Cryopreservation Methods

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Thursday 17 May 2012, 10:51

Written by Dr. Koliakos

Monday, 01 June 2009 15:22

Methods of cryopreservation

Biohellenika follows a specific certified method. Furthermore Biohellenika uses special material and reagents certified from the relevant organizations (FDA, EC.) IVD and for human use. The process of the stem cells isolation and cryopreservation is performed in a closed system (sterile), so the cells do not come in contact with the environment. Only specified from ΕΟΦ materials are used and prior to the storage the process of exclusion of the red blood cell from the finale volume of storage is performed.

The results of the exclusion of the red blood cells are certified with tests that are performed with a hematology analytic device and a flow cytometer at the beginning and the end of the process. For the count of the hemopoetic stem cells of the sample a package of reactants designated for clinical use from the European Union is used.

The storage of the stem cells can be performed in cryovials or cryobags. For the storage to be performed in cryovials a small volume of cryopreservation is required that is reached with full exclusion of the red blood cells from the initial collection volume. The cryovials are stored in liquid nitrogen were the temperature is preserved stable at -196°C during the whole duration of the storage of the cells. The storage in cryovials gives the possibility of storage in two separate locations and thus rises the safely of the sample in cases of natural disasters and provides the possibility for more than one uses without defrosting and refreezing of the whole sample.

The storage in sacs is preferred mainly by the public bank were the stem cells are used one time only and only for the treatment of diseases of the blood. Due to the fact that a large number of red blood cells remain in the sample the donor and the patient must have the same blood type and so within the family they can be used only by members that share the same blood type. The sacs are stored only in liquid nitrogen steams due to the problem of fragility that they present were the temperature fluctuated and for this reason the public bank recycles its samples every 5 years. The preservation in sacs does not require a specially trained staff, it is a simplified method and is addressed more to the public storage and for the use in diseases of the hemopoetic system and is not recommended for applications of regenerative medicine that are considered treatments of the future.

Scientists have observed the sacs of cryopreservation that are defrosted for transplantation in a public bank in the United States as to their credibility from January 2000 until May 2006. At the same time they recorded the results of the transplantations after the administration of the samples that came from sacs that were ruptured, at the cases were the doctors decided to administer the sample ignoring this fact. The results showed that the 3,5% up to 9,6% presented signs of rupture. Most of the ruptures (75%) were found in sacs that had been cryopreserved for more than 2 years. In spite of the increased administration of antibiotics no other serious side effect was observed.

Biohellenika has the know-how and the special equipment of the storage either in cryobags or cryovials. After carefully informing the parents, it follows the way of storage that they have selected.

The process in Biohellenika is performed with the special device ΑΧΡ Autoexpress Platfrom of Thermogenesis USA, the only in Europe that results in 98% retrieval of the whole quantity of the stem cells of the initial sample.

In cases of a large initial sample Biohellenika after consultation with the parents can store the stem cells in two cryobags to assure storage in two different locations.

The containers were the samples are stored are equipped with an automatic system of liquid nitrogen intake and therefore remain always full and there is never danger of damage in the level of the liquid nitrogen falls.

Furthermore in Biohellenika the containers are checked on a 24 hour base for the level and the temperature of the surface with a special surveillance and alarm system. The company has also two generators of electric current (main and spare).