If you actually go to the ATCC web site page FOR Y79 CELLS (ATCC HTB-18) and not just an irrelevant generic protocol, as posted above, you will find that the subculturing procedure for Y79 does not involve trypsinization, since Y79 don't adhere significantly to plastic. The clumps are a normal feature of Y79 and you will see a photomicrograph of this at the same site. The ATCC subculturing protocol for Y79 is as follows (copied from the site and pasted here):

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium Renewal: Twice per week

Allow aggregates to settle to the bottom of the flask. Remove supernatant and discard. Add fresh medium, aspirate and dispense into new flasks.

THERE IS NO TRYPSINIZATION INVOLVED.

You can look it up yourself at:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Retina/HTB-18.aspx?slp=1#11F1A8C17A7A4FB8A36940C154A9B585

You may have to register first (free), then under documentation go to PRODUCT SHEET and download it. You will also find references describing the cell line. Anybody using a cell line they are unfamiliar with should be doing this basic literature research before starting a project, not collecting uninformed opinions from people who have never seen the cells. That's just good science!

To more generally address your question: There are many reasons why cells might clump, ranging from divalent cations in the medium to cell adhesion molecules, serum contaminated with cross-linking antibodies, contamination with mycoplasma or fungi, etc. However, Y79 normally clump! Presumably they do not have adhesion molecules that allow attachment and spreading on plastic, but are able to adhere to each other. I have worked with CHO adhesion mutants (unable to attach to plastic because of fibronectin mutation) years ago that clumped in suspension. We selected non-clumpers by simply transferring the cells to a 15 ml conical centrifuge tube in 10 ml of medium and letting the clumps settle under gravity for 5 minutes, then taking the top 1 ml and subculturing that. After about 20 passages using exactly the same technique the cells were entirely a single-cell suspension. Unfortunately, the project was shelved without investigating what the difference was between the clumpers and non-clumpers. You may be able to do this with Y79. A WARNING however: this is a SELECTION process and whatever features of Y79 cells that are actually important to your work may also be altered as a result of the selection. Similarly, if when you subculture you discard loosely attached cells and only passage cells that are somewhat adherent to the plastic (and require trypsin to remove them) you will eventually end up with cells that are highly adherent to plastic. Researchers experienced with tissue culture are always wary of changing the properties of their cells by subtle selections due to technique and are always going back to frozen stock at regular intervals. The literature is replete with examples of cell lines that have diverged significantly in different labs because the methods for cell culture were different and put divergent selective pressure on the cells. Y79 are a (typical) karyotypically screwed up cancer cell line (hypertryploid with a lot of fragmentation anomalies) and would be VERY susceptible to such divergence.

Add trypsin for enough time that makes the cell separate from each other when you store the cells. Once you want to use the cells again, you will through away the old media and add new media to a tube and shake your cells gently before you culture them. You can look under the microscope to make sure that they are separated.

If you think that the stored cells were not separated enough before they were stored or before you culture them, you can add some trypsin to them for few seconds and add media to stop trypsin , throw away the media and then add new media and shake the cells gently.

I hope this will solve your problem

Thanks for answer. I would like to keep cells separated during the culture on 96-well microplates in FBS supplemented medium for 48h for colorimetric assay. Won't trypsin be inactivated by FBS in medium?

Yes it would. What you do, you add trypisn for maximum 1-2 minutes. Then you add media to stop the action of trypsin. You must throw away the media that has trypsin after you centrifuge your cell with the media at low speed 1200 g for 3 minutes. Then you add new media with FBS and shake it gently. Then add the media with the cells to you plates.

You may need to count then before you add them to your plates and therefore you justify the amount of media depending on the # of cells you want.

FBS can inactivate tripsin. and long time incubation with tripsiin can kill the cells.

Make sure that you use 10% trypsin.

I can separate cells before seeding on plates by simply pipetting or vortexing and it works just fine in serum-free medium. If I want to use serum-supplemented medium, cells form new clusters once they are seeded on the plate.

