Cryopreservation | Open Access Journals

e-ISSN No.:2581-3897


Ankita Dey*

Department of Biotechnology, Haldia Institute of Technology, West Bengal

*Corresponding Author:
Ankita Dey
B-Tech in Biotechnology
Haldia Institute of Technoogy
West Bengal
Tel: +918961770956

Received date: 25/07/2016 Accepted date: 26/07/2016 Published date: 28/08/2016

Visit for more related articles at Research & Reviews: Journal of Veterinary Sciences


The capability to preserve human or animal oocytes, blastocysts, fruitful organ/gonad/sex gland tissue and spermatozoa is a crucial tool in animal assisted reproductive techniques. This preservation permits patients undergoing therapy or radiation therapy to preserve their fertility, and helps to achieve all advantages from the pricey sex gland superovulation therapies before ART (Assisted Reproduction Techniques). The first goal in establishing Associate in nursing applicable phase change protocol is to try to as very little injury as doable whereas exposing specimens to non-physiologic radical low temperatures. Today two techniques square measure utilized in cryopreservation: The slow cooling technique and also the more modern speedy procedure of vitrification. Vitrification is straight forward, needs no costly programmable phase change instrumentation, economical and value effective thanks to improve additive gestation rates per cycle. Oocytes, blastocysts, sex gland tissue and spermatozoa may be appropriate for vitrification and so cryopreservation. Vitrification well-tried to be the longer term of cryopreservation and vital progresses square measure achieved every day during this active domain during a trial to line the best protocol for cryopreservation of various varieties of gametes, embryos and tissue. The cryopreservation is additionally wont to preserve alternative natural materials adore spore grains, a number of the foremost vital and nutrient seeds and plenty of a lot of. There are several natural materials that are becoming extinct. These materials are often preserved for the long run use by subjecting it to cryopreservation.


Oocytes; Blastocysts; Superovulation; Vitrification; Amnionic


Cryopreservation also called as cryoconservation may be a method wherever organelles, cells, tissues, extracellular matrix, organs or the other biological constructs at risk of harm caused by unregulated chemical dynamics area unit preserved by cooling to terribly low temperatures (typically -80°C victimization solid greenhouse emission or -196°C victimization liquid nitrogen) [1]. At low enough temperatures, any catalyst or chemical activity which could cause harm to the biological material in question is effectively stopped. Cryopreservation strategies get to achieve low temperatures while not inflicting extra harm caused by the formation of ice throughout temperature reduction. Ancient cryopreservation has relied on coating the fabric to be frozen with a category of molecules termed cryoprotectants. New strategies area unit perpetually being investigated because of the inherent toxicity of the many cryoprotectants [2]. By default it ought to be thought-about that cryopreservation alters or compromises the structure and performance of cells unless it's established otherwise for a specific cell population. Cryoconservation of animal genetic resources is that the method within which animal genetic material is collected and holds on with the intention of conservation of the breed.

Preservation of Amniotic Membrane

There has been an excellent attention to the capacities of the amniotic membrane and its stem cells within the recent years. This membrane has been shown to own many distinctive capacities reminiscent of anticancer effects [3,4] additionally as development and vascular formation capacities [5]. It's vital to note that so as to contemplate the amniotic membrane as a possible choice that's sensible to be used in clinical settings, it's essential to create certain that this tissue may be preserved and transferred to totally different patients required as required pro re nata PRN and once needed, whereas keeping its capacities once the mandatory modifications. The current study aims to summarize in short what has been done to assess the chance that the amniotic membrane may be preserved keeping its viability and specific properties even once freeze and thaw procedure is performed on the membrane.

The amniotic membrane is that the personal layer of the human placenta, secrets numerous substances and has distinctive characteristics reminiscent of immunoregulatory, antiangiogenic, and pro-apoptotic activity, additionally as antimicrobial properties, antifibroblastic activity, cell migration, and cell growth-promoting activity. The amniotic membrane (AM) acts as a biological barrier that supports the fetus by making ready associate degree anatomically, physiologically and immunologically privileged house [6,7]. The AM consists of a thick basement membrane with one layer of animal tissue stem cells (ESCs) associate degreed an avascularstroma containing mesenchymal stem cells (MSCs). Studies have shown that the amniotic membrane owes its distinctive characteristics to special stem cells that it harbors on its either side. One of the numerous properties that the AM has shown to own is development capacities within the presence of MSCs. Studies have evidenced a pro-angiogesis result for these stem cells [5,8] whereas ESCs of the AM square measure shown to own anti-angiogenesis and pro-apoptosis properties [3,4]. Pro-angiogenesis effects of AM will facilitate in dominant vascular diseases [5] whereas anti-angiogenesis and pro-apoptosis effects of this membrane and its stem cells may be applicable in cancer medical aid [9-15].

