Serum-free cultivation of adult normal human choroidal melanocytes

Valtink, Monika; Engelmann, Katrin

doi:10.1007/s00417-007-0588-3

Cited by 10 publications

(10 citation statements)

References 24 publications

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“…NCM cultures were established from 3 donors as described under Material and Methods section ( Figure S1 and Table S1 ). Geneticin was used to suppress growth of retinal pigmented epithelium (RPE) cells and fibroblasts, thus ensuring pure NCM cultures, as previously described [ 49 ]. The majority of NCM cells displayed a spindle morphology with brown pigmented cytoplasmic granules ( Figures S1 and S2A ).…”

Section: Resultsmentioning

confidence: 99%

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Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation

Tsering

Laskaris

Abdouh

et al. 2020

Cancers

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Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.

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Section: Resultsmentioning

confidence: 99%

“…The majority of NCM cells displayed a spindle morphology with brown pigmented cytoplasmic granules ( Figures S1 and S2A ). In addition, these cells stained positive for MART-1/Melan A, vimentin, S100 and HMB45, which is an indicator of melanogenesis [ 49 ] ( Figure S2B,C ). This suggests that our purification protocol yielded pure NCMs.…”

Section: Resultsmentioning

confidence: 99%

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation

Tsering

Laskaris

Abdouh

et al. 2020

Cancers

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“…Melanocytes are found at higher densities in the foreskin and scalp, so samples from these tissues have previously been used in attempts to culture melanocytes. Some researchers have also successfully cultured hair follicle melanocytes and choroidal melanocytes (Valtink and Engelmann 2007;Zhu et al 2004). The pigmentation diseases that affect appearance, such as melisma and freckles, occur in the face and can often be improved with laser treatment (Wang and Chen 2012).…”

Section: Discussionmentioning

confidence: 99%

Primary culture of human face skin melanocytes for the study of hyperpigmentation

Tang

Jing

2013

Cytotechnology

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Facial epidermal pigmentation and skin tumors can be caused by UV exposure and other physical and chemical irritations. In this report we describe the primary culture of melanocytes from human face skin. The ability to culture these melanocytes will enable their morphological and biological properties to be investigated. Skin specimens were obtained from patients who had undergone lower blepharoplasty procedures. Digestion with neutral protease and trypsin was used to obtain single cell suspensions of epidermal cells. The cells were cultured in M254 medium supplemented with human melanocyte growth solution. Cell morphology was observed using inverted microscopy. Melanocytes were positively identified using both L-DOPA staining and S-100 protein immunohistochemical staining. Immunofluorescence was used to confirm the expression of tyrosinase-related protein-1, a melanocyte-specific protein. The cellular ultrastructure of the melanocytes was observed by transmission electron microscopy. The cultured human melanocytes from face skin were multi-dendritic, and many mature melanosomes were observed. Therefore, using a specific culture medium, melanocytes from face skin can be successfully cultured and made available for further investigations.

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“…PM were isolated from porcine eyes provided by a slaughterhouse. The preparation and primary cell cultivation were performed as described (Valtink and Engelmann, 2007 ). In brief, eyeballs were cut into two pieces.…”

Section: Methodsmentioning

confidence: 99%

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells

et al. 2018

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In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 μM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 μM menthol and 20 μM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 μM) but not by a highly selective TRPM8 blocker, AMTB (20 μM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 μM) and menthol (100 μM) increased the whole-cell currents, whereas 20 μM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.

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Serum-free cultivation of adult normal human choroidal melanocytes

Abstract: The cultivation protocol yields pure cultures of NHCMs that can successfully be maintained for several months without the use of serum, tumour-inducing substances, or toxins.

Cited by 10 publications

References 24 publications

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation

Primary culture of human face skin melanocytes for the study of hyperpigmentation

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells

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