Cryobiology - Science topic

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Hi! Did you get high-quality DNA after storage of disrupted plant sample at -20? We will have the same situation and should keep it at -20 for one day.

It sounds like you expect the cells to remain intact upon lyophilization, except for dehydration. I would expect the cells to burst when frozen because of the expansion of water when it freezes into ice, as well as formation of ice crystals if the freezing process is not super-fast.

In the process of lyophilization, ice sublimes because of the low pressure at which lyophilization is performed. It does not first melt, then evaporate. If the membranes of the cells somehow remain intact during the freezing and lyophilization procedure, my guess is that the water molecules pass through the membrane as a gas.

I used Axiovision - works well. Try it.

what is the optimal concentration to induce cutaneous melanoma please ?

@Bruce

Thanks for your answer. I am aware of the protocols needed.

I am looking for a theoretical model on how the biochemical processes unfold.

-80 c is not for long term storage of the cells, however it will be ok to transfer your cells to liquid nitrogen after 6-8 months storage at -80 c.

Regards

we ended up going with the zebra printer. We don't really use the barcode, but we print the stickers all the time.

As you rightly say, autoclave water is not RNAse free, nevertheless RNAses will resist your autoclaving process if present in your plastic. Doing this you'll eventually disrupt DNases and other proteins and you'll kill bugs if present. This will not ensure the elimination of pyrogens such as LPS and also RNAses. I normally buy PCR-clean, RNAse/pyrogen free bags of vials and PCR-clean RNAse/pyrogen free pipette tips at least for RNA-related procedures. I always use this plastic with gloves previously treated with RNAse ZAP by ambion. I treat the surfaces where I extract with the same solution and periodically I also treat the RNA-extraction dedicated pipette sets with the same solution and the centrifuge rotor. I also never add my extraction reagents into a glass bottle, bottles even autoclaved are not RNAse free. I prefer 50 mL sterile tubes and then I periodically discard them for clean ones. Finally get DEPC water for your RNAs resuspension.

If you see someone gloveless with hands inside your RNAse free plastic bags then use a tanto knife for an emergency amputation (It's a joke but often this could happen, specially with thesis students).

Hi Daniel, you get dry and bad tasting fish with freezer burns.

Yes it does!

It all depends on the protein stability, some like Lysozyme and Ubiquitin can undergo several freezing cycles, others just can't. This has to do with the so called cold denaturation. Hydration of proteins changes in freezing condition and can affect the 3D fold, sometime in an irreversible way. If your protein is particularly unstable you might think of using cryo-protectant.  

We use these Qiagen products:

https://www.qiagen.com/qdm/aw/rna/stabilization-reagents?cmpid=Qven10GARNAstabilizationGA_SEM_&kwid=rnalater&akamai-feo=off 

We have validated that they yield high quality RNA nearly as good as immediate snap freezing (within 3 min of removal from the body). 

Naval Blood Research Laboratory (NBRL) method include developing the procedure to freeze red blood cells with glycerol. Melting point (crystallization of glycerol 17.9 0 C. Glycerol can be supercooled to a temperature of minus 70-100 0 C. This is its main property in this method. Crystallization of any substances is accelerated if there are crystallization centers, ie, glycerin crystals. To not have them Glycerol is used warm with a temperature above 18 ° C, but less than 37-40 ° C. At a higher temperature, the protein loses its properties.

Thanks alot dr

Hi I normally dissociate the spleen tissue.  Suspend the cells in 2.7 ml cold medium containing 10 % FBS.  Finally add 300ul DMSO (10 % final concentration).  Mix and freeze at -80 C as 3x1 ml aliquots.  Try to work as quickly as possible to minimize time between adding DMSO and freezing.

For recovery thaw cells rapidly at 37 C. Add 9 ml medium at 37 C.  Mix and centrifuge (this step removes DMSO).  Add 10 ml medium containing 10 % FBS to cells and incubate overnight at 37 C in CO2 incubator.  We found that this gives us better colony numbers than doing the fusion immediately after thawing cells.  What we do routinely to improve no of positive clones is secondary in vitro immunization for 3 days after thawing.  The medium added to the cells after suspension then contains 1 ug/ml antigen, thymocyte conditioned medium and 10 % FBS.

