Storage and skin metabolism

British Journal of Plastic Surgery (x97z), 25, 440-453

STORAGE AND SKIN METABOLISM

By J. C. LAWRENCE

M.R.C. Industrial Injuries and Burns Unit, Birmingham Accident Hospital, Bath Row, Birmingham Bz5 zNA

SPLIT-THICKNESS skin is commonly stored at 4°C for short periods of time wrapped in saline moistened gauze. Skin stored in this way has been reported to remain viable for 3 to 4 weeks (Flatt, 1948 ; Pepper, I954) but others doubt whether the tissue is clinically useful beyond 2 weeks (Bondoc and Burke, I971).

Using suitable procedures it is possible to store skin in the viable state for very long periods in liquid nitrogen (Cochrane, I968; Bondoc and Burke, I97I). For many years homografts have been used in the treatment of extensive burns when sufficient autografts are not available (Jackson, I95 I) and recently they have been claimed to debride or cleanse contaminated wounds (Morris et al., I966 ; Burke and Bondoc, I968 ; Shuck et al., I969).

It was therefore of interest to obtain a more precise evaluation of both these storage methods to discover what degree of damage was sustained by split-thickness skin at different stages of storage and whether there was scope for improving or simplifying either of these methods.

Suitable techniques for investigating various aspects of skin metabolism such as respiration (Cruickshank, I954), the incorporation of suphate ions into ground substance (Lawrence and Ricketts, 1957) and the ability of skin to reduce tetrazolium salt (Hershey et al., I958) have been developed and used to investigate the effect of thera- peutic materials (Lawrence, I959, I969), heat (Lawrence and Ricketts, I957 ; Cruick- shank and Hershey, 196o) and non-ionising radiation (Carney, Lawrence and Ricketts, 197o ) on skin in vitro. The response of skin stored at 4°C and in liquid nitrogen has now been studied.

METHODS

Skin. Experiments were made with guinea-pig ear skin obtained as described by Lawrence (1961). Red-eyed white animals of the Dunkin-Hartley strain aged 4 to 6 months were used.

Some of the experiments were repeated with human skin ; this was split-thickness skin surplus to requirement in grafting operations.

Skin Metabol ism. The following standard culture medium was used unless indicated otherwise: homologous serum, Krebs-Ringer phosphate 5 per cent w/v glucose and dihydrostreptomycin sulphate, 5 ° /zg./ml. mixed 5, 3, I, I, by volume (Lawrence, 1969).

Skin respiration was measured with the differential microrespirometer described by Cruickshank (i954). To determine the effect of storage the respiratory activity of skin slices was measured for a 2-hour period then the skin transferred for the required time to the storage system ; after this the respiration of the skin was measured again for a 24-hour period.

The ability of skin to incorporate sulphate ions was measured by the method of Lawrence and Ricketts (I957). A series of control cultures was incubated for 24 hours on medium containing Na2~sSO~ (0.03 mC./ml.) and further series of explants from the

STORAGE AND S K I N M E T A B O L I S M 441

same skin donor were stored. After storage these skin samples were incubated for 24 hours on the radio sulphate labelled medium. The radioactivity of all the skin samples from an experiment was determined on the same occasion ; to obviate variation between the radioactive labelling of different batches of medium care was taken to prepare sufficient for one complete experiment on each occasion.

The ability of skin to reduce tetrazolium salt to an insoluble formazan was tested by a method based on that used by Hershey et al. (1958). The tetrazolium salt reductase TSR activity was measured on a series of control cultures and on further series of skin explants from the same donor after storage. In other experiments the skin was incubated on medium for 24 hours before determining TSR activity.

Storage at 4°C. A domestic refrigerator was used ; a temperature of 4°C could easily be obtained in the lower half by suitable adjustment of the thermostat.

The skin explants were stored in 5 ° × 12- 5 mm. glass tubes with a rubber enclosure, the tissue being placed on small pieces (15 × 15 ram.) of sterile filter paper. Monitoring the temperature within one of these containers showed that this varied + I°C during normal running conditions and, when the refrigerator door was opened, the temperature might rise as much as 3°C. These temperature fluctuations within the specimen tube were eliminated by enclosing the specimen tubes in a box made of 15 mm. thick expanded polystyrene. These polystyrene containers take some hours to cool to the operating temperature but once cooled the temperature in the specimen tubes varied less than + o.i°C.

S torage in Liquid Nit rogen. Specimens were stored in the vapour phase of a large liquid nitrogen container (Union Carbide LD 4o), the nitrogen being replenished weekly. Skin for storage was treated with a cryoprotective agent and placed in polythene bags (3o5 × 155 mm.) and the bag heat sealed. A thread, about 5o0 mm. in length, was attached to the bag by heat sealing and a label identifying the contents fastened to the other end of the thread. The labelled end of the thread was hung over the rim of the nitrogen container thus facilitating rapid identification and removal of a particular specimen.

