It is important with Caco-2 not to let them reach confluence before subculture. If they grow to confluence they adhere very tightly, are difficult to disaggregate, and do not plate well.

Make sure you know the number of viable cells that you are plating after subculture. 2x10e4 or 5x10e4/ml should be about right. Do not rely on split level.

DMEM/F12 should be OK (we used DMEM/F10). Not sure you need 20% serum or heat inactivation. We used 10% FBS, not heat inactivated.

I agree with your other respondents -- make sure they are mycoplasma free.

It is better to grow them without antibiotics to ensure that you have no low level contaminants in there. You MUST leave out antibiotics for at least one week before you test for mycoplasma.

I looked at the American Tissue Type Collection, and they have information on media and propagation. One thing I noticed is that they recommend 20% FBS, higher than many standard media. Also, one thing I found is that their in house media often differs in glucose levels than found in Life Tech media. I would suggest that you consider investing in some of their media. You also probably need to lot test FBS for them. Here's the link to the information: http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=htb-37&Template=cellBiology#aProp0b7c6

You may use DMEM (high glucose) [from Sigma; Cat No. D7777] + 10% FBS. The high glucose aids the cells to proliferate easily. However, do be wary of contamination problems.

Hi Claudia,

As Phyllis mentioned, make sure you are following ATCC recommendations for its culture. IMPORTANT: please note that ATCC reports a doubling time for CACO cells of about 62 hours. This is three times higher when compare to the reported doubling time, for example, of a lung carcinoma cell line as A549 (22 hours). So, the slow growth that you are observing for these cell may be perfectly normal

Also, did you test for mycoplasma?

Hi, I used 20% serum in the past. There were happy growing cells. Good luck.

you may use DMEM + 20% FBS .(Change the medium in alternate days).

Hi Claudia,

Use high glucose culture media, 20 % serum (for short period) and the most important thing to do is to split the cells more offen, this should boost your to grow rapidly. Good luck.

Usually Caco-2 grow fairly fast. Did you check if they are contaminated with e.g. mycoplasm. That is the most often reason for slow growing cell cultures.

I believe these cells are very sensitive to density for good growth. When passing the cells be sure you are not passing them too thin. If confluent, pass at 1:3 -> 1:5 dilution at most.

Hello,

I routinely grow Caco2 and Caco2-Bbe cells in the lab with DMEM 10%FBS and they grow fine. It's important to be sure if you have no mycoplasma. Plus always try to be thorough when you trypsinize. If they are too clumpy when you pass them they will not attach and will die. Also don't do very diluted passages (I always do 1:2 dilutions when I pass them), since this cells really don't grow very well when they are diluted. Another thing is to always freeze them in FBS with 10%DMSO. Some people freeze them in DMEM 10%DMSO and they don't really thaw out very well.

Good luck.

Use of 20% de-complimented FBS is necessary. I too faced the same problem with Caco-2 as they are indeed slow growing cells. Besides, there growth was enhanced with MEM (with Sodium bicarbonate + Non essential amino acids) + Sodium Pyruvate + L-Glutamine + 20% FBS. Please do not dilute the sub culturing titer to more than 1:4. One way to speed up your planned experiments is to first do not panic and try to make a good quantity of 90mm petri plates and use 2-3 plates for sub culturing and others for experimentation. Do remember that after plating the cells for further experimentation you must leave them for more than 24 hrs in 12-, 24- or 96-well plates which so ever you would use as they are slow growing so they would take time to over come the pipetting errors. Good luck.

I would use Eagle’s Minimum Essential Medium (MEM) for maintaining the coltures, and I would implement medium with Earle’s Balanced Salt Solution (BSS), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, 1.0 mM sodium pyruvate (for non-ATCC MEM),20% Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin (pen/strep). Then I don't know precisely which kind of experiments you are going to perform, but I would not use high glucose medium: it could change proteins quantitations and pathways closely related to cellular metabolism. You could also try DMEM-F12 Mediun: it should be a more enriched medium.

Good luck!!

Hi Claudia

All suggestion and comment are right.

But are you sure in Day 0 of your cell culture , your cell viability was more than 80%.

If your cell viability had been less than 60%, it takes time little bit more than usual.

Good Luck

I usually grow caco2 in 10%FBS ( heat inactivated) , change media 3X per week , split when confluent @ 1:3- 1:4 dilution. They grow very well .

1) How many passages have those cells been gone through already? They may be old and thus losing vigor... why don't you try a vial with cells with fewer passages from your liquid nitrogen container?

2) Check that you are using the right medium or if it is the right one, check the day it was opened or the colour; it might be a bit too old

Good luck!!

I do not think you need 20% FBS; 10% FBS and 1% P/S usually works well for Caco-2 cells (make sure FBS is heat inactivated, though) and pass 10% as the minimum; less than that, too thin

It is important with Caco-2 not to let them reach confluence before subculture. If they grow to confluence they adhere very tightly, are difficult to disaggregate, and do not plate well.

Make sure you know the number of viable cells that you are plating after subculture. 2x10e4 or 5x10e4/ml should be about right. Do not rely on split level.

