There are several possible explanations:

(1) The cells are dead because of not being properly stored (problems with liquid N2 container, problems with cryoprotective medium, etc).

(2) Lack of some additives in the medium. E.g. did you add double amount of fetal calf serum? Do your cells need any special additive not commonly used?

(3) For revival, cells were seeded at too low densities. To avoid this, use smaller flasks/plates.

It could be usful if you could give some details about which type of cells you want to revive. My answers refer to common mammalian cell cultures, but if you are working with bacteria, insect cells, etc, there are other possible explanations.

There are several possible explanations:

(1) The cells are dead because of not being properly stored (problems with liquid N2 container, problems with cryoprotective medium, etc).

(2) Lack of some additives in the medium. E.g. did you add double amount of fetal calf serum? Do your cells need any special additive not commonly used?

(3) For revival, cells were seeded at too low densities. To avoid this, use smaller flasks/plates.

It could be usful if you could give some details about which type of cells you want to revive. My answers refer to common mammalian cell cultures, but if you are working with bacteria, insect cells, etc, there are other possible explanations.

Other than the points above:

you may have frozen the cells too rapidly- directly in liquid nitrogen (highly unlikely, but possible). Ideally you should freeze them in a freeze box with isopropanol in -80C overnight, then move to liquid nitrogen.

you could also be thawing them too slowly. Contrary to popular belief you must thaw cyropreserved cells at room temp for 1 min followed by incubation in water bath until you see the freezing medium turn liquid. Do not thaw on ice.

Also, when cryopreserving, it is important that the cells are of high viability, and are in exponential growth phase, and not at plateau.

In addition to the above, some sensitive cells could be frozen in 90% FCS. The higher the FCS ratio, the higher the recovery upon thawing. Also, make sure to get rid of all the DMSO after thawing by spinning the cells down and carefully removing all supernatant before suspending in fresh medium and plating (DMSO is toxic for cells). I usually freeze 5x10^5 cells per vial and find that works pretty well. Putting too few or too many cells per vial will also reduce your recovery upon thawing. It would indeed be useful if you would tell us how you're freezing and thawing your cells exactly to help pinpoint the problem. Good luck!

Also a little note to the mentioned above - there are some indications of rather harmful centrifugation step during cell thawing procedure, which may also cause even higher cell death in some cases, than DMSO present in the medium, so, the medium could be changed the next day or after cells are attached..

this is my protocol for cryopreservstion:

cryopreservant: 70% FCS+ 5% DMSO+ Incomplete media (remaining)

i usually make 10 ml of this cryopreservant.

trypsinized confluent flasks, using 200ul of trypsin, incubated them for 5 mins, after incubation 800ul of complete media was added to it. Of this 1ml, 200 ul was used for cryopreservation, by adding this 200ul into the cryovials containing 1 ml of cryopreservant. After this the vials were sealed with parafilm and kept at 4 degree for 20 mins, they were then transferred to minus 20 freezer for 1 hour, and finally kept at minus 80 deep freezer after that for at least 2 months. My cells are of Neuroblastoma.

for reviving them... I quick thaw them at 37 degree in a water bath, then centrifuge it at 1500rpm for 5 mins, suspend the pellet in fresh media, this entire suspension is put into a T25 flask with 4ml of fresh media.

There you are then, best not to centrifuge after thawing.

Centrifuge after thawing should be avoided but it is important to use the correct method (you should follow the recommendation of the cell line provider) for freezing the cells as well.

ok ok I will check it!! Can anyone provide me with standard protocols.

In the web you can find many standard protocols and here (http://www.abcam.com/index.html?pageconfig=resource&rid=11742) you can find one, although I would recommend to follow the protocol from the cell line provider

There are several problems with you protocol.

1. cells should be only 70-80% confluent NEVER use 100% confluent cells for freezing.

2. You don't state the size of the flask used. However, if your cells are in a t-25 flask, 200 ul of trypsin is not enough (in 5 min most of cells will dry out). Rinse cells with PBS (ca+2 and Mg+2 free), remove PBS and add 1ml. observe cells under a microscope., when cells are detached add 10 ml of medium, centrifuge cells and resusspend cell pellet in x vol of COLD freezing medium.

3. As stated above cells should then be placed in a a freeze box with isopropanol in -80C overnight, then move to liquid nitrogen.

4. keeping the cells a -80o C for also did not help cell survival.

5. When thawing you cells, thaw quickly, add cells to flask containing 2x the vol of medium normal used (this is to dilute out the DMSO), place the flask in the incubator for 3-4 hours. Observe cells for attachment. Once the cells have attached, gently remove medium and gently add fresh medium.

