Low temperature over extended periods

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Cells are inclined to many possible hazards when they are preserved at low temperature over extended periods. Such potential hazards could be the possibility of differences in the temperature in which they are stored and also, contamination.

Contamination arising from accumulation of microbes brought about by environmental agents has also been seen to take place (Fountain et al.1997). This contamination can be brought about by the presence of sludge of ice depositions at the lowermost part of the vessels. Liquid nitrogen behaves not only as a coolant but acts similar to water in that it may also act as a vector for transmitting virus, fungi, bacteria and cells from animals. Fountain et al. (1997) investigated a study in which haematopoietic stem cells were preserved in liquid nitrogen freezers that were contaminated with bacteria and fungi. 583 cultures were tested; Microorganisms infected 1.2% of it. 80% of the freezers investigated possessed minimal contamination by microbes while Aspergillus was found to mightily infect the remaining 10% of the freezers investigated. The contamination discovered in these freezers were comparable to those noticed in the cultures. This report does not say authoritatively that the liquid nitrogen was the source of microbial contamination but there are also similar reports showing cryopreserved cells with microbial infection (Stroncek et al., 1991; Prince et al., 1995; Webb et al., 1996; Lazarus et al., 1991) When the plastic tubes have been capped and immersed into liquid nitrogen, it can bring about contamination between the sample and liquid nitrogen. Also, when there is condensation in the atmosphere within the tubes, liquid nitrogen may be drawn in and contaminants present in the liquid nitrogen may also contaminate the sample (Burden, 2002)

Another challenge noticed in freezers used for storage containing liquid nitrogen is the unevenness of the temperature inside the chamber. When the vessels in which they are kept are examined, or periodically checked, it causes variations in the temperature cycle which may be particularly damaging to the cells. If the vessels used to store nitrogen are not maintained properly or filled properly, this could have a huge and possible damaging effect on the viability of the cells that are stored to be used. In such cases, when the cells where casually observed, there was noticed to be a huge loss of valuable material resulting from an improper filling protocol or a malfunction of the instruments employed. (Stacey, 2004) Systems employing the use of liquid nitrogen work by saturating the entire bottom of the vessel with liquid nitrogen and allowing it to diffuse upwards, cooling the upper chamber. When this is done, the cells present in the higher stands of the chamber begin at -196°C. A temperature gradient is generated with a reduction in the vapour level.( Wolfinbarger 1998), Gradients across the vapour phase are known to exceed the glass transition temperature of water, sometimes getting to -70°C, White and Wharton (1984) suggested -72°C and Rowley and Byrne ( 1992), said -95°C, in big freezers utilizing liquid nitrogen. These variations in temperature of these systems are characteristic to their functioning and when stored for a long period of time leads to destruction of their viability. Temperatures below -130°c encourage even very sensitive cells to live for up to five score years and more but above this, the life of cells get reduced by months.

Another possible issue for long term preservation of cells could be the emission of rays, waves in forms of particles. Although this has not been evidential in nitrogen containers that have been properly maintained but these emissions have been thought to cause a permanent change in the DNA sequence of a gene. (Glenister et al, 1984).

When cells are stored for extended periods of time, unforeseen risks may occur. This includes damage or irregularities in the storage vessels. Fire may occur, electric power supply may be sporadic, nitrogen may not be filled properly and possibly damage to the equipments

After a research or experiment has been carried out, cell lines and tissues which were used in the laboratories have been considered to be in the public domain. Historically public collections maintain a good stock control of the experimented cells for many years and the ownership of the tissues and cells have not been claimed nor requested for over the years, until of recent due to potential use of the researches and experiments in biomedicine and biotechnology, has brought about much attention which has been directed to the ownership of the cells and the tissues. Several people seem to be more interested including the donors. The donor may want to claim the possession of the cells with an interest of exploitation. Gold (1986), described that this kind of situation may arise when accurate and informed knowledge and understanding both by the physician and donor have not been made.

In order to achieve an appropriate method of practice, a variety of guidelines have been put in place (Stacey et al, 1998; Doblhof - Dier and Stacey, 2000) for the use of cells , cell bank and cell lines. A code of practice was drafted by the Stem Cell Steering Committee. It gave advice on the appropriate way to work with stem cell lines and defined methods for studies with the use of embryonic stem cell lines from humans. This code is transparent and clear and performed with great ethics thus, reassuring experts and members of the public. The Steering Committee distinguished stem cells in 3 categories; somatic, embryonic and embryonic germ cells in order to establish a proper code of practice. In the UK, this body is responsible for the ethics regarding import and export and use of human embryonic stem cell lines

Also, governing the setting up human stem cell lines, the Human Tissue Act was established in 2004 to regulate the extraction, use of materials from humans including tissues, cells and organs. This Act takes seriously consent gotten from donors regarding storage and use of materials from humans and clearly states the detriment of inappropriate consents. Under this is the Human Tissue Authority that designs regulations, licenses regulated acts and disseminates Codes of Practice giving real guidance on the way these activities are carried out. This law pertains to all human cells (excluding nails and hairs), embryos and gametes which is governed by the Human Fertilization and Embryology Act (HFEA).

