Garcia Fernàndez E.,(1) Bejar J.M.(2) Maruri M.,(1) Garcia Masdevall M.D.,(1) Camarero C.(3) Gabilondo FJ .(2)

(1) Department of Immunology, Hospital cle Cruces, Baracaldo, Vizcaya, Spain
(2) Department of Plastic Surgery and Burns, Hospital cle Cruces
(3) Department of Pathological Anatomy, Hospital cle Cruces (Basque Health Service)

SUMMARY. The cutaneous coverage of large and deep skin defects secondary to burn wounds or certain diseases is a complex surgical problem. Normal human keratinocytes can be serially cultured in vitro, and in appropriate culture conditions give rise to a stratified squamous epithelium. Although no stratum corneurn is developed in vitro, these cultures enable us to study early events in the development, proliferation, and differentiation of human keratinocytes. For a better understanding of these findings, a study was made of cell viability and the histological (morphological analysis) and immunohistochemical picture in epidermal sheets, tested at different culture times, as compared with normal human skin from foctuses, newborns, and adults. It was observed that cultures a few days old are similar to foetus and newbom skins as regards immunohistochemical expressivity (but not the number of cell layers). Adult skin is more similar to secondary cultures as regards its superficial differentiation, slight positivity to involucrin, and larger number of cell layers, although the cultures never present such a high number of cell layers as adult skin.

The cutaneous cover of large and deep skin defects secondary to burn wounds and certain diseases presents a complex surgical problem. Normal human keratinocytes can be serially cultured in vitro,' and in appropriate culture conditions give rise to a stratified squamous epithelium.' Cultured human epithelium possesses a site-specific differentiation programme which is expressed after grafting, independently of the receiving body site. The growth and differentiation of keratinocytes and the development of a new dermo-epidermal junction are influenced by culture techniques and epithelial sheet handling. For a better understanding of the development, proliferation, and differentiation of in vitro cultured human epidermal cells, a study was made of cell viability and the histological (morphologic analysis) and immunohistochemical picture in epidermal sheets, tested at different culture times.

Culture of human epidermal cells
The skin samples, biopsies, and skin grafts were placed in a culture medium and transferred to the laboratory for cultivation. They were washed twice in DMEM (Gibco BRL Co.) with 10% bovine foetal serum (Gibco BRL Co.) and antibiotics (penicillin 100 u/ml, streptomycin 0.1 mg/ml and Fungizone 0.25 ug/ml) (Sigma Chemical Co.). After removal of as much subcu-aneous tissue and dermis as possible, the tissue was cut into smaller fragments, which were digested overnight with 0.17% trypsin solution (Seromed. Biochrom KG). This was followed by treatment with trypsin-EDTA (25% - 0.02 mM) solution (Seromed. Biochrorn KG) to ensure dissociation into single cells. The single cells were then washed, and after centrifugation and suspension in complete medium the viability of epidermal cells was determined by trypan blue (Sigma Chemical Co.) dye exclusion.
Plastic tissue-culture 75 cm' flasks (Costar Co.) already containing 2.5 x 10' lethally irradiated murine 3T3 fibroblasts (European Collection of Animal Cell Cultures) were inoculated with over 3 x 10' epithelial cells/cml. The cultures were fed with a 3:1 mixture of the Dulbecco-Vogt modification of Eagle's medium and Ham's F-12 medium (Gibco BRL Co.). Supplements were as follows: foetal calf serum 10% (Gibco BRL Co.), adenine 24,ug/ml (Sigma Chemical Co.), epiden-nal growth factor 10 ng/ml (Sigma Chemical Co.), hydrocortisone 0.5 ng/ml (Sigma Chemical Co.), insulin 5 i)g/i-nl (Gibco BRL Co.), cholera toxin 6 ng/ml (Gibco BRL Co.), transferrin 10 i)g/nal (Gibco BRL Co.) and 3,3',5-triodothyronine 1.3 ng/ml (Sigma Chemical Co.). The cultures were incubated at 37 'C in a humid 95% C02 environment (Selecta Co.); the medium was changed every third day.
To prepare secondary cultures, subconfluent primary cultures were trypsinized and the cells were transferred to flasks of the same size containing irradiated 3T3 cells. These cultures were maintained in the same way as the primary cultures.

