Cryotube Freezing

Cryotube freezing is a process that enables cell culture by freezing the culture medium to a sub-zero temperature. It is also called a Hawking chamber, freezer-tube, or cryo-doodle. 

Thermal profiles of cryotube freezing

In the present study, we measured the thermal profiles of cryotube freezing using two-mL Corning cryovials containing cryoprotectant. Several K-type thermocouples were attached to at least five cryovials, and the temperature was measured by using software.

The initial phase of freezing is initiated by ice nucleation events, which may occur at -10 degC. The temperature at which this transition begins is known as the threshold nucleation temperature. The next step in the freezing process is the thawing process. The fastest thaw results in the least amount of post-thawing metabolic activity, while a slow thaw creates the most stable freezing condition.

Temperature changes in cryovials have a profound effect on their cryosurvival properties. Too-fast freezing results in excessive formation of intracellular ice, which causes cytoplasmic cryoinjuries. In addition, too-slow freezing can result in toxic concentrations of electrolytes. It is therefore important to use an optimal freezing rate that limits intracellular ice formation while minimizing solution effects.

Comparison of cryotube freezing with MTG-straw freezing

The process of freezing embryos and egg mother cells is known as cryotube freezing. The freezing liquid is composed of hyclone and DMSO. After freezing, the cell is transferred to liquid nitrogen. This process helps to increase the recovery rate of the cell, and totipotency remains intact. In some cases, this method can achieve a survival rate of 97% or higher.

This technique has been used to freeze murine, bovine, and human eggs. However, the size of the eggs affects their perfusion and permeability. Hence, cryotube freezing is not recommended for superovulating females. However, this method is widely used and is becoming the standard for embryo freezing.

Cryopreservation significantly reduces total motility and progressive motility in sperm, but increases DFI in mutant mice. It also improves fertilization rates. The MBCD-GSH and MTG-straw methods also improve fertility.

Sucrose-thawing increases cell viability

Slow freezing was the first method used in infertility clinics to preserve embryos. However, the viability of embryos varied significantly between clinics, making vitrification a better and more reliable method. Nevertheless, slow freezing remains an effective method to preserve embryos.

A new polymeric cryoprotectant developed by Bailey and colleagues enhances cell viability post-thaw by preventing damage to the cells. This polymer is compatible with RBCs and can be produced in one step from a commercially available bulk polymer.

The process of cryotube freezing requires the use of sucrose as a critical ingredient. Sucrose is a key component in cryopreservation media and in rehydration. Earlier studies showed that slow freezing with higher sucrose concentrations significantly improved survival rates. The same method can be used to thaw embryos after vitrification. During thawing, the temperature of the oocytes is reduced by a stepwise decrease in extracellular cryoprotectants. The most common rehydration methodology uses a sucrose gradient.

The process of thawing has become a real challenge in embryology laboratories. Ideally, the same protocol and brand of cryoprotectant products should be used for cryopreservation and thawing.

Storage of cryotubes

When storing cryotubes for freezing, make sure to follow the following steps. First, do not overfill the cryotubes. The cells need space to expand during freezing. For this reason, it is recommended to fill the tubes 0.2 ml less than the maximum graduation.

Second, use an appropriate cryoprotectant. The most commonly used cryoprotectant is DMSO, and its concentration should be about 10%. Some researchers also add FBS or Ficoll to the freezing medium to improve the recovery efficiency. The best time to prepare cryoprotectant mixture is on the day of the experiment. This will minimize the risk of sample loss if the cryoprotectant solution deteriorates during the experiment.

Third, add cryoprotective compounds to minimize the potentially lethal effects of cryopreservation. Cryoprotectants can be added to the culture before freezing to protect the cells from possible damage.