Expansion Of Keratinocytes Research Articles

Management of the burn patient with human epidermal allograft cultured in vitro

In Mexico a biological dressing was developed based on the culture of human keratinocytes in vitro. As a result, these procedures allow the expansion of keratinocytes in approximately 10,000 times their initial number, used in the treatment to cover the areas affected by burns, being a more important factor in obtaining this adequate result in the healing with its respective advantages both in social and economic terms.

Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro.

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

566 DermaCult™ Keratinocyte Expansion Medium, a Serum- and BPE-free Medium Supporting Long-term Feeder-free Expansion of Primary Human Epidermal and Pluripotent Stem Cell-Derived Keratinocytes

Human epidermal keratinocytes (HEKs) typically undergo growth arrest after 15 - 30 population doublings (PDs) when maintained in traditional feeder-free culture medium formulations. To circumvent this early senescence, we developed DermaCult™ Keratinocyte Expansion Medium (KEM), a serum- and bovine pituitary extract (BPE)-free medium that promotes long-term growth (> 30 PDs) of HEKs while maintaining their air liquid interface (ALI) differentiation potential. To measure expansion over the lifespan of the culture, HEKs derived from neonatal (HEKn) or adult (HEKa) skin were thawed and seeded onto uncoated tissue cultureware at a density of 1 - 2.5 x 103 cells/cm2, and passaged once the cultures reached approximately 50 - 70% confluence.

Keratoacanthoma, Verruca Vulgaris Arising over Porokeratosis of Mibelli : A New Association

Introduction: This case report highlighting the presence of keratoacanthoma and verruca vulgaris over the lesion of porokeratosis of mibelli. Case Report: In this case a 50-year-old housewife came with large well-defined asymptomatic plaque over the upper part of back of the trunk for last 10 years. After evaluation we got the presence of keratoacanthoma and verruca vulgaris on the plaque of porokeratosis of mibelli. Discussion: Porokeratosis is a clonal expansion of keratinocytes. Among the neoplasm squmous cell carcinoma (SCC) is the most commonly reported malignancy in porokeratosis. But in our case we didn’t find lesion of SCC. Porokeratosis of Mibelli, Keratoacanthoma and Verruca vulgaris have an association with human papilloma virus(HPV). This eithopathogenesis may be related to our clinical finding. Conclusion: There were reporting of squmous cell carcinoma or keratoacanthoma on Porokeratosis of Mibelli. But in our case we found both Keratoacanthoma and Verruca Vulgaris over Porokeratosis of Mibelli. Medicine Today 2021 Vol.33(2): 172-175

Abstract 2675: Clonal expansion of skin keratinocyte

Abstract Skin is the largest organ system and is exposed to ultraviolet (UV) or sun light, a well-known mutagenic insult in various skin cancers, depending on the body site. A previous study showed that keratinocytes in sun-exposed eyelids harbor a high number of somatic mutations affecting a number of common driver mutations found in skin cancers, including NOTCH1, NOTCH2, NOTCH3, FAT1, TP53, and RBM10, suggesting pervasive expansion of clones before the development of skin cancer. However, clonal expansion in other skin sites in association not only with UV but also other insults, such as chronic inflammation is yet to be evaluated. Thus, to fully characterize the clonal expansion in the skin, we performed sequencing analysis of apparently normal skin, as well as that affected by chronic inflammatory conditions such as psoriasis and atopic dermatitis. We analyzed small fragments (0.2mm2) of epidermal samples accrued from surplus biopsy materials using exome sequencing. Analysis of bulk epidermal samples revealed a median of ~30 mutations/exome. Sun-exposed skin was notable for a high mutation burden (>200 mutations/exome). On the basis of the analysis of their variant allele frequencies, the median clone size was estimated to be ~0.1mm2. Several cases harbored large of clones that spanned multiple samples, which were invariably found in sun-exposed skin. In one of such cases, a clone spanned a region having more than 2mm in diameter. Skin affected by chronic inflammation showed a higher number of mutations. dN/dS analysis revealed positively selected driver genes that were similar to the previous report. Diseased skin showed a similar mutation profile that is seen in apparently normal sun-exposed skin. Analysis of mutational signature disclosed signatures that are related ageing (SBS1) and UV light exposure (SBS7). The contribution of SBS7 was prominent in the sun-exposed area but also detected in areas not exposed to sun light, such as the back, buttock, or thighs. By contrast, inflammation-affected skin samples are characterized by a large contribution of the SBS1 signature, likely reflecting increased cell cycles. In conclusion, our analysis shows UV light is a relevant driver of clonal expansion of skin keratinocytes. Chronic inflammation accelerates an accumulation of somatic mutations in and/or clonal expansion of keratinocytes. Citation Format: Yoshihiro Ishida, Nobuyuki Kakiuchi, Yoichi Fujii, Tomonori Hirano, Yoshikage Inoue, Tomomi Nishimura, Tatsuki Ogasawara, Hirona Maeda, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Atsushi Otsuka, Kenji Kabashima, Seishi Ogawa. Clonal expansion of skin keratinocyte [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2675.

