Cryobiology: Low temperature studies of biological systems
Cryobiology: Low temperature studies of biological systems
By
Krista Nussbaum (1999)
Thesis: Low temperature biology, or cryobiology, has the possibility, and may very easily and very drastically affect everyones' lives in the future. Through rapidly approaching cryobiological medical procedures and techniques, the presence of intense ethical issues may play an important role in many everyday decisions that do not now exist.
Outline:
I. Introduction
A. What is cryobiology?
1. definition
2. synonyms
3. general information
II. Cryobiology Techniques
A. cryosurgery
B. embryo and gamete preservation
C. tissue and blood cryopreservation
D. cryotransport
III. Support of Cryobiology
A. Medical gain
1. techniques
2. knowledge
B. Time gain
1. increase actual length of life
C. having biological children when you are ready to have them
D. Dispersal of Information
IV. Opposition to Cryobiology
A. interfering with the life cycle
B. medical complications
1. physical damage
C. extremely high priced
D. potential law suits over paternal rights to frozen embryos
E. people may very easily get carried away with preserving things
V. Conclusion
A. overview of methods and techniques
B. overview of ethical issues
C. question for the readers
Introduction
What is cryobiology? It is "the study of the effects of freezing and low temperatures on living organisms" (Read, 1999). While the main focus of cryobiology is on living organisms (as suggested above), cryobiological techniques have been expanded to include treatment of nonliving things as well. This addition of non-living objects has expanded the focus to not only include plants, animals, and humans, but also to include machinery and other metal objects.
A common synonym of cryobiology is "cryogenics". According to the New American Desk Encyclopedia (1989), cryogenics is defined as "the branch of physics dealing with the behavior of matter at very low temperatures, and with the production of those temperatures." As can be seen, cryogenics is a slightly more focused study of the cryobiological field dealing primarily with production methods of those freezing temperatures. Another sub-study field, if you will, is that of cryonics. Cryonics is defined as
the practice of freezing humans who are not curable by current medical technology, in the hope that ways may be found to bring them back to life at some future time when ways of repairing the damage caused by the freezing process have been developed, as well as cures of the diseases or other causes of death which necessitated their cryonics suspension (Cryogenics International, 1999).
As suggested above, cryobiology is primarily centered on the effects on those living organisms or non-living objects, although the methods and procedures are also very important. All around the world new and improved techniques are being tested to make this a better medical and preservational procedure. Not only are the different techniques and procedures getting increased attention, but also many accompanying controversial issues are as well. Low temperature biology, or cryobiology, has the possibility to very easily and very drastically affect everyoneís lives in the future. Through rapidly approaching cryobiological medical procedures and techniques, the presence of intense ethical issues may play an important role in many everyday decisions that do not now exist.
Cryobiology Techniques
There are many different cryobiological techniques that exist and are being used today. Such techniques include cryosurgery, embryo and gamete preservation, tissue preservation and transplantation, blood and blood product preservation, and cryotransport. Some of these have been around for so long that they are now considered commonplace medical procedures, while others are still considered experimental in the medical world.
Cryosurgery
Cryosurgery, a technique that has been in operation for years, still has some aspects that are being extensively tested. Much of the questions and continual testing revolves around the injury or infection that needs to be treated and hopefully cured through cryosurgery. Many of these uses of cryosurgery are widely accepted from a medical standpoint while others are not widely accepted to the point that in some clinics and medical facilities they are not provided. According to Gage (1992), "commonly accepted uses of cryosurgery are for the treatment of many types of skin lesions (including cancer), for benign and dysplastic mucosal lesions, for uterine cervicitis and intraepithelial neioplasia, and for cardiac surgery in the treatment of tacchyarrhythmias". Studies in all of these areas can easily be found and researched.
Cryosurgery involves different techniques in which selective necrosis of tissues is achieved, or hoped to be achieved, through freezing at extremely low temperatures. The achievement of these low temperatures is through a very precise cooling sequence, using a specific solution called cryogen. Each of the separate techniques involves its own specific procedure in order for the surgery to be successful. Also the goal of treatment is a determining factor on which cryosurgical technique is used. According to Kuflik (1994), the three major goals of treatment to choose from are improvement, cure, or palliation. Once these initial factors are chosen, the technique to appropriately achieve the goals is used.
One of these cryosurgical techniques uses a dipstick apparatus. The dipstick, is simply a cotton-tipped applicator that is dipped into liquid nitrogen and applied to a lesion until adequate necrosis has occurred. Another technique involves an open-spray apparatus by which a fine spray of cryogen is applied to the lesion. Solidified carbon dioxide can also be applied directly to the skin with a mix of acetone to treat certain types of acne. This type of treatment is called slush therapy. Perhaps the most popular technique used in cryosurgery is the cryoprobe. This technique involves a precooled metal accessory that is directly applied to the lesion. The cryoprobe is also frequently accompanied by liquid nitrogen (Kuflik, 1994).
In most medical facilities, these procedures are routinely done and have shown both positive and negative results. Both the positive and the negative results have been reported on numerous accounts in scientific research papers and journals. An example of negative research results comes from a study done by B. Rubinsky, et. al., of the University of California-Berkeley (1990). This study involves cryosurgical procedures done on the liver as well as analysis of damage done. They found that the formation of ice in the hepatic sinusoids expands the sinusoids, dehydrates the hepatocytes, and then leads to possible structural damage. This particular study shows that not all cryosurgical procedures are effective, at least, without causing irreversible damage to the operation subject in the process.
An example of positive research results comes from a study of other surgical techniques in comparison to cryosurgery, by A. Hohki (Gage, 1992). In this study the other treatment techniques were laser, ultrasonic aspiration, microwave surgery, scalpel, and electric scalpel. The major finding was that while all techniques have advantages and disadvantages, cryosurgery was the best method for cosmetic effect and economy. In agreement with these findings, Emmanuel G. Kuflik, MD (1994) has concluded that cryosurgery has many added benefits besides just cosmetic and economic advantages. Examples include, suitability for office, nursing home, or outpatient facility; the lack of need for general anesthesia; optional need for local anesthesia; the lack of need for operative suits; the simplicity and safety of the procedure; the lack of physical restrictions afterward; availability for pregnant patients; and suitability for those patients who are fearful of surgical procedures.
Not only has there been research-based proof that cryosurgery is only positive or only negative, but also there can easily be a mixture of both. A study by Vellet AD, et al., (1997) shows that cryosurgery for prostate cancer can have both positive and negative results. "Hypothermic injury of the rectal wall and bladder base was demonstrated" after prostate cancer treatment with cryosurgery. However in addition to this, "no residual prostatic tissue or tumor was found on follow-up biopsy".