Most authorities quote I°-5°C/min. drop in temperature as the best freezing rate for tissue storage (Parkes and Smith, 196o ) ; to achieve this a special freezing box was devised. This was a box made of 25 mm. thick expanded polystyrene, internal dimen- sions 3oo × 41o × 8o mm. The base of this container was covered to a depth of about IO mm. with crushed solid carbon dioxide. The packets of skin to be frozen were placed between two sheets of 3 mm. thick " Perspex" sheet 355 × 235 mm. and secured with elastic bands. This package was then placed in the box which has an internal support arranged to keep the lowest piece o f " Perspex" 2o ram. from the box base. An alumi- nium sheet 360 x 25o mm. was placed on the upper "Perspex " sheet and this sprinkled with crushed solid carbon dioxide to a depth of 2- 3 mm. The lid was then replaced. This container froze skin within the specified rates and could accommodate up to 20 bags of skin. When the skin had reached - 65°C the polythene bags were removed and rapidly transferred to the liquid nitrogen storage container.

When required the skin was thawed rapidly by removing the plastic bag from the nitrogen container and plunging it into a water bath at 37°C.

T e m p e r a t u r e Moni tor ing . Temperatures between 37°C and - 8 o ° C were measured where required with a copper-constantan thermocouple and an electric thermometer (Comark, Type 1625). Where continuous observation of temperature was needed the output from the electric thermometer was fed into a chart recorder (Telsec Type 7oo).

442 B R I T I S H JOURNAL OF P LAS TI C SURGERY

Cryoproteefive Agents. For most experiments on liquid nitrogen storage 15 per cent glycerol in physiological saline was used as the cryoprotective agent. Some tests were made using 15 per cent dimethyl sulphoxide in physiological saline. Other experiments were made in which serum-buffer mixtures replaced the physiological saline.

Experimental Skin Graf t ing. The test animals were the same red-eyed white guinea-pigs from whom grafts had been taken for storage.

The animals were anaesthetised with ether and an area about 60 × 4o mm. Shaved on the flank opposite the original donor area. Split thickness skin was excised as described previously then the area was excised a second time so that most of the dermis was removed The stored skin grafts were then applied to the bed thus exposed.

The donor area that had provided the skin for storage was then excised similarly and the excised tissue discarded. This area was then grafted with the fresh skin obtained from the previous excision.

For grafting purposes both the stored and fresh skin were divided into 6 to 8 pieces. The animal was finally dressed with a crepe bandage and plaster of Paris. The

grafted areas were inspected every 3 days until the wounds had fully epithelialised. A positive graft take was assumed if the grafts were pink, adherent and blanched under pressure ; all other appearances were assumed to be failures.

Surv iva l o f Skin on the Body after Death . Experiments were carried out to see how the tetrazolium reductase activity of guinea-pig skin altered with the passage of time between the death of the donor and excision. The animal was killed and skin excised from one ear. A copper-constantan thermocouple was inserted into the centre of the cartilage at the midpoint of the ear and the temperature recorded until skin was excised from the other ear. This second excision was made at 6 hours, 18 hours, or 24 hours after death, two animals being used for each time interval. In one series of experiments the cadavers were kept at room temperature (2o°C_+ 2 °) and in a further series the cadavers were kept in the refrigerator at 4°C.

Sterility Checks. All cultures of skin were checked for sterility at the end of each procedure. Occasionally guinea-pigs carry a fungus which can be seen on the surface of the tissue during incubation ; bacteriological tests were made by spotting loopfuls of the culture medium on to the surface of 4 per cent blood agar plates and incubating these overnight. All contaminated cultures were discarded. The incidence of contamina- tion was very low in these experiments, less than o" 5 per cent.

• RESULTS

Storage at 4°C. The respiration of guinea-pig skin and its ability to incorporate 35SO~ were investigated for storage periods of up to 42 days at 4°C. The activity of the skin was taken as IOO per cent at the time of excision and subsequent changes in activity were related to this. The results are shown in Fig. I. Respiration results were based on values obtained from at least 3 separate experiments each being duplicated at each selected storage period ; in the asSO~ uptake study at least 3 experiments were made at each interval of storage time and there were usually 5 explants used in each experiment. As might be expected the respiratory activity and the ability of skin to incorporate 35SO~ decreased with storage time. After 14 days storage skin respiration was reduced by 50 per cent ; sulphate incorporation showed a greater reduction, showing only 3o per cent of the initial activity. Measurements made after 28 days storage indicated that the activity of the skin had decreased by 92 per cent and 97"5 respectively. This decrease in

STORAGE AND SKIN METABOLISM 443

metabolic activity was logarithmic so that transferring the percentage data to a log. scale yielded a straight line.

