Elsevier

Burns

Volume 31, Issue 1, February 2005, Pages 25-29
Burns

The Viennese culture method: cultured human epithelium obtained on a dermal matrix based on fibroblast containing fibrin glue gels

https://doi.org/10.1016/j.burns.2004.06.015Get rights and content

Abstract

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin glue gel containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on to the gel. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained. After 10 days an irregular multilayer could be observed. The cells showed active interaction with the fibrin support, presenting as cell formations projecting into the matrix.

After 15 days a regular epithelial sheet consisting of three to four layers of cells was formed. A limiting membrane demarcating the keratinocytes from the fibrin matrix was discernible. Squamous differentiation similar to Strata reticulare and corneum found in vivo could be observed. Nuclei of basal cells were regularly spaced from each other and the chromatin was of homogeneous appearance without prominent nucleoli. The last time point (20 days) showed signs of disintegration of the epithelial sheet. A basement membrane-like structure could not be seen any more. Detachment of the basal cells was associated with subepithelial vacuoles. Basal cells contained irregular nuclei. Therefore, we conclude that 15 days of culture were optimal for the generation of a keratinocyte layers with signs of differentiation; this new culture system could be an important step forward in covering severely burned patients due to a number of advantages, as for example a large expansion factor, the shortening of the optimal culture time to 15 days, the usage of commercially available fibrin glue gels and the versatile manipulation of composite cultures.

Introduction

Since the development of the method for growing epithelial sheets with the support of lethally irradiated 3T3 cells in submerged culture conditions in 1975 [1], cultured epithelium has been used as grafting material in different clinical situations such as the treatment of burn wounds [2], chronic skin ulcers [3] and oral mucosal defects [4]. This method has gained particular attention in the treatment of seriously burned patients, since it is impossible to obtain, in a short period of time, an epithelium surface, large enough to cover the needs of a patient whose skin is largely damaged [5]. Rapid and effective burn wound closure is one of the most important aspects in the treatment of burn patients, because the patient is in a sub-septic condition until all skin defects are closed.

The sheet grafts used clinically consist of cultured epidermal cells attached to gauze with surgical clips. They are usually available three to five weeks after initial biopsy. The grafts consist of three to five cell layers [6]. The surgical handling with secondary devise is delicate [7], [8]. Moreover, it has been found out that keratinocyte grafts still have persistent problems: blistering and a lack of stability. These drawbacks have limited their clinical application and provided further stimulus to the development of keratinocyte culture methods on surfaces that mimic the human dermis (dermal equivalent). Several dermal equivalents of diverse composition have been reported [9], [10], [11]. In addition to the dermal bed that is often damaged by the burn, dermal substitutes increase the possibility of graft take [12]. However, the keratinocyte expansion factor found under these conditions (i.e. the total surface of keratinocyte culture that can be obtained in a given period) is lower than that obtained by the traditional culture in the presence of 3T3 cells [13]. So far, this restriction and the elevated cost of materials precluded the use of dermal equivalents for seriously burned patients, who require a large skin surface within a short period. Data from several laboratories indicated the possibility of growing fibroblasts and endothelial cells on/in fibrin gels [14], [15], [16]. Moreover, autologous keratinocytes in fibrin glues suspension have been employed in the treatment of burned patients [6], [17], [18], [19] and acellular fibrin gels have been used as biological support for keratinocyte cultures [20]. Based on these reports, we investigated the possibility that fully commercially available fibrin gels enriched with human fibroblasts could act as a dermal substitute for keratinocyte culture. Bearing in mind the possible use of this type of culture for the treatment of seriously burned patients, we have evaluated our system in regard to architecture, expansion rate and handling of the composite culture for grafting purposes.

Section snippets

Primary keratinocyte culture

To obtain primary keratinocytes, normal human keratinocytes derived from adult skin donors or from burn patients, where a small particle of skin is harvested within first the 3 days (at the first operation), were isolated following previously described methods [5]. The skin should be harvested as soon as possible in order to avoid hospital acquired bacterial infection. The skin particle is incubated in trypsin-EDTA for 18 h at 4 °C in order to obtain individual cells. Keratinocytes were cultured

Fibrin/fibroblasts gels

Within 10 days of culture a confluent epithelium multilayered was achieved. In all cultures, it was possible to peel off the fibroblast/fibrin glue/keratinocytes sheets from the culture flask without the use of enzymes.

Morphological and differentiation features of keratinocytes growing on fibrin fibroblasts gels

At time point one (10 days) an irregular keratinocyte multilayer could be observed. A delicate extracellular structure was deposited between cells and matrix. Mitoses were regular and frequent. Nuclei were located centrally and nucleoli easily visible. The cells showed active

Discussion

At the moment, several technologies are under active development as aids in cutaneous wound repair. However, many of them deal with a single aspect of the healing process that does not necessarily serve as primary source of new tissue. The biochemical and cellular composition of the composite graft described in this paper closely resembles that of skin during the first steps of wound healing. In fact, during the healing of a skin wound, the defect is temporally plugged with the formation of a

References (21)

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