Vasiliev A.V.,¹ Kiseliov I.V.,² Ivanov A.A.,³ Fedorov D.N.,³ Smirnov S.V.,² Terskikh V.V.¹

¹ Institute of Development Biology, Russian Academy of Sciences, Moscow, Russia
² Sklifosovsky Research Institute of Emergency Care, Moscow
³ Institute of Molecular Medicine, Moscow Medical Academy, Moscow

SUMMARY. The aim of this study was to evaluate the initial viability of cadaver skin that was sufficient for the cryopreservation of viable grafts. We also studied the effect of cryopreservation on the pre-graft viability of skin on the basis of the viability of isolated keratinocytes. Various methods of evaluation of skin viability were compared: trypan blue exclusion, incorporation of labelled leucine, and growth of isolated keratinocytes in culture. The histology of the skin samples was also assessed. The results presented show the correlation between trypan blue counts and the capacity of epidermal keratinocytes to grow in culture. We suggest that within certain limits of trypan blue counts, cadaver skin retains viability sufficient for the cryopreservation of viable grafts. We also found that short-term banking is possible in clinics at about -70 °C.

Introduction

A major goal in the treatment of burn wounds is the creation of a protective skin barrier that provides hydro-, gas-, and heat-exchange. Among the numerous artificial and biological wound dressings, skin allografts are the most widely used. The general recognition of the need for viable human allografts has stimulated the establishment of skin banking facilities and research to improve methods of harvesting, processing, storing, transporting, and evaluating subsequent performance. The development of reliable methods to cryopreserve viable skin grafts is important for the capacity to develop tissue banks for the ready supply of skin grafts.

The term “viability” is widely used in assays for preserved cells, tissues, and organs. Viability may be defined as the ability of preserved skin samples to exhibit some specific functions expressed as a proportion of the same functions in the same samples before treatment, or in identical samples before treatment.1 Many studies emphasize the importance of using a multiparametric approach to assess the viability of cryopreserved biological wound dressings.2,3

The aim of the present study was to evaluate the initial viability of cadaver skin that was sufficient for the cryopreservation of viable grafts. We also studied the effect of cryopreservation on the pre-graft viability of skin on the basis of the viability of isolated keratinocytes.

The viability of skin samples was determined by three methods: trypan blue exclusion, incorporation of labelled leucine, and growth of isolated keratinocytes in culture. The histology of the skin samples was also assessed. In the present study we evaluated the viability of cadaver skin obtained at different terms post mortem as well as fresh skin preserved with several methods. We suggest that the ability of keratinocytes to proliferate in culture is the main criterion of skin viability.

Materials and methods

Preparation of skin samples and graft preservation

Human skin obtained by plastic surgery and cadaver skin were used in the experiments. The viability of fresh skin was assessed on the day of sampling. If necessary, excessive dermis was removed by scalpel before the skin was preserved. The viability of the preserved skin was studied at different terms from 5 to 90 days after initiation of preservation. The skin was stored in glass flasks according to different protocols: in 199 medium with glycerol (10%, 15%, and 20%) and EGF (from 10 to 100 ng/ml) at -18 and -70 °C. Gentamycin (0.16 mg/ml) was added to solutions to prevent bacterial contamination. Before skin was frozen to -70 °C the flasks were placed in foam plastic boxes with walls 1 cm thick. The samples were thawed at 40-42 °C and washed with Eagle’s medium.

Evaluation of skin viability

1. Keratinocyte cultures

Keratinocytes were obtained and cultured according to the method described by Rheinwald and Green4 with some modifications. In brief, epidermal keratinocytes were isolated by overnight trypsinization at 4 °C, followed by additional treatment with diluted trypsin to allow dermo-epidermal separation. Epidermal suspension was obtained by pipetting after inhibition of trypsin activity by 5% bovine serum. Keratinocytes were resuspended in the growth medium and seeded at a concentration of 106/ml. The growth medium consisted of DMEM:F12 (1:1) mixture supplemented with 10% foetal calf serum, 10 ng/ml EGF (Sigma), 5 ng/ml insulin (Sigma), 10-6 M isproterenol (Sigma), and 5 ng/ml sodium selenite (Sigma). Keratinocytes were cultivated without a feeder layer in 35 cm2 flasks (Costar) coated with collagen. The medium was changed every other day and cultures were kept at 37 °C in a humid atmosphere incubator containing 3% CO2.