6. cell should be good to go!

Thanks a lot!!

But why does centrifuging cells affect, i cannot understand??

I don't see a problem with your freezing, you are using an older method that worked before newer protocols with 100% isopropanol-containing freezing caddies were available. Am I correct that you are leaving the trypsin in the cells you freeze? That doesn't sound appropriate. I am familiar with spinning down the cells into a pellet, removing the trypsin/media mixture, and resuspending (gently) your cells in freezing media (my best result is with 90% FBS/10% DMSO or 80/10/10 FBS/Media/DMSO) that is a little cooler than room temperature, then slowly freezing. I also don't centrifuge upon thawing, I wait up to 24 hours for the cells to recover then replace the media as soon as the cells are well-spread.

How you fridge and thaw the cells?

Slow freezing rate 1-2 degree cooling rate and rapid freezing is desirable for all mammalian cells. What cells you like to stock?

@Ashok Mukhopadhyay....Neuroblastoma cells.

Whenever I don't know the specific storage/freeze protocol for a cell line I use the following:

1. Take cells in "exponential" growth phase (You will need at least 500 000 cells in most cases - I do not recommend making aliquots containing less than 200 000 cells (unless you have no choice) - aim for 500 000 per aliquote -the more the better as there will always be cells loss in freeze/thaw cycles)

2. Rinse with PBS-- (w/o Ca Mg)

3. Add trypsin solution (200ul doesn't sound enough for any culture vessel?) - more appropriate is minimum 500ul (or 1ml) for T-25. If possible start with T-250 flask format (trypsin 2.5ml)

4. Put cells into incubator - occasionally observe until cell detach

5. Add minimum 5 vol of Complete medium with 5%-10% FCS to inactivate trypsin and spin down cells (3min ~150-200g (=) 1000rpm standard 15ml tabletop))

6. remove supernatant (yes, you must get rid of the trypsin even if "inactivated") and gently resuspend into cold "freeze solution" (90%FCS, 10% DMSO) - aliquote into cryotubes

7. immediately transfer cryotubes into isopropanol cooling box or into a styrofoam box containing ice

8. Put the box into -80 freezer (you could use the -20oC in between but I've seen no benefit from that - you should certainly skip the fridge step!)

9. Next day transfer into N2-tank for prolonged storage

10. Prepare 10ml of prewarmed complete medium into 15ml falcon tube

11. Partially thaw a cryotube quickly in your hands and before it is completely thawn pipette it gently (using the help of the prewarmed medium) into the falcon containing the 10ml

12. Spin down 150g for 3min - remove supernatant - add fresh medium

13. When reseeding - make sure that you have high enough cell density - seed an aliquote into area such that you end up with an cell/area ratio that is approximately the same when the cells where harvested (eg. do NOT seed 1/5 aliquote from t-25 directly into T-25!!) many cell types will not proliferate very well after thawing and they need friends around!

Good luck!

Great answer from Aki but I would beware of putting cells in trypsin in the incubator. If your cells are less than 100% confluent or have been on the plate less than one week, you should be able to remove them at room temperature within 15 minutes. If you forget and leave them out for 30 minutes they should still be ok. I don't remember exactly but there is a problem with trypsinization in the incubator, the cells get sick. I have never had problems with human fibroblasts and keratinocytes using room temp. trypsin.

Dear Himanshy

Before you freeze the cell, it has been better to have the growth crave of your cell. The cell should be freeze at log phase. Otherwise may be you freeze the cell in stable or death phase and it hard for you to recover the cell

kindly let me know how to draw growth curve? is cell viability assay is not enough (tryphan blue method)?

@Ramani, there is a thread on researchgate I assume would answer your question.

https://www.researchgate.net/post/How_to_properly_do_a_population_doubling

There are many reasons why frozen cultures do not revive properly.

1, How you freeze the cells (composition of the cryo-mixture, rate of addition and cooling rate).

2. Storage temperature. For long term storage it is recommended to stock in LN2 rather than -80 oC. Up to 60 oC of freezing the cooling process is critical. There should not be function of storage temperature if stocks  are kept in mechanical freezer.

3. Finally how you thaw the vial. It is recommended that just after LN2 evaporation, the vials are immediately transferred to 37 oC, Once thawed the cryo-mixture is slowly diluted with PBS or plain medium over the period of 10 min.

4. The recovery is cell line dependent, solid tumor cells can be maintained highly viable whereas PB-MNCs recovery is always low.