Quality of matrigel, contamination by mycoplasms

We already tried two new batches of Matrigel, without succes.

Hi Katerina,

The quality of endothelial cells is crucial and most likely to be the issue in your case.

Do cells adhere to Matrigel after 30-60 min? Do they start spreading after 1 hours? If they don't then the problem is most likely to be their quality, i.e. loss of properties.

Please check two parameters:

1). Cell viability after trypsinization (smaller amount of viable cells - less tubes).

2). Passage (late passage cells loose the expression of endothelial cell-specific genes and hence won't form tubes).

Best wishes,

Leonid

Hi, I had the same problem. Now I use accutase instead of trypsin, and cells are in better condition after passage.

P.S.

1). Donor's age is important and worth checking.

2). Primary cultures can indeed be contaminated by mycoplasms (as well as incubator), as mentioned by Gilles.

Cell isolates ability to proliferate in culture would be a good test to check their viability.

Contaminated cells grow very slowly even as a monolayer.

3). Expression of endothelial-cell markers and cell-cell adhesion molecules is worth checking (CD31 and VE-cadherin) in these two isolates too. These molecules play role in tube formation.

Thank you a lot. We will test the cultures for mycoplysms, first of all.

But they proliferate normally (even maybe better, than others), look like normally, migrate etc.. And they indeed start spreading shortly after plating on Matrigel - but the complete tubes are not formed.

Concerning the passage, the problem is, that one is already passage six, but the other one is passage two-three.

From your updated information my conclusion ("diagnosis", if you like) would be that mycoplasma contamination is less likely cause (they would grow slower) but that the cells from these patient have partially lost their endothelial properties (quick check for CD31 expression by FACs would be good) due to late passage (first case) or age of the patient (second case).

Hi,

If youve already tried 2 different lots of matrigel then I suspect its your ec isolation as some else has also said. Have you recently got a fresh batch of collagenase? the specific activity and the trypinase activity of collgenase can vary widely. What else is in your enzyme mix. New BSA ? Sure its cell culture grade and tested for endotoxin. I can send you our enzyme mix if you want. Good luck caroline

Hi! Katerina!

I think you must check these stages:

1. If you add any additives recently you must be sure it's quality for example Cell tracker or any coloring agent

2. be sure there was no any transformation in your applying cells culture

3. I advice you to detach cell by collagenase not trypsin (CD31 is susceptibe to trypsin)

4. Check CD31 expression

Best

Just wondering why the donor's age is important with HUVECs. Why would it affect umbilical ECs so significantly?

Re: To Jaako Kangas

The statement was based on a practical/laboratory observations from comparison of the properties of HUVECs isolated from several patients. Unfortunartely, we didn' follow up and test whether this was due to the difference in replicative potential of the cells etc.

I agree with most of the answers above, mainly:

1. HUVEC quality.

2. Use of alternatives to trypsin.

And following up:

- Did you try different dilution of cells? Our experience tell us that this is also a relevant issue for success.

- What about the cell culture media that you use? Surely you look at that, but just in case!

Hi Katerina,

I noticed one interesting detail about your cells. If they proliferate faster than usual, these cells may be very "active", less differentiated and may contain a population of progenitor-like cells. Just for the interest, you may check a percentage of CD34+ and CD133+ cells and compare with the percentage of CD31+ cells. From our experience with angiogenically active microvascular endothelial cells, these populations exhibit early (within 2-4 hrs) tube-like structures that collapse faster. If you are planning to continue this stock, try them again for tube formation at later passages. You may also apply some relevant agonists. Our cells have a perfect morphogenetic response to ATP (100 uM). Good luck!

Totally agree with Evgenia. Great suggestion!

Just wanted to add to my previuos comment. I would suggest that you have overdigested your HUVEC and stripped them of their CD31 expression. This means that they are dedifferentaited and most probably fibroblast like. I agree with the above stain for both CD31, and vimentin (fibroblast). And in your new isolates try to limit the collegenase activity. we use trypsin Inhibitor and BSA in our enzyme mix.

Thank you. We will try the maximum. For the moment, we will test 2-3 isolates we had frozen and by the beginning of January, do a new isolation. Unfortunately, I am very limited by the end of year concerning finances, but we will do our best.

Caroline, can you write me what trypsin inhibitor and what quantity of BSA do you use?

We want to try centrifuge the cells after trypsinisation and then dissolve in new batch of medium. Do you think, that this procedure want might solve the problem with insufficient formation of tubes ? Because trypsin could affect adhesive molecules (CD31,...), i think.

Hi Katerina Hi Daniel, Please find attched my endothelial isolation enzyme mix and a short protocol. In your last comment are you talking about passaged cells if so then yes you should always 1) neutralise the trypsin activity follwing detachment of the cells by adding 1 vol of the media plus 10% FCS. Then spin down remove the supernatent and resuspend the pellet in fresh growth medium. What do you grow your cells in ?. hope this helps caroline

Thank you for the protocol. In terms of growth, we had no problems, just maybe one

batch of cells grew very quickly. Btw. we are using Endothelial cell basal medium from PromoCell with supplements for growth of cells and we add always gentamycin, tetracyklin anf FCS.