Respiration tests were also made on human skin stored at 4°C for 7, I4 and 2I days ; the results were very similar to those obtained with guinea-pig skin. The comparative results are shown in Table I.

In a further series of tests the tetrazolium salt reductase capacity of guinea-pig skin was measured for storage periods of up to 7 days at 4°C. The activity of this enzyme declined more rapidly in these conditions than either respiration or 35SO~ uptake; these results are compared in Table II.

Previous experiments with skin stored up to 4 days at room temperature (2o°-2z°C) showed that culture medium resulted in least loss of viability, saline was poor and the use of buffered saline with and without glucose did not appreciably improve viability

100 ~

80 •

'~ ~ o •

60

~ ~ 20

i ~ , , . , i i i i i i i i i i I i '

0 7 14 2 l 28 35 42

Storage Time, Days

FtG. I. The respiration and sulphate uptake of guinea-pig skin stored in physiological saline at 4°C. Q • respiration ; © & sulphate uptake

Each respiration mean was based on 6 observations (3 separate experiments). The means for sulphate uptake were based on values obtained from 15 explants (3 separate experiments).

(Lawrence, unpublished observations). Experiments were made with some skin slices stored at 4°C on culture medium and with other slices on medium 199 containing 5 per cent foetal calf serum (B.D.H.) instead of saline. The results (Table III) showed that the standard culture medium gave excellent results for storage periods up to 7 days and that medium I99 + 5 per cent calf serum was considerably better than saline.

S torage in Liqu id Nit rogen. Respiration, the incorporation of sulphate ions and the tetrazolium reductase activity of slices of guinea-pig skin were measured before and after storage in liquid nitrogen. The results are shown in Table IV. These experiments were made using 15 per cent glycerol in physiological saline as a cryoprotective agent, freezing at I ° to 5°C per minute and thawing the tissue rapidly on removal from storage. The metabolic activity of skin was reduced by about 4 ° per cent after storage in liquid nitrogen ; all three tests used to measure viability yielded a similar result. Human skin subjected to the same procedure showed a similar survival (Table IV). These losses of activity were present at 7 days and did not decrease further by longer storage; the majority of experiments to date have been made with skin stored up to 28 days. Two

4G

444 BRITISH JOURNAL OF PLASTIC SURGERY

Storage time

(days)

7 I4 2 1

TABLE I

The Respiration of Guinea-pig and Human Skin stored on Saline at 4°C

Guinea-pig skin Human skin

Oxygen uptake, Oxygen uptake ~l./mg./hr. ~l./mg./hr.

c - - - - - - - - - - -~- - - - - -~ Number Percentage c . . . . . ~-- -----~ Number Percentage At After of initial At After of initial

excision storage observations activity excision storage observations activity

0.92 0'68 IO 74 o'51 0"36 4 7I o'96 o'45 6 47 0'48 o'24 4 50 I'OI 0"35 6 36 0.42 o.12 4 29

TABLE I I

T h e Respirat ion, Sulphate Uptake and Tet razo l ium Salt Reductase Guinea-pig Skin stored on Physiological Saline at 4°C

Percentage activity relative to fresh skin Storage time c ---~

(days) Respiration ~5SO~' uptake TSR 0 I00 I00 IO0

2 88 84 65 4 79 72 53 7 74 67 25

Activity of

T h e

TABLE I I I

Te t razo l ium Salt Reductase Activity of Guinea-p ig Skin stored at 4°C on Physiological Saline, Standard M e d i u m and M e d i u m 199 plus Foetal Calf Se rum

Percentage activity relative to fresh skin Storage time r •

(days) Physiological saline Skin cultllre medium Medium I99 0 I O O I00 I00

i 99 lO5 95 2 65 ioo 82 4 53 95 64

Each percentage value was the mean of at least 12 explants obtained from 2 separate experiments

different samples of human skin have been stored for 6 months ; bo th of these showed 5 ° per cent survival after this t ime as measured by tetrazolium reductase activity.