DMEM/F12 should be OK (we used DMEM/F10). Not sure you need 20% serum or heat inactivation. We used 10% FBS, not heat inactivated.

I agree with your other respondents -- make sure they are mycoplasma free.

It is better to grow them without antibiotics to ensure that you have no low level contaminants in there. You MUST leave out antibiotics for at least one week before you test for mycoplasma.

I agree with some of the other respondent about a possible contamination with mycoplasma, so make sure they are myco-free. After that, if could help you we have recently published a paper and a protocol about the influence of cell maintainance on Caco-2 cell growth. You you can find the pdf in my publication list.

Hello,

You should check your medium.

I think it needs some essential nutrients, growth factors,...

Just, look in literature and adjust if necessary.

It may also be due to CO2, temperature or pH problems.

GOOD LUCK

Try starting the cultures with 20% FBS-DMEM, check confluence and morphology after 24hrs. If cells are growing well, switch to 10% FBS-DMEM. Pancreatin can be used in place of trypsin in case trypsin is being used to detach cells from flask since trypsin is quite harsh on cells as compared to pancreatin.

Also remember that RPMI is not conducive for Caco-2 cells as DMEM is recommended by Riken Inc.

Also check the passage number may be the Caco-2 cells have gone through enough passages and lost potency.

we tried some kind of serums, including 8866, transgene or wisent. I think serum is very important. we once fed caco2 with 10%trans+DMEMF12, and caco2 grew well, but when trans serum changed to other batch number, it grew slowly. Now we use 10% wisent serum, and caco2 grows faster as before. also growth speed depends on cell type.I think that MDCK and Hela grow faster than Caco2.

also, we use NUNC instead of NEST plates to culture Caco2.

I agree with Michael Fenn completely. Take the advice of Ian Freshney, I have used his book as a reference "bible" for cell and tissue culture for the past 25 years.

One thing not mentioned is the "quality" of the FCS/FBS i.e. New Zealand FCS/FBS is the best ....European sourced serum the worst. This reflects in the price, NZ (£230) and European (£40). We look at sensitive cellular mechanism and we see massive differences in the results we get between the serums.

I am not sure with colon cancer cell lines, but my friend is well known on this cell line. He works always with this cell line,You can contact him : [email protected]

I hope you will get the right answer.,.,

If your problem is still there after trying the tipps above - and best anyway: test for mycoplasma.

How long take Caco-2 cells to get confluent?

Please note that the Population Doubling Time for this cell line is about 62 hours

(http://www.atcc.org/Products/All/htb-37.aspx#A7931A04156C4C7FA40828AEF707302F )

This is more than three times of the average doubling time for many of the colon cell lines. Please see the attached table.

Good luck!

@Rolando-Garcia-Milian: Thank you very much for the helpful information!

Dont worry this is normal they grow in colony and very slow I grow them in DMEM (L-glucose) medium and change medium after every 3 days

i think you can increase the seeding density whenever you need to split the cells. Caco-2 cells grow quite slowly when they are at low density.

All researcher`s suggestion are correct. By increase the percentage of FBS may cell growth little fast. If you decide to increase FBS percentage, I suggest you, use the some FBS by different Batch number and mix them after that, added to media. Because it possible one batch has unknown inhibitor.

Please be careful the character of caco-2 cell is growing slowly and it will better to arrange your experiments base on normal cell growth. If you induce proliferation of caco-2, may be it will be hard to reach reproducible results.

Good luck

I use MEM(Eagle) + 10%FBS , replate at 1:3-1:4 ratio . Usually , the first 24hrs Caco-2 grow slowly,but they pickup the pace afterward. Do not plate at a low density.

Hi,

I am growing them in EMEM +10%FBS+5ml L-glutamine and 5ml NEAA.

The cells are growing fast in this media ,but insure when you split them you don't split into low ratio because it will affect their growth. 

Thanks

Hi,

.

A lot of publications people use DMEN with high glucose and according to ATCC  I should use EMEM. Which medium is better?

help me 

Hi, I have grown Caco-2 cells in 20% FBS, very happy cells. Good luck. :-)

I use DMEM high glucose with 10% FBS, They grow slow, you can't do much about it.

Don`t worry, This kind of slow growing is the character of Caco-2 cell line. May be by adding more FBS (20%), will have little bite more proliferation.

 i want to know how you decide the time when you do transport assay?after the  TEER stable or 21 days?

Full differentiation and expression of transporters is normally reached after 18 to 21 day of confluence. TEER stabilisation is reached earlier but it only represents closure of tight junctions. Therefore, if you are interested in mostly passive or paracellular permeability, 14 days of confluence should be sufficient. If you are looking for active transport, I would suggest using cells seeded on filters at high density (3.5 x 105 cells/cm2 , so that they reach confluence earlier) and assay transport after 21 days. See ref. Ferruzza S et al  Toxicol In Vitro. 2012 Dec;26(8):1252-5. doi: 10.1016/j.tiv.2012.01.008. 

Hello!

I used RPMI 1640 as a cell culture medium without Penestrep and it helped its growing instead of waiting to adhere on to the culture flask between 4-7 days and also the method affected on the cell freezing. Wish you luck and happiness