what did you cryopreserve them in? It may well be they are no longer viable. Did you follow standard cryopreservation protocols? including slowly freezing the cells prior to cryppreserving with something like DMSO to prevent ice crystals?

eg Sigma Aldrich recommend:

1.Cultures should be healthy with a viability of >90% and no signs of microbial contamination.

2.Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures i.e. cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).

3.A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.

4.Use a cryoprotectant such as dimethyl sulphoxide (DMSO) or glycerol to help protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 10%, however, this is not appropriate for all cell lines e.g. where DMSO is used to induce differentiation. In such cases an alternative such as glycerol should be used (refer to ECACC data sheet for details of the correct cryoprotectant).

Ussualy the survival cells attached after 1-2 days. Are your cells survival after thawing?

Apparently, the cells were not frozen properly, so (MAYBE) it is not the media to blame, dear biologists, it's the physics here: slow freezing, vitrifcation etc, each type of cells needs own protocol, speed of coolling, ect to get optimal results. And what is very good for one type of cells can be detrimental for the others.

BTW, it's, not "crypreservative cells" but "cryopeserved cells". The media that "preserves" the cell during freezing is called "cryoprotector", some (wrongly) cal it a "crypreservatiive/cryopreservant". So, the media PROTECTS the cells from some bad process that occur during freezing and after thawing; in contrast, cryopreservation process per ce PRESERVES the cells from otherwise degradation or necessity to culture and duplicate them indefinitely. Two different things, they are often confused.

So, tell me HOW did you cool and warm the cells, and what CPA (cryopreotector) and in what concentrations was used. ONLY then, I can give you PROPER advise. Good luck!

Igor i. Katkov

Cryobiologist by training

P.S. Don't listen "Sigma Aldrich" or alike blindly, take with a grain of salt!!

Joanne Doleman : i want to reculture the SSN-1 that previous student did cryogenic process before but after culture it in fresh media,hey did not attach,i did the SSN-1 culture protocol but nothing...

Bui Xuan Nguyen : i did not know that i can determine the cell survival after thawing,i checked them after adding fresh media. but at my first culture, i checked the cell after 3 days but they were floating ,

Hi Mariam,

(somehow private Outbox doesn't indicate that I sent you a message, so it is a public copy:

-

How the cells WERE frozen?? That's the "64 million dollar question" (the highest level in "Who Wants To Be a Millionaire" game). If they were not cryopreserved properly and died during FREEZING or any other related pre-freezing procedure per se, there is ALMOST nothing you can do now.

Many things can go wrong, from simply forgetting to add DMSO ( or opposite, to freeze the cells in the stock 50% DMSO solution w/o dilution- as an example) to exposing frozen villas back to room temperature, etc, etc.

But, practically, you are not much interested now to do the Sherlock Holmes's work and "investigate the crime scene and interrogate the witnesses and a possible suspect", correct? Then, the only you can do is to collect attached cells every time you changed the media and replate them: if the amount of the dead cells is too much, they can release the "dead poisons" like dead bodies of peoples or animals, and that can kill those few alive cells left!

And pray, of course.

I must say: it'd need a lot of efforts to kil ALL the cells during freezing, few mistakes can be SO detrimental!

Two books are recommended for reading (browsing):

http://www.intechopen.com/books/current-frontiers-in-cryobiology

http://www.intechopen.com/books/current-frontiers-in-cryopreservation

I was planning to issue one book but the Pulishers told me split in tow books. The first one is a more of basics while the second one is rather on pareticular protocols. Feel free to upload any of them, it's indeed a free Open Acess publication. Just find what best fit you needs and background.

Good luck!

IK

P.S. If someone has difficulties to upload the Books' chapter(s), pls, contact me. Look like they have changed the security settings and it is no SO easy as it was before. Not my fault, sorry ;-))

IK

Thomas Re: "The ratio unstained cells/(unstained + stained cells) should ideally be greater than 0.75. If it is below 0.5 cells are in a very bad shape and will not attach."

In GENERAL, this statement is incorrect, several points as exmaples:

1. TB assay may give you 70-90% negative after thawing, yet the cells may die (e.g., go to an apoptotic patways) after 1-2 days of culture. See, for EXAMPLE, my 2 publications on hESCs on PumMed/Medline (type "Katkov and Snyder").

2. Similarly, TB can be good and attachment is OK but the cells may loose iimpotant functional properties suchs as plui-i or multi-potency,exitability, ect (again, my papers just as an example).

3. Just an opposite scenario the cells may demonstrate 40% TB(-) yet practically ALL of them will atatach, duplicate and go very well. I saw it in many time non-homegenous populations of cultured cells (so called "selective freezing out").

4. Moreover, non-cultured cells like sperm or eryhtrocytes are NOT equal in regards to size, shape, age, etc, on definition as they are produced in organism continiously. Thus, reaching very high TB is VERY difficult. WHAT type of cells are you exactly tlaking about? ONLY SSN-1?