A series of experiments was then carried out to see i f varying the cryoprotective :: sys tem affected the tetrazolium reductase activity of guinea-pig skin. T h e following

combinations were investigated :

I . 15 per cent w/v glycerol in physiological saline. 2. 15 per cent v/v glycerol in s tandard med ium 3- 15 per cent v /v dimethyl sulphoxide in physiological saline: 4- Physiological saline only 5- 15 per cent glycerol w/v in m ed i um 199+ 5 per cent foetal calf serum 6. 15 per cent glycerol w/v in med ium 199 + 2o per cent foetal calf serum 7- 15 per cent glycerol w/v in m ed i um 199 + 5 ° per cent foetal calf serum

T w o separate experiments were made with each preserving system using 5 explants in each experiment. Freezing and thawing were as described earlier, but the skin was


TABLE I V

(a) The Respiration, Sulphate Uptake and Tetrazolium Salt Reductase Activity of Guinea-pig Skin stored in Liquid Nitrogen

Percentage activity relative to fresh skin Storage time r •

(days) Respiration 3aSO2 uptake TSR o IOO ioo ioo

7 62 56 43 I4 53 - - 49 ~8 50 - - 50

(b) The Tetrazolium Salt Reductase Activity of Human Skin stored in Liquid Nitrogen Storage time Percentage activity relative

(days) to fresh skin 0 I00

7 50 I4 53 28 4 °

150 48

On a statistical basis it can be shown that there is a significant decrease in the metabolic activity of stored skin relative to fresh skin. There was no significant difference in the residual activity of skin stored for different periods of time.

TanLE V

The Tetrazolium Salt Reductase Activity of Guinea-pig Skin stored in Liquid Nitrogen for 2 Days using Different Cryoprotective Solutions

Percentage Cryoprotective solution control value

Controls (fresh skin) IOO

r. I5% glycerol in physiological saline 59 2. I5% glycerol in standard medium 7 ° 3. I5% dimethyl sulphoxide in physiological

saline 44 4. Physiological saline 3 ° 5. 15 % glycerol in medium 199+ 5 % foetal calf

serum 54 6. 15% glycerol inmedium 199+2o% foetal calf

serum 49 7. 15% glycerol in medium 199+5o% foetal calf

serum 65

The percentage values represent the mean obtained from 2 separate experiments (Io explants).

On a statistical basis the TSR activity of stored skin was significantly less than that of fresh skin. There was no significant variation between groups I to 7 except for skin stored in saline (4), in this instance the residual TSR activity was less than that of the other groups.

only kept at liquid nitrogen temperature for 24 hours. The results (Table V) suggest that alternatives to 15 per cent glycerol in saline (i) do not appear to have any particular advantages. The use of saline only (4) was the least satisfactory and dimethyl sulphoxide (3) was not as effective as glycerol (2) for cryoprotection.

Glycerol and dimethyl sulphoxide were investigated for possible toxicity to skin ; this was done by exposing skin slices to I5 per cent w/v glycerol in physiological saline or to 15 per cent dimethyl sulphoxide in physiological saline for periods of time up to


24 hours. The results (Fig. 2) indicate that neither substance permits particularly good survival of skin metabolism after 24 hours exposure. Glycerol however was better than dimethyl sulphoxide, the skin showing a residual activity of 20 per cent instead of 6 per cent. Glycerol was markedly superior to dimethyl sulphoxide for shorter exposure times ---up to 2 hours.

Experiments were made using guinea-pigs to check that cryoprotection with glycerol did not adversely affect the take of skin grafts. Thin slices of skin were excised from the ear and then exposed to 15 per cent glycerol in physiological saline for I hour. These slices were then transferred to an area (about 4oo mm~.) on one flank in which the skin had been excised to a depth of o'5 mm. Two animals were used ; at 4 days post- operation one animal showed 9o per cent graft take and the other IOO per cent. Both wounds were completely healed 7 days after grafting. Thus it seems that exposure to

80" - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

~ O i i • t t I . l I , I I I i i " I I I I I I I i i | l

0 2 4 6 8 10 12 14 16 18 20 22 24 Exposure Time; Hours

FIG. 2. The tetrazolium salt reductase activity of guinea-pig skin exposed to glycerol and dimethyl sulphoxide for various times at room temperature (20~-22°C).

& ~ I5 per cent glycerol in standard medium 9 - [ ] physiological saline O O 15 per cent glycerol in physiological saline O ~ O 15 per cent dimethyl sulphoxide in saline

Each point was the mean obtained from 12 explants (2 separate experiments).

15 per cent glycerol in physiological saline is unlikely to interfere with the subsequent take of split thickness skin.