Some More Examples Where The Cryopreservations Can Be Done

Indian landmass may be a storage of Bos indicus cows, one in every of the foremost widespread breed amongst them is “Ongole” that by inheritance may be a twin purpose breed accepted for its frugality, robustness and malady resistance. It’s been ascertained that in native similarly as crossbred bulls an enormous share of ejaculates were discarded thanks to low motility, high proportion of abnormal sperms and poor freezability throughout varied stages of cum temperature reduction. There can be species variations in overall sperm cell sensitivity to cryopreservation; the ejaculate was heterogeneous with a variable resistance to diffusion stress amongst the cells. Underneath tropical conditions, exotic breeds showed considerably seasonal fluctuations in cum characteristics as high close temperature throughout summer adversely affected the sex gland size, sexual desire and cum quality and epididymal spermatozoa by elevated sex gland temperature with weakened the power of spermatozoa to keep up motility and acrosomal integrity once temperature reduction. Compared to recent cum, eight times a lot of cryopreserved bovine sperms were needed to attain equivalent fertilization rates in vivo [16].

Qing et al. has investigated the impact of delipation (lipid driblet removal) on the biological process competency of porcine oocytes. Delipated (+/−) and/or glassy (+/−) oocytes were subjected to apomictic activation, the viability of the first apomictic embryos was recorded, and therefore the embryos were then transferred to recipients for more development. The results showed that the cleavage and blastula rates of the apomictic embryos from the first delipated oocytes were considerably below those of embryos from an effect cluster (undelipated oocytes) (P<0.05), once transfer to recipient pigs, these embryos were ready to more develop and to supply apomictic fetuses. Mistreatment the minimum volume cooling (MVC) technique, the apomictic embryos from early delipated oocytes can be cryopreserved by vitrification at 1-cell to early blastula stages. Once thawing, the first blastula stage was found to be optimum for vitrification. Conjointly embryos glassy at 2-4 cells derived from early delipated oocytes were transferred into 2 recipient pigs, and resulted in limb-bud stage fetuses. Lastly, the results incontestable that apomictic fetuses is made from porcine embryos before or once vitrification by a strategic combination of in vitro-matured (IVM) gametocyte delipation with vitrification at any early embryonic biological process stage. This approach could have application to cryopreservation of cloned and intra cytoplasmic spermatozoon injection (ICSI) embryos derived from delipated oocytes [17].

African catfish is a superb fish with adequate market demand [18]. However, the standard and amount of fish seeds made for pool stocking stay a significant concern for property cultivation. Therefore, cryopreservation is inspired as a way of enhancing viable seed production. Technology for cryopreservation of humor might be developed among fish place operators in African country catfish cultivation trade. The procedure includes choice of brood stock, sperm cell assortment, cold storage, freezing, thawing, rule to be used and disposal, transfer agreements and information development [19-36].

To Stop Risk Main Strategies

The main techniques to stop cryopreservation damages square measure a well-established combination of controlled rate and slow chilling and a more recent flash-freezing method called vitrification.

Slow Programmable Freezing

Controlled-rate and slow chilling, conjointly called slow programmable freezing (SPF), is a set of well-established techniques developed throughout the first Seventies that enabled the primary human embryo frozen birth Zoe Leyland throughout 1984. Since then, machines that freeze biological samples exploitation programmable sequences, or controlled rates, are used everywhere the globe for human, animal and cell biology – "freezing down" a sample to raised preserve it for ultimate thawing, before it's frozen, or cryopreserved, in nitrogen. Such machines square measure used for chilling oocytes, skin, blood merchandise, embryo, sperm, stem cells and general tissue preservation in hospitals, veterinary practices and analysis laboratories round the world

Lethal intracellular chilling is avoided if cooling is slow enough to allow comfortable water to depart the cell throughout progressive chilling of the liquid body substance to attenuate the expansion of extracellular frost mist growth and recrystallization, biomaterials reminiscent of alginates, poly vinyl alcohol or chitosan is wont to impede frost mist growth together with ancient tiny molecule cryoprotectant. That rate differs between cells of differing size and water permeability: a typical cooling rate of concerning 1°C/minute is acceptable for several class cells once treatment with cryoprotectants reminiscent of glycerin ordimethyl sulphoxide, however the speed isn't a universal optimum. The 1°C/minute rate is achieved by exploitation devices reminiscent of a rate-controlled electric refrigerator or a bench top transportable chilling instrumentality [37].

Several freelance studies have provided proof that frozen embryos hold on exploitation slow-freezing techniques could in some ways that be 'better' than contemporary in IVF. The studies indicate that exploitation frozen embryos and eggs instead of contemporary embryos and eggs reduced the chance of miscarriage and premature delivery although the precise reasons square measure still being explored.