Collect the splenocytes after the overnight/3day culture period and proceed with fusion as usual.

www.origene.com/‎▼ (888) 267-4436‎

Thanks a lot! 

Thank you for informations Ahmed Munahi !

 Regards

Your welcome!!!

Hello Zain,

To be specific. No, you can't replace -70 with -20 as even an overnight storage in -20 will result in the compromise in cell viability. So it's best that you use -70/-80 for cell storage as nicely explained by Simon but in many cell lines snap freeze (-1 per minute) is not required. I have also published a paper on the same. You can put cells directly into -70/-80 but thawing step is really important.

Hope it helps!

Hi Samuel,

which Mill dis you use and which conditions (all parameter)?

Best regards, Tanja

In that case a cryoprotectant other than DMSO could be used.  This is why more detailed information, such as the cryoprotectant used, would be helpful.

The observation f or the bull semen comes from personal observation in the industry. I am not sure if it has been published but testing before full scale harvesting is widespread practice.

Since this time I have observed patients who have responded well to a fresh treatment, nothing from a thaw and then nothing from another fresh treatment.. It may not be a problem that relates to individuals freezing abilities. It may simply reflect the fact that we get all the improvement possible for that patient with one treatment.

Thank you for your help.

Use of 80% and pure acetone as extraction solvents results in the formation of chlorophyllide

It was our objective to provide a simple and reliable method to extract chlorophyll for HPLC analysis that would be free from artifacts. In order to achieve this goal, we started with one of the simplest methods to extract pigments and attempted to improve it. We first compared two conventional methods in which pigments are extracted from Arabidopsis leaf samples by soaking them in 80% or pure acetone for 12 hours at 4°C. Since CLH has been reported to be active in aqueous acetone but precipitated in pure acetone [18, 19], it was expected that chlorophyllide would only be produced in the 80%-acetone extracts. In line with this expectation, we determined that nearly 70% of the combined chlorophyll and chlorophyllide a content was composed of chlorophyllide a in the 80%-acetone extracts (Figure 2). In contrast, only a small amount of chlorophyllide a was produced in pure acetone (Figure 2). Therefore, it is likely that most of the chlorophyllide a detected in 80% acetone was formed during extraction or after extraction. Since chlorophyll a is much more abundant than chlorophyll b and the trend of chlorophyllide b formation was similar to that of chlorophyllide a, we only describe the results on chlorophyll a and chlorophyllide a in the present study.

My paper coted second one above... will give you insight regarding many protocols.. if you still need help let me know.

Regards

If your mice was perfused with formaldehyde, you should do the antigen retrieval step.

I  microwave my slides (650 W) in Target Retrieval Solution (Dako, Denmark) twice for 3 minutes each. You can also used Tris-EDTA buffer (pH 9). It works for MMP-9 (ab76003, Abcam).

For cryosections many antibodies work well without antigen retrieval step as they are either not fixed or fixed for relatively short period of time, so that the croslinking did not occur in a significant manner. Presence of  detergent in blocking buffer and antibody diluent is enough to ensure good antibody penetration. If your staining doesn't work you can play with the percentage of Tween that you are using. If antigen retrieval is needed you can try enzymatic methods., such as proteinase K treatment. Just remember that you need to tightly control incubation times to get good, repetitive results with this approach.

Good luck.

For the frozen plant material, you can treat it the same as you would with fresh.  I place the bit of tissue in the petri dish with the extraction buffer and allow it to sit in there for a short time, as it absorbs the liquid.  After soaking in the buffer, the tissue should be more malleable and ready to chop.  You can then chop and filter just as you do with the fresh tissue.  One thing I'm not sure of is whether the state of the internal standard has an affect on relative estimates.  So it may be wise to freeze some of your internal standard as well.  I have not done anything with fixed plant tissue.  

