Maria Beatriz Puzzi

Model for human skin reconstructed in vitro composed of associated dermis and epidermis

CONTEXT AND OBJECTIVE The technique of obtaining human skin with dermis and epidermis reconstructed from cells isolated from patients can enable autologous skin grafting on patients with few donor sites. It also enables in vitro trials on chemicals and drugs. The objective of this work was to demonstrate a method for obtaining human skin composed of associated dermis and epidermis, reconstructed in vitro. DESIGN AND SETTING Experimental laboratory study, in the Skin Cell Culture Laboratory of Faculdade de Ciências Médicas, Universidade Estadual de Campinas. METHODS Cells from human fibroblast cultures are injected into bovine collagen type I matrix and kept immersed in specific culturing medium for fibroblasts. This enables human dermis reconstruction in vitro. On this, by culturing human keratinocytes and melanocytes, differentiated epidermis is formed, leading to the creation of human skin composed of associated dermis and epidermis, reconstructed in vitro. RESULTS We showed that human skin composed of associated dermis and epidermis can be successfully reconstructed in vitro. It is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with cells and extracellular matrix organized in parallel to multilayer epidermis. CONCLUSIONS It is possible to obtain completely differentiated human skin composed of associated dermis and epidermis, reconstructed in vitro, from injection of human fibroblasts into bovine collagen type I matrix and culturing of human keratinocytes and melanocytes on this matrix.

Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

CONTEXT Recent progress in the field of epithelial culture techniques has allowed the development of culture systems in which the reconstructed epidermis presents characteristics of morphological differentiation similar to those seen in vivo. Human epidermis reconstructed in vitro may be used as the best alternative for the in vitro testing of the toxicology and efficiency of products for topical use, as well as in the treatment of skin burns and chronic skin ulcers. OBJECTIVE To demonstrate a method for obtaining human epidermis reconstructed in vitro, using keratinocytes and melanocytes cultivated on dead de-epidermized human dermis. TYPE OF STUDY Experimental/laboratory. SETTING Skin Cell Culture Laboratory of the Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. PROCEDURE Human keratinocytes and melanocytes cultured in vitro were grown on a biological matrix (dead de-epidermized human dermis) and the system was kept at an air-liquid interface, in a suitable culturing medium, until a stratified human epidermis was formed, maintaining the histological characteristics of the epidermis in vivo. RESULTS It was histologically demonstrated that it is possible to reproduce a differentiated epidermis through keratinocytes and melanocytes cultured on dead de-epidermized human dermis, thus obtaining a correctly positioned human epidermis reconstructed in vitro with functional keratinocytes and melanocytes that is similar to in vivo epidermis. CONCLUSIONS It is possible to obtain a completely differentiated human epidermis reconstructed in vitro from keratinocyte and melanocyte cultures on a dead de-epidermized human dermis.

Immunoarchitectural characterization of a human skin model reconstructed in vitro

CONTEXT AND OBJECTIVE Over the last few years, different models for human skin equivalent reconstructed in vitro (HSERIV) have been reported for clinical usage and applications in research for the pharmaceutical industry. Before release for routine use as human skin replacements, HSERIV models need to be tested regarding their similarity with in vivo skin, using morphological (architectural) and immunohistochemical (functional) analyses. A model for HSERIV has been developed in our hospital, and our aim here was to further characterize its immunoarchitectural features by comparing them with human skin, before it can be tested for clinical use, e.g. for severe burns or wounds, whenever ancillary methods are not indicated. DESIGN AND SETTING Experimental laboratory study, in the Skin Cell Culture Laboratory, School of Medical Sciences, Universidade Estadual de Campinas. METHODS Histological sections were stained with hematoxylin-eosin, Massons trichrome for collagen fibers, periodic acid-Schiff reagent for basement membrane and glycogen, Weigert-Van Gieson for elastic fibers and Fontana-Masson for melanocytes. Immunohistochemistry was used to localize cytokeratins (broad spectrum of molecular weight, AE1/AE3), high molecular weight cytokeratins (34betaE12), low molecular weight cytokeratins (35betaH11), cytokeratins 7 and 20, vimentin, S-100 protein (for melanocytic and dendritic cells), CD68 (KP1, histiocytes) and CD34 (QBend, endothelium). RESULTS Histology revealed satisfactory similarity between HSERIV and in vivo skin. Immunohistochemical analysis on HSERIV demonstrated that the marker pattern was similar to what is generally present in human skin in vivo. CONCLUSION HSERIV is morphologically and functionally compatible with human skin observed in vivo.

Evaluation of Culture Medium for Human Keratinocytes

Human keratinocytes are needed for tissue engineering of skin applied in tissue repair and regeneration aimed at clinical application. The need to have high cell concentrations within a short time when performing cell proliferation is vital. The growth and maintenance of these cells commonly involve the use of MCDB 153 medium, which is supplemented with animal-derived components, such as growth factors and fetal bovine serum (FBS). The aim of this work was to evaluate different formulations based on MCDB 153 medium for keratinocyte proliferation. For that, several experiments were realized to define which supplements are important for skin cell culture using 24 factorial design. Skin samples were obtained from four consenting patients submitted to dermolipectomy, the plastic surgery operation. These fragments were treated with 0.25% trypsin-EDTA for four hours, at 37oC. Furthermore, the epidermis was separated from the dermis, providing skin cells. Batch cultures were performed in 6 and 96-well plates, in addition to 25 cm2 flasks. Results concerning cell growth and metabolism showed that keratinocytes were full grown up in MCDB 153 medium containing insulin (7.5 µg.mL-1), bovine pituitary extract-BPE (80 µg.mL-1), epidermal growth factor-EGF (0.08 µg.mL-1), hydrocortisone (0.63 µg.mL-1) and glutamine (1 g.L-1). On the other hand, culture media proposed in the experimental design did not resulted in satisfactory cell growth. In addition, although, the initial glucose concentration was low in the most of cases, the lactate was strongly produced by cells reaching 1 g.L-1.

Tetrasomy 3q26.32-q29 due to a supernumerary marker chromosome in a child with pigmentary mosaicism of Ito

Abstract Pigmentary mosaicism of Ito (PMI) is a skin abnormality often characterized by hypopigmentation of skin, following, in most cases, the Blaschko lines, usually associated with extracutaneous abnormalities, especially abnormalities of the central nervous system (CNS). It is suggested that this pattern arises from the presence and migration of two cell lineages in the ectoderm layer during the embryonic period and embryonic cell migration, with different gene expression profiles associated with pigmentation. Several types of chromosomal aberrations, with or without mosaicism, have been associated with this disorder. This study comprised clinical description and cytogenetic analysis of a child with PMI. The G-banded karyotype analysis revealed a supernumerary marker chromosome in 76% of the analyzed metaphases from peripheral blood lymphocytes. Array genomic hybridization analysis showed a copy number gain between 3q26.32-3q29, of approximately 20.5 Mb. Karyotype was defined as 47,XX,+mar[38]/46,XX[12].arr 3q26.32-3q29(177,682,859- 198,043,720)x4 dn. Genes mapped in the overlapping region among this patient and three other cases described prior to this study were listed and their possible involvement on PMI pathogenesis is discussed.