Eur J Plast Surg (1991) 14:232-234 European l r~ ' l l ~ o x oa'o' lff.lfllStlC Sur-g ¥Y © Springer-Verlag 1991

Prolonged skin graft preservation with keratinocyte culture medium I.J. Peled, E. Notea and E. Lindenbaum

Department of Plastic Surgery, Rambam Medical Center and Medical School, Technion Institute, Haifa, Israel

Summary. A method of prolonging skin viability for de- layed skin grafting is reported, Saline and skin bank fluid were compared with keratinocyte nutrient MCDB 153medium and studied histologically. The results showed a significant difference in favour of the keratino- cyte nutrient medium where the skin maintained its structure for up to I 0 weeks.

Key words: Skin graft - Culture - Preservation

Skin grafting is one of the most common procedures in plastic surgery. It usually implies a donor area, a recipient area and the healing period of both. In many cases, however, a possibility of a delay or late skin graft procedure is necessary or planned beforehand. In these circumstances as well as in multiple stage reconstructive procedures, skin storage and preservation of its vitality are needed.

Several methods of ensuring viability and prolonga- tion of storage time of the skin autografts were reported, combining the use of low temperature (refrigeration) and enriched medium.

This work presents a histological study of skin au- tografts preserved in different solutions at 4 ° C which shows and confirms our clinical experience.

Skin immersed in tissue culture media retains its structure for a longer period of time, 4 times longer as compared to skin immersed in saline and double com- pared to "skin graft fluid" at the same refrigerating temperatures.

Materials and methods

Human split skin samples obtained during routine surgical proce- dures were used. For each experiment the split skin graft section used was from one patient. The skin was cut into 3 equal sized segments, immersed in 1:10 v/v medium and stored at 4 ° C temper- ature.

Three types of media were used: 1. Sterile normal saline 0.9% v/v isotonic solution at pH 4.5-6, supplemented with antibiotics (Penicillin 100 u/ml, Streptomycine 100 pg/ml and Amphotericine B 2.5 lag/ml). 2. Skin graft fluid (SGF) at pH 6.4 was prepared using: • Concentrated balanced salt sol. 8.0 ml which was composed of: NaC1 8.0 g/l, KC1 0.4 g/l, dibasic sodium phosphate 7H20 USP 0.0875 g/l, nonbasic potassium phosphate 0.0625 g/l, magnesium sulphate 7H20 USP 0.2 g/l, dextrose anhydrous USP 1.0 g/1 all dissolved in H20 and sterilized. • Normal human plasma USP AB or A 20.0 ml. • Neomycine sulphate 0.5% sterile in distilled water 72.0 ml. 3. MCDB 153, W L-Glutamin W 25 mm Hepes, W.L. Cysteine (Hazelton Biologics Inc. Kansas City, Missouri). Supplemented with antibiotics as above; 1.176 g/1 NaHCO3 adjusted to pH 7.4 with NaOH 4 N, and 1% NBCS (new born calf serum). The medi- um was prepared in buffered solution. The ph was measured weekly and remained stable throughout the experimental period. After every week from the 1st to the 10th, a small skin sample was taken from each of the 3 different media and prepared for histologi- cal examination using paraffin embedding and Hematoxylin Eosin stain as well as the Masson trichrome stain for collagen. The histo- logical examination and photography were performed using the Nikon Optiphot light microscope.

Results

The criteria for determining the "viabil i ty" of the skin grafts was based on the histological examination in which the following tissue parameters were observed: • The extent of the epidermal/dermal attachment at the interface line. • Appearance of intracellular vacuoles in the germinal layer of the epidermis. • Staining characteristics of the epidermal layer. All skin samples were observed at zero time i.e. on the day the samples were taken and immersed in the media. The intactness of the skin was identical in the 3 samples demonstrating continuous attachment at the epidermal/ dermal interface, light eosinic stain of the epidermal cells cytoplasm and no vacuolization at the germinal layer.

Of the 3 skin samples immersed in the 3 media tested, the sample in the saline solution exhibited the fastest

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Fig. 1. Skin sample immersed in saline for 2 weeks. Note the exten- sive detachment at the epidermal-dermal interface (H & E x 533)

Fig. 2. Skin sample immersed 2 weeks in skin graft fluid (SGF); (H & E x 533). Note the continuous attachment at the epidermal- dermal interface

Fig. 3. Same as Fig. 2, at 6 weeks. Note scattered discrete areas of detachment at the epidermal-dermal interface. (H & E x 533)

Fig. 4. Skin sample immersed 2 weeks in MCDB 153 (H & Ex 533). Note the intact junction line at the epidermal-dermal interface

Fig. 5. Same as Fig. 4 at 6 weeks. Interface between epidermis and dermis completely intact (H & E x 533)

Fig. 6. Same as Fig. 5 at 10 weeks. Note discrete localized areas of detachment at the epidermal-dermal interface (H & E x 533)

m a c e r a t i o n a n d de te r io ra t ion . As ear ly as the 2nd week the skin sample exh ib i ted extensive areas o f d e t a c h m e n t t h r o u g h o u t the e p i d e r m a l / d e r m a l in ter face and some va- cuo l i za t ion o f the germina l layer o f the ep idermis

(Fig. 1), Subsequen t ly the areas o f d e t a c h m e n t were ex- t ended t h r o u g h the 3rd and 4th week when large seg- ments o f the ep idermis were comple te ly de t ached and the vacuo l i za t ion in the r ema in ing areas was widespread .