Hi Amir,

It is believed that CLA increases sperm cell viability by reducing the oxidative stress, thus decreasing lipid membrane peroxidation which eventually preserves membrane stability and mitochondrial potential.

For your information here is a recent study you can look at:

Effect of Conjugated Linoleic Acid on Boar Semen Quality After Long-term Refrigeration at 17°C http://www.ncbi.nlm.nih.gov/pubmed/25976112

Hope this helps. Norbert

Dear Mustafa,

I only met articles concerning the use of hydroxyapatite nanoparticles in oocyte vitrification. The authors suppose the role of nanoparticles in the protection of the system against the ice crystals formation during the warming. Second article is oriented on the warming rate improving using the electromagnetic field and the ferromagnetic nanoparticles. See below.

Best regards

Pavel

Zhou X, Li W, Zhang D, Dai J. Hydroxyapatite nanoparticles improved survival rate of vitrified porcine oocytes and its mechanism. Cryo Letters. 2015 Jan-Feb;36(1):45-50.

Eisenberg DP, Bischof JC, Rabin Y. Thermomechanical Stress in Cryopreservation Via Vitrification With Nanoparticle Heating as a Stress-Moderating Effect. J Biomech Eng. 2016 Jan 1;138(1). doi: 10.1115/1.4032053.

I am not sure what is most effective with insects (flash freezing or RNALater), but when we flash freeze mammalian tissue for RNA extraction we freeze it in liquid nitrogen in a tube by itself or in Trizol, then store at -80 °C.  We do use RNALater, but you need to treat the tissue with RNALater first, then refrigerate or freeze for longer storage.  The American Natural History Museum has a collection and storage protocol online suggesting "When collecting very large arthropods breaking open the exoskeleton just before submerging the insect may promote greater profusion of RNAlater through the tissue."  

Thank you Beric, For the answer and for the literature tip, seems very helpful indeed. We are currently trying some standard embeddings, but if it does not work out, we will dig deeper into the methods you suggest.

BW /ERik

I work with bovine A.I. station Natural. We use  dry shippers (from China) very often - for transport in the Czech Republic, as well as abroad. Their advantage is that the transport needn´t be done under ADR principles.... You only have to refill the NL into the material when reusing and it will keep  the required temperature for 3 - 4 days.

Here's a fairly recent paper I found on the subject (file attachment).

I have a link to another article, but  was unable to purchase it.

Hope this helps.

Hey in case you don't have access to mr. frosties you can use polystyrene e.g. the plates cuvettes for photomoeters were delivered with. You take the cryo tupe, wrap it with paper towl and put in one of this polystyrene slots.

further more it is of interest which cell type you freeze and if the cells are potential sensitive for DMSO. What is the recommedation from your supplier?

Regards

Sven

This is an interesting question because many groups use fresh PBMCs due to concerns about the quality of cryopreserved PBMCs. We routinely use a 10% DMSO with 10% FBS protocol and obtain 50-65% PBMC viability by Trypan Blue exclusion. We use these cells in one way mixed lymphocyte cultures and they behave in a manner similar to fresh PBMCs. The protocol suggested by Andrei incorporates using human serum albumin, a thorough thawing protocol and slow dilution of the cryoprotectant solution. These are all excellent methods suggested by the literature for minimizing damage to cryopreserved PBMCs that are not necessarily needed for most of the relatively hardy cell lines that we work with. We will give this protocol a comparison trial next time we freeze back a batch of PBMCs.

One can use the CD103 that binds to blood basophils when activated: FACS must compare the marking before and after activation to obtain the percentage of basophils.

The antibody 212H6 is a good marker of basophils. This antibody is specific for blood basophils, which works by FACS and which moreover do not porvoque basophil degranulation.

This antibody is recommended to isolate basophils without induced histamine release by the selection, for use in the degranulation assays.  

 

Do RBC lysis with RBC lysis buffer than centrifuge it and wash with RNAse free PBS  twice and isolate RNA with trizol or any preferable method... Hoping you will get the real expression.....

