Basics of Cryopreservation

Dr. Dharmendra Kumar, Scientist CIRB, Hisar (Haryana)- India & Dr. Taruna Anand, Scientist VTC, NRC Equine, Hisar (Haryana) India

Definition

A state of suspended animation from which it can be revived to continue it s normal growth

Embryo cryopreservation: First of all in 1971, David Whittingham in London obtaine live mice pups after transfer of frozen-thawed embryos.

Cont..


Pregnancy rates with fresh IVP blastocysts 50-60% but this percentage is halved with frozen-thawed embryos. In sheep it was shown that freezability of IVP sheep embryos was markedly below that of their in vivo- produced counterparts.

The Dilemma
 

must remove water from blastomeres ice crystal formation within embryo is lethal if remove too much water, cell damage will result must remove just enough water but not too much, and not too fast

Types of freezing


According to the cooling rates



equilibrium (slow freezing) or non-equilibrium (vitrification and ultra rapid freezing)

Conte..


Slow freezing is a method that requires a long slow controlled cooling before the sample is stored in liquid nitrogen (LN2 -196rC). Vitrification, by contrast, enables rapid cooling of samples by direct plunging into LN2.

Slow freezing


The slow freezing method (also known as programmable freezing) involves cooling the cells at a slow controlled rate before these are plunged and stored in LN2. Slow freezing is a conventional equilibrium method using permeating cryoprotectants. In this method, the samples are suspended in 1-2 mol/L cryoprotectant dissolved in a physiological solution, ice seeded, and cooled very slowly so that the cellular contents become concentrated by gradual dehydration in response to the concentration of the extracellular unfrozen fraction during the growth of extracellular ice.

Serial dilutions- slow freeze



add cryoprotectants in steps to minimize shrinkage 0, 3, 6, 10% gylcerol with sucrose added the last step (10% + sucrose) slow, to allow water to leave cells

The slow freezing method has the following distinctive steps:



 

 

The oocytes are exposed at room temperature to an appropriate concentration of a cryoprotectant until equilibrium is reached between the cryoprotectant solution and the oocytes. The oocytes are loaded in 0.25 ml French plastic straw. The straws are kept in the programmable freezing machine where the temperature is lowered @ 0.3 to 0.5C/min to around -5rC at a controlled fashion. Seeding (induction of ice crystals) is carried out at 5rC to 7rC. The temperature is reduced to around 30 to -70rC at rate of around 0.2 to 2rC/min. Finally the sample is plunged into LN2 for storage.

Vitrification


Vitrification is a physical process by which a solution is transformed into a stable amorphous glass by rapid cooling bypassing ice crystal formation while maintaining the properties of a liquid in a solidified form. In most of the studies on vitrification of oocytes or embryos, a 0.25 ml straw is used. The straw is cooled by direct plunging in LN2. In this case the cooling rate is as high as >2500C/min.

Vitrification method has the following distinctive steps:



 

The oocytes are exposed at room temperature to a high concentration of a cryoprotectant until equilibrium is reached between the cryoprotectant solution and the oocytes. The oocytes are exposed to a high concentration of the cryoprotectant for a very brief period. Oocytes are loaded in French straws. In last, sample is plunged into LN2 for storage.

Conte..


Though vitrification gives better result than slow freezing still it is associated with poor survivability rates when compared with the latest ultrarapid vitrification protocols.

Ultrarapid Vitrification


The container for conventional vitrification is a plastic insemination straw (0.25 mL). This straw is (2.0 mm in outer diameter and 13 cm long). When it is cooled by direct plunging in LN2 and warmed by immersion in water (Thawing), the cooling and warming rate is ~2500C/min (between -25 and -175C). Nevertheless, cells that are sensitive to chilling still suffer from chilling injury. An effective way of obtaining a much higher cooling rate and warming rate would be to minimize the volume of the solution and the container. In order to increase the survivability rate and handling oocytes with a minimal volume of solution, various methods have been devised. These include vitrification by open pulled straw (OPS), Cryoloop, microdrops etc. These are now a day followed widely in a lot of labs in the world.

Open pulled straw (OPS) Vitrification



The insemination straws (0.25 mL) are heat softened and pulled manually until the inner diameter and the wall thickness of the central part is decreased from 1.7 to ~0.8 mm and from 0.15 to ~0.07 mm, respectively. Then the straws are cut at the narrowest point with a razor blade. Cells are loaded by means of the capillary effect by placing the narrow end in a drop of vitrification solution containing cells, and aspirating the solution in a 2 3 cm long column (1.0 1.5 QL). Although it would be difficult to measure the precise rates of cooling and warming, it is estimated that the liquid column in OPS is cooled at 22,500C/min (between -25 and -175C), whereas the rate in the conventional straw is 2500C/min.

