 - you need to shake/rock the dish in a star-like pattern. Rock it back and forth in at least 3 different directions. This is most conveniently done when they are already placed in the incubator. Then let rest for at least 1-3 hours before you change the medium the first time.

Ze rocking, it does nothing! (with apologies to The Simpsons)  In my experience (over 10 years in cell culture), rocking the plates doesn't do anything much, the cells will be floating around before attaching for at least an hour or two and transferring them to an incubator will cause just as much shaking as knowingly rocking them around.  Just make sure that you don't swirl them so that they get pulled into the centre by the vortex that forms, and make sure that your incubator isn't shaking slightly, which will cause patterns to form.

[leelee]

Totally agree with bob1 on the rocking. In my experience (from observing the plates of those who insist on using this "technique"), it actually usually makes things worse.

(also, why would you change the media after only 3 hours? Seems a mighty waste to me!)

[steinpdh]

That star-pattern rocking is still the best way to get them as evenly distributed as possible. The swirling due to the transport to the incubator indeed distributes them too, but just not as uniformly.

And these cells are huge. They will pretty much drop fast right where they are. They will adhere well if you coated the dish appropiately (ie with laminin). After 3 hours they are attached strong enough to not disturb them when you change the medium. And changing the medium is necessary then, because the medium has turned almost yellow by that time (due to the stress the cells had through the digestion). After that change the medium is very stable for long periods of time.

These are my experiences with rat cardiomyocytes. They are handled very differently than any other cell type I have ever worked with.

[steinpdh]

I just noticed that "rocking" may actually be the wrong word..

The motion I am trying to explain rather is a sharp back-and-forth sliding in several directions in the same plane. I'm sorry for my english skills and not being able to describe this any better :/

[lydialing]

Dear all,

Thank you for your reply and suggestions.
I feel there’s a need to describe my question more clearly.
I’m doing primary cardiomyocyte culture using either embryos or neonates. The wells or chambers are coated with rat tail collagen I. The collagenase digestion is about 35 min but no less than 30min and no longer than 40 min at warm room for somehow the cell survival rate is low if the digestion time is out of the range. (Any indicators to show that the digestion is just good enough/ complete and not over-digested?) When seeding, I was pipetting in the middle of the chamber to disperse the cells and gentle shake back and forth, but the cells ended up to form a line in the middle of the chamber and around the chamber.

The cells attach well and they can beat so the coating is ok I think.
I attached 2 pictures for your reference.

Any idea I can do to get cell distribution more even and in a way that the outline of each cell is easier to tell? Thank you.

Attached Thumbnails

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[bob1]

Hmmm, do the cell distributions look anything like the ones in figs 10,11, or 13 in the attachment?

Attached Files

•  cc_guide_identifing_correcting_common_cell_growth_problem_5_2_03_cls_cc_013w.pdf   493.25K   106 downloads

[leelee]

To me it looks like those clumps of cells (in your second picture) are still all stuck together and were that way before you seeded the plate. Do you examine them under the microscope just after seeding (i.e. before they have had time to settle). You'll be able to see the clumps then, if that is your problem.


During your digestion step, are you stirring?


After you filter your cells, do you mix them by pipetting up and down several times? (say 20 or so times?)

[leelee]

Also, when you are pipetting into the chamber, are you adding a small volume of cells to a greater volume of media?

If so, instead I would suspend the cells in the total volume first- then add the whole lot to the chamber at once. This eliminates the need for mixing/shaking/rocking/whatever completely- as the cells are already evenly mixed in your suspension.

[lydialing]

Thanks for your attached file, bob1. Since this unevenly distribution is only seen in cardiomyocyte culture but not in my other cell cultures in the same incubator, I feel it may not be due to the mechanic vibration. I attached a sketch of the uneven cell seeding I encountered, trying to show the two problems when using a 4-chamber slide, tangled cells in patches (or maybe not digested completely? How do I know the digestion is just enough?)  most observed in the middle of the chamber, and cells landed heavily around the well ( Was the way I shake the chamber slide wrong? It seems hard to handle containers with small wells, such as 24 well plates.)
Leelee, I use an intelli-mixer to rotate the tissuses at the speed of 4 RPM. After filtered the cells, however, I didn’t do so many times of pipetting as you said. I’ll pay attention to this next time and see if it helps, thanks.
And thanks you all, dear forum fellows.

Attached Thumbnails

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