2. Trypan blue test

Equal volumes of cell suspension and 0.4% trypan blue solution were mixed and, under a light microscope, blue-stained cells were counted as nonviable. This test was performed before cultivation of keratinocytes.

3. Protein synthesis

Keratinocyte suspension was incubated with C14-leucine (4uCi/ml) for 3 h. The cells were then twice washed with Hanks solution and fixed by incubation in 5% solution of trichloracetic acid for 10 min followed by two washings with Hanks solution. After this the cells were lysed in 0.1 N NaOH and the radioactivity of incorporated leucine was determined by counter.

4. Histological analysis

Skin samples were fixed in Karnovsky mixture (2.5% glutaraldehyde and 4% paraformaldehyde), post-fixed in 1% osmium tetroxide, and embedded into araldite-M. Semi-thin sections of 1 um thickness were stained with methylene blue, azure II, and basic fuchsin.

Results

We subdivided the patterns of culture formation into three types.

The first type was characterized by certain stages. The adhesion and spreading of single cells and small clusters of keratinocytes were completed within some hours of inoculation. The migration of cells from the clusters was then observed: small keratinocyte colonies were formed by day 2. On days 4-6, some cells detached owing to differentiation. By days 8-10, active growth of colonies was observed. The colonies finally fused, and multilayered confluent keratinocyte sheets were formed by days 16-20.

This pattern of culture formation is typical of cells obtained from fresh skin and is used for the production of keratinocyte sheets for subsequent transplantation.

In the second case, the formation of keratinocyte sheets was not completed. The number of attached cells was less than in the previous case, few colonies were formed, and less than 50% of the flask area was covered with cells. During the subsequent cultivation, necrotic foci were observed in the centre of the colonies and the culture was involuted by days 7-10.

This pattern of culture growth probably depends on the small number of cells capable of colony formation. It is possible that in this case the majority of keratinocytes are committed to terminal differentiation. Cell death thus prevails over growth. It is however be possible that the inoculation of higher cell numbers might result in the formation of multilayered sheets.

In the third variant, single cells and cell clusters attached to the plastic, and migration of some cells from the clusters, were observed. By day 5 of cultivation all the keratinocytes had detached. We believe that this pattern of growth may indicate that the skin samples were non-viable. However, primary attachment and migration of some cells showed that a certain percentage of cells were viable in these samples.

In dead skin samples no attachment of cells was observed.

To quantify the viability of skin samples, we compared the patterns of keratinocyte growth in culture and the results of the trypan blue exclusion test in the cell suspension used for cultivation.

A correlation between the percentage of viable cells and the results of cultivation is presented in Table I. We found that multilayered confluent epithelial sheets were formed when the average number of non-stained keratinocytes was 83 ± 8% (type I). When non-stained cells averaged 65 ± 10%, less than 50% of the flask area was covered with cells (type II). Statistical analysis of these two samples showed that the difference between them was not significant (p > 0.05).

When the number of viable cells was 53 ± 4%, some cells attached to the plastic but died by day 5 (type III). If the number of non-stained keratinocytes amounted to 23 ± 18%, the cells did not adhere to the flask surface. Statistical analysis of these samples indicated their reliable difference from each other (p > 0.05) and from the above two groups.

The effectiveness of cell cultivation from cryopreserved skin samples in repeated experiments depended on the initial number of viable cells. The higher the initial number of viable cells, the better the results were.