Pseudocarcinomatous changes in giant porokeratosis of mibelli’: Histopathology as a valuable asset for early recognition of neoplastic changes – A case report with review

Porokeratosis is defined as a rare disorder of keratinization caused by clonal expansion of keratinocytes and characterized by an expanding thread-like border of hyperkeratotic plaques or papules with atrophic center. It is a genetically heterogeneous disorder that is mostly inherited as autosomal dominant trait. It is characterized histologically by the presence of a thin column of parakeratotic cells “cornoid lamella” representing the active border. This is a case report of a patient suffering from giant porokeratosis of Mibelli who developed secondary changes over the lesion in due course of time arousing suspicion of malignancy which turned out to be benign histopathologically. The risk factors for malignant changes are usually persistence of the lesion for long time, radiation therapy, long-term exposure to ultraviolet radiation, electron beam therapy, and immunosuppression associated with malignancies such as lymphoma, HIV infection, or due to immunomodulatory drugs.

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

Transcriptional activity of epigenetic remodeling genes declines in keratinocytes after in vitro expansion

PurposeIn vitro expansion is an invaluable method to obtain keratinocytes in amounts necessary for effective transplantation therapies. In vitro cell culturing provokes questions concerning potential epigenetic alterations occurring in expanded cells in the context of usefulness for transplantation and safety. The purpose of this study was to investigate as to whether keratinocyte expansion is associated with changes in the activity of genes responsible for the maintenance of epigenetic stability. Materials and methodsWe focussed on the transcriptional activity of genes involved in different epigenetic mechanisms including DNA methylation and histone modifications. We used quantitative real-time PCR to determine transcript levels of 16 epigenetic remodelling markers in 14 patients in the epidermal cells directly after collection and after in vitro expansion. ResultsWe observed a remarkable decline in the transcriptional activity of the epigenetic remodelling genes following in vitro expansion, while no further fall of expression with passaging. In whole skin, we found even higher expression levels of the epigenetic markers. ConclusionsTransmission to in vitro environment challenges cellular signalling and metabolism. The regulation of epigenetic remodelling maintains the balance between cellular plasticity and phenotype deviation. This preliminary research demonstrated reduced activity of genes responsible for epigenetic modifications of DNA and histones in in vitro expanded epidermal cells. This observation indicates that epigenome re-patterning in cultured epidermal cells is significantly less intensive than in the skin. Also, this observation may imply that after adaptation to in vitro conditions, the epigenome does not undergo extensive transformation during further cultivation.

Promotion of Human Epidermal Keratinocyte Expansion in Feeder Cell Co-culture.

Co-culture of human epidermal keratinocytes with mouse 3T3-J2 feeder cells, developed by Green and colleagues, has been used worldwide to generate skin autografts since the early 1980s. In addition, co-culture with 3T3-J2 cells has served as a fundamental tool in skin stem cell biology as it allows the evaluation of self-renewal capacity of epidermal stem cells. This chapter describes a recent improvement in the Green method to promote further the expansion of human epidermal keratinocytes utilizing a small-molecule inhibitor of TGF-β signaling. This new protocol enables more rapid expansion of human epidermal keratinocytes in co-culture with not only 3T3-J2 cells but also other feeder cells including human dermal fibroblasts and human preadipocytes, two major alternatives to 3T3-J2 cells, with a long-term goal of developing customized skin autografts.

245 - Development of a keratinocyte-specific defined media suitable for self-assembled bilayered skin substitute production

Skin epithelial cell (keratinocytes) culture and self-assembled skin substitutes (SASS) have greatly improved treatments for patients with severe burns. To properly culture human keratinocytes, fetal bovine serum (FBS) must be added to the medium. However, FBS exact biological composition is undefined and is known for batch-to-batch variations. FBS is also associated with a risk of xenogeneic pathogen transmission. For these reproducibility and safety reasons, discarding FBS usage in both research and clinical contexts is desirable. Although keratinocyte-specific serum-free (defined) media are commercially available, they are not effective in culturing keratinocytes for more than a few passages and are not suitable for SASS production. Therefore, our objective is to develop a defined medium optimized for keratinocyte expansion and SASS production. Three candidate molecular factors capable of substantially promoting keratinocyte growth were initially identified from an experimental screening on a single cell population. This led to the development of an effective defined medium for culturing keratinocytes (DMK). DMK was then optimized in terms of cost-effectiveness by testing different concentrations of the factors previously identified, and three DMK derivatives (α, β, and γ) were formulated. Using two additional populations, DMK and its derivatives were ranked over their ability to sustain culture quality. Daily population doublings, passage count, and cell size were used as proxies. Keratinocytes cultured in DMKβ were smaller and could be grown over at least as many passages as in FBS-containing medium and more passages than those cultured in DMK or its other derivatives. Also, keratinocyte population doubling rates were greater in DMKβ. Keratinocyte cultured in either DMKβ or FBS-containing medium were morphologically comparable. SASS were then produced with both DMKβ and FBS-containing medium using four anatomically distinct keratinocyte populations. A portion of the amplified keratinocytes were also used to compare complete transcriptomic profiles. Substitute histological analyses showed no clear differences between those produced with DMKβ or FBS-containing medium. Substitute similarity was further evaluated with immunolabelling of K10, K14, K19, Ki67, filaggrin, and type IV collagen. In conclusion, this new defined medium is promising for keratinocyte culture and skin substitute production for research and clinical purposes.