I would like to add something to the medium to prevent the formation of clusters in microplate wells after I seed separated cells.

Vortexing cells and even pipettiing harm the cells.

I think, it's not a substance that let the cells grow within clusters. The factors promoting this phenomenon are adhesin molecules, such as integrins or cadherins. And these are differently expressed on different cell types.

I ( or another internet user; I just googled) also found another protocol from the ATCC webpage:

Protocol:

Remove and discard culture medium.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.

Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.

Add appropriate aliquots of the cell suspension to new culture vessels.

Incubate cultures at 37°C.

(ATCC website)

Jabcub, what is your research question? From my experience, less differentiated and more "primitive" cell tend to grow as grape-like aggregates. If growing as clusters is a feature of Y79 cell line, why do you want to change their phenotype? Cell adhesion is regulated by integrins, cadherins, and selectins. If you want to modulate their adhesion, try different physiological concentrations of Mn2+, Mg2+, and Ca2+. Generally, Mn2+ increases affinity to integrin ligands, Mg2+ promotes adhesion to cells, and Ca2+ decreases cell adhesion.

If you actually go to the ATCC web site page FOR Y79 CELLS (ATCC HTB-18) and not just an irrelevant generic protocol, as posted above, you will find that the subculturing procedure for Y79 does not involve trypsinization, since Y79 don't adhere significantly to plastic. The clumps are a normal feature of Y79 and you will see a photomicrograph of this at the same site. The ATCC subculturing protocol for Y79 is as follows (copied from the site and pasted here):

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended

Medium Renewal: Twice per week

Allow aggregates to settle to the bottom of the flask. Remove supernatant and discard. Add fresh medium, aspirate and dispense into new flasks.

THERE IS NO TRYPSINIZATION INVOLVED.

You can look it up yourself at:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Retina/HTB-18.aspx?slp=1#11F1A8C17A7A4FB8A36940C154A9B585

You may have to register first (free), then under documentation go to PRODUCT SHEET and download it. You will also find references describing the cell line. Anybody using a cell line they are unfamiliar with should be doing this basic literature research before starting a project, not collecting uninformed opinions from people who have never seen the cells. That's just good science!

To more generally address your question: There are many reasons why cells might clump, ranging from divalent cations in the medium to cell adhesion molecules, serum contaminated with cross-linking antibodies, contamination with mycoplasma or fungi, etc. However, Y79 normally clump! Presumably they do not have adhesion molecules that allow attachment and spreading on plastic, but are able to adhere to each other. I have worked with CHO adhesion mutants (unable to attach to plastic because of fibronectin mutation) years ago that clumped in suspension. We selected non-clumpers by simply transferring the cells to a 15 ml conical centrifuge tube in 10 ml of medium and letting the clumps settle under gravity for 5 minutes, then taking the top 1 ml and subculturing that. After about 20 passages using exactly the same technique the cells were entirely a single-cell suspension. Unfortunately, the project was shelved without investigating what the difference was between the clumpers and non-clumpers. You may be able to do this with Y79. A WARNING however: this is a SELECTION process and whatever features of Y79 cells that are actually important to your work may also be altered as a result of the selection. Similarly, if when you subculture you discard loosely attached cells and only passage cells that are somewhat adherent to the plastic (and require trypsin to remove them) you will eventually end up with cells that are highly adherent to plastic. Researchers experienced with tissue culture are always wary of changing the properties of their cells by subtle selections due to technique and are always going back to frozen stock at regular intervals. The literature is replete with examples of cell lines that have diverged significantly in different labs because the methods for cell culture were different and put divergent selective pressure on the cells. Y79 are a (typical) karyotypically screwed up cancer cell line (hypertryploid with a lot of fragmentation anomalies) and would be VERY susceptible to such divergence.

