Abstract
Objective
Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4°C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32°C, air–liquid interface and physiological skin tension.
Design
Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4°C and organ culture.
Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining).
Results
In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4°C, as compared to organ culture.
Conclusion
These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CFE:
-
Colony Forming Efficiency
- CFU:
-
Colony Forming Unit
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- FBS:
-
Fetal Calf Serum
- MTT:
-
Methyl Thiazolil Tetrazolium
- PBS:
-
Phosphate Buffered Saline
- PD:
-
Population Doubling
References
Alexander JW, Mac Millan BG, Law E, Kittur DS (1981) Treatment of severe burns with widely meshed skin autograft and meshed skin allograft overlay. J Trauma 21:433–438
Alotto D, Ariotti S, Graziano S, Verrua R, Stella M, Magliacani G, Castagnoli C (2002) The role of quality control in a skin bank: tissue viability determination. Cell Tissue Bank 3(1):3–10
Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84(8):2302–2306
Black AF, Bouez C, Perrier E, Schlotmann K, Chapuis F, Damour O (2005) Optimization and characterization of an engineered human skin equivalent. Tissue Eng 11(5–6):723–733
Borderie VM, Laroche L (1998) Microbiologic study of organ-cultured donor corneas. Transplantation 15;66(1):120–123
Boucher F, Poumay Y, Degen A, Paye M, Leloup R (1991) Utilization of human cultured epidermal keratinocytes: irreversibility of the inhibition of proliferation induced in stored detached cultures. Burns 17(3):205–208
Bravo D, Rigley TH, Gibran N, Strong DM, Newman-Gage H (2000) Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns 26(4):367–378
Builles N, Perraud M, Reverdy ME, Burillon C, Crova P, Brun F, Chapuis F, Damour O (2006) Reducing contamination when removing and storing corneas: a multidisciplinary, transversal, and environmental approach. Cornea 25(2):185–192
Bullock AJ, Higham MC, MacNeil S (2006) Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes. Tissue Eng 12(2):245–255
Castagnoli C, Alotto D, Cambieri I, Casimiri R, Aluffi M, Stella M, Alasia ST, Magliacani G (2003) Evaluation of donor skin viability: fresh and cryopreserved skin using tetrazolium salt assay. Burns 29(8):759–767
Chang P, Rosenquist MD, Lewis II RW, Kealey GP (1998) A study of functional viability and metabolic degeneration of human skin stored at 4°C. J Burn Care Rehabil 19(1Pt1):25–28
Cuono C, Langdon R, McGuire J (1986) Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1(8490):1123–1124
Delbosc B, Naegelen J, Herve P, Carbillet JP, Montard M (1987) Preservation of human corneas in an enriched culture medium at +37 degrees Centigrade: histological and biochemical analyses. J Fr Ophtalmol 10(10):547–549
Doughman DJ, Van Horn D, Harris JE, Miller GE, Lindstrom R, Good RA (1974) The ultrastructure of human organ-cultured cornea. I. Endothelium. Arch Ophthalmol 92(6):516–523
Fabre JW (1991) Epidermal allografts. Immunol Lett 29(1–2):161–165
Fahmy FS, Navsaria HA, Frame JD, Jones CR, Leigh IM (1993) Skin graft storage and keratinocyte viability. Br J Plast Surg 46(4):292–295
Flaxman BA, Harper RA (1975) Organ culture of human skin in chemically defined medium. J Invest Dermatol 64(2):96–99
Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636
Kratz G (1998) Modeling of wound healing processes in human skin using tissue culture. Microsc Res Tech 42:345–350
Laroche L, Borderie V, Lopez M, Saraux H (1994) Microbiological safety and endothelial quality control during preservation of corneal grafts at +31 degree C. J Fr Ophtalmol 17(5):314–320
Messager S, Hann AC, Goddard PA, Dettmar PW, Maillard JY (2003) Assessment of skin viability: is it necessary to use different methodologies? Skin Res Technol 9(4):321–330
Pels E, Schuchard Y (1983) Organ-culture preservation of human corneas. Doc Ophthalmol 15;56(1–2):147–153
Poumay Y, Boucher F, Degen A, Leloup R (1991). Inhibition of basal cell proliferation during storage of detached cultured epidermal keratinocyte sheets. Acta Derm Venereol 71(3):195–198
Prunieras M, Delescluse C, Regnier M (1976) The culture of skin: a review of theories and experimental methods. J Invest Dermatol 67(1):58–65
Prunieras M, Regnier M, Woodley D (1983) Methods for cultivation of keratinocytes with an air–liquid interface. J Invest Dermatol 81(1Suppl):28s–33s
Rheinwald JG, Green H (1975) Serial cultivation of human epidermal keratinocytes: the formation of keratinising colonies from single cells. Cell 6:331–344
Rennekampff HO, Kiessig V, Loomis W, Hansbrough JF (1996) Growth peptide release from biologic dressings: a comparison. J Burn Care Rehabil 17(6 Pt 1):522–527
Sarkany I, Grice K, Caron GA (1965) Organ culture of adult human skin. Br J Dermatol 77:65–76
Webster JP (1944) Refrigerated skin grafts. Ann Surg 120:431–449
Yang H, Jia XM, Acker JP, Lung G, McGann LE (2000) Routine assessment of viability in split-thickness skin. J Burn Care Rehabil 21(2):99–104
Acknowledgements
We thank all the participants for their contribution to our study. We thank Odile Damour and the Laboratoire des Substituts Cutanées, Lyon, France for technical support. This work was supported by the Agence de Biomédecine and the Assistance Publique-Hôpitaux de Marseille.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hautier, A., Sabatier, F., Stellmann, P. et al. Assessment of organ culture for the conservation of human skin allografts. Cell Tissue Banking 9, 19–29 (2008). https://doi.org/10.1007/s10561-007-9042-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10561-007-9042-3