Some experiments were made to see whether altering the freezing rate influenced the damage sustained by skin during storage. The procedure was identical to that described earlier except that the controlled freeze in solid carbon dioxide was omitted, the skin being placed directly in the nitrogen container. Thermometry showed that these specimens cooled quickly, taking about IOO seconds to cool within 2o°C of the storage container ( - I96°C) . Some samples were treated with 15 per cent glycerol in physiological saline, others were frozen in physiological saline only. Viability was assessed by respirometry and by measuring the tetrazolium salt reductase activity. These measurements were made immediately after storage and also after 24 hours incubation on culture medium. The results are shown in Table VI. These findings suggest that this quick freeze yields similar results to those obtained by the slower method. Skin stored with no cryoprotective agent shows an appreciable viability immediately


TABLE VI

The Tetrazolium Salt Reductase Activity of Guinea-pig Skin Cooled to Liquid Nitrogen Temperature at Different Rates with and without Cryoprotecfion

Percentage activity relative to control skin

Skin explants Skin explants not incubated incubated 24 hr. at 37°C

Cooled at I ° to 3 ° per minute Physiological saline 9 15 Physiological saline + I5 % glycerol 64 7o

Cooled rapidly (see text) Physiological saline 34 Physiological sa l ine+ 15% glycerol 69

4 82

Mean percentage values were obtained from the results of 3 separate experiments ( i8 skin explants).

TABLE V I I

The Take of Guinea-pig Skin Autografts stored in Liquid Nitrogen with and without the Addition of a Cryoprotective Agent (i5 per cent Glycerol)

lqo. of grafts Percentage No. of No. of showing typical Percentage epithelial cover

Treatment animals grafts take at 4 days graft take at 7 days Fresh autografts 4 24 22 92 Ioo Autografts stored

with cryoprotection 3 19 16" 84 1oo Autografts stored

without eryoprotection 3 21 6~ 28"5 85

* Take not significantly less than fresh skin autografts ; X ~ = 0'078, 0"9 < P > 0-8 ~ Graft take significantly less than fresh skin autografts; x z = 16"38, P > o.ooi mid also significantly

less than skin autografts stored with cryoprotection, X 2 = lO-33 , o.or < P > o.ooi

TABLE VIII

The Tetrazolium Salt Reductase Activity of Guinea-pig Skin excised at Various "rimes after Death

Percentage activity relative to control skin of excision of skin c __h____

Tim(hrs . e after death) Explants Explants incubated not incubated 24 hr. at 37°C

Cadaver stored at 2o°C 0 IO0 IO0*

6 7 ° 5 ° 18 50 35 24 45 32

Cadaver stored at 4°C 0 IO0 I00"

6 9 ° 85 18 83 85 24 81 84

* Incubated skin explants show only 75 per cent of the T S R activity of freshly excised skin.

Mean percentage values were obtained from the results of 2 separate experiments (Io skin explants),


after storage but this decreased considerably when the tissue was incubated for 2 4 hours. A similar result was noticed with the slower freezing method.

Grafting experiments were made with guinea-pig skin stored in liquid nitrogen with and without cryoprotecfion; the results are shown in Table VII. Assessment of graft take was made 4 days post-operation.

The success of graft take was in approximate agreement with the measurements of metabolic activity (e.g., respiration) of skin similarly stored. Skin treated with 15 per cent glycerol in physiological saline and stored in liquid nitrogen showed a considerably better take than skin without cryoprotection. The take of fresh skin grafts was slightly better than that of skin stored with cryoprotecfion°

1 0 0 ~ _

8 0 ~ ~ 0 .~

4 0

20

. 8

Time; Hours

Fla. 3. The tetrazolium salt reductase activity of guinea-pig ear skin excised at various times after death. © TSR test on tmincubated skin • T S R test after 24 hours incubation at 37°C

Cadaver stored at 4°C Cadaver stored at 2o°C - 22°C

Each point was the mean obtained from xo skirt explants (2 separate experiments).

Viabili ty o f Skin Left on Cadave r a f te r Death. Tests were made on skin removed from guinea-pigs at the time of death and 6, 18 and 24 hours post mortem. The tetrazolium reductase activity of the skin was measured immediately, 5 explants being used, and also after incubating a further 5 explants for 24 hours on the culture medium. The results are shown in Table VIII. There was a 2o per cent decrease in the tetrazolium reductase activity of the freshly removed skin incubated for 24 hours compared to that of freshly removed skin tested immediately after removal. Skin removed from cadavers kept at 4°C for 24 hours showed little decrease in activity relative to fresh skin, but the skin from guinea-pig cadavers kept at room temperature (~-~ 2o°C) for 24 hours showed only half the activity of fresh skin. These changes in tetrazolium reductase activity are illustrated graphically in Fig. 3. The decrease seems to begreatest within the first few hours following death.

The rate of cooling of guinea-pig cadavers in air at 2o°C and 4°C is shown in Fig. 4- The cooling rate is greatest during the first 2 to 4 hours following death, then decreases as the environmental temperature is approached.