Thanks for your answers. I asked this question because I noticed that adding one compound to cell culture on 96-well microplate promotes the formation of clusters. While in control wells cells form medium-size aggregates dispersed evenly across the well, treated cells form only one gigantic glump in a center of the well. So do you think this compound may promote the activity of cadherins or integrins? Maybe adding some simple chelator like EDTA will abolish this activity?

Yeah, and I have never added trypsin to this cell line :)

Jacub, I agree with Evgenia Gerasimovskaya's answer. some cell lines show a tendency to aggregation; or even a tendency to grow in suspension despite being adherent, thus promoting a cluster behaviour. you can partially solve this problem by better pipetting when you detach cells, or by trying to change centrifugation speed/time during propagation also (if this is required), always remaining in a "gentle" range...

What a strange question, since you do not explain why you would like them to grow a part. Y79 normally clump! Presumably they do not have adhesion molecules that allow attachment and spreading on plastic, but are able to adhere to each other. The other aspect of the question is that cells normally forms tissues and that they goes towards this type of organization since the cells form an ecological network and that they exchange products (like vitamins, cofactors, ions) by proximity. I don't see any problem in having them in clamp, if a simple addition of fresh medium and effective checking allow them to separate. You maintained our cells in their growing conditions (clamps) and them use them separate...

But I also agree with the following sentence of Norbert Kartner

• "Anybody using a cell line they are unfamiliar with should be doing this basic literature research before starting a project, not collecting uninformed opinions from people who have never seen the cells". That's just good science!

Work on its one is the start of learning! the second point is to present your question at whole and to explain the purpose of your question. We might use this site for more problematic and specialize questions instead.

But if you still wanted to grow them separate, you can buy special flash which allow to grow cells under gentle stirring. We have done that with primary hepatocytes, and that's works... So... Just go for it.

Patrice

That's an interesting observation (depending to some extent on what the "compound" actually is... not so interesting if it's a lectin or antibody). EDTA may have an effect, but it will not be possible to distinguish whether this is because it is acting directly on the adhesion process by sequestering divalent cations, or indirectly by interfering with Ca2+ metabolism/signalling causing shifts in gene expression. Multiplex ELISA or qRT-PCR for an array of adhesion/ECM proteins or their mRNA might be a place to start if you really want to pursue this. I would watch that well that is aggregated for a while just to make sure that it is not clumped for the most common reason for cell clumping... contamination with a microorganism... before investing a lot of time and money.

Suspended cell lines grow in grape like clusters and if you like to make is a single cell suspention grow cell in shaker state

If you want them to grow seperately, then i guess they would rather die or some catastrophic events may occur? So, it may be better to keep them as they are. And it is highly possible that they may be quite primitive (or stem cell-like) cells! So, Evgenia must actually be right!

Clumps in suspension cultures of some cell lines indicate a normal healthy state and don't create any problems in many functional assays. For the purpose of counting, single cell suspension can be obtained by gentle pipetting just before seeding the plates.

Hi Jakub, If you see a one big clump in the "treatment" well than control well - it could be the results of overgrowth of cells. If the growth factors in the medium favour growth, cells can divide much faster and and form a bigger clump in the centre. I have noticed even disturbances from adding media to the well can cause this. If possible, you can start with much less number of cells.

http://www.sparks-of-light.org/hela-50years%20on.html

Hi, I agree with the others that many cell types, such as immune cells, grow like this naturally. Check information on uropods, which are the trailing end of some cells that are used for attaching to other like cells but not plastic. However do check out the link above which predicts that many cells are contaminated. However you are right to ask your question and should not just accept that thats the way it has always been like some researchers are suggesting. You should make sure that your cells are true, and are also a true model of the tissue/malignancy/disease that you wish to study.

It sounds like you have an interesting observation, now you need to determine whether it is interesting enough that you wish to follow it up.

Simply, you have to shake using finger tips after washing and after centrefugation steps and also, you have to take into your account the cellular density.

Try growing them in serum free medium with a commercial serum substitute supplement. They will attach to the bottom of the flask. Regards, Marshall Kadin