Researchers Greg Fahy and William F. Rall helped introduce vitrification to fruitful cryopreservation within the mid-1980s. As of 2000, researchers claim vitrification provides the advantages of cryopreservation while not injury because of frost mist formation, true became a lot of complicated with the event of tissue engineering as each cells and biomaterials got to stay unfrozen to preserve high cell viability and functions, integrity of constructs and structure of biomaterials. Vitrification of tissue designed constructs was 1st rumored by Lilia Kuleshova, World Health Organization conjointly was the primary soul to realize vitrification of woman’s eggs (oocytes), that resulted in nascency in 1999. For clinical cryopreservation, vitrification sometimes needs the addition of cryoprotectants before cooling. The cryoprotectants act likeantifreeze: they decrease the chilling temperature. They conjointly increase the body. Rather than crystallisation, the syrupy answer becomes associate degree amorphous ice- it vitrifies. Instead of a natural process from liquid to solid by crystallization, the amorphous state is sort of a "solid liquid", and therefore the transformation is over atiny low temperature vary delineate because the "glass transition" temperature.

Vitrification of water is promoted by fast cooling, and might be achieved while not cryoprotectants by an especially fast decrease of temperature (megakelvins per second). The speed that's needed to realize glassy state in pure water was thought of to be not possible till 2005.

Two conditions sometimes needed to permit vitrification square measure a rise of the body and a decrease of the chilling temperature. Several solutes do each, however larger molecules usually have a bigger impact, significantly on body. Fast cooling conjointly promotes vitrification.

For established strategies of cryopreservation, the substance should penetrate the cytomembrane so as to realize hyperbolic body and reduce chilling temperature within the cell. Sugars don't without delay permeate through the membrane. Those solutes that do, reminiscent of dimethyl sulfoxide, a typical cryoprotectant, square measure usually nephrotoxic in intense concentration. one amongst the troublesome compromises of vitrifying cryopreservation issues limiting the injury created by the cryoprotectant itself because of cryoprotectant toxicity. Mixtures of cryoprotectants and therefore the use of ice blockers have enabled the ordinal Century medication company to vitrify a rabbit excretory organ to -135°C with their proprietary vitrification mixture. Upon rewarming, the excretory organ was transplanted with success into a rabbit, with complete practicality and viability, able to sustain the rabbit indefinitely because the sole functioning excretory organ [37].

Autologous transplant of alloperipheral cells in myeloma is profit most of the patients. By dispensing cryopreservation, the procedure may be performed with lower prices in additional hospitals with scarce technological instrumentation, safely and effectively. In associate degree age within which the value analyses gain importance within the higher cognitive {process} process of health programs involving cancer, it's important to acknowledge that survival of patients accumulated and want to come them to their work surroundings as shortly as doable may be a priority, for quality of life, and to avoid work days absences that ought to be summed up to the full value of antineoplastic treatments.

In a country as Mexico, wherever the access to trendy and effective medicine against millimeter is scarce and restricted (bortezomib, lenalidomide, carfilzomib, pomalidomide, etc.) it's of high importance to develop methods to widen hematogenic transplantation programs through its simplification [38].

Cryopreservation is associate degree integral a part of motor-assisted replica. For years the applying of slow freeze has created the cryopreservation of zygotes and embryos triple-crown, giving to thousands of patients the chance to extend the effectiveness of in vitro fertilization (IVF) cycles. Recently, the introduction of vitrification within the everyday clinical apply entailed a better success within the cryopreservation of embryos and principally of oocytes. Vitrification is taken into account to be a lot of economical methodology than the slow freeze. This is often a faster and less complicated technique that achieves higher survival rate of embryos and oocytes .Thus, vitrification gave an extra impulse to IVF attempts [39-43].

The introduction of vitrification into IVF came together with associate degree sweetening of the relevant analysis. As a result, new vitrification protocols and devices have appeared attending to improve any the success rates of cryo cycles .In the gift study, rat oocytes were vitreous with a replacement vitrification protocol that was compared with another well-established and commercially accessible protocol. The most outcome live was the survival rate in real time once thawing and forty eight hours later [9,44-52].


As it has been observed that in maximum cases the before and after the subjection to cryopreservation of an organ, both are having the same characteristics and there is a very little or no loss in the qualities. As cryopreservation have the capability to preserve human or animal oocytes, blastocysts, fruitful organ/gonad/sex organ tissue and spermatozoa may be a crucial tool in animal motor-assisted fruitful techniques. This preservation permits patients undergoing medical aid or irradiation to preserve their fertility, and helps to realize all blessings from the dear gonad superovulation therapies before ART (Assisted copy Techniques) which is the primary goal in establishing Associate in nursing applicable action protocol is to do to as little injury as achievable whereas exposing specimens to non-physiologic radical low temperatures. There are several natural materials that are becoming extinct. These materials are often preserved for the long run use by subjecting it to cryopreservation.