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FogeltheVogel t1_ixl9uro wrote

Reply to comment by Magnetic_Syncopation in If freezing tissue generally damages the cells, how are we able to freeze human eggs and embryos for birthing later? by badblackguy

Lots of cryoprotectants are toxic to the cells we add to. The trick is that, after adding it to the sample, the sample quickly goes in the liquid nitrogen to freeze. This stops biological activity, which is why the toxic properties of the cryoprotectant don't damage the cells.

After thawing a sample, the cryoprotectant is quickly replaced with regular media before it can damage the sample

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ebfortin t1_ixlvn08 wrote

What happen when you thaw the sample? How do you make sure it thaw relatively evenly? You can freeze pretty quickly but thawing is another matter.

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FogeltheVogel t1_ixlwx8v wrote

The cells won't instantly die. We're talking about a window of (up to) hours here. And we're also talking about vials containing 1 millilitre of liquid.

In general, you thaw such a sample by simply placing it in liquid water. It'll be thawed in minutes.

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aTacoParty t1_ixlx6cp wrote

Depends on the size of the sample. Cells are really small so the volume they are frozen in can be very small too. I work with cells (stem cells, neurons, and others) and cryopreserve them regularly often at 100,000 cells in 500 microliters (0.017 fl ounces) using DMSO as a cryopreservent. When I thaw them, I'll place the vial in a 37C water bath which will thaw them in about 30 - 45 seconds. Then the cells are quickly diluted in media without DMSO to reduce the concentration. The solution is spun so the cells all pellet in the bottom of the tube, the media with DMSO is removed and replaced with fresh media for plating.

Larger volumes are generally not used precisely for the reason that they do not freeze and thaw evenly (IE the interior freezes/thaws slower than the outside). It can be done with special plasticware that increases the surface area but I've never seen it done in routine tissue culture as there is no need.

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ebfortin t1_ixm0kad wrote

And a cell needs oxygen and fuel to stay alive. After you thaw them what happen? How do they stay alive? Are they in some kind of stopped state?

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Farts_McGee t1_ixm16ut wrote

So a single cell's oxygen requirement is trivial. Atmospheric diffusion is more than enough to supply the required amount.

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Saccharomycelium t1_ixmfysi wrote

Cells are ok in liquid cultures, as long as the amount is right. The majority grows fine at 5% carbondioxide and for some types, 20% oxygen. Typicalls the cells are kept in wide surface containers instead of tubes, so there can be some gas exchange passively. If the volume is too large, it may be insufficient. But also if there's too little liquid medium, the nutrients get exhausted faster and cell waste piles up faster, which is toxic and will kill cells again. And one of the main wastes is carbondioxide again.

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FogeltheVogel t1_ixmhkkp wrote

The media contains nutrients (fuel), that's (part of) its purpose. As for oxygen, it is dissolved in the liquid and the cells get their oxygen from that. Just like how, in our body, oxygen is dissolved in blood.

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aTacoParty t1_ixn3ctv wrote

The cells resume their cell functions almost immediately after thawing. You'll hear the term "media" a lot when talking about cell culture. Media is a general term for liquid food for cells. It contains macronutrients (carbs like sugar, protein usually in the form of amino acids, and fats), micronutrients (vitamins/minerals), some other components (hormones/growth factors), and a buffer to maintain a physiological pH.

There are a ton of different types of media for different cells but the most commonly used is DMEM (Dulbecco's modified Eagle's medium) supplemented with 2-10% fetal bovine serum (FBS) for additional nutrients. Often researchers will also add antibiotics such as penicillin and streptomycin to prevent bacterial growth.

The media provides the fuel while atmospheric dissolved oxygen provides oxygen. Once the cells return to 37C, they spend a little time recovering as generally cells undergoing stress such as temperature changes and exposure to organic solvents like DMSO will stop dividing. This recovery period can take between 2-48 hours depending on cell type. For commonly grown cells like HeLa (from Henrietta Lacks) or HEK293, they take about 6 hours to recover and begin dividing.

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Tiny_Rat t1_ixn2c85 wrote

You freeze in small volumes and thaw them as fast as possible. Partly thawing 1ml or so of media in a waterbath, and then adding saline to thaw it the rest of the way only takes a minute or two if you do it right.

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