Cryoloop Vitrification:
Another strategy to increase the cooling rate is to use a refined system called the cryoloop, which consists of a minute nylon loop (20Qm wide, 0.50.7 mm in diameter) mounted on a stainless steel pipe inserted into the lid of a cryovial. Oocytes are placed on loops that have been loaded with a thin film of vitrification solution, the amount of which is< 1 QL. The cryovial is submerged in LN2, the loop containing the oocytes is immersed in LN2 in the cryovial, and then the cryovial is screw sealed. As the sample is enclosed in a cryovial, it can be labeled and stored easily.

Microdrops
 

This approach is a completely containerless method called microdrops (or microdroplets). Originally, this method was reported by Landa and Tepla in 1990. It reported that this method is more effective than conventional straw vitrification for bovine embryos at early cleavage stages, which are sensitive to chilling. With this method, oocytes suspended in vitrification solution are aspirated in a pipette and expelled onto the surface of LN2 as a microdrop. Then, the 4 8 QL microdrop vitrifies.

Advantage of ultrarapid vitrification



One advantage of ultrarapid vitrification is that the formation of intracellular ice can be prevented with a smaller amount of intracellular cryoprotectant. Therefore, the use of a lower concentration of the permeating cryoprotectant, thus a less toxic solution, is possible. In actual fact, the concentration of the permeating cryoprotectant in ultrarapid vitrification is lower than that adopted for conventional vitrification. In conventional vitrification, solutions with 40% (or more) permeating cryoprotectant(s) are widely used, whereas in ultrarapid vitrification, those with ~30% cryoprotectant are more frequently used.

In cattle, cryopreservation of embryos is highly successful. The success of cryopreservation is dependent on the stage of the embryo; that is, especially good results are obtained with blastocysts. Cryopreservation of embryos resulting in live offspring has been reported for important (mammalian) livestock species. Cryopreservation of pig embryos has long been quite problematic, due to extreme chilling sensitivity and high lipid content of the pig embryos.

Advantages
   

shipping don t need to synchronize recipients Diseased stage - cancer, age

Disadvantages
  

decrease viability ~ 50% survival cost time and management

Cryoprotectants


Cryoprotectants are compounds that are used to reduce the cellular injury when the cells are subjected to low temperatures. The presence of cryoprotectants in the freezing solution is necessary to prevent cell damage during freezing and thawing of the samples. The cryoprotectants used can be broadly classified into the following categories  Sugars as cryoprotectants  non-glycerol cryoprotectants  Glycerol

The cell membrane is the site of most freezing damage. Therefore good cryoprotectants may not only perform the anti-freeze function of preventing ice formation, but protect cell membranes as well. Cryoprotectant toxicity, however, can potentially affect any organelle or macromolecule with proteins being the most vulnerable.

Sugars
  

 

Are polyhydroxyl aldehydes or ketones. Glyceraldehyde is a simple sugar. High levels of sugars and sugar alcohols are found in many polar plants, insects, fungi, etc. as non-toxic cryoprotectants. Sucrose Sucrose and trehalose inhibit the membrane mixing associated with chilling. Both sugars fit well in cell membranes, binding to phospholipid head groups.

Conte..


Disaccharides like trehalose and sucrose do not cross cell membranes, however, and thus only protect the inner cell membranes of organisms that synthesize them (not vertebrates). Many strategies have been attempted to get trehalose inside of vertebrate cells so that its cryoprotective, protein-protective and membrane-protective properties can be of benefit on the inside as well as on the outside of cells. Microinjection, for example, has been used to get trehalose into human oocytes which improve cryopreservation.

Non-glycerol cryoprotectants


Although glycerol has been used for years in cryonics, other cryoprotectants (such as methoxlylated compounds ie, CPAs in which a methyl (-CH3) group is added to an alcohol to make an ether) now being considered are less toxic and more penetrating.

Conte..
     

Glycerol DiMethyl SulfOxide (DMSO) Ethylene glycol Propylene glycol Methoxylated compounds Glycerol is, however, biochemically toxic (denaturing one or more enzymes)

Cryoprotectants


Intracellular cyroprotectants
   

1,2 propanediol dimethyl sulfoxide DMSO ethylene glycol glycerol sucrose and raffinose

Extracellular cryoprotectants


Seeding
 

forceps dipped in LN2, and touch straw at -5oC cause media to become solid before freezing pt.