The decrease in metabolic activity of guinea-pig cadaver skin appeared to be related to the temperature of the cadaver; when these were stored at 4°C there was a minimal

, i J ! | i ! t t t ! ! t . . d ! ! ! ; ~ . . F t t _ |

0 6 12, 18 24


decrease in activity, with storage at room tempera ture this decrease was more pronounced. At bo th storage tempera tures the greater par t o f this decrease in metabolism occurred during the first few hours following death, before the cadaver had cooled to the t empera - ture of the environment.

e~

36

3

28

24

20

16

12

] ~ I I I ! I I I I _ I I ' i L ~

0 2 4 6 8 10 12 14 16 18 20

Time; Hours

FIG. 4. The rate of cooling of guinea-pig ears after death. • • Stored at ambient temperature © ~ 0 Stored at 4°C

Each point up to 6 hours was the mean of 6 observations, subsequent points were the mean of 4 observations. Ambient temperature was usually about 2o°C ; the low value at Io hours occurred

during the night, room heating being discontinued during the overnight period.

DISCUSSION

T h e living body can be regarded as an indefinite storage system for skin and it seems reasonable to suppose that freshly harvested skin is metabolising normally and that it will continue to do so when regrafted immediately. However situations arise in which it is either undesirable or impracticable to use fresh donor skin to cover denuded areas and in this circumstance recourse has to be made to artificial storage.

The re is a current t rend towards establishing banks of stored cadaver skin (Bondoc and Burke, I971 ; Cochrane, I968) intended as a source of living homograf t for the treatment of extensive burns (Bondoc and Burke, I97 I) or to cleanse contaminated wounds


(Morris et al., 1955). It is therefore pertinent to consider the survival of skin on a cadaver since it is rarely possible to harvest this tissue immediately fbllowing death.

Most authorities (e.g. Cochrane, 1968) suggest that cadaver skin should be obtained within = 4 hours of death. The experiments made with the guinea-pig show that skin metabolism decreases at least 5 ° per cent within 24 hours of death if the cadaver is kept at room temperature (about 2o°C). If the cadavers are kept at 4°C there is a marked improvement ; metabolic studies suggest a survival of about 8o per cent of the initial activity of the skin after z4 hours storage. The tissue of the guinea-pig ear cooled sur- prisingly slowly and an animal cadaver transferred to 4°C within 3o minutes of death took 5 hours to cool within 1°C of the refrigerator temperature. As might be expected the decreases in the metabolic activity of the skin were related to storage temperature (Figs. 3 and 4) ; the greater part of this decrease in metabolism occurred during the first few hours following death. When the cadaver had attained environmental temperature only slight further decreases in skin metabohsm occurred. It seems possible that skin viability could be improved by rapidly cooling the donor; the earlier that donor skin can be removed from a cadaver the better.

Detached from the host and without special protection skin can only be kept for a limited time though for longer periods than on the cadaver; for instance, there is little loss of respiratory activity of skin slices incubated on a serum medium at 37°C for 24 hours (Lawrence, I969) ; however, if a mineral salt'solution is used and no substrate included considerable loss of respiratory activity occurs (Brooks et al., 1959). Similar results have been obtained with skin kept at room temperature (2o-22°C) for up to 4 days; metabolic activity after storage on a serum medium was good; if physiological saline or a buffer solution was used residual metabolic activity was poor (Lawrence, unpublished observations).

Pepper (1954) reported survival times of skin slices kept at various temperatures ; as might be expected the cooler the environment the longer the tissue survives, provided that the tissue does not freeze. The clinical use of skin stored at 4°C for relatively short periods of time is well-established; previous experimental work (Brown et al., 1955 ; Flatt, 1948, 195o ; Gemmell, Laing and Veall, 1954) suggests that skin stored in this way remains viable for up to 28 days. Other workers (e.g., Bondoc and Burke, 1971) consider that skin stored more than 14 days at 4°C has little clinical value. The experiments described in this paper show that the metabolism of both human and guinea-pig skin declined rapidly when the tissue was stored at 4°C; after 14 days storage in saline respiratory activity was halved. Replacing the saline with a mineral salt-serum medium resulted in some improvement, but this might not be acceptable for clinical use. The synthetic medium 199 which is often used in tissue storage systems did not improve the survival of metabolic activity of stored skin nor did the use of a physiological buffer solution nor the inclusion of glucose.

Skin detached from its host can be regarded as an organ culture system, and many of the precautions normally observed when handling skin in vitro for experimental purposes apply to skin that is to be stored. The possible toxicity of the container and the support used for the skin should be considered ; for example, saline extracts of filter paper or surgical gauze can be slightly toxic to skin (Lawrence, 1972 ) though in the present study the volume of saline used to moisten the support fo r the skin was such that any potentially toxic material eluted would be very dilute.

It is possible that bacteria on the skin could have some effect on storage; the skin used in the current experiments was sterile on removal from storage, but human skin often has a small residual bacterial content even after pre-operative preparation (Lawrence and Lilly, 1972 ) and even though the number of organisms is low these could increase considerably over a storage period of 2 to 3 weeks. It has yet to be shown if this is an important factor in practical storage systems.

