Assessment of skin viability - Wiley Online Library

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respiration rates. Fresh and thawed samples stored for a long period of time had a significantly lower (sometimes non-existent) oxygen consumption rate.

Skin Research and Technology 2003; 9: 321–330 Printed in Denmark. All rights reserved

Copyright & Blackwell Munksgaard 2003

Skin Research and Technology

Assessment of skin viability: is it necessary to use different methodologies? Syndie Messager1, A.C. Hann2, P.A. Goddard3, P.W. Dettmar4 and J.-Y. Maillard4 1

Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, UK, 2School of Biosciences, Cardiff, UK, 3Reckitt Benckiser, Healthcare UK Ltd., Hull, UK and 4School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK

Background/aim: Skin is complex and may display variable structural and metabolic change ‘ex vivo’. The present study aimed to follow measures of skin viability and evaluate their usefulness as markers of viability. Materials and methods: We evaluated the viability of skin samples fresh or after being frozen and subsequently thawed. Assessments included histopathological appearance, lactate dehydrogenase (LDH) activity, oxygen consumption and skin pH. Results: Morphological investigations of fresh and frozen skin samples using light and electron microscopy showed samples with relatively well-defined epidermis and dermis. Frozen samples showed some sign of stratum corneum fragmentation, although this was not obvious. LDH activity measured in fresh samples kept at 4 1C was low, but it was stable up to 7 days. Fresh samples kept at 32 1C had a comparable LDH activity to the ones kept in the fridge up to 4 days. Frozen samples, thawed and then kept at 4 1C showed a stable LDH activity after 24 h of incubation. However, frozen samples incubated at 32 1C demonstrated a high variability in results, with up to 800 U/L of LDH activity after 5 days of incubation. Freshly excised as well as freshly thawed samples showed the highest respiration rates. Fresh and thawed samples stored for a

& Blackwell Munksgaard, 2003 Accepted for publication 28 May 2003

‘viability’ corresponds to the ability of a biological unit to remain alive. The ‘ex vivo’ test used in our laboratory for the assessment of antiseptic activity against microorganisms requires the use of excised skin samples, mainly from breast reduction. Skin harvested from living or dead persons retains life function. Skin Harvested from living persons or post mortem is utilized in hospitals and research laboratories for various reasons. For nearly four decades, skin has been used in hospitals as an effective temporary covering for burn wounds and research laboratories have used excised human skin to assess the percutaneous absorption of drugs (2) and the effects of hazardous

chemicals of environmental concern (3, 4). Since human skin is not easily obtainable, skin storage is commonly performed prior to utilization. Thus, skin viability has become a concern. It is a well-documented fact that skin harvested from living or dead persons retains life function (5, 6). There are several methods available to evaluate skin viability: tryptan blue dye exclusion (7), oxygen consumption (8, 9), lactate dehydrogenase (LDH) activity (10), lactate production (6, 11), cellular activity in tissue (7), histopathological studies (11) and pH changes (12). Understanding and maintaining human skin viability places the skin use closer to the ‘in vivo’ situation (6). Skin viability being a difficult task to assess, it is not easy to choose the right protocols to perform. As

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long period of time had a significantly lower (sometimes non-existent) oxygen consumption rate. Our results also showed an increase in the oxygen consumption rate of fresh samples being incubated at 32 1C for 24 h. The oxygen consumption rate for all samples reached a plateau within the 15-min measurement period and even the fresh samples did not deplete all the oxygen from the medium. Skin samples ex vivo showed a significantly higher pH than human skin in vivo, and when incubated for 46 h at 32 1C, fresh samples had a significantly lower pH than frozen samples. All protocols were reproducible and freshly excised and freshly thawed skin samples showed the highest rates of viability. Conclusion: ex vivo skin shows variation of several parameters over time. It is recommended to use two or three techniques for evaluation of skin viability including at least oxygen measurement and an enzyme assay. Key words: human skin – viability – ex vivo – EM – oxygen consumption – histology – LDH activity

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previously described in a report by Malinin and Perry (7), the multiplicity of processes that occur in a living cell forces the conclusion that no single laboratory approach can be useful by itself. However, the collection of chemical, structural and functional data may be meaningful. In our laboratory, fresh and frozen samples of skin are used for the testing of antiseptics efficacy directly on human skin. Hence, it was necessary to evaluate the viability of these samples by observing the histopathological appearance, by measuring the LDH activity and by assessing the oxygen consumption as well as the pH of the skin.