Embryo and gamete cryopreservation
Although cryosurgery may easily be the most frequent cryobiological technique, embryo and gamete preservation comes in a close second. This particular technique has been used for many species of animals, starting with the mouse in 1972, to the goat in 1976, and the human in 1983 (Leibo, 1999). Not only has this technique been used for these types of animal species, in each it has been a successful operation; successful meaning that live young have been produced from frozen embryos. In fact, this type of procedure has been so successful throughout many species of the animal kingdom, the only species that has still been unable to undergo this procedure with success is swine (Leibo, 1999). So many animals from all different species have been born through this process of embryo cryopreservation that it can be estimated that tens of thousands individual organisms have survived the storage in liquid nitrogen at ñ196 degrees Celsius (Leibo, 1999).
Along the same lines as for cryosurgery, embryo and gamete cryopreservation involves a very precise and straightforward method of freezing, unique for each organism. Also along the same lines, many of the methods involve general steps in the freezing process. These general steps were outlined by Leibo in his study of preservation of mammalian embryos (1999). The first step is to expose the embryo to some sort of cryoprotective solution, for it has been shown that this is an essential step in successful cryopreservation. Also, this step is where a lot of further testing has occurred, and disagreement has been found, on which cryoprotective solution is the best. The next step is the first step in the actual cooling process. In this step, the embryo is cooled to slightly lower than zero degrees Celsius, followed by a seeding procedure. Seeding is used for inducing ice formation under controlled conditions. There is much disagreement on whether seeding is actually necessary, although most procedures require it. The next step, is to control the cooling of the embryo to an intermediate subzero temperature. Many studies have shown successful cryopreservation at temperatures as low as ñ80 to ñ100 degrees Celsius and temperatures as high as ñ20 to ñ40 degrees Celsius. As mentioned before, what is being preserved and who was performing the procedure would determine this temperature. The final step in the freezing process involves a very rapid cooling to ñ196 degrees Celsius for long-term storage. It has been estimated that if the proper freezing steps have been taken, the embryo can remain "alive" for thousands of years (Leibo, 1999).
Now that you have successfully cryopreserved an embryo, you must be able to thaw it out in a proper way too. In general, the warming and thawing of the frozen embryo is directly related to the amount of freezing that had initially been done; this is specific for each organism. After warming and thawing of the embryo is complete, the cryoprotective solution must be removed, because in most cases, if handled improperly, this solution is toxic to the embryo (Leibo, 1999).
A new method of embryo cryopreservation has become a recent cause for study. Katrina Forest and her colleagues of the University of Wisconsin have been studying the effects of the cryoprotective solution on the successful preservation of embryos, and have come up with some striking possibilities and observations (Bubble Wrap, 1999). They have observed that immersion in large quantities of cryoprotective solution has caused the unsuccessful cryopreservation of many of the test organisms, for example the hamster. As a result, in their study, they used a technique in which they dipped a thin nylon loop into the cryoprotective solution and suspended the embryo on this thin layer of liquid. This allowed them to simply dip the loop containing the embryo into liquid nitrogen for instantaneous freezing. Using this method, they have been the only group to successfully get a hamster birth from a frozen embryo. They hope that this method would help improve the successfulness of human embryo preservations in the future.
As mentioned above, most scientists disagree upon the ideal makeup of the cryoprotective solution. Traditionally, the cryoprotective solution that has been used was supposed to simulate the body fluids of the womb, consisting of a sodium ion- rich saline solution. Until recently, this was a standard procedure. A study done by James Stachechki (Boyce ,1999) points out that maybe the success of any cryopreservation is directly related to the amount of sodium ions in the cryoprotective solution. In his study, he tested a new solution containing choline ions (a less abrasive ingredient) on mouse eggs, and got a survival rate of 90%. This compares to the 50% survival rate of the use of conventional saline solution. Numerous other studies on improvements of the cryoprotective solution have been made and many alternate choices are now available.
Tissue and Blood Cryopreservation
Cryobiology technology has recently been focused and manipulated to aid in the care and well being of ill patients, in a different way than cryosurgery. Tissues and blood of the body can be removed, frozen, stored, and then later reintroduced into the body when they are needed or desired. One particular use of this technology is done for bone marrow transplants. Until this technology, these transplants relied on time to be effective, but now cells can be frozen and stored for years and still be effective. The Cryobiology Laboratory located at the Fred Hutchinson Cancer Research Center deals directly with this type of cell storage (Center News, 1997). According to the laboratory with this technology, "donor and recipient need no longer be the same ABO blood type [because] the red blood cells can be removed from the marrow". In this laboratory, the cells, stem cells or bone marrow cells, are stored for short-term in a controlled freezer that can cool at a rate of one degree per minute, or for long-term in a large, liquid nitrogen freezer at ñ180 to ñ195 degrees Celsius (Center News, 1997). In this same article, the laboratory has reported storage of about 1200 patientsí cells, giving a total of 4500 cell packets that are labeled and ready to use when needed. Since this article was published, the numbers have most likely increased.
Another form of tissue preservation is that of blood. In the same manner, blood or blood products can be stored in freezers and then used when needed by a patient. Current technology has designed methods of recycling placentas and umbilical cords, that would normally be discarded after birth, to be used as blood and hematopoietic stem cell sources. According to Lifebank Cryogenics Corporation (1999), this procedure of saving the placenta and umbilical cord at birth is a very simple procedure that only takes minutes, and in turn could possibly save your life or the life of your child. These cells are used for many possible reasons including stem cell matching for your child, treatment of life-threatening diseases, or even gene therapy (Lifebank, 1999). Umbilical cord blood transplantation also has been shown to be an acceptable alternative to bone marrow transplantation.
CryoTransport
Cryotransport is the newest procedure done in cryobiology. As defined earlier cryotransport, also known as cryonics, is the freezing of the entire body. Cryotransport also can be the freezing of any organ, for example the brain. This procedure is also known as neuropreservation. A clear-cut method of how cryonics is done is not in existence, because every cryonics company or industry has their own specific procedure. One of these cryonics companies is the Alcor Life Extension Foundation. They have not only precise methods of preservation, but also precise methods of training and requirements for training. Alcor's main objective is to "give people a plausible, 'if-all-else-fails' pathway to what they have always wanted, something likely to be achieved in any case, over the next century: an indefinitely long, healthy life" (Alcor, 1999).
