The production of serum biomarkers by CAISMOV24 cells in culture was confirmed by the detection of CA125 (135.80?U/ml 56.26) and HE4 (811.45?pmol/ml 53.53) in the supernatant of CAISMOV24 cell cultures. Open in a separate window Fig. neutral loss of heterozygosity or cnLOH) in chromosome 16 of CAISMOV24 cell line. (XLSX 1750?kb) 12885_2017_3716_MOESM4_ESM.xlsx (1.7M) GUID:?C2355E71-1E3A-46B5-BFA7-9CC840A5F106 Additional file 5:Table S5: CAISMOV24 PanCancer BMS-687453 transcriptome. Transcriptome data of CAISMOV24 cell line. (XLSX 531?kb) 12885_2017_3716_MOESM5_ESM.xlsx (531K) GUID:?097AD660-4950-4A72-8195-7979DA3F7CDC Data Availability StatementThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Abstract Background The spontaneous immortalization of primary malignant cells is frequently assigned to their genetic instability during in vitro culturing. In this study, the new epithelial ovarian cancer cell line CAISMOV24 was described and compared with its original low-grade serous ovarian carcinoma. Methods The in vitro culture was established with cells isolated from ascites of a 60-year-old female patient with recurrent ovarian BMS-687453 cancer. The CAISMOV24 line was assessed for cell growth, production of soluble biomarkers, expression of surface molecules and screened for typical mutations found in serous ovarian carcinoma. Additionally, comparative genomic hybridization was employed to compare genomic alterations between the CAISMOV24 cell line and its primary malignant cells. Results CAISMOV24 has been in continuous culture for more than 30?months and more than 100 in vitro passages. The cell surface molecules EpCAM, PVR and CD73 are overexpressed on CAISMOV24 cells compared to the primary malignant cells. CAISMOV24 continues to produce CA125 and HE4 in vitro. Although the cell line had developed alongside the accumulation of genomic alterations (28 CNV in primary cells and 37 CNV in CAISMOV24), most of them were related to CNVs already present in primary malignant cells. CAISMOV24 cell line harbored mutation with wild type (exon 2), (exon 2) and (exon 2C11) genes by Sanger sequencing with the BigDye Terminator v3.1?Cycle Sequencing Kit (ThermoFisher Scientific) and capillary electrophoresis in an Applied Biosystems automated sequencer. Primers used for PCR are provided in Additional file 1: Table S1, and were based on the previously described by Arcila et al. . Additionally, TruSight RNA Pan-Cancer Panel (Illumina, Inc., USA) was employed for transcriptome analysis of 1385 genes and 21,043 exons regions implicated in hotspot cancer pathways, following the manufacturers protocol. Briefly, targeted RNA-seq libraries were prepared using 50?ng of total RNA. The sample was subjected to RNA sequencing on an Illumina MiSeq (Illumina, Inc.) at 8 samples per circulation cell (~3?M reads per sample). Go through mapping, gene manifestation information, variant phoning, and fusion detection were performed using the RNA-Seq Positioning App with Celebrity aligner on BaseSpace Sequence Hub. Results CAISMOV24 cell collection establishment and in vitro growth kinetics Primary tradition with cells from ascites was primarily composed of epithelial cells, and a small number of fibroblasts. However, the number of fibroblasts decreased until disappearing along with the initial in vitro passages. As previously mentioned, Mouse monoclonal to RBP4 the 1st 9 to 12 initial subcultures were performed without a regular period of BMS-687453 time (among 3 to 4 4?weeks), the period in which cell proliferation was slow and unable to cover the entire tradition flask surface. After this period, cell proliferation became quicker and in vitro passages for the maintenance of cell tradition became regular (every 2?weeks). To evaluate the reproducibility of the cell tradition transformation from main cells into the cell collection, this procedure was repeated using cells from ascites which were managed and cryopreserved. As a result, the same pattern of development was observed. CAISMOV24 has been in continuous tradition for more than 30?weeks and more than 100 in vitro passages. Number ?Number2a2a shows the typical epithelioid morphology of the established cell collection. After cell plating, CAISMOV24 cells require 2C3?days to exhibit their fully proliferation ability, when their common doubling population time was calculated to be 71.2?h. Even though growth rate of the cell tradition diminished from your 10th day time, the cells continue to proliferate until covering the entire surface of the tradition flask, reaching approximately 100,000 cells/cm2 with 96% viability (Fig. ?(Fig.2b).2b). VPD450-stained CAISMOV24 cells assessed by circulation cytometry allowed the mean proliferation index to be determined as 3.94??0.94 times (Fig. ?(Fig.2c2c). Open in a separate windows Fig. 2 a Different time points of the in vitro growth of the CAISMOV24 cell collection. CAISMOV24 cells were launched at 104 cells/cm2 in HAM F10 medium supplemented with 2?mM L-glutamine and 10% fetal bovine serum. b Representative growth curve BMS-687453 for the CAISMOV24 cell collection, assessed from your.