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Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

Blum W, Pecze L, Felley-Bosco E, Worthmüller-Rodriguez J, Wu L, Vrugt B, de Perrot M, Schwaller B - In Vitro Cell. Dev. Biol. Anim. (2015)

Bottom Line: All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Fribourg, Route Albert-Gockel 1, 1700, Fribourg, Switzerland.

ABSTRACT
Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

No MeSH data available.


Related in: MedlinePlus

Immortalized mesothelial cell morphology and cell growth in DMEM + 10% FBS. (a) Brightfield images were taken with the Incucyte Live Cell Imaging system (FLR 10×) and show immortalized mesothelial cell clones derived from WT and Nf2+/− mice. The typical “cobblestone-like” morphology is conserved; all clones show a higher proliferation rate than their nonimmortalized primary counterparts (scale bar: 100 μm). (b) Representative Incucyte growth curves of WT and Nf2+/− clones. (c) MTT signals obtained at 144 h postseeding using all cells and cell lines as in Fig. 1 and 2a. (d) MTT data grouped for three clones each of the genotypes WT and Nf2+/− in comparison to the primary mesothelial cells (*p < 0.05; ***p < 0.0005).
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Fig2: Immortalized mesothelial cell morphology and cell growth in DMEM + 10% FBS. (a) Brightfield images were taken with the Incucyte Live Cell Imaging system (FLR 10×) and show immortalized mesothelial cell clones derived from WT and Nf2+/− mice. The typical “cobblestone-like” morphology is conserved; all clones show a higher proliferation rate than their nonimmortalized primary counterparts (scale bar: 100 μm). (b) Representative Incucyte growth curves of WT and Nf2+/− clones. (c) MTT signals obtained at 144 h postseeding using all cells and cell lines as in Fig. 1 and 2a. (d) MTT data grouped for three clones each of the genotypes WT and Nf2+/− in comparison to the primary mesothelial cells (*p < 0.05; ***p < 0.0005).

Mentions: Of note, primary mesothelial cells (initial density, 1500 cells/cm2) from Nf2+/− mice showed slightly enhanced proliferation compared to the cell lines generated from WT mice (Fig. 1b). From the initial batch of immortalized cells, by limited dilution, clones derived from three colonies for each genotype, likely originating from a single cell, thus representing single clones, were isolated, expanded, and stored for further usage. Unlike the primary mesothelial cells, SV40-immortalized mesothelial cell clones (iMeso-WT, iMeso-Nf2) grew rapidly in DMEM supplemented with 10% FBS (Fig. 2). Cells from both genotypes showed persistent cell proliferation even under low serum conditions (2%) and all survived for prolonged periods of time (>120 h) under very low serum conditions (0.5%), however without signs of proliferation evidenced by lack of mitotic cells (data not shown). All cultured mesothelial cells showed the typical cobblestone-like morphology like primary mesothelial cells from WT and Nf2+/− (Fig. 2a). All clones irrespective of genotype maintained their typical pavement-like morphology, and moreover, immortalized cell lines of both genotypes showed an increased cell proliferation rate when compared to the initial primary mesothelial cells (Fig. 2b) evidenced from real-time growth curves, as well as from the results of an independent proliferation assay, the MTT assay, carried out 144 h post-plating (Fig. 2c). Of note within one genotype (WT and Nf2+/−), growth rates of the various clones were considerably different, e.g., WT clones 1, 2, and 3 reached the plateau (close to 100% confluence) within 100, 80, and ≈140 h, respectively (Fig. 2b); similar proliferation rates were also observed for Nf2+/− clones. This was also confirmed using the MTT assay (Fig. 2c), when clones were grouped according to their genotype. No significant differences in growth rates were evident between the group of WT and NF2+/− clones (Fig. 2d). All SV40-immortalized mesothelial cell lines (clones) were cultured for more than 40 passages without showing any signs of a decreased growth and/or changes in cell morphology. A stock was produced at passage number 7–10 and frozen for long-term storage.Figure 2.


Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

Blum W, Pecze L, Felley-Bosco E, Worthmüller-Rodriguez J, Wu L, Vrugt B, de Perrot M, Schwaller B - In Vitro Cell. Dev. Biol. Anim. (2015)

Immortalized mesothelial cell morphology and cell growth in DMEM + 10% FBS. (a) Brightfield images were taken with the Incucyte Live Cell Imaging system (FLR 10×) and show immortalized mesothelial cell clones derived from WT and Nf2+/− mice. The typical “cobblestone-like” morphology is conserved; all clones show a higher proliferation rate than their nonimmortalized primary counterparts (scale bar: 100 μm). (b) Representative Incucyte growth curves of WT and Nf2+/− clones. (c) MTT signals obtained at 144 h postseeding using all cells and cell lines as in Fig. 1 and 2a. (d) MTT data grouped for three clones each of the genotypes WT and Nf2+/− in comparison to the primary mesothelial cells (*p < 0.05; ***p < 0.0005).
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Fig2: Immortalized mesothelial cell morphology and cell growth in DMEM + 10% FBS. (a) Brightfield images were taken with the Incucyte Live Cell Imaging system (FLR 10×) and show immortalized mesothelial cell clones derived from WT and Nf2+/− mice. The typical “cobblestone-like” morphology is conserved; all clones show a higher proliferation rate than their nonimmortalized primary counterparts (scale bar: 100 μm). (b) Representative Incucyte growth curves of WT and Nf2+/− clones. (c) MTT signals obtained at 144 h postseeding using all cells and cell lines as in Fig. 1 and 2a. (d) MTT data grouped for three clones each of the genotypes WT and Nf2+/− in comparison to the primary mesothelial cells (*p < 0.05; ***p < 0.0005).
Mentions: Of note, primary mesothelial cells (initial density, 1500 cells/cm2) from Nf2+/− mice showed slightly enhanced proliferation compared to the cell lines generated from WT mice (Fig. 1b). From the initial batch of immortalized cells, by limited dilution, clones derived from three colonies for each genotype, likely originating from a single cell, thus representing single clones, were isolated, expanded, and stored for further usage. Unlike the primary mesothelial cells, SV40-immortalized mesothelial cell clones (iMeso-WT, iMeso-Nf2) grew rapidly in DMEM supplemented with 10% FBS (Fig. 2). Cells from both genotypes showed persistent cell proliferation even under low serum conditions (2%) and all survived for prolonged periods of time (>120 h) under very low serum conditions (0.5%), however without signs of proliferation evidenced by lack of mitotic cells (data not shown). All cultured mesothelial cells showed the typical cobblestone-like morphology like primary mesothelial cells from WT and Nf2+/− (Fig. 2a). All clones irrespective of genotype maintained their typical pavement-like morphology, and moreover, immortalized cell lines of both genotypes showed an increased cell proliferation rate when compared to the initial primary mesothelial cells (Fig. 2b) evidenced from real-time growth curves, as well as from the results of an independent proliferation assay, the MTT assay, carried out 144 h post-plating (Fig. 2c). Of note within one genotype (WT and Nf2+/−), growth rates of the various clones were considerably different, e.g., WT clones 1, 2, and 3 reached the plateau (close to 100% confluence) within 100, 80, and ≈140 h, respectively (Fig. 2b); similar proliferation rates were also observed for Nf2+/− clones. This was also confirmed using the MTT assay (Fig. 2c), when clones were grouped according to their genotype. No significant differences in growth rates were evident between the group of WT and NF2+/− clones (Fig. 2d). All SV40-immortalized mesothelial cell lines (clones) were cultured for more than 40 passages without showing any signs of a decreased growth and/or changes in cell morphology. A stock was produced at passage number 7–10 and frozen for long-term storage.Figure 2.

Bottom Line: All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Fribourg, Route Albert-Gockel 1, 1700, Fribourg, Switzerland.

ABSTRACT
Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

No MeSH data available.


Related in: MedlinePlus