Alcor's cryotransport procedure follows these steps when a person who has joined Alcor's Life Plan, dies. First, a standby team immediately starts cooling the body and giving intravenous medications. The medications are circulated through the body by external chest compressions or by a specialized circulation apparatus. This standby team includes four technicians, one of whom is certified as an EMT, and another who is an Alcor physician. From the site of death, the cryotransport patient is then moved to the site of surgery. Here a solution cooled to around 5 degrees Celsius is circulated through the body. This procedure is known as "washout", and is performed by a team augmented by a surgeon. This washout procedure must begin within 30 minutes after cardiac arrest and end within 2 hours to ensure decreased probability of brain damage to the patient. Once the body has been stabilized at 5 degrees Celsius, the body is transported to Alcor's main medical facility where it undergoes the remaining steps. Here the body's water is replaced with a cryoprotective agent (glycerol). Over a period of a few hours the concentration of the cryoprotective agent is increased from 4% to 75%. Upon completion of this cryoprotection procedure, the body is then placed into a dewar. A dewar is essentially a large "thermos flask", which is filled with liquid nitrogen. Liquid nitrogen is used because it is relatively inexpensive, but it also requires no refrigeration, thus cutting down on energy needed. The body can then be kept, preserved in the dewar, for years (Alcor, 1999).
The resuscitation of preserved people has not yet been successful, although much testing has been done. "The main problem is that when cells are frozen, water seeps out of them and collects between them. As this water turns to ice, it forms crystals which puncture cell membranes" which cause irreversible damage (Alcor, 1999). The Alcor Foundation, as well as their patients, believe that there is a way in which to revive these preserved people and that it will be found in the near future. For this reason, Alcor's preservational procedures are so precise and specific.
Although, there are many other cryonics companies, Alcor is the largest in the world in both popularity and number of patients. This is why much of the general cryonics procedures are based on their research.
Support of Cryobiology
Cryobiology techniques have achieved much support, especially in recent years. This support has come from the public, the industries dealing with cryobiology, and also from the medical field in general. For the most part, cryobiology has achieved many things in each of these areas. Not only has cryobiology given new and effective treatments for patients who need them, it has also provided a substantial gain of knowledge in the medical field. And this, in turn, allows industries to make more money and to grow.
The medical gain of what cryobiology has put into our grasp is sufficient enough for many people to support this with not only their money, but with their participation. New techniques would not be effective or even operational if it wasn't for the persons using the service, or those having faith in the possibilities presented by them.
The knowledge gained through these newly evolved procedures in the medical field has been phenomenal. For example, the different types of cryoprotective solutions has given the medical field a better handle on how to properly treat certain tissues and cells in other areas of medicine. New techniques have been developed, based on prior cryobiological work, which have also improved the quality of life, and aided in the treatment of certain illnesses. Many new transplantation techniques and processes have been gained. The applications of these processes are getting well known as cryobiology is being further explored. Many other medical procedures would not even be possible if it hadn't been for cryobiology.
For many people, the major "plus" about cryobiology is its ability to lengthen a person's life. The Full Length Life Society (1999) is a prime example of this point of view. They have six major purposes for supporting cryonic suspension of a person. The first purpose is to give a child who has "died" a chance to grow. The Society believes that science is on the road to major discoveries in child illnesses and the treatment and cure of them, and for this reason they believe that it is a gift to the family to put a child in cryonic suspension. The second purpose is a chance to continue parenthood. This purpose comes into play when a parent "dies", but is not ready to go or to be left go. The third purpose is a chance to see great grandchildren. They point out that the scientific process of increasing the maximum life span is a very time consuming process, and, at times, in order to meet great grandchildren, you would have to undergo cryonic suspension. The fourth purpose is a chance for an extended life span. Many people, just feel that they are not ready to leave this world, even though their bodies have given out. The Society says that by undergoing cryonic suspension, one can literally lengthen their life span. The fifth purpose is a chance to be born. By this they actually mean that every person should have the right to be born into a loving and caring family and at the right time. Cryobiology has allowed unborn fetuses be kept under cryopreservation for years, until they are desired by the parents. The Society also believes that this procedure can easily take the place of many abortion problems, both physical and emotional. The sixth, and last, purpose is a chance to be whole. This purpose is geared towards those who have some sort of major disability, such as, the loss of a limb, the loss of eyesight, or being a quadriplegic. Through this purpose, as with the others, the Society feels that science will eventually come up with solutions for every medical condition (including the ones mentioned above), and cryonic suspension will allow these new technologies to affect everyone's lives.
Another form of support for cryobiology is the dispersal of cryobiological information. The Society for Cryobiology is a group with more than 400 members, whose main goal is to do just that (1999). This organization achieves this goal in two primary ways. Yearly, a scientific meeting is held, which focuses specifically on cryobiology and related topics. Also, the Society for Cryobiology has its own journal entitled Cryobiology. These types of informative measures not only inform the public, but also those who are involved in cryobiological science.
Opposition to Cryobiology
Not only is there much support of cryobiology, there is also much opposition. This opposition comes in all shapes and sizes. Many people oppose cryobiology in some forms, but not in others. An example of this would be, when a person undergoes cryosurgery to repair a skin lesion, but then opposes cryonics of a human being. Not only is this type of point of view common, it is understandable. The parameters of cryobiology and its different procedures are so far apart that they are hard to lump together as being similar.
One of the major oppositions to cryobiology is one of the same reasons why there is much support for it. This factor would be the ability to increase the length of a life. Many persons believe that the length of life that we are destined to should not be tampered with, and this is exactly the reason why they oppose cryobiology techniques. The ability to live "forever" is one of the major points of emphasis by cryonics and other cryo-manipulative techniques, and this brings about much of the controversy. The definition of "death" is the backbone of this controversy, because everyone has their own definition. According to the Alcor Life Extension Foundation (1999), "death" for the whole body is not what actually happens when a person is pronounced clinically dead, because the individual cells and whole organs are still alive. Using this definition of "death", they have justified cryonics as a life-lengthening process. Those with other definitions, feel that cryonics does not extend life, but rather may even extend death. Since a successful method has not yet been found to resuscitate cryonics patients, persons opposing this technique believe it is not actually a "life-lengthening" procedure.