5. For some so called "unfreezxeable" type cells even 50% TB(-) would be a treasure!

So, It DEPENDS. My current line of reserach is exactky to address this issue and develop a "unified" cell type insensitive (I called it originally "the universal" but some of my grant reviwers did not like it ;-) cryopreservation platform platform (equipment, cryocarries/ containers, a cryo-medium, etc). But this is deifinitely an irrelelavat to the Maryam's Q topic, don't want to use this thread to promote my RG-score ((I guess I already did ;-)))

IK

Shiv: why only CRYOPRESERVED cells were affected by this supposedly "heavy "AB load"? IMHO, bacterial contamination is even a greater disaster than a bad/improper freezing. In nay case, your suggestion doesn't address why only frozoen cells were affected: it should've killed the fresh cells as well.

Yesterday i cultured SSN-1 for third times ,but i didn t do centrifuge and just added the contents of vail after thawing into fresh media (offred by one of RG member), today i cheched the cell but most of them are floating,he told me replace the media every day,but i think it is better to change the media just today and waiting to be confluent

Really i don t know bcoz cryopreservation was done with another student who she was graduated

Shiv: then i should change the media today or wait for some days?

Thom: So, we are talking about SSN-1 here. OK. In that narrow pespective, you might be correct.

Shiv: the cell will be "weakened" after re-warming (NOTE: I don't use terms "freezing" and "thawing" if no ice were formed during cooling) if you used an improper method of cryopreservation. LN2 per see doesn't hurt, the coid does (you can equally 'sucessfully" kill al the cells in an industrial electrical freezer' on he other hand, the cells are not exposed directly to LN2 in so called "closes" containers such as straws used for sperm and oocyte/embryo cryopreservation. Otherwise, I agree, in general, slow freezing and equilibrium vitrfication (with LARGE amounts of permeable vitrficants and relatively "slow" cooling rate), both can damage cells on a sub-lethal level and massive antiobiotcs' overload can "finish" themter re-warming af. Thus, the only good way is kinetic (very fast) vitrfication: it doesn't produce mechnical stress of the cell and organelle membranes, neither the lethal ice inside the cells is formed. But (!): it is not an easy method. We are working on it as we speak so hopefuly, it'll be much easier one day. ;-))

Hi Maryam, I agree with the answers that the freezing procedure is the bottle neck. The cooling down procedure depends on the "cryoprotecion-reagent" you use.

But this does not help you now. I did not found anything about SSN-1 cells in cell databases ex from ATCC, can you tell me where these cells come from ?

In general I would always spin the cells after thawing. Maybee it would help if you thaw 2 vials and put them in a smaller flask or dish to increase the amount of viable cells.

Thomas: I checked cells today,around 10% of them were attached , then I changed the media. I m in virology lab but the stuff didn t have any experience about fish cell line or fish virology.

Thank you for all of the informative replies :) , i want to measure the degree of some cell lines susceptibilities ( SSN-!, FHM, BF-2 and 2 other cell lines that i should prepare myself ), SSN-1 and FHM were cryopreservation before but i doubt start this experience for FHM too.

Good to know Maryam, as I said, it is very difficult to kill ALL the cells. Unfortunately, it is practically IMPOSSIBLE to get them all alive after CP. You are lucky because your type of cells can grow and duplicate, 10%- you will regain the initial population relatively fast.

You can do thawing control in 3 HRS after placing the sampeles in LN2 tank (UNLESS, you are using a special cryo container "Mr. Frosty" or smth specifically designed for slow freezing in a -80C freezer overnight..

If you used another method of slow CP using e.g, a Planer (UK) programmed freezer (CRF) with a multi-step program (which is MUCH better than uncontrolled cooling in cryo containers but even the chepaest CRF is quite expensive), and when the sample has reached approx -85 -90C, you can PLUNGE the sample in LN2 then.

The samples in a cryorack placed a bit above the survace LN2 (to avoid cross contamination) will reach the water glass transition temeprature (Tg(H2O) is around -130C) in around 5-6 hrs (do you use 1..5 mL cryovials?), and practically NOTHING will happen after that. You can freeze a bulk of cells in the morning and check them late afternoon. Youu don't wantto wait 2-3 days and wander "how'd go?!". It's nerve-breaking and unnecessary. And you will know right away what to do..

Good luck with the cell recovery and with the first freezing. Let me know when you will go, we'll rehearse everything again.

Hello Maryam, you are getting good advice here already, but I would add that you definately need to spin the cells down to remove them from the crypopreservation media prior to bringing the pellet up again in the 15% serum based fresh media. I would leave them in this media alone for at least a few hours before you add in the antibiotics. Also, check your CO2 source with the CO2 indicator to make sure the CO2 is not more than 5% in your incubator and make sure you have not run out of CO2 either. I am assuming this cell type normally plates well and this is a first time event? If your plates are not normally "coated" with a matrix, then it may be exposure to DMSO in plating media or lack of CO2 or excessive CO2 which has resulted in cell death. And you should definately change out the plate after 24 hours, gently, and then every other day. Your media may also need growth additives added to it to assist in optimum cell health and attachment.

Hello Maryam, I'm not sure whether someone already talked about it but could you please add a little about the method used for the freezing of your cells. It may very well show the problem.