We also investigated incorporation of 14C leucine to evaluate skin viability. When cells covered less than 50% of the flask area, the level of the incorporated leucine was four times lower than when a multilayered epithelial sheet was formed (Table II). These groups were reliably different (p < 0.05).

We found no differences in skin histology, irrespective of the storage solution and the cryopreservation protocol. The number of viable keratinocytes and the results of their cultivation were however different. In cryopreserved skin, keratinocytes with lysed cytoplasm and centred nuclei surrounded by light thin rim were found, which was not the case in fresh skin (Figs. 1-3). The number of such cells varied in different loci irrespective of the preservation protocol used.

As cadaver skin is the main source of viable grafts, we evaluated skin viability at different terms post mortem. We also took into account the fact that cadaver skin can be set up in culture followed by transplantation.

We compared the viability of skin from 20 donors harvested between 3 and 21 h post mortem (Table III) with that of skin obtained during plastic surgery (Table IV).

Table III shows that multilayered epithelial sheets can be obtained from skin taken within 17 h post mortem. Isolated keratinocytes had a high level of 14C leucine incorporation and the number of trypan blue excluding cells was not less than 70%. The formation of these keratinocyte cultures was similar to that of cultures obtained from fresh skin. Comparison of the data presented in Table IV shows that cadaver skin taken within 17 h post mortem corresponds to fresh skin and can be used for the banking of viable skin grafts.

Discussion

The clinical use of cryopreserved human allograft skin has expanded dramatically in recent years. Although it is unclear whether viability of the allograft is really necessary for its function as a biological dressing, it is nonetheless widely accepted that living allograft is superior to all other dressing materials. It has been suggested5 that allogeneic skin graft may be used as “pharmacological agent”.

Strict viability criteria are necessary for the storage and utilization of viable skin grafts. This problem is still urgent as the various criteria for the evaluation of skin viability prevent comparison of the various methods of preservation (tissue culture, vital dye exclusion, radiolabelled nucleotide uptake, enzymatic assays, oxygen consumption, nitroblue tetrazoleum absorption, histological assay). The reliability of some methods, that have been used in the past is debatable.

The general disadvantage of viability tests is that they measure only a single function. Many studies consequently emphasize the importance of using a multiparametric approach to assess the viability of cryopreserved biological wound dressings.1

The trypan blue dye exclusion test is a proven method for determining cell viability. Rapid intracellular blue staining is considered a sign of membrane damage and cell death. But it does not provide exact information concerning the proliferative potential of keratinocytes. That is why we used keratinocyte growth in culture as a complex index of cell viability. We therefore compared the results of the trypan blue test with the capacity of cells to grow in culture. The keratinocyte population is highly heterogeneous and consists of a small fraction of clonogeneic stem cells, transit-amplifying cells, and differentiated cells, while the formation of keratinocyte cultures depends in the first place on stem cell proliferation. The high viability determined by the trypan blue test in the whole population does not correspond to the high proliferative potential of keratinocytes in separate fractions. The use of this test in combination with keratinocyte cultivation provides more adequate information about skin viability levels. On the basis of the relationship between the percentage of cells excluding trypan blue and cultivation effectiveness, we obtained a reliable proportion of viable keratinocytes that could indicate skin viability. We believe however that actual trypan blue counts may be higher because the trypsinization of skin takes many hours and increases cell damage.

We showed that multilayered confluent epithelial sheets formed when the average number of keratinocytes excluding trypan blue was high (not less than 70%). If the number of viable cells was in the range of 50-70%, the cultures formed were not confluent. We consider that both types of culture pattern indicate the viability of skin samples. We believe that in the latter case the lower percentage of stem cells capable of proliferation was retained.

The samples in which the number of viable cells was less than 50% were unable to produce keratinocyte cultures, and we considered these samples to be nonviable. The presence of cells not stained with trypan blue also indicates that there were some viable cells in these samples. In this case we observed culture involution after initial attachment and migration of a small proportion of the seeded cells. It is possible that in this case the majority of keratinocytes are committed to terminal differentiation. Such skin cannot however be considered to be completely nonviable.

