Vitreous cryopreservation maintains the function of vascular grafts
Although successful human cryopreservation and revival likely won’t be available in the near future, it’s amazing to think that nowadays, living cells can be frozen for over a year before being thawed and re-cultured. As engineered vascular grafts increase in demand, a method is needed to store tissue constructs for indefinite periods of time, they will be of limited practical use. This need is the basis for work on cryopreservation.
Cryopreservation so far has mainly involved simply placing cells and solvent in low-temperature freezers in vials, cryoprotectant optional. Ice crystals formed through this method, however, cause damage to the tissue, and blood vessels do not maintain the same mechanical properties as when they are first obtained. To avoid this problem, scientists have developed an alternative technique for cryopreservation called vitrification. Rather than relying on the slow freezer, this process involves an extremely fast temperature decrease (e.g. plunging the sample into liquid nitrogen). This reduces the formation of ice crystals and instead causes the solution to become increasingly viscous and glass-like.
In this paper’s study, researchers tested the effects of freezing on the mechanical properties of rabbit blood vessels. Blood vessels were placed into one of three categories: fresh (control), slow-frozen (the original method for cryopreservation), or vitrification. After being cut into cross-sectional vein rings and undergoing their respective procedures, the vessels were treated with various drugs such as histamine or acetylcholine. Contractile tensions were measured to indicate response to these drugs.
The results show that slow-frozen blood vessels show a marked decrease in contractile response to the drugs compared to fresh tissue, while vitrified vessels mostly maintain this contractile response.
The graphs to the left show the differences in contractile response to various drugs among control (C), slow-frozen (F), and vitrified (V) vessels.
Microscope images of these tissues also reveal the extensive damage in slow-frozen tissue due to ice crystal formations, a feature largely absent in vitrified blood vessels.
Microscope images of slow-frozen (A) and vitrified (B-D) tissues are shown on the right. Slow-frozen tissue shows extensive ice crystal formation, while vitrified tissue remains mostly free of this damage.
The ultimate goal for cryopreservation is, as previously mentioned, storage and revival of complete organisms. The possibilities of such a technique are so varied that I won’t attempt to go into the details, lest I lose sight of the line dividing the real world with science fiction (Is cryopreservation of astronauts as in the movie 2001: A Space Odyssey possible?). Even nowadays, cryopreservation is used in such areas as sperm banks, where sperm can be frozen for long periods of time until someone wants them. There is a large need for effective storage of tissue for future use, and I predict that cryopreservation will become increasingly important in the medical and in the bioengineering fields.
7 comments:
It's neat how the vitrified vessels were almost able to retain properties observed in fresh tissue. Could vitrification be used for all types of cells/tissues? Also, if these vitrified tissues were used for transplantation, do you think the tissues would be affected by the body's immune system?
@ elena: I think that in terms of individual cells, pretty much any type can be cryopreserved; if we simplify the concept, each cells is just a tiny factory with enzyme workers and substrate products, and freezing one would be like stopping time within that factory (maybe a lame analogy, but it makes sense).
Tissues are a bit more complicated because of two issues. First, vitrification requires high amounts of cryoprotectant solution, yet the protection that this solution can provide is limited if the tissue is a bit thicker (the solution needs to perfuse throughout the tissue). Second, this process involves a very rapid cooling rate; this is more difficult to achieve in thicker tissues and organs, where the cells on the inside may cool at a slower rate than at the outside.
That said, research is being conducted to perform vitrification on organs; I've also encountered several papers testing this type of cryopreservation on rabbit kidneys and other small organs.
As for the immune system, I do not think vitrification is going to cause any immune reaction that wouldn't have occurred in the fresh tissue. After all, the goal of this process is to preserve the tissue, ensuring that it returns to its original state after it's thawed. Although, if there is an excess of dead cells, I suppose the immune system would do something about that...
Your article interested me very much since I had worked on cryopreservation before. I wonder what makes you think that vitrification will be more "increasingly important" since the cryoprotectants we currently use is pretty good already. From what I know, vitrification usually needs cryoprotectants as well. In other words, what makes vitrification more practical and useful in industrial or clinical use?
There are also a number of genes associated with cryopreservation in fish and...frogs, though I'm working for memory here. So it may be possible to take advantage of cryopreservative proteins in the future as well.
If you were to introduce DNA encoding these cryopreservation proteins into your cells and got them to produce the proteins, would this be more effective in preservation that just using cryoprotectants like DMSO? Adding DNA to your cells seems like it is more likely to cause side affects in cells (in function, growth, ect) than just adding a cryoprotectant to the solution the cells are in. I wonder if a study comparing the two (cryoprotectants vs cryopreservation proteins) has been done?
@Merline: Scientists are interested in vitrification because ideally, it would freeze the sample fast enough to prevent the formation of ice crystals, which damage the cells/tissue. It's true that both the regular cryopreservation and vitrification require high amounts of cryopreservatives, but this ability to store tissue at low temperatures without worrying about ice damage is still a compelling reason to take that path.
@Terry: Yes, I've heard of those natural cryopreserving proteins/chemicals in frogs and fish. I also vaguely remember reading about genetically modified tomatoes that could withstand frost and extreme cold due to some sort of similar cryopreserving gene.
@Shannon: As far as I know, there have been no studies comparing the two. The two areas are somewhat different; I think the natural cryopreserving proteins serve more as an antifreeze than as something that protects cells during freezing. I suppose eventually we could try incorporating these genes into regular tissue, but since the current goal of vitrification is just for tissue storage and eventual implantation or other purpose, I don't know if we have to go as far as making it a permanent part of the tissue. Although, if you were trying to genetically engineer an Iceman or something like that, I can see how this would be an attractive option.
Don't forget the cryonics people! I think companies like Alcor are currently using this vitrification method to preserve their customers.
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