A modified ex vivo skin organ culture system for functional studies

Abstract

To investigate the immunological function of cells in normal and diseased skin under conditions approximating the in vivo situation, it is necessary to maintain the structural integrity of the tissue. To achieve this, freshly isolated skin has to be cultured ex vivo, or an in vitro-constructed complete skin equivalent may be used. Different skin organ culture systems have been described. Basically two systems prevail: submerged or air-exposed skin organ cultures. The former model has been used for measuring cytokine secretion by skin cells in the medium, and the latter to study the expression of cell membrane proteins in situ and the kinetics of epidermal Langerhans cells. Here we present a modified ex vivo skin organ culture system which approaches the in vivo situation by maintaining the normal skin architecture without spontaneous induction of the regenerative maturation markers. This method allowed the expression of cell membrane proteins in situ to be measured, and the cytokine secretion by skin cells in the culture medium to be quantitated in the same experiment. In this system, both general and specific stimuli (LPS and IL-1beta) upregulated the expression of skin-derived cytokines IL-8 and IL-6 in the medium and different markers (ICAM-1, CD40 and CD86) on cells in the tissue in a 24-hour culture-formed. Elevation of both cytokine and cell marker expression could be blocked by dexamethasone and by IL-1ra which acts specifically on IL-1beta-mediated responses. The system presented here is both quick and simple and can be used as a model to study the behaviour of skin cells in their natural microenvironment.

FcR-Independent Effects of IgE and IgG Autoantibodies in Bullous Pemphigoid

“…The human organ culture is valuable as a readout for studies investigating mechanisms of other autoimmune blistering diseases because the architecture of human skin is maintained (43,44). For our purposes, the absence of infiltrating immune cells is an added benefit.…”

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model

“…The mechanism of re-epithelialization in mammalian skin has been studied by using a variety of in vitro culture systems, including skin explants (Lu and Rollman 2004;Onuma et al 2001;Stoll et al 2003), skin substitutes (Boyce et al 2002;Companjen et al 2001), and monolayers of several cell lines (Sharma et al 2003;Turchi et al 2002;Zhang et al 2005). Although skin organ cultures are suitable for examining which factors are important for epidermal migration and where those factors are expressed and distributed in situ, cellular responses are difficult to investigate, because of the complexity of mammalian skin.…”

Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy

“…This is caused by the loss of blood supply after surgical excision, hypoxia, and the accumulation of toxic metabolites. 8 Methods commonly used to assess the viability of ex vivo skin include the morphology of the tissue, 9 metabolic activity by MTT, [10][11][12] lactate dehydrogenase ͑LDH͒ activity assays, 13 skin pH, 14 and oxygen consumption. 11,15,16 Several studies have used the MTT assay to measure the decreasing viability of ex vivo skin.…”