Cell and Tissue Banking

, Volume 3, Issue 1, pp 3–10

The role of quality control in a skin bank: tissue viability determination.

  • D. Alotto
  • S. Ariotti
  • S. Graziano
  • R. Verrua
  • M. Stella
  • G. Magliacani
  • C. Castagnoli
Article

Abstract

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider line arrange, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4°C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative,whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.

Allografts Cryopreserved skin Quality control Skin storage Tissue viability 

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References

  1. Alexander J.W., MacMillan B.G., Law E. and Kittur D.S. 1981. Treatment of severe burns with widely meshed skin autografts and meshed allografts overlay. J. Trauma. 21: 433–438.PubMedGoogle Scholar
  2. Bank H.H. and Schmehl M.K. 1989. Parameter of evaluation of viability assays: accuracy, precision, specificity, sensitivity, and standardization. Cryobiology 26: 203–211.PubMedCrossRefGoogle Scholar
  3. Bravo D., Rigley T.H., Gibran N., Strong D.M. and Newman-Gage H. 2000. Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns 26: 367–378.PubMedCrossRefGoogle Scholar
  4. Chang P., Rosenquist M.D. II, Lewis R.W. and Kealey G.P. 1998. A study of functional viability and metabolic degeneration of human skin stored at 4 8C. J. Burn. Care. Rehabil. 19: 25–28.PubMedGoogle Scholar
  5. Cuono C.B., Langdon R., Birchall N., Barttelbort S. and McGuire J. 1987. Composite autologous-allogeneic skin replacement. Development and chemical application. Plast. Reconstr. Surg. 80: 626–637.PubMedCrossRefGoogle Scholar
  6. Fahmy F.S., Navsaria H.A., Frame J.D., Jones C.R. and Leigh I.M. 1993. Skin graft storage and keratinocyte viability. Br. J. Plast. Surg. 46: 292–295.PubMedCrossRefGoogle Scholar
  7. Hira M., Taijma S. and Yamamoto Y. 1992. The metabolism of skin grafts stored with excess carbon dioxide. Plast. Reconstr. Surg. 89: 1122–1128.PubMedGoogle Scholar
  8. Ingham E., Matthews B., Kearney J.N. and Gowland G. 1993. The effects of variation of cryopreservation protocols on the immunogenicity of allogeneic skin grafts. Cryobiology 30: 443–458.PubMedCrossRefGoogle Scholar
  9. Kearney J.N., Wheldon N.A. and Gowland G. 1990. Cryopreservation of skin using a murine model: validation of a prognostic viability assay. Cryobiology 27: 24–30.PubMedCrossRefGoogle Scholar
  10. Klein M.B., Shaw D., Barese S., Chapo A. and Cuono C.B. 1996. A reliable and cost-effective in vitro assay of skin viability for skin bank and burn centers. J. Burn. Care. Rehabil. 17, n.6: 565–570.PubMedGoogle Scholar
  11. May S.R. and DeClement F.A. 1981. Skin banking III. Cadaveric allograft skin viability. J. Burn. Care. Rehabil. 2: 128–140.CrossRefGoogle Scholar
  12. May S.R. and Roberts D.P. 1988. Development of a passive device for freezing large amounts of transplantable skin at one time in a-70°Cmechanical refrigerator. Cryobiology 25: 186–196.PubMedCrossRefGoogle Scholar
  13. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55–63.PubMedCrossRefGoogle Scholar
  14. Pegg D.E. 1989. Viability assays for preserved cells, tissues, and organs. Cryobiology 26: 212–231.PubMedCrossRefGoogle Scholar
  15. Scudiero D.A., Shoemaker R.H., Paull K.D., Monks A., Tierney S., Nofziger T.H. et al. 1988. Evaluation of a soluble tetrazolium / formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48: 4827–4833.PubMedGoogle Scholar
  16. Vistica D.T., Skehan P., Scudiero D., Monks A., Pittman A. and Boyd M.R. 1991. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res. 51: 2515–2520.PubMedGoogle Scholar
  17. Yang H., Jia X.M., Acker J.P., Lung G. and McGann L.E. 2000. Routine assessment of viability in split-thickness skin. J. Burn. Care. Rahabil. 21: 99–104.CrossRefGoogle Scholar
  18. Zieger M.A.J., Tredget E.E. and McGann L.E. 1993. A simple, effective system for assessing viability in split-thickness skin with the use of oxygen consumption. J. Burn. Care. Rehabil. 14: 310–318.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • D. Alotto
    • 1
  • S. Ariotti
    • 1
  • S. Graziano
    • 2
  • R. Verrua
    • 2
  • M. Stella
    • 1
  • G. Magliacani
    • 1
  • C. Castagnoli
    • 1
  1. 1.Ospedale CTO, Dept. of Plastic Surgery and Burn Unit “Skin Bank”TurinItaly
  2. 2.Piedmont Foundation of Studies and Researches on BurnsTurinItaly

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