We define skin graft viability as the capacity to give rise to keratinocyte culture. The method of keratinocyte cultivation is in our opinion the most appropriate for skin viability evaluation because keratinocyte is the major element of the skin. This method is a reliable indicator of skin viability in the course of preservation.

It has been shown that the proliferative potential of basal cells in cultured keratinocyte sheets decreases in the course of preservation.6 As these cells are the main clonogeneic population of cultured keratinocytes, it may be supposed that different variants of culture growth are also caused by a decrease in the proliferative potential of keratinocytes in skin after preservation.

As demonstrated in Table II there is a relationship between trypan blue data, cultivation reliability, and the incorporation of labelled leucine in keratinocyte cultures. However, when nonconfluent keratinocyte sheets covered less than 50% of the flask area, the level of protein synthesis was almost three times lower than when a multilayered epidermal sheet was obtained. At the same time, the number of viable cells decreased, but not significantly.

It is known that the parameters of viability based on a determination of metabolic processes can be measured more accurately, even if in this case the prediction of cell survival is somewhat indefinite.7 We suggest that a reduction in protein synthesis is not a direct indication of cell viability since many forms of metabolic repression are reversible.

Our data also indicate that histological inspection of skin samples is insufficient to determine their viability. This is consistent with the data of Richters et al.,8 who studied skin preserved in 85% glycerol and showed that its structure did not change. The keratinocytes were however dead.

The post-mortem delay period may need to be set at a pragmatic level until there is more evidence regarding effects on clinical performance. Our results regarding the evaluation of cadaver skin viability are consistent with the data of Wester et al.,9 who showed high skin viability determined by glucose consumption within 18 h post mortem. Also according to the data of May,10 skin can be used within 18 h post mortem. The Michigan University Skin Bank programme recommends that to obtain viable cells, skin should be removed within 12 h of death,11 while the recommendation of the British Association of Skin Banks is that cadaver skin should be taken within 48 h post mortem, but that it is better to reduce the time to 24 h.12

We presume that skin viability gradually decreases in time after death. However, it is not always possible to determine accurately the time interval between death and its medical registration. Another consideration is that the duration of the period when skin remains viable depends on the rate of cadaver cooling, which is determined by ambient temperature and differs according to the season and other conditions. Individual differences should also be taken into account.

Conclusion

Epidermal keratinocyte cultivation, together with the trypan blue test, thus provides adequate evaluation of skin viability. These methods complement each other and provide both a qualitative and a quantitative evaluation of skin viability. The correlation between cultivation results and the number of live keratinocytes makes it possible to use the figures of live cells obtained as criteria for skin viability. We also conclude that the number of live keratinocytes determined by the trypan blue test makes it possible to predict the viability of skin grafts after preservation.

RESUME. Les Auteurs dans cette étude ont évalué la viabilité initiale de la peau cadavérique qui était suffisante pour la cryopréservation de greffes viables. Ils ont en outre étudié l’effet de la cryopreservation sur la viabilité pré-greffe de la peau sur la base de la viabilité des kératinocytes isolés. Diverses méthodes pour l’évaluation de la viabilité de la peau ont été confrontées: l’exclusion du bleu trypan, l’incorporation de la leucine étiquetée et la croissance de kératinocytes isolés en culture. L’histologie des prélèvements a été aussi évaluée. Les résultats présentés démontrent la corrélation entre les comptes du bleu trypan et la capacité des kératinocytes épidermiques de croître en culture. Les Auteurs expriment l’hypothèse que, entre certains limites des comptes du bleu trypan, la peau cadavérique maintient une viabilité suffisante pour la cryopréservation de greffes viables. Selon les Auteurs, la conservation à court terme dans la banque de la peau est possible dans les cliniques à environ -70 °C.

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