Cryobiology

Cryobiology has tremendously advanced. It exhibits this in its ability to cryopreserve reproductive cells and, in fact embryos, which actually formed the basis of many research done to initiate studies in cryobiology. Cryopreservation presents a technique in which these livings cells can be kept for long periods of time while still maintaining their intrinsic properties. Oocytes from humans can be utilized in various ways which include inducing pregnancies and actually causing births to take place. In animals, cryopreservation is also utilized when there is a possibility of extinction or a disease condition. The make- up of these cells (oocytes and embryos) play a major role in determining their ability to be cryopreserved. Factors include the zona pellucida, membrane permeability, size and cytoskeleton.

At birth, Oocytes are present in primordial follicles while embryos exist after fertilization in the pre and post implantation stages(early stage) .

The oocyte is one of the largest cells in the body, being significantly larger than the mouse oocytes(lect blue)

It is a lot easier to cryopreserve oocytes than embryos( lect note- vitri)

Large, low surface are/ vol ratio, low permea, high intracellular nucleatn temp

It has been more victorious to cryopreserve early- cleavage embryos than mature oocytes; showing that the reproductive stage has great significance. (Fuller et al, )It has also been found that embryos are more successfully preserved than oocytes ( Fuller and Paynter, )

The mature oocyte has a low surface area to volume ratio which makes it difficult for it to absorb cryoprotectants and lose water. Because it retains water when supercooled, intracellular ice formation results. Oocytes also exhibit different permeability's for cryoprotectants and water depending on the degree of maturation, strain and the species. In order for oocytes to be cryopreserved , they need to be dehydrated but also kept in a state that their internal water component are intact to avoid denaturation.

These cold dehydrative states could be done by controlled slow cooling or vitrification. The two methods are equally beneficial in crypreservation (vitri)

The temperature gradient while cooling is going on is dependent upon the housing compartment and the thermal characteristics, also the comparative volumes to the coolant can control the manner in which heat is transferred. An end temperature of -100C is sought after, . Linear cooling is preferable over passive cooling because it stands advantageous in the fact that samples can be monitored more easily with it, and linear slow cooling is preferentially used because it can prevent intracellular ice formation that take place at sub- zero temperature if the intracellular surroundings of the oocyte takes place near the place the ice originates from.(19).Equipments can be used to perform slow cooling which stand as an advantage because they can monitor the temperature, and keep records of the ‘dummy' as well as the recording chambers which could be used for to ascertain the degree of safety of the samples and also quality assurance checks.

Some laboratories use baths in dewars containing mixed alcohol suspended in liquid nitrogen to achieve slow cooling in oocytes but Hayes et al, in 2000(20) showed that controlled slow cooling of temperatures of -100C and below could be achieved by using an electric heater to balance freezing nitrogen vapour in a concealed surrounding. This is the principle that governs most recently made nitrogen based cooling machines. Recently, another method based on the free piston Sterlings cooler method was developed using a linearized motor capable of delivering cooing rates of -0.5C/min utilising a Sterling cooler cryocooler as its cooling source where helium gas is continually expanded and contracted in the cylinder. THE EF600 as it is called, can be controlled over cooling rates of +30C to -100C.

To obtain the highest yield results, seeding (ice nucleation) can be used.....

Ice can be nucleated by the use of forceps or identical equipments immersed in liquid nitrogen to clip the top of the cooled straw or a tiny is size nitrous oxide tool that is capable of expanding N20 very quickly from local cold spot. This device can be used where liquid nitrogen is unavailable because it uses a battery source and not a cryogen (22). Oter machines have been developed for oocyte cropreservation which function by lowering the oocytes into straws then a carrier moves it into ...by a stepper motor into nitrogen vapour(23)

The developmental stage of the oocyte is very important because it dertermines to a large extent the degree of survival of cell. For example, unfertilised oocytes are more susceptible to osmotic stress. Also, bovine immature oocytes are more prone to osmotic damage than mature ones when they are put in hypertonic solutions(24).Also, pocine oocytes have been noticed to their spindle structures altered....(25) Human oocytes developed in vitro also exhibited post ICSI when exposed to osmolarities at 37C for 5 mins(26). This can be controlled by determining the knowing to what degree cryoprotectants as well as water pass through

Movement of water out of the embryos can be determined by the temperature, volume of the cell, osmotic potential and water permeability of the cell.

A cryoprotectants/ vitrifcation medium was done by Mukaida et al that involved an 8 cell mouse embryo. Ehylene glucol, DMSO , glycerol in glucose and a 30% Ficol when applied to humans resulted in healthy twins

The make up and concentration of the vitrification solution plays an important role in influencing the survival of the embryo, the procedure used to equilibrate embryos in solution, cooling and warming process, the method used to dilute embryos from the vitrification solution

Talk about the optimal conditions for equilibration in and dilution of the vitrification media.--

Slow cooling can also be dictated by the large size of the oocytes as water can only leave slowly during dehydrative freezing.

Slow freezing does not bring about any real damage and osmotic effects on cells ( Mazur, 1990).it also uses makes use of low concentrations of cryoprotectants , it involves a lot of time and the freezing machine utilized is expensive. (Rama Raju et al., 2005; Al-Hasani et al., 2007; Balaban et al., 2008; Kuwayama et al., 2005)

Recently, Moose oocytes and embryos were first successfully vitrified in 1985 (Rall and Fahy, 1985)

Another method for achieving cryopreservation without crystallization is called vitrification . ( Luyet, 1937) (Vajta & Nagy, 2006),it is inexpensive and unlike controlled slow cooling, it does not involve a lot of time According to a research study done in Iran, vitrification is preferable to slow freezing for the human cleaved embryo. The survival rate is high, and upon warming, the morphology of the embryo has little damaged effects.(Valojerdi et al, 2009)

Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes," wrote Valojerdi et al. ("Vitrification Versus Slow Freezing Gives Excellent Survival, Post-Warming Embryo

 

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