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In addition, the staining of the epidermal cells was darker indicating death of cells. By the 5th week the epidermis was completely detached from the dermis.

The samples immersed in SGF were unaffected after 2 weeks (Fig. 2) but began to show deterioration on the 3 week which started as areas of vacuolization of the germinal layer. By the 5th-6th week, in addition to the widespread vacuolization there were small loci of detach- ment at the epidermal/dermal interface (Fig. 3). On the 7th week and onward the detachment continued to ex- tend throughout the whole sample and the staining of the epidermal epithelial cells was darker, nuclei became pyknotic indicating death of cells.

The integrity of the skin sample immersed in MCDB 153 (Fig. 4), lasted longer (Fig. 5) and only at the 8th week vacuolization began to appear at the ger- minal layer of the epidermal layer and small loci of de- tachment of the epithelium can be seen. By the 9th-I 0th week the vacuolization and detachment of small areas became more elongated along the epidermal/dermal in- terface (Fig. 6).

Comparison of the rate of deterioration of the three samples showed: 1. The saline sample started to decompose on the 2nd week while both the SGF and MCDB 153 samples were still intact. Complete maceration of the saline sample was observed by the 5th-6th week when the SGF sample exhibited only small segments of detachment and the MCDB 153 was still intact. 2. The SGF sample started to macerate by the 5-6th week and it was completely deepithelialized by the 7th week, while the MCDB 153 sample was still intact. 3. The MCDB 153 sample started to deteriorate on the 8th week after both the saline and the SGF samples lost their epidermis. The MCDB 153 continued to deteri- orate through the 10th week. We can summarize that MCDB 153 preserved skin "survived" twice longer than SGF and at least 3 times longer than saline.

Discussion

In theory, the viability of any tissue separated from the donor is limited by the rate of utilization of available nourishment and by the rate of build-up of toxic meta- bolic products [4].

For many years, it has been known that storage for preservation of viable skin should include low tempera- tures and enriched medium. Therefore, freezing tempera- tures are utilized for skin homograft banking and refri- geration at 3M ° C was found to be suitable for autograft preservation [6].

The initially used saline solution was improved by adding serum [1] resulting in longer preservation with good skin take of up to 3 weeks. Similar results were reported with the use of "skin bank f luid" [3], and a combination of tissue culture medium RPMI-1640, plas- ma and antibiotics [2]. For more than 10 years, the se- nior author (IJP) used a modified "skin bank fluid"

for skin autograft preservation with good take of the grafts after 4-5 weeks skin storage time. During the last year the medium was changed to MCDB 153, with fur- ther improvement of the preservation time of the skin.

The fact that the skin is preserved longer in MCDB 153 indicates that this medium, which was de- signed specifically for keratinocyte culture [5] contains the essential requirements of the cells and therefore was able to sustain their morphology longer than saline which contains no nutritional value. The MCDB 153 medium was specifically designed to support the growth and proliferation of the germinal cells of the skin [7]. It contains all the essential nutritional requirements of the keratinocytes and was used to culture keratinocytes in laboratory conditions. Therefore this medium can and should be used as a preservation solution to maintain the viability of the cells by supplying them with their specific requirements. Our experience in culturing kera- tinocytes indicates that skin cells can be grown in culture for long periods using this medium. The histological study doubtlessly points to longer morphological preser- vation of skin autografts in MCDB 153 medium. The prolongation of the viability and take of skin graft is most helpful in multi-staged procedures as well as homo- graft preservation when the patient (donor) does not need it any longer.

References

1. Allg6wer M, Blocker TJ Jr (1952) Viability of skin in relation to various methods of storage. Tex Rep Biol Med 10:3

2. Cram AE, Domayer AA (1953) Short-term preservation of hu- man autografts. J Trauma 23 : 872

3. Feller I, De Weese MS (1958) The use of stored cutaneous au- tografts in wound treatment. Surgery 44:540

4. Hemphill JE, Brown JB (1954) Skin storage in tissue banking. Plast Reconstr Surg 13 : 118

5. Pheel DM, Ham RG (1980) Clonal growth of human keratino- cytes with small amounts of dialyzed serum. In Vitro 16:526

6. Webster JP (1944) Refrigerated skin grafts. Ann Surg 120:431 7. Wille J J, Pittelkow MR, Shipley GD, Scott RE (1984) Integrated

control of growth and differentiation of normal human prokera- tinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol 121:31

I.J. Peled