The HFEA established in 1990 does not allow for the embryo to be taken likely. This includes making it or destroying it. It gives licenses to researchers using embryos of humans. These licenses are only given for the purpose of increasing knowledge on how to treat infertility, causes if inborn diseases and miscarriages, establishing improved system for contraception, discovering abnormalities in chromosomes or genes before they are implanted.

The Medicines and Health Care Products Regulatory Agency (2003)was established to regulate the use of blood products and blood, medical devices and medicines and also the use of cell lines as part of an IMP.

The Gene Therapy Advisory Committee (GTAC) was initiated in May 2008 to monitor gene therapies to cause minimal genetic risks. In pre-clinical trials, the researcher needed to demonstrate therapies, toxicity level and potential damage. They gave ethical advice.

Ethical issues are also involved, which differ with the place and time involved. What obtains in one decade might be differ in the next decade and it might be a criminal offence to still adopt the same style of doing things

REFERENCES

  • Burden, D. E (2002) Issues in Contamination and Temperature Variation in the cryopreservation of animal cells and tissues Available from: http://www.btc-bti.com/applications/cryogenicstorage.htm [Accessed: 26
    January, 2010]
  • Doblhoff-Dier, O and Stacey, G (2000). Cell Lines: Applications and Biosafety, In Biological Safety - Principles and practices, 3rd edition, Fleming, D.O and Hunt, DL, Eds, American Society for Micro Biology,
    Washington, D.C. pp221 -241.
  • Fountain D., M. Ralston, N. Higgins, J. Gorlin, L. Uhl, C. Wheeler, J. Antin, W. Churchill, and R. Benjamin. (1997). Liquid nitrogen freezers: A potential source of microbial contamination of hematopoietic stem
    cell components. Transfusion 37: 585-591.

    http://www.ncbi.nlm.nih.gov/pubmed/9191818
  • Glenister, PH, Whittingham, D.G, and Lyon, M.F (1984) Further studies on the effect of radiation during storage for frozen 8 - cell, mouse embryo at -196 degree, C J. Reprod. Fert. 70, pp 229- 234
  • Gold, M (1986), A Conspiracy of Cells, state university of New York press, Albany
  • Lazarus H., M. Magalhaes-Silverman, R. Fox, R. Creger, and M. Jacobs.(1991). Contamination during in vitro processing of bone marrow for transplantation: Clinical Significance. Bone Marrow Transplant 7:
    241-246.

    http://www.ncbi.nlm.nih.gov/pubmed/2059759
  • Prince H., S. Page, A. Keating, R. Saragosa, N. Yukovic, K. Imrie, M. Crump, and A. Stewart. 1995. Microbial contamination of harvested bone marrow and peripheral blood. Bone Marrow Transplant 15:
    87-91.

    http://www.ncbi.nlm.nih.gov/pubmed/7742761
  • Rowley S. and D. Byrne. 1992. Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: Decreased temperature gradients with an aluminum racking system. Transfusion 32: 750-754.

    http://www.ncbi.nlm.nih.gov/pubmed/1412683
  • Stacey G (2004) Fundermental issues for cell- line banks in biotechnology and regulatory affairs: In Fuller BJ, Lane N, Benson EE. Life in the Frozen State. New York. CRC Press pp506-516
  • Stacey G, Doyle A and Hambleton, PH (1998), Safety in Cell and Tissue Culture, Kluwer Academic Publishing, Dordracht, Netherland
  • Stroncek D., S. Fautsch, L. Lasky, D. Hurd, N. Ramsay, and J. McCullough. (1991). Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 31: 521-526.

    http://www.ncbi.nlm.nih.gov/pubmed/1853447
  • Webb I., F. Coral, J. Anderson, A. Elias, R. Finberg, L. Nadler, J. Ritz, and K. Anderson. (1996). Sources and sequelae of bacterial contamination of hematopoietic stem cell components: Implications for the safety
    of hematotherapy and graft engineering. Transfusion 36: 782-788.

    http://www.ncbi.nlm.nih.gov/pubmed/8823450
  • White W and K. Wharton. (1984). Development of a cryogenic preservation system. American Laboratory Oct. 65-76.
  • Wolfinbarger L. (1998). The basics of laboratory-scale mammalian cell cryopreservation. BioPharm October pp: 35-39.

    http://www.axeb.dk/pdfs/application_briefs/Heraeus/4_Laboroatory_cooling_systems/Cryogenic%20Application_e.pdf
  • Websites

  • The Human Tissue Act (2004):

    http://www.legislation.hmso.gov.uk/acts/acts2004/20040030.htm
  • The Human Fertilisation and Embryology Act (HFEA)(1990):

    http://www.hmso.gov.uk/acts/acts1990/Ukpga_19900037_en_1.htm
  • Steering Committee (2002)

    http://www.mrc.ac.uk/index/strategy-strategy/strategy-science_strategy/strategy-strategy_implementation/strategy-government_spending_review_initiatives/strategy-stem_cells/strategy-stem_cell_governance.htm

    http://www.ukstemcellbank.org.uk/documents/Code%20of%20Practice%20for%20the%20Use%20of%20Human%20Stem%20Cell%20Lines.pdf

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