Do not forget that, apart from ice or osmotic shock, spermatozoa undergo important changes during the extension/cooling/freezing/thawing/redilution processes. Free radicals, reorganization of the plasmalemma, loss of cholesterol and phospholipids, increased plasmalemma permeability, ionic changes... some authors have combine part of these changes in a term: cryocapacitation. You can obtain viable spermatozoa with compromised functionality, already "capacitated", with DNA damage, etc.

About choosing "good freezers", it might be feasible in some cases, but it might not be a good strategy. Good freezing might be associate to traits other than you are looking for in the offspring. Or you might select good freezer males only to realize, generations later, that you also were losing genetic variability or alleles that could be desirable at the present. In some cases, bad freezer males are genetically important in your program, and you just cannot afford to discard them. That is why an important line of research nowadays is to distinguish good and bad freezers when optimizing protocols.

Hello Andriy Korbetskyy, thanks so much the article was a great help. Stay blessed

I would recommend to try to add both an intra- and extracellular stabilizing agent, e.g. 5% DMSO with 5% HES (hydroxyethylstarch). This combination is in general used in many PBMC cryopreservation protocols.  

Products can be used depending on the Dhopte

Hi! First correct the spelling it is "psychrophilic".

check the following literature you get some useful information.

Hi Benjamin, I can suggest to use less glycerol, 15-25% in a v/v of a fresh culture, mix well and then to freeze in LN2 or dry-ice, before long storage at -80°C.

Ha-ha-ha! Do viruses move themsleves (if animation means that)? One of the usaes:

The term "suspended animation" is a "poetic" expression of the glassy state where a molecule is basically "stuck" and move ver-very little around its close vicinity. As the result, practically ALL chemical reactions are effectively stopped (see the refrence to my paper as an example, in my previous ansswer about prediction of Tg.

Your question is quite different and rather philosophical, related to hypernation, wintering, etc. It's a diferent field, a very undermined so anybody can mean anything by that term. For exapmpe. sperm in bee's spermatheca:is supended animation or not? The delayed implataion of the zygote in Mustelides: suspended development or not? We are entering very murky waters there!

BTW, cryonics is not a science, it's a cult and a total bogus!! Again, not for the sake of promotion, but read Chapter 1 here:

What's bothering me: a good Q about Tg prediction and THIS!! Run from those cryonic charlatans with the speed of light, this is my advise. It can be good and quick money at first but you will damage your scoentific reputation irreparably in long run. Ask Greg Fahy about it ;-)))

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):

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

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.

I have kept cultures in glycerol at -20ºC of actinobacteria for years (at least 4), but they were sporulated. It is going to be really dependant of what do you want to keep.

Hi Patrick, Checkout E-bay. There is a FTS MC855A1 BIO-COOL CONTROLLED RATE FREEZER available for $2,800. Regards, Kelvin

Hi Sudipta, I've been working with flies for almost 15 years now, and this question always comes up. While I've never tried it myself, I think the consensus is that if it could be done, it would have been done. It's one of the real drawbacks of flywork (though far outweighed by the benefits!!). That's not to say if you have an idea of how to make it work you shouldn't try! Who knows? It would be a HUGE boon to the fly community if someone could find a way...

Those containers are dewars, that means they are double-walled with a vacuum in the gap. Stainless steel is just a convenient material for it. Corrosion-resistant, reasonably strong and tough, weldable, bad thermal conductor, reasonably cheap, reflecting like a mirror. Aluminum alloys would perhaps be an alternative. But they couldn't be anodized to protect from wear and corrosion, because they would in that case absorb and emit far infrared radiation and increase the loss of nitrogen. Aluminum alloys are also better thermal conductors. And strong aluminum alloys are usually not weldable without a heat treatment afterwards. Easily weldable alloys are often too soft.

Glass dewars were used for smaller quantities in the past. They are better insulators, but they are also a real hazard in the lab because they tend to implode and send glass shrapnel all around. Stainless steel is really an ideal choice.

Update:

The material has to be tough and strong *at the temperature of liquid nitrogen*. That excludes a lot of steels, and you'l have to select your stainless steel carefully. It should have a lot of nickel to stabilize the austenitic phase, if I recall correctly.

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