Role of geneticin in isolation and culturing of skin melanocytes and melanoma cells
, , , , , , , , oraz | 14 wrz 2023
Article Category: Original Study
Data publikacji: 14 wrz 2023
Zakres stron: 72 - 81
Otrzymano: 12 mar 2023
Przyjęty: 06 lip 2023
DOI: https://doi.org/10.2478/ahem-2023-0014
Słowa kluczowe
© 2023 Aneta Ścieżyńska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Introduction
Development of an effective, suitable, and reliable method for both the isolation and culturing of melanocytes is crucial for studies on pathomechanisms of skin diseases originating from melanocyte disorders. In this study, we have investigated the influence of geneticin (G418), a substance used for melanocyte selection, in the view of the frequency of presence of cells such as keratinocytes and fibroblasts, widely known as contaminators of melanocyte-originating cell cultures.
Materials and Methods
Study was conducted on primary, freshly isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells as well as on commercially available melanoma cell lines MeWo, G-361, and A375. Cells were cultured in different culture media supplemented with various concentrations of geneticin ranging from 0.05 to 1 mg/mL. Cell viability, proliferation rate and detection of apoptotic/necrotic cells was assessed.
Results
Choice of culture media supplemented with various concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) strongly affect viability of melanocytes, fibroblasts, and keratinocytes. Selective culture media without FBS facilitate the process of melanocytes and melanoma pure cell culture, yet without geneticin supplementation are insufficient for complete eradication of fibroblast contamination from cell culture.
Conclusions
In this study we provide, for the first time, the dose-response action of keratinocytes and fibroblasts upon geneticin stimulation in different culture media and show that a low concentration (0.05 mg/mL) of geneticin added to the selective culture media may be safely implemented to facilitate the production of melanocyte and melanoma cell cultures that are free from frequent cell contaminants.