Dear Stephan,

I have enough experience with the cryopreservation of human embryos, the problem will be similar to the your. The ice crystals damage the cells very intensively and therefore we effort control the ice crystal nucleation using the initiation of the ice crystals outside the cells due to the seeding – the rapid point cooling of the cryopreservation vessel. Recently we prefer using of the vitrification – the supercooling of the liquid with the cells without the ice crystals formation. The results are amazing.

Best regards

Pavel Travnik

Dear Pavel,

Thanks for the information sir, i have another question that bother me. in the freezing profile, there is a peak (heat latent) occur, would it effect my cell viability? i have issue with the cell viability, so i have to eliminate as much as posible variable that can make my judgement be blurred.

Regards,

Stefan

Dear Stephan,

I do not know, how is your exact procedure. Describe it please (apparatus, steps), it is possible, I will have some improovements.

Best regards

Pavel

Dear Stephan,

I was working for a long time with a programmable freezer for sturgeon fish sperm cryopreservation. As you can see from the image, the critical point for having a good viability after thawing is making concise of the program cooling rate with the actual temperature inside the straws.This need so time to make try and error tests to find the best cooling rate. But be aware, adjusting the cooling rate is completely depends on cell type, cell density,  % of extender and also the kind of extender. Each of these has their specific effect on changing the heat latent point in the actual temperature curve.

During my experiences, I have find no deleterious effect  about "a little out of curve" fluctuations, especially at  heat latent point of fish sperm cells viability.

So, I recommend you try other cryoprotectants, other extenders and other % cryoprotectants.

Good luck

Dear Pavel and Shahrouz,

After trials and eror i can supress the heat latent by gradient temperature, some journals say that heat latent can induce ice nucleation and damage cells. in the image below, i think the cell's temperature go down by -1 to -3 C. the problem i'm facing right now is the viability of cell is different for each vials and some vials have more than 100% viability. do you think this is because inhomogeneous suspension?

i really appreciate any advice from you.

Best Regards,

Stefan

Dear Stefan,

if some of vials have 100% viability, the method you use is in principle good. The cryopreservation is never absolutely reproducible process, the result depends on small changes of the conditions and the quality of the frozen cells. Inhomogeneity of the cell suspension may be one of the factors influencing the result.

Best regards

Pavel Travnik

Dear Stephan

From the freezing chart, I can say that you are in the right way, but I think it could be better especially at the beginning. In fish, we sometimes have the semen and extender in refrigerator to be reached to a same temperature (4 C) for 15-30 min (called equilibration time)and then start the protocol. This may not be mentioned else, but I believe this time has a great importance for later conditions. Your different results ,may be because of not to be at the same temperature between treatments at the beginning.So, again from my point of view try to repeat the treatments in completely same condition to have more equal results.

One more suggestion, take a calibration for all your volumetric devices or repeat experiments with new sets. It may works.

Truly

Shahrouz

 Dear Pavel,

It drives me crazy sometimes to have different result even the cell and protocol is same. do you have any suggestion how to eliminate inhomogeneity in large scale cells?

Regards,

Stefan

Dear Shahrouz,

I tried it too before i get this result from new protocol, but the chart didn't seem good, the heat latent occured because the temperature in chamber were too high and it couldn't suppress the heat, so i have to make the temperature in chamber down. I think I will go with your advice, I hope the result would be better.

Regards,

Stefan

Hi, stefan

The chriztalization heat latent  does not occur because of temperature inside the cooling chamber, It will occur in every king of water solutions, as you can see from the attached picture, but the time of occurrence, is dependent the cell type, cryoprotectant and .......

Good luck

Truly

shahrouz

another advise,

may be your cells are very sensitive to cooling rate procedure, so try to prolong the protocol between critical temperatures (eg -10 to -70). as you can see a work done on fish blastomer which prolonged for about 100 minutes.

shahrouz

Dear Stefan,

May these article also help you

Dear Shahrouz,

Thank you so much for the information, it help me to improve my experiment. i have another question, how long you have to wait to thaw thce cell that you cryopreserved? some journal said you can thaw it after a week, but if we frozen the cell in -196 C it shouldn't have any different because the cell metabolism is stopped, kindly correct me if i'm wrong.

Best Regards,

Stefan

Dear stefan

You are right. I have read somewhere cryopreserved cells could have their viability for more than 200 years. In my field of study , I have some sturgeon samples from 12 years ago and still they have their motility, but the quality would not be the same as the first days of preservation, because of ROS, and other internal and external enzymatic factors.

So you can have your cryopreserved cell for a long time, but it referred to the aim of your study, the expenses and laboratory facilities.For me, it was a laborious work in such a long time!!!!!!

Best

Shahrouz

Dear Shahrouz,

For 12 years? Sure it is ! i can't imagine how many times and work you have done to maintain the cells. 

I encounter another problem, when i cryopreserve 48 vials and observe the temperature drop, i find that the temperature drop at the center of rack and outer rack is so different and i think it will be a problem. do you have any experience for that problem?

Best Regards,

Stefan

 In sperm cryopreservatio, working with less volume makes better results, because of unified temperature exchane. Also making cell cryopreserved in a uiform cylenderical straws make best in comparison with other vials with conical or other ununiform shape vessels. So i can say the less volume to be cryopreserved,the better results achieved. Make sure to fill the vials with exactly same volumes and spread them in a equal distance from each other and from the edge of cooling chamber.

Best regards 

Shahrouz