Optimización del cultivo de queratinocitos humanos para el desarrollo de un modelo de piel artificial humana: alternativas celulares como capa alimentadora

Purpose: This study aims to optimize keratinocyte culture to develop an artificial human skin model. For this purpose, human cells are used as feeder layer: human dermal fibroblasts and adipose derived mesenchymal stem cells. The results obtained are compared with 3T3 fibroblasts, murine feeder layer used for decades. Methods: We conducted an experimental study using human and murine sub-lethally irradiated cells as feeder layer for the establishment of keratinocyte culture. Cell expansion rate and doubling rate were evaluated in the keratinocyte cell passage and in the final cell recovery (was carried out at 3 weeks). The yield and viability of keratinocytes were also evaluated in the initial processing. Results: The results determine that irradiated human dermal fibroblasts and irradiated adipose derived mesenchymal stem cells can act as feeder layer promoting adhesion and expansion of keratinocytes. Human dermal fibroblasts provide comparable results to those obtained with murine 3T3 fibroblasts. Conclusions: Irradiated human dermal fibroblasts provide a functional feeder layer which allows effectively in vitro expansion of keratinocytes to be used for clinical purposes for the development of an artificial human skin model.

Human adipose-derived stromal cells as a feeder layer to improve keratinocyte expansion for clinical applications

The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

In vitro keratinocyte expansion for cell transplantation therapy is associated with differentiation and loss of basal layer derived progenitor population

An alternative approach for traditional clinical mesh grafting in burn wound treatment is the use of expanded autologous keratinocytes in suspension or sheets that are cultured over 2–4 weeks in a remote service facility. While a wound reepithelialization has been described, the functional and aesthetic outcome is under debate. Cell isolation from split-skin donor tissue aims to preserve the valuable stem cell progenitors from the basal epidermal layer and to provide patients with a rapid wound reepithelialization and a satisfying outcome.While the presence of epidermal progenitors in the cell graft is thought to enable an improved epidermal surface post reepithelialization, we investigated a feasible clinical approach involving cultured versus noncultured epidermal cells comparing the α6inthigh/K15high/FSClow/SSClow and α6inthigh/K5high/FSClow/SSClow keratinocyte progenitor subpopulations before and after in vitro culture process. Our results show a significant increase of cell size during in vitro passaging and a decrease of progenitor markers linked to a gradual differentiation. A provision of the regenerative epidermal progenitors, isolated from the split-skin biopsy and applied directly onto the wound in an on-site setting of isolation and cell spray grafting in the operation room, could be of interest when choosing options for skin wound care with autologous cells.

The effect of isolation and culture methods on epithelial stem cell populations and their progeny—toward an improved cell expansion protocol for clinical application

Background aimsThe use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. MethodsWe compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. ResultsDuring the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. Discussion.Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.

Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

IntroductionThe development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment.MethodsIn this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry.ResultsPolybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene.ConclusionsThis study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Rho Kinase Inhibitor Y-27632 Prolongs the Life Span of Adult Human Keratinocytes, Enhances Skin Equivalent Development, and Facilitates Lentiviral Transduction

The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.

Entwicklung eines transplantierbaren Hautäquivalentes auf Basis von Matriderm®mit menschlichen Keratinozyten und Fibroblasten

A cell-based wound coverage with keratinocytes and fibroblasts on the basis of a commercially available dermal substitute (Matriderm ((R)), Kollagen/Elastin matrix) was generated, in order to treat wide burn wounds. First the expansion of keratinocytes was optimised and the culturing time was minimised. Raw material was 1-2 cm (2) split skin. Dermis and epidermis were separated by enzymatic treatment with thermolysin. After treatment of both compartments with trypsin and collagenase I, keratinocytes and fibroblasts were isolated and expanded in collagen I coated dishes. After 10 days fibroblasts were seeded on Matriderm ((R)). After cultivation of the fibroblasts-containing matrix for one week keratinocytes were seeded on top. After an additional week of submersed cultivation the matrix was lifted up to the air-liquid interface to initiate epidermal cell differentiation. After 16 days in the air-liquid interphase the matrix was fixed and underwent immunohistochemical and electron microscopic analysis. Histological analysis showed a regularly stratification of the epidermal part. We observed collagen IV, a marker for the basement membrane, between epidermis and dermis. Desmoglein and the differentiation markers involucrine and cytokeratin 10 were found in the suprabasal layers of the epidermis. Electron microscopic analysis showed the basement membrane in the epidermal junction zone as well as cell-cell connections in the form of desmosomes. Late differentiation characteristics, like granular structures and the cornified layer, were found in the stratum granulosum and stratum corneum. Our results demonstrate that a skin equivalent can be generated by using a collagen/elastin matrix, with an expansion rate of 50-100-fold. This skin equivalent may be useful for covering deep wounds.