Another very important reason for opposing cryobiology is the potential damage that may occur. Any type of cryobiological procedure affects the cells and the surrounding areas, and, thus, could cause damage. In cryosurgery, damage has been reported right along with the success of treatment for the problem, as mentioned earlier. In most cases, this damage can cause future problems that will need to be treated, producing a viscous cycle. Damage may also occur in embryo or gamete cryopreservation. Viable births from cryopreserved embryos are, by far, the minority of all births (Leibo, 1999). This minority is because of poor technique, simply inactive embryos, or damage sustained by cryoprotective solutions or the freezer. Birth defects have been known to be caused by cryopreservation techniques, and the long-term implications are still unknown to us. The fact that no successful resuscitations have been performed on cryonics patients shows that damage has occurred somewhere along the way, or else that resuscitation of a frozen human being is, in fact, impossible.
The overall price of most cryobiological procedures is very expensive for the average person to afford. At the same time, cryobiological procedures are becoming more of the "norm" in many medical clinics or facilities. This has the potential for great financial problems when payment is due, because most people would not have the money. On the other hand, this great risk of financial burden on those using cryobiology has been addressed by some of the cryobiology industries. For example, Alcor Life Extension Foundation (1999) has formulated payment plans suitable to those inquiring their services. Alcor has also provided the "patients" with a fund known as the Patient Care Fund, which is for the benefit of the "patient" and their family. It seems that not only has Alcor made payment plans suitable for the "patient" and their family, but they have also increased the cost of these procedures in recent years, by considerable amounts. Alcor, in specific, claims to be a non-profit organization, while at the same time is charging a fee for cryonic suspension between $50,000 and $120,000. On top of these prices, a whole-life (insurance) policy costs around $500 a year, $150 for a sign up fee, and $360 per month for annual dues. These prices, along with normal living expenses, add up to a lot of money to pay, not to mention that the annual fees extend through the length of cryonic suspension (Alcor, 1999). These seemingly large prices, are, yet, another reason why not everyone is supportive of cryobiology.
Legal issues may also arise from some of these techniques and procedures. For example, what if a married couple decides to have embryos cryopreserved for later, and more convenient, use, but before that time comes, they get a divorce. This introduces the problem of who gets the embryos that used to be "theirs", but no longer can be? The custody of frozen embryos is a growing issue, and really is a legitimate question. Who is in control of stored, frozen embryos and gametes? And who gets legal rights to them?
There is also the possibility of people going to extremes with cryopreservation. By this, I mean that perhaps people will begin preserving inanimate objects such as their favorite stuffed animal or their lucky shirt, using up valuable and expensive time, energy, and space. Perhaps this is a very far-fetched idea, but it is, in fact, possible. Many other concerns for the effects of cryobiology and cryopreservation exist, but all can not be mentioned in one short paper.
Conclusion
As described above, cryobiology can be many different things, including treatment, preservation, as well as, controversy. The different cryobiological techniques each have their own equipment, procedures, and ethical issues associated with them, yet are so similar. While there is much unrest about whether cryobiology is justifiable, a personal choice is still the governing force in the matter. For example, my personal choice can not be influenced by anyone else's. I believe that some cryobiological procedures I would have no problem participating and trusting in (i.e. cryosurgery), and others i would never involve myself or anyone close to me with (i.e. Cryotransport). The theory of Cryotransport and embryo cryopreservation have very high expectations for the technology of the future, and that is too much of a risk for me to take. I guess you could say that I, personally, just don't have the faith it takes to accept that these theories will hold true. I believe that cryobiology does have the possibility for many important medical contributions, but what are we, as human beings and the inventors of these technologies, willing to give up in order to achieve them?
Works Cited
Alcor Foundation. (1999). "What We Do: Cryotransport and the Alcor Life Extension Foundation." URL: http://www.alcor.org/01b.html WWW document. 11/3/99.
Boyce, Nell. (January 16, 1999). "Itís the Solution: A womanís eggs can be freezer friendly, if you go easy on the salt." New Scientist. Page 10.
"Bubble Wrap". (January 23, 1999). New Scientist. Page. 14.
Cryogenics International. (1999). "Deep Cryogenic Treating-Tempering-Processing Terms." URL: http://www.goodnet.com/~cryo/drfreeze.htm WWWdocument. 11/3/99.
Fred Hutchinson Cancer Research Center. (1997). "Cryo handles cells between harvesting, transplant." Center News. URL: http://www.fhcrc.org/about/CenterNews/1997/Jul17/Cryobiology.htm WWW document. 10/30/99.
Full Length Life Society. (1997). URL: http://morelife.org/purpose-details.html WWW document. 10/30/99.
Gage, Andrew A. (1992). "Workshop Report: Progress in Cryobiology." Cryobiology. Vol. 29. Pages 300-304.
Kuflik, Emanuel G., MD. (1994). "Continuing Medical Education: Cryosurgery Updated." Journal of the American Academy of Dermatology. Vol. 31. No. 6. Pages 925-946.
Leibo, S.P. (1999) "Cryobiology: Preservation of Mammalian Embryos." Pages 251-272.
Lifebank Cryogenics Corporation. (1999). "Cord Blood Banking." URL:
http://www.lifebank.com/why.htm WWW document. 11/3/99.
New American Desk Reference Encyclopedia. (1989). Concord Reference Books, Inc.
Read, Allen Walker, Chairman. (1999). The New International Websterís Comprehensive Dictionary of the English Language. Trident Press International.
Society for Cryobiology. (1998). URL: http://sapphire.surgery.wisc.edu/cryobiology/about.html WWW document. 10/30/99.
Vellet AD, et al. (1997). "MRI to Evaluate the Success of Cryosurgery for Prostate Cancer." American Family Physician. Vol. 56. No. 7. Page 1868.
Other References
Lincoln, Tim. (Aug. 28, 1997). "Cold comfort for anglers and toxicologists." Nature. Vol. 388. Page 833.
McCaffery, Margo. (March 1999). "Pain Control: Using superficial cooling for pain relief." American Journal of Nursing. Vol. 99. No. 3. Page 24.
Menezo, Yves J. R. and Moncef Ben Khalifa. (1995). "Cytogenetic and Cryobiology of Human Cocultured Embryos: A 3-year Experience." Journal of Assisted Reproduction and Genetics. Vol. 12. No. 1. Pages 35-40.
Sikorski, Robert and Richard Peters. (August 21, 1998) "A Frankenstein Experiment." Science. Vol. 281. Pages 1163-4.
Taylor, Michael J., Ph.D. (1977). Cryobiology Research Program. URL: http://www.auhs.edu/medschool/bgs/resumes/taylor.htm WWW document. 10/30/99.
