Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems

Abstract

Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study.To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (−log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.

Mapping Cell-in-Cell Structures in Oral Squamous Cell Carcinoma

“…Despite bioprinting spheroids having been used to study various cancer types such as glioblastoma [53,77], breast cancer [78], pancreatic duct adenocarcinoma [55], osteosarcoma [79] and ovarian cancer [80], there is a lack of studies that validate this model for OSCC. The spheroid bioprinting method might represent an advantageous approach compared to hanging drop cultures [81], organ-on-a-chip [82], and organoids [83] for allowing higher reproducibility, technical simplicity, and the ability to construct heterotypic spheroids with well-nutrient distribution and tumor zone formation [84][85][86]. This approach cooperates in dealing with the lack of appropriate methods to study CIC structures [11,18,87].…”

Approaches for in vitro culture of granulosa cells and ovarian follicles

“…In this study, the GCs were cultured with a density of 20,000 cells/10 μl in a six-well culture plate. The 3 μl of media regularly added to each HD nutritional losses replenish losses due to evaporation and cell's metabolism (after every 48 h) [72].…”

Three‐dimensional culture of rat ovarian granulosa cells shows increased SPP1 and FGF7 expression through the PI3K/Akt pathway

“…Although many studies have stressed the importance and superiority of 3D culture, there are few studies on its effects on OGCs. Buffalo OGCs cultured using the hanging drop method mimic the intrafollicular environment (Yadav et al, 2018), and this system was found to be better than the other methods for simulating the inflammatory milieu required for ovulation (Pandey et al, 2021); however, no such studies have been performed on rat OGCs (rOGCs). In our study, we investigated the effects of 2D and 3D cultures of rOGCs on their proliferation using RNA‐seq to analyze the transcriptome differences between two groups, to investigate the effects of 3D culture on the transcript levels of OGCs, and to provide a theoretical basis for attaining the best OGCs for clinical application.…”

“…Although many studies have stressed the importance and superiority of 3D culture, there are few studies on its effects on OGCs. Buffalo OGCs cultured using the hanging drop method mimic the intrafollicular environment (Yadav et al, 2018), and this system was found to be better than the other methods for simulating the inflammatory milieu required for ovulation (Pandey et al, 2021); however, no such studies have been performed on rat OGCs (rOGCs).…”