hello,

from my experiance.....

about picture...I think left picture can be about 50 % and right about 90% confluency, when you have 80-90% its better for cells and better for you to trypsinize them for next seeding....

when I have overconfluency it hard to trypsinize them (it takes too long to do it). In the 75 cm2 flask I have around 1-2 mnl cells (fibroblas-like cells)......

I think if cells not grow, you shoud pass them 1:3 or 1:2 and look at cells after 24-48 h, or maybe add some more serum (to final 20% if it not affect you cells) and wait for a week.... or maybe you cells too old....

1. Trypsinize and ask a colleague to grow some of your cells. If it grows with them, there is some problem with your handling (since you use the same flask/media/serum/incubator etc.)

2. Are you screwing the flasks too tightly?

3. Try using more serum.

The real problem here is that whilst knowing what split ratio they use is useful in the lab as a ready reckoner, you need to know the number of cells they passage. For example if someone thinks the flask is 80% confluent and splits it 1:8, but it is only 40% then the split is actually 1:16, then there may not be enough cells to give you an exponentially growing culture within a few days, it may take a week. Why don't you ask your colleagues for a small sample of their cell suspension when they passage their cells, and they can tell you what the total volume of cell suspension they have. If you then count the cells, then you will know how many cells they passage for 48h and over a weekend. Do not forget to adjust your calculation for the area of the vessel you plate them into. You could also plate cells out at different densities on a 6 or 12 well plate and watch them grow and see which is best. You could even plate out multiple plates and count them to get a good growth curve see what happens at confluence.

good luck

Andy

I forgot: my cells are before passage 25, so they are not too old. Regarding the corks, it's a good suggestion, but ours have filters, so we can screw them on tightly!

More serum? I'm at 10%, which is what everyone else uses...

I think I should ask the colleagues for cells when they split, so I know exactly how many they use. Also if someone will be kind and exchange cells with me - so that I pass a batch of his and he passes a batch of mine.

Next question will be: if it's my handling, how do I find out what to change, when apparently I'm doing the right gestures?

Hi Andrea! Are you using the same exact media as the other folks? When I worked with RPE cells, the cells were very picky as to growing in their environment. I remember that I had to add Sodium Pyruvate and Hepes Buffer into a High Glucose media solution to make the cells happy enough to grow. I would still use 10% FBS serum as this is the recommended dosage.

Are you seeing a lot of dead cells, infected cells, or just very few cells? If you're noticing cells are constantly dying off, then maybe it's a sign that the cells are not very good anymore and you should thaw a new batch of cells. Also, this is such a small matter, but if I ever forgot to remove all the DMSO from the frozen cells, they wouldn't grow properly. So little things like that always help. If cells are just not growing after passaging, maybe it's a sign that you are not passing enough cells and so just up the ratio.

**Another thing you can do, that I've tried and worked, if you are seeing so many dead cells versus live cells, is a Ficoll Paque centrifugation method. This will separate the dead cells from the live, and then you will be able to seed only live cells. I've noticed a boost in growth after doing this method.

Good luck and please let us know what worked and what didn't!!

Shahila

I would approach it as follows.

Ask a colleague to split one of your cultures using your supplies and reagents. Ask a colleague for one of his/her cultures and split it using your colleague's supplies and reagents. Keep the two sets of cultures in the same incubator - preferably on the same shelf.

If the cultures that were handled by your colleague grow well, but the cells handled by you do not grow, the problem is YOU. One former member of my lab had this problem. It turned out to be caused by rough handling of single cell suspensions (pushing and pulling them through narrow gauge-needles with excessive force.) By the time the cells were aliquoted into new culture plates, most of them were dead. Therefore, if the first round of testing indicates that YOU are the problem, determine how many of the cells are still excluding trypan blue when you get ready to plate and how many are adhering to the surface of the culture flask after a reasonable period of time.

If, on the other hand, your colleague's cells perform well in your hands but your cells perform poorly in your colleague's hands, something is obviously wrong with the cells and/or the reagents/supplies. If the experimental design rules out incubator problems and further testing suggests that the problem is with the cells rather than the supplies, go back to a trustworthy fresh stock such as cells from ATCC and your problems will probably go away - at least for a while. Cell lines can change with time, especially if they have been passaged extensively and/or have been passaged in ways that deviate from the recommended procedures.

Problems with supplies may be tedious to sort out. Don't forget to double-check the plasticware. You may have heard the story of the lab that accidently used gas-sterilized plasticware instead of tissue culture-grade plasticware. There can be enough residual ethylene oxide in the gas-sterilized plasticware to prevent cell growth.

Best of luck!

Don't plate the cells too sparsely, they won't like it. Try plating 1,000,000 cells per 10 cm plate (that's about 55 cm squared). You will need to split them more often - every two days perhaps if they start growing again. Have they been tested for mycoplasma infection? Other infections should be obvious when you look at them down the microscope or by the colour of the medium.

Another thing I would mention is the batch of serum - is it the same batch as your colleagues are using? Maybe try an aliquot of theirs in case of batch-to-batch variation.

I think someone else might have mentioned this, but are your flasks ventilated?! You can get two kinds of caps for flasks - solid ones and ones with a membrane that allow movement of gases - make sure you have the ventilated ones.

Are you using your trypsin at the right concentration?

Those are the most obvious things I can think of - good luck.

Found Fritz's suggestion very useful. I think you must try it out.

I remember someone saying once that you must treat your cells like babies. Must be handled very gently - especially adherant cells, when they are detached.