Materials and methods Transmission electron microscopy (TEM) investigation Skin samples used were taken from breast reduction with the consent of the donor. Two different defrosted skin samples and one freshly excised sample were used for this experiment. The first frozen sample was kept in Earle’s balanced salt solution (EBSS, Sigma, Poole, UK) for 3 days prior to testing. The second one was left to thaw at room temperature and used the same day. Thin sections Skin samples of 0.50.5 cm were cut and prefixed for 24 h in 4% paraformaldehyde12.5% glutaraldehyde (GTA, Johnson & Johnson, Irvine, CA, USA) in 0.1 M sodium cacodylate buffer (Agar Scientific), pH 7.2. Samples prepared for EM were based on methods by Glauert (13) and Hayat (14). After preparation, blocks were thin sectioned on a Reı¨chert Ultracut microtome (Reı¨chert-Jung, Austria) with a glass knife and mounted onto polioform-coated copper grids. Sections were counter-stained first with 2% uranyl acetate for 10 min in the dark, washed twice with water and then stained with Reynolds lead citrate (Agar Scientific, Stansted, UK) for 5 min and washed twice in water. Thin sections were examined and photographed using a Philips EM208 transmission electron microscope operating at 60 kV under standard conditions. Histological study Histological study was performed with the frozen sample left to thaw at room temperature

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and the fresh sample described in the EM study above.

Paraffin wax method Both samples (i.e. frozen left to thaw at room temperature and fresh) were fixed in 3% GTA overnight; this slow process aiming to protect cells structure. They were then rinsed three times in cacodylate buffer (pH 7.4) and gradually dehydrated in 70% ethanol overnight, in 95% ethanol for 2 h and in 100% ethanol for 2 h; this last step was repeated twice. Samples were then left to clear overnight in chloroform. The following day, the samples were deepened in paraffin wax for 2 h, twice. This step was then repeated under vacuum. Finally, samples were embedded in a glycerol–gelatin mold and allowed to harden in a fridge. Once hard, the molds were cut using a microtome. Once on glass slides the thin cut sections were stained in 1% eosin for 2 min, rinsed in H2O for 10 s and dehydrated as follows: 70% ethanol for 10 s, 95% ethanol for 10 s and 100% ethanol for 10 s; this latest step was repeated once. The samples were then dipped in xylene (Agar Scientific) twice 10 s and finally fixed with distrin acylique plastic xylene (DPX).

Enzyme activity: LDH Activity The epidermis is the major site of metabolism in the human skin. The enzymatic reaction, which leads directly to the production of lactate from pyruvate is catalyzed by the enzyme LDH and requires the presence of reduced disphosphopyridine nucleotide as the hydrogen donor (NADH) (10). During reduction of pyruvate, an equimolar amount of NADH is oxidized to NAD. The oxidation of NADH results in a decrease in absorbance at 340 nm. The rate of decrease in absorbance is directly proportional to LDH activity in the sample: pyruvate þ NADH þ Hþ L !lactate þ NADþ : Fresh skin samples were kept in EBSS at 4 1C directly after excision. Immediately after collection from the hospital, samples were mounted onto diffusion cells (JB&DW Jones; Loughborough, UK) (4) previously filled up with EBSS as the sampling fluid so that it was in direct contact with the dermis. A magnetic stirrer was also added in the recipient compartment. The samples were left to stabilize and after approximately

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30 min, the first measurement of LDH activity was performed as described below. Samples were kept at 4 1C between each measurement. After mixing the sampling fluid, 200 mL was removed through the sampling arm and replaced with 200 mL of fresh EBSS and mixed. The sampling procedure was performed at 25 1C as follows:

kept at 32 1C instead of 4 1C. Experiments were carried out three to six times for up to 7 consecutive days. As controls, the LDH activity of EBSS media only and of autoclaved (metabolically inactivated) skin samples was assessed.

Oxygen consumption The oxygen-measuring system used in this study was similar to that described by Zieger and colleagues (8). A polarographic oxygen electrode (DO-166MT-1, Lazarlab, Los Angeles, CA, USA) was connected to a digital pH meter (EDT instrument, GP 353) for interfacing with a PC for data collection, display and storage (Pico Technology Products Limited, Cambridge, UK). In this experiment, the sample-containing chamber (Fig. 1) was made of Delrin (polyoxymethylene) for its low O2 permeability. Before measurement, the oxygen electrode was zeroed using a solution of 1.6% w/v sodium bisulfite (Sigma) in water and calibrated as described by the manufacturer (Lazarlab). The electrode was placed in the chamber in a bottle (Fig. 1) containing a skin sample of thickness 11 cm, stratum corneum facing down in 1 mL of EBSS medium continuously stirred by a magnetic bar. The electrode was not in direct contact with the tissue but with the medium, hence measuring the oxygen concentration in the medium. The medium was pre-equilibrated at atmospheric O2 and room temperature (21 1C) and was supplemented with 80 mg/mL gentamicin to prevent bacterial contamination. The electrode measured the decline over time in oxygen concentration in the medium as the skin cells absorbed it. Data were collected automatically every 5 s for 15–20 min. The slope of the curve obtained for each experiment represented the rate of ‘consumption’/absorption of O2 by the skin