By
Krista Nussbaum (1999)
Thesis: Low temperature biology, or cryobiology, has the possibility, and may very easily and very drastically affect everyones' lives in the future. Through rapidly approaching cryobiological medical procedures and techniques, the presence of intense ethical issues may play an important role in many everyday decisions that do not now exist.
Outline:
I. Introduction
A. What is cryobiology?
1. definition
2. synonyms
3. general information
II. Cryobiology Techniques
A. cryosurgery
B. embryo and gamete preservation
C. tissue and blood cryopreservation
D. cryotransport
III. Support of Cryobiology
A. Medical gain
1. techniques
2. knowledge
B. Time gain
1. increase actual length of life
C. having biological children when you are ready to have them
D. Dispersal of Information
IV. Opposition to Cryobiology
A. interfering with the life cycle
B. medical complications
1. physical damage
C. extremely high priced
D. potential law suits over paternal rights to frozen embryos
E. people may very easily get carried away with preserving things
V. Conclusion
A. overview of methods and techniques
B. overview of ethical issues
C. question for the readers
Introduction
What is cryobiology? It is "the study of the effects of freezing and low temperatures on living organisms" (Read, 1999). While the main focus of cryobiology is on living organisms (as suggested above), cryobiological techniques have been expanded to include treatment of nonliving things as well. This addition of non-living objects has expanded the focus to not only include plants, animals, and humans, but also to include machinery and other metal objects.
A common synonym of cryobiology is "cryogenics". According to the New American Desk Encyclopedia (1989), cryogenics is defined as "the branch of physics dealing with the behavior of matter at very low temperatures, and with the production of those temperatures." As can be seen, cryogenics is a slightly more focused study of the cryobiological field dealing primarily with production methods of those freezing temperatures. Another sub-study field, if you will, is that of cryonics. Cryonics is defined as
the practice of freezing humans who are not curable by current medical technology, in the hope that ways may be found to bring them back to life at some future time when ways of repairing the damage caused by the freezing process have been developed, as well as cures of the diseases or other causes of death which necessitated their cryonics suspension (Cryogenics International, 1999).
As suggested above, cryobiology is primarily centered on the effects on those living organisms or non-living objects, although the methods and procedures are also very important. All around the world new and improved techniques are being tested to make this a better medical and preservational procedure. Not only are the different techniques and procedures getting increased attention, but also many accompanying controversial issues are as well. Low temperature biology, or cryobiology, has the possibility to very easily and very drastically affect everyoneís lives in the future. Through rapidly approaching cryobiological medical procedures and techniques, the presence of intense ethical issues may play an important role in many everyday decisions that do not now exist.
Cryobiology Techniques
There are many different cryobiological techniques that exist and are being used today. Such techniques include cryosurgery, embryo and gamete preservation, tissue preservation and transplantation, blood and blood product preservation, and cryotransport. Some of these have been around for so long that they are now considered commonplace medical procedures, while others are still considered experimental in the medical world.
Cryosurgery
Cryosurgery, a technique that has been in operation for years, still has some aspects that are being extensively tested. Much of the questions and continual testing revolves around the injury or infection that needs to be treated and hopefully cured through cryosurgery. Many of these uses of cryosurgery are widely accepted from a medical standpoint while others are not widely accepted to the point that in some clinics and medical facilities they are not provided. According to Gage (1992), "commonly accepted uses of cryosurgery are for the treatment of many types of skin lesions (including cancer), for benign and dysplastic mucosal lesions, for uterine cervicitis and intraepithelial neioplasia, and for cardiac surgery in the treatment of tacchyarrhythmias". Studies in all of these areas can easily be found and researched.
Cryosurgery involves different techniques in which selective necrosis of tissues is achieved, or hoped to be achieved, through freezing at extremely low temperatures. The achievement of these low temperatures is through a very precise cooling sequence, using a specific solution called cryogen. Each of the separate techniques involves its own specific procedure in order for the surgery to be successful. Also the goal of treatment is a determining factor on which cryosurgical technique is used. According to Kuflik (1994), the three major goals of treatment to choose from are improvement, cure, or palliation. Once these initial factors are chosen, the technique to appropriately achieve the goals is used.
One of these cryosurgical techniques uses a dipstick apparatus. The dipstick, is simply a cotton-tipped applicator that is dipped into liquid nitrogen and applied to a lesion until adequate necrosis has occurred. Another technique involves an open-spray apparatus by which a fine spray of cryogen is applied to the lesion. Solidified carbon dioxide can also be applied directly to the skin with a mix of acetone to treat certain types of acne. This type of treatment is called slush therapy. Perhaps the most popular technique used in cryosurgery is the cryoprobe. This technique involves a precooled metal accessory that is directly applied to the lesion. The cryoprobe is also frequently accompanied by liquid nitrogen (Kuflik, 1994).
In most medical facilities, these procedures are routinely done and have shown both positive and negative results. Both the positive and the negative results have been reported on numerous accounts in scientific research papers and journals. An example of negative research results comes from a study done by B. Rubinsky, et. al., of the University of California-Berkeley (1990). This study involves cryosurgical procedures done on the liver as well as analysis of damage done. They found that the formation of ice in the hepatic sinusoids expands the sinusoids, dehydrates the hepatocytes, and then leads to possible structural damage. This particular study shows that not all cryosurgical procedures are effective, at least, without causing irreversible damage to the operation subject in the process.
An example of positive research results comes from a study of other surgical techniques in comparison to cryosurgery, by A. Hohki (Gage, 1992). In this study the other treatment techniques were laser, ultrasonic aspiration, microwave surgery, scalpel, and electric scalpel. The major finding was that while all techniques have advantages and disadvantages, cryosurgery was the best method for cosmetic effect and economy. In agreement with these findings, Emmanuel G. Kuflik, MD (1994) has concluded that cryosurgery has many added benefits besides just cosmetic and economic advantages. Examples include, suitability for office, nursing home, or outpatient facility; the lack of need for general anesthesia; optional need for local anesthesia; the lack of need for operative suits; the simplicity and safety of the procedure; the lack of physical restrictions afterward; availability for pregnant patients; and suitability for those patients who are fearful of surgical procedures.
Not only has there been research-based proof that cryosurgery is only positive or only negative, but also there can easily be a mixture of both. A study by Vellet AD, et al., (1997) shows that cryosurgery for prostate cancer can have both positive and negative results. "Hypothermic injury of the rectal wall and bladder base was demonstrated" after prostate cancer treatment with cryosurgery. However in addition to this, "no residual prostatic tissue or tumor was found on follow-up biopsy".