Thanks a lot for all these great suggestions. Unfortunately I have already thought of most of them:

- same serum batches for everyone (we keep common stocks of everything)

- same trypsin solution (commercialised)

- same flasks with ventilated caps

- no mycoplasma infection

- different dilutions of seeded cells (counting before seeding)

- The DMSO in the freezing medium (10% DMSO, 90% FBS) isn't a problem, since they always grow when I seed them directly into the DMEM/F12. I've even heard that the centrifugation to remove the DMSO could even be worse (since they stay in the DMSO for longer, too!)

I asked ATCC, who says that different batches of cells should be seeded at specific densities, and gave me an example (I don't know which batch we bought), of 8.0 x 10(3) viable cells/cm^2, which means 2*10^5 cells in a T25. Lately I've been seeding approximately ten times as many (!) which is more even than you suggest, Nicola. In theory I should pass them at 80% conflueny, at a 1:10 dilution and have 80% confluency again 48 h later.

It's as if I do something between counting and seeding that makes the cells vanish! I take the aliquot for counting after seeding, so if there's sedimentation, I'd rather under- than overestimate the number of cells. The other option would be that I'm rough on them, and kill them off, but I've had a colleague look over my shoulder, and she said everything was fine in that aspect... (Although she did have an objection as to how I unscrewed the flask, and said I might contaminate it by blocking the air flow with my hand over the opening. So she really looked at how I worked.)

Shahila, what do you mean by "RPE1 cells are picky"? Everyone I've ever talked to say that these cells grow like crazy! And that these cells aren't very fragile at all, still, I do try to take care of them gently.

Maybe it's a good suggestion to colour the cells when I count them. If I count dead cells, I'd know where the problem is. In the flask, before trypsonising them, they do look good and healthy, however, just as on the ATCC-reference picture I linked to in the question, but a little bit less confluent than the right image. No dead cells or debris floating around. And even when they are few, if I change the medium regularly, they (most often) eventually grow and fill the flask. So they seem happy in the flasks. Could I kill them during trypsonisation or resuspension?! Or lose them in the pipette tips (everyone using the same...) Or just magically teleport them to another planet? Maybe I should become entertainer or physicist ;-)

Rajalakshmi, what do you mean by over trypsinisation? Why should I change the medium next morning? I usually don't change the medium at all, since the cells should be passaged every 2-3 days anyway. It's only when I have too few, that I change for fresh medium.

A few additional suggestions:

1) Check the pH of cultures in the incubator. I rely mostly on the color of the (phenol red-containing) culture medium. If the color is off, I adjust the CO2 flow no matter what the incubator gauges indicate. Many cells do not do well if the medium is too alkaline.

2) If cell viability is poor after trypsinization or cells do not attach readily and spread after seeding, consider the following the following old tricks:

Stop trypsinization by the addition of some serum (about 10% final concentration). Do this before you start dispersing the cells into a single cell suspension by repeated aspiration into a pipet. Cells tolerate physical abuse better in the presence of serum (the higher viscosity helps). They will also survive the centrifugation step better. Make sure there is serum in the medium when you re-suspend the pelleted cells.

Use warm medium to re-suspend cells after trypsinization. It prevents extensive breakdown of the cytoskeleton and allows cells to attach quickly.

3) Back to the incubator: Low humidity can be a problem, especially if cultures have a high surface-to-volume ratio or are not split for extended periods of time, because the medium becomes hypertonic.

Try to be a part of research team of your coworkers to reproduce whole procedure with the same cells and chemicals or to ask your coworkers to perform the same procedure with your cell samples.

Greetings

Hello Andrea,

Sorry for my later answer. You should know I am not a specialist, only I have a some experience. I haven´t worked with RPE. I worked with MSC, mesenchymal stem cells. Also I had a lot of problems with my cells. When I sure that incubator and culture medium were perfect I thought the problem was the pass to cell (cell in vitro are too old). The confluence are very important, because if density are very low, MSC don´t grow. The stems cells need her sister and growns factor which they are producing. I was almost mad, when my colleage recomended me to start de experiments with another cells more young.

I expect you will have luck!!

Regards,

Sonia

Above comments are excellent. I found that Trpsinization for ARPE-1 cells should be done as follows: Remove media, and wash twice with sterile PBS. Add trypsin (warmed to 37degC) for a minute, and discard it. Now keep cells in CO2 incubator for 3-4 minutes. You may need to tap cells to detach them. For confluent flasks, I needed almost 7-8 minutes of incubation. I usually stopped at 5 minutes, and took whatever cells I could get (usually anout 60% of what was in original flask). I used a wide pipette tip (sterile cut blue tip or 5ml pipette) to take up cells gently. I also used to add new media to original flask. They always grew back in a day--so I knew my trysinization was not too harsh. Hope this helps!

Thanks for all the answers! Before the holidays my cells were doing fine. I'm not sure in what way I changed the way I treated them, but I hope I'll be able to keep them growing this autumn as well.

RPE1 cells seem to be sensitive to pH and in our hands they only grow well if we supplement DMEM-F12 medium with sodium bicarbonate.

My answer is probably too late for you Andrea, but hopefully it may help somebody else.

If you are using freezing media as 60% FBS, 10%DMSO with 30%DMEM. Make sure you change the media next morning and alternative day to ensure the complete removal of DMSO. It will inhibit the growth of cells in general. Similar problem I solved in J744.A1 cell lines. RPE1 cells should grow easily till 25-30 passage. 

I plate 1-2x10^6 cells in 10 cm dish in High glucose-DMEM+10% FBS (10mL). After 48-72h I split them (3 min trypsin at 37°C) and plate in 15cm dish, 20mL (1 or 2 dishes, depending when I would use them). I do not let them go over 90% confluency. Passage number does not exceed 6. I hope it helps.

These cells are not easy! They growth much better on dmem than dmemF12 in which start to transform in undifferentiated cells. However, in this moment my cellls are not growing neither dyeing