1. 1 mL of LDH reagent A (Lactate Dehydrogenase Kit, Sigma Diagnostics, Poole, UK) was added to the test cuvette (Fisher, Loughborough, UK), 2. 40 mL of sampling fluid was added to the test cuvette and mixed by inversion for 1 min, 3. 40 mL of LDH reagent B (Lactate Dehydrogenase Kit, Sigma Diagnostics) was added to the test cuvette and mixed by inversion for 30 s. 4. Following the initial absorbance reading at 340 nm vs. water as a reference, absorbance was measured every minute for 3 min using a spectrophotometer (Ultrospec II 4050, LKB Biochrom, Cambridge, UK). LDH activity was calculated using the following equation: LDH activity (U/L) 5 (DA per minTV1000)/(6.22LPSV), where DA per min is the change in absorbance per minute at 340 nm, TV the total volume (mL), SV the sample volume (mL), 6.22 5 millimolar absorptivity of NADH at 340 nm, LP the light path (1 cm) and 1000 5 conversion of units per mL to units per L. The same experiment was carried out with frozen skin samples. Samples from breast reduction had been frozen immediately after surgery. Before use, samples were left to thaw at room temperature typically for 3–4 h. Samples were then mounted onto diffusion cells and kept at 4 1C between each measurement, as described above. LDH activity was measured as previously described. Finally, these experiments were repeated with fresh and frozen skin samples that had been

PC serial port Oxygen electrode Delrin (O2 proof cylinder) Skin sample

input Small amount of media

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Fig. 1. Set up of oxygen-measuring system. Protocol based on Zieger (8).

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cells from the medium. Experiments were carried out with fresh and frozen skin samples. Oxygen consumption of the medium only and of ‘dead’ skin (i.e. autoclaved skin) samples was assessed as controls. Tests were performed in triplicate. As a control of skin viability, the oxygen consumption of fresh and frozen samples of skin incubated for up to 46 h at 32 1C was also measured. Experiments were performed in triplicate.

Skin pH Skin surface pH was measured potentiometrically using a flat-ended electrode (Lazarlab, USA) and an ATC pH meter (EDT Instruments GP 353). The pH meter was calibrated daily by measuring buffer solutions (Sigma) with known pH values of 4.0, 7.0 and 10.0. The pH electrode was applied directly on the skin surface. In order to maintain contact between the probe and the skin the electrode was dipped in distilled water immediately prior to application. Experiments were carried out at room temperature with six fresh samples of skin as well as 15 frozen samples of skin. The pH of the excised skin was also compared with the ‘control’ pH measured in vivo on 10 healthy female volunteers aged between 22 and 60 years old. They were asked not to apply any detergent or cosmetic to the hands, forearms and chest for 12 h before measurement. Volunteers were also asked to rest for 15 min before the test to become accustomed to the environment. The average temperature in the test room (21.57 0.2 1C) was monitored prior to each measurement. Samples of fresh and frozen skin were prepared and incubated at 32 1C and skin pH was measured up to 46 h. This experiment was carried out with six different frozen samples and four different fresh samples.

Results TEM investigation Electron micrographs of fresh and frozen samples that were left to thaw at room temperature and used the same day generally appeared intact (Figs 2 and 3). Well-defined epidermis and dermis could be observed (Fig. 2). The surface contour of the stratum corneum generally appeared smooth. Some layers of the stratum corneum appeared detached from the rest of

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epidermis dermis epidermis * *

desmosome

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Fig. 2. Thin cut sections of a fresh sample of human skin showing a well-defined stratum corneum, epidermis and dermis.

the epidermis (thick arrow in Fig. 2), hence some intercellular spaces between the corneocytes could be observed (asterisks; Figs 2 and 3a). The stratum corneum of these skin samples consisted of keratinized stratified cells connected by desmosomes (arrow, Figs 2 and 3a). Frozen samples that had been defrosted and left in EBSS for 3 days prior to testing appeared slightly different from the other two samples. The surface contour showed a more undulated surface and the layers of the stratum corneum appeared mainly fragmented (Fig. 3b). Some parts of the stratum corneum were completely detached from the rest of the epidermis leaving wider intercellular spaces (asterisk; Fig. 3b). The residual culture medium appeared as an amorphous substance between the stratum corneum layers of the frozen sample (arrow; Fig. 3b). Moderate to severe autolysis of the epidermis could also generally be observed (data not shown).