Embryo and gamete cryopreservation
Although cryosurgery may easily be the most frequent cryobiological technique, embryo and gamete preservation comes in a close second. This particular technique has been used for many species of animals, starting with the mouse in 1972, to the goat in 1976, and the human in 1983 (Leibo, 1999). Not only has this technique been used for these types of animal species, in each it has been a successful operation; successful meaning that live young have been produced from frozen embryos. In fact, this type of procedure has been so successful throughout many species of the animal kingdom, the only species that has still been unable to undergo this procedure with success is swine (Leibo, 1999). So many animals from all different species have been born through this process of embryo cryopreservation that it can be estimated that tens of thousands individual organisms have survived the storage in liquid nitrogen at ñ196 degrees Celsius (Leibo, 1999).
Along the same lines as for cryosurgery, embryo and gamete cryopreservation involves a very precise and straightforward method of freezing, unique for each organism. Also along the same lines, many of the methods involve general steps in the freezing process. These general steps were outlined by Leibo in his study of preservation of mammalian embryos (1999). The first step is to expose the embryo to some sort of cryoprotective solution, for it has been shown that this is an essential step in successful cryopreservation. Also, this step is where a lot of further testing has occurred, and disagreement has been found, on which cryoprotective solution is the best. The next step is the first step in the actual cooling process. In this step, the embryo is cooled to slightly lower than zero degrees Celsius, followed by a seeding procedure. Seeding is used for inducing ice formation under controlled conditions. There is much disagreement on whether seeding is actually necessary, although most procedures require it. The next step, is to control the cooling of the embryo to an intermediate subzero temperature. Many studies have shown successful cryopreservation at temperatures as low as ñ80 to ñ100 degrees Celsius and temperatures as high as ñ20 to ñ40 degrees Celsius. As mentioned before, what is being preserved and who was performing the procedure would determine this temperature. The final step in the freezing process involves a very rapid cooling to ñ196 degrees Celsius for long-term storage. It has been estimated that if the proper freezing steps have been taken, the embryo can remain "alive" for thousands of years (Leibo, 1999).
Now that you have successfully cryopreserved an embryo, you must be able to thaw it out in a proper way too. In general, the warming and thawing of the frozen embryo is directly related to the amount of freezing that had initially been done; this is specific for each organism. After warming and thawing of the embryo is complete, the cryoprotective solution must be removed, because in most cases, if handled improperly, this solution is toxic to the embryo (Leibo, 1999).
A new method of embryo cryopreservation has become a recent cause for study. Katrina Forest and her colleagues of the University of Wisconsin have been studying the effects of the cryoprotective solution on the successful preservation of embryos, and have come up with some striking possibilities and observations (Bubble Wrap, 1999). They have observed that immersion in large quantities of cryoprotective solution has caused the unsuccessful cryopreservation of many of the test organisms, for example the hamster. As a result, in their study, they used a technique in which they dipped a thin nylon loop into the cryoprotective solution and suspended the embryo on this thin layer of liquid. This allowed them to simply dip the loop containing the embryo into liquid nitrogen for instantaneous freezing. Using this method, they have been the only group to successfully get a hamster birth from a frozen embryo. They hope that this method would help improve the successfulness of human embryo preservations in the future.
As mentioned above, most scientists disagree upon the ideal makeup of the cryoprotective solution. Traditionally, the cryoprotective solution that has been used was supposed to simulate the body fluids of the womb, consisting of a sodium ion- rich saline solution. Until recently, this was a standard procedure. A study done by James Stachechki (Boyce ,1999) points out that maybe the success of any cryopreservation is directly related to the amount of sodium ions in the cryoprotective solution. In his study, he tested a new solution containing choline ions (a less abrasive ingredient) on mouse eggs, and got a survival rate of 90%. This compares to the 50% survival rate of the use of conventional saline solution. Numerous other studies on improvements of the cryoprotective solution have been made and many alternate choices are now available.
Tissue and Blood Cryopreservation
Cryobiology technology has recently been focused and manipulated to aid in the care and well being of ill patients, in a different way than cryosurgery. Tissues and blood of the body can be removed, frozen, stored, and then later reintroduced into the body when they are needed or desired. One particular use of this technology is done for bone marrow transplants. Until this technology, these transplants relied on time to be effective, but now cells can be frozen and stored for years and still be effective. The Cryobiology Laboratory located at the Fred Hutchinson Cancer Research Center deals directly with this type of cell storage (Center News, 1997). According to the laboratory with this technology, "donor and recipient need no longer be the same ABO blood type [because] the red blood cells can be removed from the marrow". In this laboratory, the cells, stem cells or bone marrow cells, are stored for short-term in a controlled freezer that can cool at a rate of one degree per minute, or for long-term in a large, liquid nitrogen freezer at ñ180 to ñ195 degrees Celsius (Center News, 1997). In this same article, the laboratory has reported storage of about 1200 patientsí cells, giving a total of 4500 cell packets that are labeled and ready to use when needed. Since this article was published, the numbers have most likely increased.
Another form of tissue preservation is that of blood. In the same manner, blood or blood products can be stored in freezers and then used when needed by a patient. Current technology has designed methods of recycling placentas and umbilical cords, that would normally be discarded after birth, to be used as blood and hematopoietic stem cell sources. According to Lifebank Cryogenics Corporation (1999), this procedure of saving the placenta and umbilical cord at birth is a very simple procedure that only takes minutes, and in turn could possibly save your life or the life of your child. These cells are used for many possible reasons including stem cell matching for your child, treatment of life-threatening diseases, or even gene therapy (Lifebank, 1999). Umbilical cord blood transplantation also has been shown to be an acceptable alternative to bone marrow transplantation.
CryoTransport
Cryotransport is the newest procedure done in cryobiology. As defined earlier cryotransport, also known as cryonics, is the freezing of the entire body. Cryotransport also can be the freezing of any organ, for example the brain. This procedure is also known as neuropreservation. A clear-cut method of how cryonics is done is not in existence, because every cryonics company or industry has their own specific procedure. One of these cryonics companies is the Alcor Life Extension Foundation. They have not only precise methods of preservation, but also precise methods of training and requirements for training. Alcor's main objective is to "give people a plausible, 'if-all-else-fails' pathway to what they have always wanted, something likely to be achieved in any case, over the next century: an indefinitely long, healthy life" (Alcor, 1999).