STO~AGE AND SKIN METABOLISM 45I

The temperature of the storage system should be reasonably low; Pepper (I954) showed that storage at 4°C permitted maximum survival of skin; higher temperatures resulted in a more rapid loss of viability. The data available in her paper suggests that there is a logarithmic relationship between storage time consistent with viability and storage temperature.

Domestic refrigerators set for normal operating condkions usually achieve 4°C in the lower compartment. I f the refrigerator is also used for other purposes or is opened frequently it is probably better to keep the skin containers in an insulated box within the refrigerator ; expanded polystyrene serves this purpose well and prevents large temperature fluctuations.

Skin and other tissues can be stored for much longer periods by treatment with a suitable cryoprotective agent (usually glycerol) followed by controlled freezing of the tissue to very low temperatures. Until comparatively recently solid carbon dioxide was the method of choice for long term storage of tissues (Ciba Symposium, I954) but most long term storage systems for skin now use liquid nitrogen (Cochrane, I968 ; Bondoc and Burke, I97I).

In the experiments described in this paper the technique of storage in liquid nitrogen was similar to that used in Boston (Bondoc and Burke, I97I). This appeared practical for clinical use ; the metabolic activity of human and guinea-pig skin after storage was 6o per cent to 7o per cent of its pre-storage rate as measured by three different tests. This decrease in metabolic activity noted after storage did not vary with storage time, at least for periods between I and 28 days. A more crucial test, skin grafting, showed that skin slices stored in liquid nitrogen exhibited a mean graft take of 8o per cent using the guinea-pig as the test subject. This compared with a mean take of 92 per cent for fresh skin autografts and 2o per cent take for grafts stored in liquid nitrogen without cryoprotection. Despite the difficulties encountered in making an accurate long term assessment of graft take in the albino animals used, results obtained by measurements of residual skin metabolism after storage appear to be related to the success rate of com- parable skin used for grafting as judged within a few days of operation. Thus it seems reasonable to accept the hypothesis that measurements of skin metabolism provide an indication of the viability of this tissue. However further experiments are needed to confirm the relationship between survival of in vitro metabolic activity of skin and the take or failure of skin autografts.

It was found that glycerol was preferable to dimethyl sulphoxide for cryoprotection and the latter was also more toxic to skin after prolonged contact. Some investigations were also made to see whether modifying the fluid containing the cryoprotective agent had much effect on skin viability after storage. The use of a medium containing 5o per cent serum showed a marginal improvement compared with saline, medium I99 or medium I99 plus serum. This small improvement in skin viability probably does not justify the use of serum in a routine storage system and it might have other undesirable features for clinical purposes.

The observation that the freezing rate was apparently not critical is of interest but further experiments are needed to see if the simpler method (allowing the skin to cool by suspension in the storage container) would be satisfactory for clinical use. It is generally accepted that a controlled freeze of I ° to 5°C per minute is necessary unless a very rapid freezing rate is achieved (Robinson, I966). More sophisticated systems employ a servo mechanism so that this steady decrease is maintained until liquid nitrogen temperature is reached (Cochrane, I968) ; such equipment was not available for this study. It would be interesting to discover what improvement in skin viability might be gained by its use ; Robinson (I966) reports 8o per cent recovery of cells using such a system.

Though a storage system for skin or other tissues could probably be devised which


would cause minimal loss of viability~ such a system may not be suited to applications outside the research laboratory. From the clinical viewpoint a storage system should be simple in use and require a minimum of manipulative procedures consistent with obtain- ing acceptable results. To achieve these ends some sacrifice in tissue viability may be unavoidable and for practical purposes this may not matter. For instance, the simple method of storing skin at 4°C for short term use only involves wrapping the tissue in gauze moistened with saline and is successfully used by many centres. This skin, if it is to be used at all, is in most instances applied to the recipient area during the first week of storage. Recently Bondoc and Burke (I97 I) suggest that skin stored more than 14 days at 4°C is no longer useful ; measurements of skin metabolism after such storage confirms this view. By using serum in the skin storage medium the metabolic activity might be prolonged but it is doubtful whether doing this would have sufficient practical advantage.

The same considerations apply to the storage of skin in liquid nitrogen. Ideally the viability of stored skin should be as high as possible ; to achieve this may require expensive equipment and considerable supervision ; a simpler system has been used here and at other centrcs (Bondoc and Burke, I97 I) and has given acceptable results. It seems possible that this method can be further simplified consistent with achieving a good viability of the stored tissue. This possibility is being investigated further, particular attention being given to the success rate of theoskin grafts.

