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Chapter 3 - Part 1: Solutions: myths and truths

What was first, the egg or the chicken? I can’t answer this ancient question, but I definitely know that the first successful cryopreservation was done on poultry… no, not egg, but sperm. A remarkable story, rather typical than exceptional in reproductive biology.

Just after World War II, Christopher Polge, an ambitious young English scientist, started to work on cryopreservation of poultry spermatozoa in London. Based on some previous research, they added fructose to the medium in various concentration. They could improve the morphological survival, but the seemingly viable sperm was unable to enter the egg, and no fertilization occurred. So, Christopher went on holiday.

When he returned, he restarted the work with the same old bottle on the shelf and made another attempt. To his greatest surprise, things started to work beautifffully! He was thrilled, but also curious – why?

What was the difference? Asked everybody around, compared the content of the bottle with new fructose solutions – it was different, the consistency, solvent effect, everything.

Finally, it has been revealed. While he was on holidays, one of his colleagues did an entirely different work at his bench, microscopic evaluation of sperm. The old bottle was used to prepare fixatives for slides. It was not a fructose solution inside. It was a mixture of egg white and – glycerol, an organic solvent, a kind of alcohol (in chemical terms, do not drink it!).

Soon, it has been revealed that it is an excellent cryoprotectant – the late Professor Polge could freeze chicken sperm, then bull sperm with it, and produced offspring in both species.  A complete industry has emerged from this discovery, artificial insemination (AI)  resulting in billions of cattle born from frozen semen. In cell and tissue culture, in bacteriology, mycology, glycerol is still the most common cryoprotectantl used. After several decades, it was also the main component of solutions used for traditional freezing of domestic animal, and even human embryos.

All this has happened because of a stupid mistake.

I can’t tell you my stories where an unplanned event (electric blackout, failure of a machine, bad timing, laziness to return to the lab at midnight, miscalculation, etc.) resulted in a breakthrough in a specific problem.  I can’t tell you because it happened so many times. And I can’t tell you, because you would say I am the Mr Bean of science.

But – as for Professor Polge – the outcome is the important part, not the story. Accordingly,  please be aware of your mistakes. Check what happens after with your tortured oocytes or embryos. Maybe a better outcome. Maybe a breakthrough. Maybe that bad experiment will determine your entire future!

Regarding the solutions, media used for cryopreservation have several essential components including

1.  a holding medium consisting of a physiological salt solution and a few (5-7) or a lot (20-40) of components, i.e. energy sources, vitamins, etc., used for various purposes in tissue culture and embryo work,

2. a buffer that helps to stabilize the pH at a physiological level in air,

3. macromolecules (organic or non-organic ones, with defined, semi-defined or non-defined,  composition)

4.  cryoprotectant agents.

1. Very few comparative studies have been published about the effect of various holding media on the outcome of vitrification. Most scientists regard them just passive components of the procedure, like figurants in a movie, or wheels in a race car. However, as wheels and especially tires may have a crucial role in some situations, the role of holding media should not be underestimated, either.

According to our experience, successful embryo vitrification can be done in cattle in a simple phosphate balanced salt solution (PBS). However, most companies offer a more complex holding medium for their vitrification kits. These media are usually the analogues of their culture media; just the bicarbonate buffer is replaced with another one that can be used without carbon dioxide (see later).

As embryo culture media have been simplified significantly in the late ’90s, early 2000s, these holding media aren’t complicated, either, especially if we compare them with the first generation of embryo culture media used in the 80’s, and used up till today frequently for somatic cell or tissue culture works. Embryos usually do not need such a complex solution. Before the implantation they live in the oviduct, that is ‘per definiitionem’ not inside of the human body, it is an open channel to the outer world, with much simpler constituents in the secreted solution that surrounds the embryo.

Obviously, what is suitable for the embryo culture should also be good for vitrification.

Or, maybe not? Our “logical” theories aren’t always applicable to the biological processes.

Domestic animal embryologists have much more freedom for experimentations. Also, pioneers of human vitrification had a lot of valuable information about fundamental issues, that seems to be forgotten today.

Regarding holding media, we started with our “old school” experiences, used a highly complicated media called Tissue Culture Medium 199 (TCM 199). Then, we compared it with the new generation of holding media, with much simpler composition. No drastic difference was found between the two, so we obeyed the word of time, and started to use the new, simpler holding media. We stored these new media at 4⁰C, as instructed, for one month, two months… then survival rates started to decline. At around the expiration date, or a bit earlier. A situation we never experienced with TCM-199.

This finding was never published – but on conference dinners, we talk about. The same result in the USA, in Australia, in Europe. So – we slowly returned to TCM-199…

2, And the same thing has happened with the good old Hepes buffer. Many companies offer alternatives; the most popular is the MOPS. However, any composition except for the HEPES-buffered TCM-199 may have some negative effect after months of storage. Maybe only after the expiration date – but shortly after. In contrast, Hepes-buffered TCM-199 has a generous backup; it maintains its consistency for long, it is reliable.

“Every gypsy praises his own horse” – a proverb from Hungary, my late homeland.

Yes. VitaVitro’s holding medium and buffer is TCM-199 and Hepes, respectively.

VitaVitro wanted to have absolutely the best components for our media, and we definitely selected this solution, as it was found the very best, by us, and by our colleagues-friends worldwide.