Organotypic Keratinocyte Cocultures in Defined Medium with Regular Epidermal Morphogenesis and Differentiation

Abstract

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.

Id proteins: Novel targets of activin action, which regulate epidermal homeostasis

“…The preparation of the dermal equivalent based on type I collagen hydrogels has been described previously (Smola et al, 1998;Stark et al, 1999;Maas-Szabowski et al, 2003). Briefly, 80% (vol.)…”

Early Keratinocyte Differentiation on Micropillar Interfaces

“…5 In squamous epithelia, differentiation occurs during keratinocyte migration from the basal to the uppermost cell layers, including morphological and biochemical changes. The biochemical changes are reflected by the synthesis of certain molecules indicating early and terminal stages of differentiation.…”

Fibroblasts facilitate re-epithelialization in wounded human skin equivalents

“…These findings indicate that the epithelial-mesenchymal interaction plays an important role in establishing the profile of released factors regulating proliferation and differentiation of keratinocytes. This interaction can be based on (i) production of soluble factors by either epithelial or mesenchymal cells that exhibit autocrine and/or paracrine activities, 24,27,[36][37][38][39][40] (ii) cell-matrix interactions 41 (iii) signaling by direct cell-cell contact. 42 The lower re-epithelialization rate in full-thickness wounds is indicative that in addition to paracrine cytokine and growth factor action, the crosstalk between keratinocytes and fibroblasts are important regulators of keratinocyte proliferation and differentiation.…”

“…This model consists of a three-dimensional human skin equivalent (HSE) model with epidermal architecture closely mimicking that of the native tissue. [21][22][23][24][25][26] The full-thickness or superficial epidermal wounds were induced by freezing with liquid nitrogen or by linear incision with a scalpel, respectively. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and deposition of basement membrane proteins.…”