Histological studies Pictures obtained with the paraffin wax method showed well-defined dermis and epidermis (Fig. 4a, b). The different parts of the epidermis of fresh and frozen skin samples could be observed,

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dermis

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SC

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Fig. 3. Thin cut section of frozen samples, (a) frozen sample defrosted the same day (b) frozen sample of human skin, defrosted and left in EBSS for 3 days showing the stratum corneum (SC) appearing fragmented and wide intercellular spaces, observed between the layers of the SC (asterisk).

Fig. 4. Skin samples prepared with the paraffin wax method and stained with hematoxylin and eosin. (a) fresh skin samples showing stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS), stratum basale (SB) and the dermis and (b) frozen sample (left to thaw at room temperature) showing some damaged layers of the stratum corneum.

stratum corneum, stratum granulosum, stratum spinosum and stratum basale (Fig. 4a, b). Slight morphological differences were apparent in the frozen skin samples; i.e. some parts of the stratum corneum appeared fragmented (Fig. 4b).

Enzyme activity The measurement of LDH activity has been used as an indicator of skin viability. In this study, background LDH activity was measured with both controls: the EBSS media alone (7.117 0.34 U/L) and the autoclaved sample (‘dead skin’; 2.9370.34 U/L). A significant enzymatic activity was demonstrated with the fresh and frozen samples, since LDH measurements were always significantly higher (Po0.05) than that of the controls.

Freshly excised skin samples kept at 4 1C showed a rather stable LDH activity (P40.05) over a 7-day period (i.e. 168 h, Fig. 5). When kept at 32 1C, the LDH activity was constant up to 96 h (4 days) and then increased to approximately 160 U/L after 144 h incubation (i.e. 6 days, Fig. 5). The LDH activity measured from frozen samples kept at 4 1C and 32 1C showed a high variability in results (i.e. values between 50 and almost 800 U/L, Fig. 5). The LDH activity of frozen samples kept at 4 1C appeared to be stable after 2 h of incubation. Frozen samples incubated at 32 1C showed the most variability in result, especially after 48 h of incubation (Fig. 5). Overall, the LDH activity of fresh samples tended to be lower than that of frozen samples. Enzyme activity also appeared to increase with samples incubated at 32 1C.

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LDH activity (U/L)

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Fig. 5. Mean LDH activity (U/L)7standard deviation of fresh and frozen samples kept at either 41C or 321C.

Oxygen consumption In this experiment, metabolic activity as an indicator of skin viability was measured by a gradual decline in oxygen concentration. Results presented in Fig. 6 showed that measurements of oxygen concentration of autoclaved skin (metabolically inactive) samples remained constant after an initial sharp decline, thus illustrating an absence of metabolic activity. However, it was noted that oxygen measurements of the medium alone showed a small gradual decline after an initial decrease of approximately 1 ppm (Fig. 6). Oxygen concentration measured over 15 min for the 1 h and 1-day-old fresh skin samples kept at 4 1C was shown to decrease gradually (by a maximum of 5–6 ppm) for about 3–4 min before leveling off for the rest of the experiments. The defrosted skin samples kept at

4 1C for 10 days did not show any metabolic activity after an initial sharp decrease in oxygen concentration. The other fresh skin samples kept at 4 1C for 4 and 5 days as well as defrosted samples kept at 4 1C for 2 days also showed an initial rapid decrease in oxygen concentration followed by a smaller gradual decline (Fig. 6). The frozen sample left to thaw for 2 h presented the highest decrease in oxygen concentration by overall approximately 10 ppm within 20 min. The incubation of fresh and defrosted skin samples at 32 1C for various periods of time showed in general the same pattern of decline in oxygen concentration: a sharp initial fall within 2–3 min followed by a more slower gradual decrease over the remaining 15–20 min (Fig. 7); the exception being the fresh and defrosted samples incubated for 46 h that presented a short

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autoclaved samples fresh 5 days fresh 4 days defrosted 2 days fresh 5h fresh 1 day

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Fig. 6. Average oxygen consumption by skin samples for 15–20 min; i.e. fresh samples kept at 41C for 5 h, 1 day, 4 days, and 5 days post mortem; frozen samples left to thaw for 2 h, and defrosted samples kept at 41C for 2 and 10 days, and control samples; i.e. negative control: autoclaved skin sample, and medium alone.

