Methyl Green-Pyronine Staining of Porcine Organotypic Skin Explant Cultures: An Alternative Model for Screening for Skin Irritants

Abstract

We describe a new alternative method for screening for skin irritants by using fresh intact porcine skin biopsies. Test chemicals were applied to the epidermis of the biopsies, which were then incubated for different times in tissue culture medium at 37°C and with 5% carbon dioxide. A decrease in epidermal keratinocyte RNA staining, visualised in frozen sections by using a modified methyl-green pyronine (MGP) staining procedure, was employed as a marker of irritancy. If a decrease in epidermal RNA was observed after incubation for 4 hours (strong irritant), the chemical had an MGP score of 3; if after incubation for 24 hours (moderate irritant), the MGP score was 2; and if after incubation for 48 hours (weak irritant), the MGP score was 1. If no keratinocyte cytotoxicity was observed after incubation for 48 hours, the chemical was classified as non-irritant (MGP score = 0). At least three ears were used per chemical. The average MGP score was used to classify the chemical. Based on the MGP score for 20% sodium dodecyl sulphate, chemicals classified as strong or moderate irritants by using the MGP test were grouped together as category R38 chemicals. Weak irritants or non-irritants were not classified (NC). The MGP staining correctly identified 23 of 25 skin irritants for which reference data were available.

An in vitro Model System to Study the Damaging Effects of Prolonged Mechanical Loading of the Epidermis

“…8 The decreased cytoplasmic RNA also indicates initial cell damage, which can eventually lead to necrosis. 23,24 Severe epidermal damage was only observed in the HP groups ( Fig. 2g and h and Table 2).…”

Prolonged viability of human organotypic skin explant in culture method (hOSEC)

“…Some authors have used the hOSEC model to analyze cell migration into the skin after chemical sensitization; others evaluate it through keratinocyte cytotoxicity by measuring the disappearance of keratinocytic RNA, which is visualized using a modified methyl green-pyronine (MGP) staining. 7 - 10 Although these studies have demonstrated the viability of this model, in none of them was such viability longer than 3 weeks, and neither was the explanted skin's proliferative capacity.…”

“…By presenting an air-liquid interface, the hOSEC system allows the application of creams, ointments and lipid-soluble substances on the horny layer, whereas in a cell culture system it is possible to add substances that are soluble in its medium due to the absence of a stratum corneum. 7 - 10 …”

Ex vivo Model of Human Skin (hOSEC) as Alternative to Animal use for Cosmetic Tests

“…Such features are become essential for its differentiation from the existing three-dimensional validated models, which are made only of keratinocytes and fibroblasts on a devitalized extracellular matrix. By presenting an airliquid interface, the system hOSEC allows the application of creams, ointments and lipid-soluble substances on the horny layer that keeps the different cell culture systems that only allow the use of soluble substances in the culture medium and the absence of stratum corneum [6][7][8][9].…”

“…Some authors have used hOSEC model to analyze cell migration into the skin after chemical sensitization, and others evaluate it by keratinocyte cytotoxicity measuring the disappearance of keratinocyte RNA, which was visualized using a modified methyl green-pyronine (MGP) stain [6][7][8][9]. Although these studies have showed the viability of the model, none of them have showed the viability longer than 3 weeks neither the proliferative capacity of the explanted skin.…”