Alcor's cryotransport procedure follows these steps when a person who has joined Alcor's Life Plan, dies. First, a standby team immediately starts cooling the body and giving intravenous medications. The medications are circulated through the body by external chest compressions or by a specialized circulation apparatus. This standby team includes four technicians, one of whom is certified as an EMT, and another who is an Alcor physician. From the site of death, the cryotransport patient is then moved to the site of surgery. Here a solution cooled to around 5 degrees Celsius is circulated through the body. This procedure is known as "washout", and is performed by a team augmented by a surgeon. This washout procedure must begin within 30 minutes after cardiac arrest and end within 2 hours to ensure decreased probability of brain damage to the patient. Once the body has been stabilized at 5 degrees Celsius, the body is transported to Alcor's main medical facility where it undergoes the remaining steps. Here the body's water is replaced with a cryoprotective agent (glycerol). Over a period of a few hours the concentration of the cryoprotective agent is increased from 4% to 75%. Upon completion of this cryoprotection procedure, the body is then placed into a dewar. A dewar is essentially a large "thermos flask", which is filled with liquid nitrogen. Liquid nitrogen is used because it is relatively inexpensive, but it also requires no refrigeration, thus cutting down on energy needed. The body can then be kept, preserved in the dewar, for years (Alcor, 1999).
The resuscitation of preserved people has not yet been successful, although much testing has been done. "The main problem is that when cells are frozen, water seeps out of them and collects between them. As this water turns to ice, it forms crystals which puncture cell membranes" which cause irreversible damage (Alcor, 1999). The Alcor Foundation, as well as their patients, believe that there is a way in which to revive these preserved people and that it will be found in the near future. For this reason, Alcor's preservational procedures are so precise and specific.
Although, there are many other cryonics companies, Alcor is the largest in the world in both popularity and number of patients. This is why much of the general cryonics procedures are based on their research.
Support of Cryobiology
Cryobiology techniques have achieved much support, especially in recent years. This support has come from the public, the industries dealing with cryobiology, and also from the medical field in general. For the most part, cryobiology has achieved many things in each of these areas. Not only has cryobiology given new and effective treatments for patients who need them, it has also provided a substantial gain of knowledge in the medical field. And this, in turn, allows industries to make more money and to grow.
The medical gain of what cryobiology has put into our grasp is sufficient enough for many people to support this with not only their money, but with their participation. New techniques would not be effective or even operational if it wasn't for the persons using the service, or those having faith in the possibilities presented by them.
The knowledge gained through these newly evolved procedures in the medical field has been phenomenal. For example, the different types of cryoprotective solutions has given the medical field a better handle on how to properly treat certain tissues and cells in other areas of medicine. New techniques have been developed, based on prior cryobiological work, which have also improved the quality of life, and aided in the treatment of certain illnesses. Many new transplantation techniques and processes have been gained. The applications of these processes are getting well known as cryobiology is being further explored. Many other medical procedures would not even be possible if it hadn't been for cryobiology.
For many people, the major "plus" about cryobiology is its ability to lengthen a person's life. The Full Length Life Society (1999) is a prime example of this point of view. They have six major purposes for supporting cryonic suspension of a person. The first purpose is to give a child who has "died" a chance to grow. The Society believes that science is on the road to major discoveries in child illnesses and the treatment and cure of them, and for this reason they believe that it is a gift to the family to put a child in cryonic suspension. The second purpose is a chance to continue parenthood. This purpose comes into play when a parent "dies", but is not ready to go or to be left go. The third purpose is a chance to see great grandchildren. They point out that the scientific process of increasing the maximum life span is a very time consuming process, and, at times, in order to meet great grandchildren, you would have to undergo cryonic suspension. The fourth purpose is a chance for an extended life span. Many people, just feel that they are not ready to leave this world, even though their bodies have given out. The Society says that by undergoing cryonic suspension, one can literally lengthen their life span. The fifth purpose is a chance to be born. By this they actually mean that every person should have the right to be born into a loving and caring family and at the right time. Cryobiology has allowed unborn fetuses be kept under cryopreservation for years, until they are desired by the parents. The Society also believes that this procedure can easily take the place of many abortion problems, both physical and emotional. The sixth, and last, purpose is a chance to be whole. This purpose is geared towards those who have some sort of major disability, such as, the loss of a limb, the loss of eyesight, or being a quadriplegic. Through this purpose, as with the others, the Society feels that science will eventually come up with solutions for every medical condition (including the ones mentioned above), and cryonic suspension will allow these new technologies to affect everyone's lives.
Another form of support for cryobiology is the dispersal of cryobiological information. The Society for Cryobiology is a group with more than 400 members, whose main goal is to do just that (1999). This organization achieves this goal in two primary ways. Yearly, a scientific meeting is held, which focuses specifically on cryobiology and related topics. Also, the Society for Cryobiology has its own journal entitled Cryobiology. These types of informative measures not only inform the public, but also those who are involved in cryobiological science.
Opposition to Cryobiology
Not only is there much support of cryobiology, there is also much opposition. This opposition comes in all shapes and sizes. Many people oppose cryobiology in some forms, but not in others. An example of this would be, when a person undergoes cryosurgery to repair a skin lesion, but then opposes cryonics of a human being. Not only is this type of point of view common, it is understandable. The parameters of cryobiology and its different procedures are so far apart that they are hard to lump together as being similar.
One of the major oppositions to cryobiology is one of the same reasons why there is much support for it. This factor would be the ability to increase the length of a life. Many persons believe that the length of life that we are destined to should not be tampered with, and this is exactly the reason why they oppose cryobiology techniques. The ability to live "forever" is one of the major points of emphasis by cryonics and other cryo-manipulative techniques, and this brings about much of the controversy. The definition of "death" is the backbone of this controversy, because everyone has their own definition. According to the Alcor Life Extension Foundation (1999), "death" for the whole body is not what actually happens when a person is pronounced clinically dead, because the individual cells and whole organs are still alive. Using this definition of "death", they have justified cryonics as a life-lengthening process. Those with other definitions, feel that cryonics does not extend life, but rather may even extend death. Since a successful method has not yet been found to resuscitate cryonics patients, persons opposing this technique believe it is not actually a "life-lengthening" procedure.