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Fig. 7. Average oxygen consumption by skin samples for 15–20 min; i.e. fresh samples kept at 41C for 5 h post mortem and then incubated at 321C for 22 and 46 h and defrosted samples left to thaw for 2 h and then incubated at 321C for 22 and 46 h.

Skin pH The breast skin pH of volunteers was 5.4070.55. The pH of fresh and frozen skin of ‘ex vivo’ samples was assessed using the same equipment as for the measurements conducted in vivo. The pH of 6 fresh samples was measured after a maximum of 24 h hours post excision and was 7.7470.20. Frozen samples were, at first, separated into two groups: nine samples frozen for more than 6 months and six samples frozen for less than 5 months. The pH of frozen samples was 7.4170.84. There was no significant difference (P40.05) between both groups of frozen samples. Thus, all the frozen sample data were pooled together for analysis and comparisons of the overall results. There was no significant difference (P40.05) in pH between fresh and frozen skin samples. Excised skin samples including fresh and frozen samples had a significantly higher pH (Po0.05) than volunteers’ breast skin measured in vivo. When changes in skin pH was investigated over a long incubation time (i.e. up to 46 h), it was observed that the pH of fresh skin samples was generally lower than that of the frozen skin samples (Fig. 8), although pH values remained alkaline.

showed samples with relatively well-defined epidermis and dermis. Frozen samples used just after thawing or after 3 days presented evidence of possible stratum corneum fragmentation. Other studies observed signs of cellular degeneration in freshly excised skin samples after 3–7 days of incubation at 4 1C (11). An investigation of the effect of freeze-thaw treatment on skin morphology using light microscopy reported necrosis of the epidermis and follicular epithelium when compared with intact fresh samples (15). However, morphological and histological studies are only reliable when clear-cut. The recognition and interpretation of abnormal conditions is only made possible by comparing with accepted standard control. Furthermore, it has to be noted that chemical fixatives used in EM protocols, immobilized tissues over a period of seconds to minutes, during which detrimental chemical and osmotic changes can occur (16). Chemical fixation has been shown to induce artifacts on various cellular structures. The three-dimensional crosslinking with glutaraldehyde may also cause a 10.5 10 9.5 9 pH

steep slope rapidly followed by a flat curve comparable to the autoclaved sample curve (Figs 6 and 7).

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Frozen skin Fresh skin

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Discussion Morphological investigation Morphological investigation of fresh and frozen skin samples using light and electron microscopy

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Fig. 8. Mean pH 7 standard deviation of four freshly excised skin samples and six defrosted skin samples incubated at 321C up to 46 h.

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condensation of cellular components resulting in shrinkage of the cells (17).

Enzyme activity LDH activity measured in fresh samples kept at 4 1C was lower than that stated in the literature, although it was stable up to 168 h (7 days); at 25 1C LDH activity of adult humans was observed to be 125–236 U/L (18). For the first 96 h, fresh skin samples kept at 32 1C had a comparable LDH activity to the ones kept at 4 1C. Frozen samples thawed and kept at 4 1C showed an overall increase in LDH activity up to 24 h after which it become reasonably stable. LDH activity of frozen samples incubated at 32 1C raised up to 48 h after which it become highly variable with increase of activity almost up to 800 U/L. This 48h rise correlated with another study that showed an increase in several enzymes activities, including LDH, in samples kept at 37 1C (15). The same study also reported a greater increase in enzyme activities over time in defrosted samples when compared with fresh ones. This, when compared with our results, showed the average LDH activity of fresh samples to be significantly lower (Po0.05) than that of frozen ones. Rises in LDH activity can be medically associated with many pathological conditions (19). In addition, surgical excision, freezing and thawing might add to the overall skin trauma. It has been observed that freezing skin for storage can destroy skin viability (6), although a clinical experience with frozen auto- or allo-graphs showed no significant difference in graft-taking when compared with the use of freshly harvested auto- or allo-graphs (20). Fresh skin samples used in this project were immediately placed in EBSS media, refrigerated and were used within 3 days. According to our results, when the viability of fresh samples kept at either 4 1C or 32 1C was assessed, there was no significant variation of LDH activity within at least the first 4 days. These results correlated with an ‘in vivo’ study that showed enzyme activity of fresh samples kept at 4 1C to be stable for up to 3 days for lactate production, and up to for 5 days for glucose utilization (11).