For the cell viability study we used 32 epidermal sheets tested at different culture times. The cells were detached with trypsin solution from 75 CM2 plastic flasks and cell viability was determined by trypan blue dye exclusion.

We studied three primary cultures after 15 days of evolution, three primary cultures after 30 days, and four secondary cultures after at least 30 days. As control samples we studied foetus (29 weeks' gestation), newborn, and adult skin obtained from the Service of Pathological Anatomy (Hospital de Cruces).
For the morphological analysis the medium was decanted off and the cultures were washed three times with sterile PBS (Gibco BRL Co.). A solution of dispase 11 (Boehringer Mannheim) was added to cover the entire cell sheet for incubation at 37 'C for 30 min. The detached epidermal sheets were placed in sterile petri dishes and the dispase was removed by washing carefully three times with PBS.
The sheets were fixed by the addition of 10% buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin-eosin for optic microscopic examination. In these sections, the number of cell layers and the basal, intermediate and superficial distribution were analysed.

The sections of paraffin-embedded were cleared in xylene, rehydrated in a graded series of alcohol rinses, and reacted with monoclonal antibodies: AEI (acidic cytokera tins of low molecular weight which mark the epidermal basal layers) (BioGenex Laboratories), AE3 (basic cytoke ratins of the suprabasal and occasionally basal layer') (BioGenex Laboratories), 5D3 cytokeratins 8, 18, and 19 (marker indicating columnar cell differentiation and glan dula,r epithelium) (BioGenex Laboratories), and involucrin(protein component of the cross-linked envelope synthesized by maturing cells of human stratified squamous epithelium, reflecting no , rmal suprabasal differentiation9) (Biomedical Technologies Inc.). The visualization system used was LSAB, marked with peroxidase and development was with AEC (AminEthyl-Carbazol).

Results

The results at different culture times by optic microscopy suggest that the greater the number of days the cells stay in culture, the higher the average number of cells after flask trypsinization. Cell viability on culture days 5 and 8 is very low (57.1% and 66.4% respectively) but on successive days viability improved in value to 80-100% in samples (Table 1).

During the early days of growth in culture, the keratinocytes proliferated as small colonies which coalesced into confluent sheets of cells within 15 days. The cultures could then be lifted from the plastic as an intact sheet. Histological sectioning of a 15-day-old epidermal cell sheet revealed that the cultures had a thickness of between one and three cell layers. The primary cultures of a 30-day-old sheet were slightly more stratified (two to four cell layers), while secondary cultures at least 30 days old attained thicknesses ranging between four and six cell layers.
During the latter stage of growth, secondary cultures were composed of small rounded basal cells attached to the plastic substrate and suprabasal layers consisting of enlarged, irregularly shaped vacuolated cells resemblimg the stratum germinativurn and spinosum. Although no stratum corneum developed in these cultures the cells retained the capacity, once grafted, to differentiate terminally.
Optic microscopy showed that epithelial thickness slightly increased in relation to the culture time and the passage number (primary and secondary cultures). The basal layer (Fig. 1) was also more evident and organized in relation to the culture time and the passage number, i.e., the epithelium appeared to have more organization. The expression of basal and immediately suprabasal keratins was however highest in 15-day-old primary cultures and decreased in 30-day-old primary cultures and in secondary cultures.

Fig. 1 - Histological sectoring: a) 15-day-old primary culture b) 30-day-old primary culture c) secondary culture at least 30 days old Note the basal layer and the keratholyne granules in intermediate layer cells and the most superficial cells

The epithelial differentiation which appeared in the expressivity of intermediate layers was highest in 30-dayold primary cultures and decreased in secondary cultures.

