Depending upon the origin of your cells and the culture conditions, these cells may either proliferate or die. Therefore, more information is needed to respond to your question. For general information, follow the link below.

Link: http://www.atcc.org/products/all/CRL-1446.aspx#culturemethod

Hi  Sivasubramanian I have used these cells. If they have not adhered, or started to adhere, in 1-2 hours then they are dead (or you have put them into non-TC flasks).

What sort of dishes flasks are you using? If you try again

a) Defrost vial at 37oC for 1-2 min.

b) Do not wash the cells but add directly into a TC coated flask containing media (DMEM + 10% FBS) the amount of media dependent upon the flask size (i.e. T25 = 5-10 ml T75 = 10-20 ml, T175 = 25-30 ml).

Note: Even using TC flasks your media MUST contain FBS - the FBS is the source of the ECM proteins (such as Fibronectin) that coats the TC plastic and the cells adhere to this (not the flask's plastic surface). In the absence of FBS the flasks will have to be pre-coated with ECM material.

Adherence should begin within ~ 1 hour and the cells should be completely spread by the following day.

If the second vial does not revive, and you are using TC flasks with FBS containing media, then it is likely that the cells were frozen down incorrectly (i.e. left too long at -80oC or they were not frozen in cryo-protectant). However even in poorly frozen cells you should see a few dozen adhere (i.e. 1-2% may survive). If you do not see any then one or more of the reasons above are likely to be responsible.

Hope this helps!

Gary

Basically, i agree with Gary, your cells are dead, thats the reason they keep floating. Except for the points Gary mentioned above, the status of these cells frozen down in liquid nitrogen decides their fate after thawing. If the cells were not very happy before they frozen, normally it is difficult to rescue them. So my point is if you try to thaw the cells as Gary's comments, but the cells still not recover, it means you have to find another source of this H9C2, such as ATCC. It may save time a lot.

I agree with Richard. Try increasing the FBS to 20 %. Also when you subculture the cells for the first time you can use half old and half new media. 

I'm currently working with H9C2, especially during first passages they don't proliferate much but I haven't experienced any problem recovering them after they've been stored in liquid nitrogen or even -80°C for long time.

Maybe your source of cell is not so good, be sure that cells have been frozen in medium containing at least 10%FBS and 5% DMSO. You can perform trypan blue analysis on thawed vials of cell in order to evaluate their viability.

"I think the process of reviving H9C2 cell is similar to general cell line. Maybe you need to confirm the reviving process and the conditions of the materials are OK. H9C2 cell line should be stored in liquid N2, not -80C. For the cell adhesion, the pretri dish can be coated with collagen before H9C2 cell culturing." From my student HY Hsieh.

Hi just to clarify - you would only need to coat a petri dish (or other surface) if:

a) you are not using Tissue Culture (TC) plastic for adherent cells (such as bacterial 10 cm petri dishes). Plastic has been pre-treated to allow the adherence of ECM from FBS.

This is a common mistake (unless it has been treated, TC plastic is only suitable for non-adherent cells and people often do not realise they are not sing the correct flasks and their cells fail to stick)

and/or:

b) you do not use FBS in your media (as this is the source of the ECM proteins - mostly Fibronectin).

H9C2 do not require the pre-coating of plastic if TC flasks/dishes for adherent cells are used.

Note: prior to plastic, when glass was used, the glass was "acid etched" for adherent cells - it gave the glass a rough surface for the ECM proteins to stick too.

Hope this helps,

Gary

I should also add (for the sake of completeness) that there is another reason to coat plastic.

If the cell type you are growing does not stick to the ECM proteins present  in FBS (such as neuronal cells) i.e. the cells do not have the integrins that bind i.e. RGD motifs, then you need to coat with a protein they can stick too (such as Laminin).

Thank you every one for helpful replies.

As said by Gary Morley I'm using TC plates which doesn't need any prior coating of FBS or collagen or matigel.

@ Gary morley :

I defrosted the cells at 37 C with in 2 mins and washed cells with FBS supplemented DMEM media for once at 300Xg for 3 mins and plated in the T50 flasks with 8 mL 10% FBS supplemented DMEM.

 @ Richard Tan Yi Jer

Thank you for the valuable suggestion. I will try to revive those cells again in the mentioned way. 

I have another doubt, kindly clarify this one too!

How should I Preserve these cells? is it I have to straightly take cells to liquid nitrogen or from -20 > -80 > Liquid Nitrogen? which way better suits this H9c2 cells?

Hi - the ATCC site suggests complete media (inc. FBS) plus 5% (v/v) DMSO.http://www.atcc.org/products/all/CRL-1446.aspx#culturemethod

However I generally freeze all of my cells in 10% DMSO in complete media-  which I did for the H9C2 and they seemed fine with that.

Ideally the cells should be harvested, resuspended in media containing 10% DMSO and placed in a Mr. Frosty (containing Isopropanol) at -80oC overnight.

The Mr.Frosty allows cells to cool down at 1oC per minute (down to -80 oC). You should not use the Isopropanol in the Mr.Frosty more than 3-5 times and it should be changed with fresh propanol after 1 month. Note: when you remove cells from the Mr.Frosty make sure you re-tighten the lid as isopropanol vapour is an explosive hazard.

If you do not have a controlled rate freezing device (like Mr.Frosty) place the cells in DMSO at -20oC for 4 -6 hours before transferring to -80oC overnight.

Cells should not be left at -80 for more than 3 days or their viability will begin to suffer. If cells are left more than 1-3 weeks at -80 (dependent upon cell type) the vast majority will die (I did this when I was a young student - argh!).

H9C2 cells are not density dependent (I have grown up many millions from a single cell for clonal analysis) so if even some stick down they should recover.

However, dependent upon intended use, if they recover very poorly you might want to buy new ones.

As mentioned, if no cells stick down after 18 hours (and it looks like you are using the correct media and TC plastic) then they are all dead and there is nothing you can do.

Don't forget that H9C2 are adherent cells - this means that they suffer from "anoikis".

When they are detached from a surface they will start to apoptose/die.

Therefore, like any adherent cell, once trypsinised they should be processed and frozen down as quickly as possible. If you Trypsinise them and leave them in suspension for too long they will die (generally 1-2 hours in suspension is ok for most cell types - you might get Annexin V positivity start to appear but this is generally a reversible process once cells re-attach).

Note: the role of the DMSO and the "controlled" freezing is to mainly prevent the formation of ice crystals in the cells (which will damage membranes etc). Also water, unlike most liquids, will expand at 4oC ("the anomalous expansion of water") before it contracts again. Part of the controlled freezing is to get passed this without damaging the cells too much.

Hope this helps,

Gary

Gary Morley Fan-Gang Tseng

Can you tell me why extracellular matrix is coming very fast ? still the passage number is very less.