Did you use trypsin to detach them? They may have lost surface adhesion molecules and don't replace them quickly without serum.

In addition to what Jason said, the cells might also need to bind to an immobilized adhesion protein.

Many tissue culture cells grown in serum adhere to vitronectin, which is a serum RGD containing adhesion protein that binds avidly to tissue culture plastic.

Do you know the amino acid composition of your medium? Do you add glutamine (GLN) to this medium?

there may be problem with the attachment of the compromized cells with no serum. The solution for you is to coat the plate with extracellular matrix such as fibronectin or even dilute matrigel bvefore plating the cells. Let me know if it worked out.

Did you compare the viabilty of the two lines after the freeze? FBS is commonly thought to be part of the external cryoprotection during freezing (some protocols use 20%for this reason). The protein is thought to block large ice crystal formation in the media as it freezes. Perhaps the KO serum replacement fails to similarly protect and as a result you are seeing differential survival of the cultures during the freeze/thaw process....just a thought.

I've also adapted cell lines to serum free conditions in the past. As you reduce the serum concentration, the cells will detach and begin grow in suspension. Reintroducing serum will cause the cells to re-attach. If you are growing cells in a serum free basic medium like DMEM, then it would be wise to enrich it by adding trace elements especially iron and a peptone e.g. soy or yeast.

Cell clumping can be an issue when growing cells serum free and in suspension. It depends on the type of media you are using. If it''s occurring, you can add ""anti-clumping agent" (Invitrogen) to enable you to achieve higher cell densities in suspension.

Thank you all for you answers: my replies:

Jason: No I use accutase for the removal of the cells

Steingrimur: we have pre-coated cell culture plates already. see answer for raphael.

Samuel: yes we add 4mM L-Glutamine to the media when completing it, along with NEAA and Serum/Replacer.

Raphael: Really interesting never thought of matrigel. I have plated them down with matrigel and will let you know how they get on,

Tim: yes we compared the viabilities and there were all >96% with PI exclusion. It was then the adhesion problems that resulting in them dying in suspension. Attachment was only 30% compared to 98% for the others.

Lisa: i need to try and stay with monolayer for the moment, so adapting them into suspension isnt an option for me. They also died when they went into suspension :( .

Further: would it be an option to look at the adhesion molecules? B1 Integrin? Does anybody know any assays for these molecules?

Thanks for the feedback.

Dear Kirsty,

what many people do not realise is that also adherent cells do not stick to plastic, They stick to proteins that themselves stick to plastic. So, all proteins that engage integrins and non-integrin receptors help the cells to find anchorage.

Serum contains fibronectin (look up Raphael Gordetsky is has done some great work on this) and vitronectin. If you seed seels in the absence of serum, ther eis no protein to make cells stick. Thats why companies are selling sticking coatings to go with serum-free media.

So, coating with fibronectin or collagen or vitronectin might help, sometimes people just use poly-D-lysine.

My group has just discovered a coating that might help you. I am willing to send you some but wil not disclose what it is and you test it and let me know how it goes. Alternatively, use fibroblasts to deposit a matrix under MAcromolecular crowding, then lyse those cells away and you now have a extracelular matrix your cells can stick to. I can get you some protocols on that.

you can contact me directly via [email protected], for a test run of our new coating, which is protein-free..

Cheers, Michael

Hello Kirsty,

You could with monolayers in 10% FBS, then gradually decrease the FBS content over 2 days instead of jumping to serum free media. Avoiding trysin (As suggested above) is a good idea..You can use Gentle EDTA treament to detach the cells and replate them. Good Luck!

Dr. VN Sumantran, IIT-Madras, Chennai, India

Venil, they where adapted like that in the media before freezing. All the cell where fully KOSR ready when frozen.

the were also adapted with FBS/KO, KO/FBS and then KO/KO. it was this section that proved to be the non-adherance step for the cells.

I dont use trypsin I use accutase which is non toxic and they release into a nice single cell suspension. they just wont adhere.

Thank you very much for your time.

Kirsty.

Thanks for clarification Kirsty, Perhaps your KO affects Integrins or Laminins?? Maybe this could lead to new data?? On the other hand, it maybe best to force the cells to attach to coated plates and finish your work.

Laminins and integrins are looking the way forward. they will adhere to matrigel quite happily and will try gelatin but they arnt proliferating like the FBS cells. i am going to try a few other sources of KOSR and see if the same things happen with those then i can further look into the adhesion problem.

Thank you for your time.

Yes

HI,

This does look like an adherence issue. As has been pointed out by Tim Lyden, FCS is thought to protect the cells during cryopreservation- indeed a lot of commercially available freezing mixtures are actually based on 90% FCS and 10% DMSO. Micheal Raghunath also makes an important point that cells do not adhere directly to the tissue culture substrate, but rather to molecules in the serum which adhere to the plastic first, and then provide a surface for the cells to stick to. In the case of some cell lines which do not recover well from freezing I often incubate a tissue culture flask with FCS for an hour, then wash it with media three times before I thaw out the cells and plate them- this enhances recovery. I would imagine that this might help your cells recover in the short term, especially if they were frozen in FCS but you may have to wean them off FCS before you actually do your experiments and that might take a long time to do.

If the purpose of culturing your cells under such conditions is to generate a cell line which has mesenchymal/osteoblastic differentiation potential, then plating you cells on a plate coated with biomolecules such as gelatin, fibronectin or even collagen should help them stick, however many of the factors regulate differentiation and you may loose differentiation potential.

One potential answer would be to use good quality BSA as your protein instead of FCS in the freezing mixture to help prevent ice crystal formation, and it might even help the cells stick down when you thaw them out, but without affecting differentiation..