Involucrin showed a similar evolution to epithelial thickness in relation to culture time, but always with slight intensity as it was observed as a weak lineal image.
An occasional finding in the first and second cultures (respectively 15 and 30 days old) obtained from the same sample was the presence of a few keratohyaline granules in the cells of the intermediate layer and in most superficial cells. This was not evident in the other samples. A slight growth of non-epithelia] dermal connective tissue was observed in one sample only (from a secondary culture). With respect to the control samples, foetus and newbom skins presented the highest immunohistochernical expressivity (always more in foetus skin) but not in the cell layers. The adult skin presents more cell layers and less expressivity in basal and suprabasal layers but more superficial differentiation, with high positivity to involucrin.

A comparison of cell cultures with control skin indicates that cultures a few days old are similar to foetus and newborn skin with regard to immunohistochemical expressivity (but not as regards the number of cell layers). Adult skin is more similar to secondary cultures in view of its superficial differentiation, slight positivity to involucrin, and greater number of cell layers (the cultures never in fact present as many cell layers as adult skin).
The number of cells derived from each primary tissue source that we obtained on day 8 was approximately I x 101 cells/cml and after at least 30 days 12 x 101 celIS/CM2. These values are in the range of cells obtained from each primary tissue source. When the epidermal sheets were detached from the flasks by dispase, they presented an organization and structure that were simpler and less differentiated than normal epidermis. The basal layer was better preserved in secondary cultures, appearing as a single line, with cubic or column-shaped cells.
The suprabasal cells were poorly organized and formed irregular layers varying in thickness in the same cell and from sample to sample (2-7 cell layers). These cells were prismatic, tending to flatten horizontally in the more superficial layers.

Fig. 2 - Histological sectoring: a) foetus skin (29 weeks' gestation) b) newborn skin c) adult skin

In the most external layer, the squamous layer, the cultures showed one or two lines of flattened cells with nuclei and organelles in variable states of degeneration.

In conclusion, cultured epithelial sheets show a similar structure to that of the epidermis from which they are derived, even when the stage of differentiation is not complete. The number of cell layers in the intermediate layer is usually lower, the granulous layer is absent, and the superficial cells show a lower level of keratinization. This partially differentiated sheet retains its capacity of further differentiation when grafted on to the wound. 12-11 This is very important for establishing a mechanical barrier and linking the grafted sheet to the underlying connective tissue. When grafted, the resulting constituted epidermis forms a barrier to bacterial penetration and dehydration.

RESUME. La couverture cutanée des défauts gros et profonds de la peau dus aux brûlures ou à certaines maladies présente un problème chir-urgique complexe. Les kératinocytes humains normaux peuvent être cultivés in vitro en manière sérielle, et dans les conditions appropriées ils produisent un épithélium pavimenteux stratifié. Bien que le stratum corneuni ne se développe pas in vitro, ces cultures nous permettent d'étudier les phases précoces du développement, de la prolifération, et de la différentiation des kératinocytes humains. Pour mieux comprendre ces données, les Auteurs ont étudié la viabilité et l'aspect histologique (analyse morphologique) et immunohistochimique des lambeaux épidermiques, testés à divers moments de la culture, en comparaison de la peau humaine normale obtenue de foetus, nouveauxnés, et adultes. Ils ont observé que les cultures de quelques jours sont similaires à la peau des foetus et des nouveaux-nés pour ce qui concerne l'expressivité immunohistochimique (mais non le numéro des couches cellulaires). La peau des adultes est plus similaire aux cultures secondaires pour ce qui concerne sa différemiation superficielle, sa légère positivité à l'involucrine, et le numéro supérieur de couches cellulaires, même si les cultures -ne présentent jamais un numéro tellement élevé de couches cellulaires comme la peau des adultes.

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