Another very important reason for opposing cryobiology is the potential damage that may occur. Any type of cryobiological procedure affects the cells and the surrounding areas, and, thus, could cause damage. In cryosurgery, damage has been reported right along with the success of treatment for the problem, as mentioned earlier. In most cases, this damage can cause future problems that will need to be treated, producing a viscous cycle. Damage may also occur in embryo or gamete cryopreservation. Viable births from cryopreserved embryos are, by far, the minority of all births (Leibo, 1999). This minority is because of poor technique, simply inactive embryos, or damage sustained by cryoprotective solutions or the freezer. Birth defects have been known to be caused by cryopreservation techniques, and the long-term implications are still unknown to us. The fact that no successful resuscitations have been performed on cryonics patients shows that damage has occurred somewhere along the way, or else that resuscitation of a frozen human being is, in fact, impossible.
The overall price of most cryobiological procedures is very expensive for the average person to afford. At the same time, cryobiological procedures are becoming more of the "norm" in many medical clinics or facilities. This has the potential for great financial problems when payment is due, because most people would not have the money. On the other hand, this great risk of financial burden on those using cryobiology has been addressed by some of the cryobiology industries. For example, Alcor Life Extension Foundation (1999) has formulated payment plans suitable to those inquiring their services. Alcor has also provided the "patients" with a fund known as the Patient Care Fund, which is for the benefit of the "patient" and their family. It seems that not only has Alcor made payment plans suitable for the "patient" and their family, but they have also increased the cost of these procedures in recent years, by considerable amounts. Alcor, in specific, claims to be a non-profit organization, while at the same time is charging a fee for cryonic suspension between $50,000 and $120,000. On top of these prices, a whole-life (insurance) policy costs around $500 a year, $150 for a sign up fee, and $360 per month for annual dues. These prices, along with normal living expenses, add up to a lot of money to pay, not to mention that the annual fees extend through the length of cryonic suspension (Alcor, 1999). These seemingly large prices, are, yet, another reason why not everyone is supportive of cryobiology.
Legal issues may also arise from some of these techniques and procedures. For example, what if a married couple decides to have embryos cryopreserved for later, and more convenient, use, but before that time comes, they get a divorce. This introduces the problem of who gets the embryos that used to be "theirs", but no longer can be? The custody of frozen embryos is a growing issue, and really is a legitimate question. Who is in control of stored, frozen embryos and gametes? And who gets legal rights to them?
There is also the possibility of people going to extremes with cryopreservation. By this, I mean that perhaps people will begin preserving inanimate objects such as their favorite stuffed animal or their lucky shirt, using up valuable and expensive time, energy, and space. Perhaps this is a very far-fetched idea, but it is, in fact, possible. Many other concerns for the effects of cryobiology and cryopreservation exist, but all can not be mentioned in one short paper.
Conclusion
As described above, cryobiology can be many different things, including treatment, preservation, as well as, controversy. The different cryobiological techniques each have their own equipment, procedures, and ethical issues associated with them, yet are so similar. While there is much unrest about whether cryobiology is justifiable, a personal choice is still the governing force in the matter. For example, my personal choice can not be influenced by anyone else's. I believe that some cryobiological procedures I would have no problem participating and trusting in (i.e. cryosurgery), and others i would never involve myself or anyone close to me with (i.e. Cryotransport). The theory of Cryotransport and embryo cryopreservation have very high expectations for the technology of the future, and that is too much of a risk for me to take. I guess you could say that I, personally, just don't have the faith it takes to accept that these theories will hold true. I believe that cryobiology does have the possibility for many important medical contributions, but what are we, as human beings and the inventors of these technologies, willing to give up in order to achieve them?
Works Cited
Alcor Foundation. (1999). "What We Do: Cryotransport and the Alcor Life Extension Foundation." URL: http://www.alcor.org/01b.html WWW document. 11/3/99.
Boyce, Nell. (January 16, 1999). "Itís the Solution: A womanís eggs can be freezer friendly, if you go easy on the salt." New Scientist. Page 10.
"Bubble Wrap". (January 23, 1999). New Scientist. Page. 14.
Cryogenics International. (1999). "Deep Cryogenic Treating-Tempering-Processing Terms." URL: http://www.goodnet.com/~cryo/drfreeze.htm WWWdocument. 11/3/99.
Fred Hutchinson Cancer Research Center. (1997). "Cryo handles cells between harvesting, transplant." Center News. URL: http://www.fhcrc.org/about/CenterNews/1997/Jul17/Cryobiology.htm WWW document. 10/30/99.
Full Length Life Society. (1997). URL: http://morelife.org/purpose-details.html WWW document. 10/30/99.
Gage, Andrew A. (1992). "Workshop Report: Progress in Cryobiology." Cryobiology. Vol. 29. Pages 300-304.
Kuflik, Emanuel G., MD. (1994). "Continuing Medical Education: Cryosurgery Updated." Journal of the American Academy of Dermatology. Vol. 31. No. 6. Pages 925-946.
Leibo, S.P. (1999) "Cryobiology: Preservation of Mammalian Embryos." Pages 251-272.
Lifebank Cryogenics Corporation. (1999). "Cord Blood Banking." URL:
http://www.lifebank.com/why.htm WWW document. 11/3/99.
New American Desk Reference Encyclopedia. (1989). Concord Reference Books, Inc.
Read, Allen Walker, Chairman. (1999). The New International Websterís Comprehensive Dictionary of the English Language. Trident Press International.
Society for Cryobiology. (1998). URL: http://sapphire.surgery.wisc.edu/cryobiology/about.html WWW document. 10/30/99.
Vellet AD, et al. (1997). "MRI to Evaluate the Success of Cryosurgery for Prostate Cancer." American Family Physician. Vol. 56. No. 7. Page 1868.
Other References
Lincoln, Tim. (Aug. 28, 1997). "Cold comfort for anglers and toxicologists." Nature. Vol. 388. Page 833.
McCaffery, Margo. (March 1999). "Pain Control: Using superficial cooling for pain relief." American Journal of Nursing. Vol. 99. No. 3. Page 24.
Menezo, Yves J. R. and Moncef Ben Khalifa. (1995). "Cytogenetic and Cryobiology of Human Cocultured Embryos: A 3-year Experience." Journal of Assisted Reproduction and Genetics. Vol. 12. No. 1. Pages 35-40.
Sikorski, Robert and Richard Peters. (August 21, 1998) "A Frankenstein Experiment." Science. Vol. 281. Pages 1163-4.
Taylor, Michael J., Ph.D. (1977). Cryobiology Research Program. URL: http://www.auhs.edu/medschool/bgs/resumes/taylor.htm WWW document. 10/30/99.
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