Oxygen consumption Aerobic respiration is a process indicative of living cells and requires complex associations of enzymes in intact mitochondria. The consump-

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tion of oxygen by the skin is a function of all cells and cell types within the tissue. Hence it is an important index of viability for preserved skin (8). From our results, freshly excised as well as freshly thawed samples showed high oxygen consumption; i.e. respiration rates. Fresh and defrosted samples that were stored for a long period of time had a significantly lower (sometimes non-existent) oxygen consumption than those used immediately. Tissue damage that occurred during thawing could have included lethally injured cells that still possessed temporarily functioning organelles, such as mitochondria, immediately after thawing (9). As previously described, a decline in apparent viability could be observed after a period of stabilization. This might be explained by the fact that some cells that were damaged during the freezing process were sill metabolically active immediately after thawing. Nevertheless, cells became metabolically inactive after 48 h of storage in media at 4 1C. This observation correlated with a previous study that showed a decline in viability of skin samples after 18–24 h (9). Our results also showed an increase in oxy-gen consumption of fresh samples incubated at 32 1C for 24 h. Fresh samples of skin have been shown to consume oxygen readily down to 1 ppm oxygen tension after 15–20 min (8, 9). However, in our study, the consumption of oxygen for all samples seemed to reach a plateau before the 15- min measurement period and even fresh samples did not deplete all the oxygen available from the medium. This might be explained by the fact that we used a larger amount of media and larger skin samples than in other studies. A decrease in the oxygen consumption rate may represent a decrease in the proportion of respiring cells or a decrease in the level of respiration of all cells within the tissue, or both (8). Divergence of the curves after the steep decline can be attributed to skin cells losing their ability to consume oxygen during the measurement period (21) and/or to the predominance of anaerobic metabolism at lower oxygen concentration (22). A vast majority of cells may survive a prolonged hypothermic stress, but may show a decreased ability to respire. This results in a decrease in the rate of oxygen consumption (8) as it was observed with the fresh samples kept in the fridge for 4 to 5 days after excision. The oxygen consumption curves observed in this study were not exponential as previously

Assessment of skin viability

described by Jensen and Alsbjorn (22). However, non-exponential results have also been reported elsewhere (8).

Skin pH This concept was created by Marchionini and Hausknecht (23) describing the importance of the acidity of the skin for its bacterial flora and is known as the ‘acid mantle’ of the skin. Normal skin pH is said to be approximately 5 (24). However, detailed studies have reported pH of 4.2–5.6 (25), which is now the range considered relevant. This is in accordance with our ‘in vivo’ results. Skin surface pH is considered as a critical parameter of skin well-being and integrity. Acidic pH is necessary to maintain bacteriological and chemical resistance of the skin (26). Our study showed that skin samples ex vivo had a significantly higher pH than human skin in vivo. This difference in pH between ‘in vivo’ and ‘ex vivo’ conditions might be explained by the deleterious effect of surgical excision and the inactivation of sebaceous and sweat glands. The pH of sweat (i.e. lactic acid system) is largely involved in skin surface pH (27). The sebaceous glands, distributed in hair follicules in sebaceous units in the dermis secrete fatty acids and lactic acid, which also lower skin pH.

Conclusion The aim of the ‘ex vivo’ test is to mimic ‘in vivo’ situations. It was therefore necessary to show that excised skin samples were still viable. Furthermore, because of the shortage in skin supply, it is more practical to carry out experiments with frozen skin samples. Therefore, it is necessary to compare the viability of fresh and frozen skin samples. From our study, it was clear that the oxygen consumption assay and pH measurements were quite simple and reproducible protocols. Only small samples of skin were required and the experiments could be repeated easily. The oxygen consumption technique evaluates an overall property of the intact tissue instead of a single isolated cell or enzyme component making it suitable for routine use in the banking of skin (8). However, our oxygen consumption results were not as reproducible as in the literature, maybe

due to the limited number of replicates. In contrast, although the measurement of enzyme activity was easy to perform and gave reproducible results, the use of single-enzyme activity (measuring only one pathway among the many metabolic pathways occurring within a cell) (28) can be criticized when used on its own. We would recommend the use of two to three techniques for the evaluation of skin viability, including at least oxygen measurement and an enzyme activity assay.

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