Thanks are due to Dr J. P. Bull for helpful advice and criticism and to Mr D. M. Jackson fo7 provision of the samples of human skin. The technical assistance of Mrs B. Griffiths is gratefully acknowledged.

REFERENCES

BO~DOC, C. C. and BtmKE, J. F. (1971). Clinical experience with viable frozen human skin and a frozen skin bank. Annals of Surgery, 174, 371-382.

BROOKS, S. A., LAWRENCE, J. C. and RICKETTS, C. R. (1959). The phosphate esters of mammalian skin maintained on glucose and various deoxyglucoses. Biochemical Journal, 73, 566-572.

BROWN, J. B., FRYER, M. P. and ZAYDON, T. J. (1955). A skin bank for post-mortem homografts. Surgery, Gynecology and Obstetrics, IOl, 4Ol-412.

BURKE, J. F. and BONDOC, C. C. (1968). A method of secondary closure of heavily contaminated wounds providing " physiologic primary closure ". Journal of Trauma, 8, 228-237.

CAR~Y, S. A., LAWRENCE, J. C. and RICKETS, C. R. (197o). Effect of microwaves at X-band on guinea pig skin in tissue culture. British Journal of Industrial Medicine, z7, 72-76.

CIBA Foundation Symposium (1954). " The Preservation and Transplantat ion of Normal Tissues ", ed. Wolstenholme, G. E. W., Cameron, M. P. and Etherington, J. London : Churchill.

COCHe~NE, T. (1968). The low temperature storage of skin : a preliminary report. British Journal of Plastic Surgery, 21, 118-125.

CRUlCKSHAI~K, C. N. D. (1954). Continuous observation of the respiration of skin in vitro. Experimental Cell Research, 7, 374-380.

CRtlICKSHANK, C. N. D. and HERSHEY, F. B. (196o). The effect of heat on the metabolism of guinea pigs' ear skin. Annals of Surgery, I51, 419-43 o.

FLATT, A. E. (1948). Refrigerated autogenous skin grafting. Lancet, ii, 249-251. FLATT, A. E. (195o). Observations on the growth of refrigerated skin grafts. British

Journal of Plastic Surgery, 3, 28-33. GEMMELL, W., LAING, J. E. and VEALL, N. (1954). The uptake of ~2p as an in vitro test of

the viability of stored skin. Radioisotope Conference, Vol. I, p. 1o7. HERSHEY, F. B., CRUICKSHANK, C. N. D. and MULLINS, L. I. (1958). The quantitative

reduction of 2.3.5.triphenyl tetrazoliu.m chloride by skin in vitro. Journal of Histo- chemistry and Cytochemistry, 6, 191-196.

JACKSON, D. M. (1951). The management of the burned patient. St Bartholomew's Hospital Journal, 55, 98-1o3.

LAWRENCE, J. C. (1959)- The comparative toxicity of arttibiotics to skin. British Journal of Pharmacology, I4, 168-I73.

LAWRENCE, J. C. (1961). Factors influencing skin metabolism as shown by tissue culture. In " Wound Healing " , pp. 32-45, ed. Slome, D. Oxford : Pergamon Press.

STORAGE AND SKIN METABOLISM

LAWRENCE, J. C. (I969). The effect of silver nitrate, sulfamylon and other antibacterial agents on the metabolism of mammalian skin. In " Pharmacological Treatment in Burns " , pp. 234-24I, ed. Bertelli, A. and Donati, L. International Congress Series I9o. Amsterdam : Excerpta Medica.

LAWRENCE, J. C. (I972). An investigation of dressing materials for possible toxicity. (In preparation.)

LAWRENCE, J. C. and LILLY, H. A. (I97z). A quantitative method for investigating the bacteri- ology of burned and normal skin. British Journal of ExperimentalPathology. (In Press.)

LAWRENCE, 5. C. and RICY.ZaTTS, C. R. (I957). The metabolic uptake of sulphate ions by skin. Experimental Cell Research, I2, 633-638.

Mom~is, P. J., BONDOC, C. C, and BtrRKE, J. F. (I966). The use of frequently changed skin allografts to promote healing in the non-healing infected ulcer. Surgery, 6o, I3-I9.

PAVd<ES, A. S. and SMITH, A. U. (Eds.) (I96o) Recent research in freezing and drying. Oxford : Blackwell.

PEPPER, F. J. (I954)- Studies on the viability of mammalian skin autografts after storage at different temperatures. British ffournal of Plastic Surgery, 6, 25o-256.

ROBINSON, D. M. (I966). Low temperature preservation of ceils in culture. Laboratory Practice, xS, 41o-412.

SCHUCK, J. M., PRUYrT, B. A. and MONCRIEI~E, J. A. (I969). Homograft skin for wound coverage. Archives of Surgery, 98, 472-479.

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Storage and skin metabolism

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