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.

中文翻译：

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水牛颗粒细胞在三维培养系统中的转录组分析

据报道，在我们之前的研究中，水牛颗粒细胞 (GC) 的悬滴 (HD) 三维 (3D) 培养模型模拟了卵巢卵泡的排卵前阶段。为了进一步验证其可靠性，本研究尝试对从卵泡（直径 <8 毫米）（FC）中新鲜分离的水牛 GC 及其在正常培养皿（ND 或 2D）、polyHEMA 涂层培养皿（PH）中的培养物进行比较转录组分析和高清培养系统（3D）。在 223 个显着（-log2 倍数变化：> 3；p  < .0005；错误发现率 [FDR]：<0.1）不同培养系统之间差异表达的基因 (SDEG) 中，发现 137 个未注释，94、29 和66 个分别在 FC、PH 和 HD 中专门表达。然而，关于消除p的不动点从整个原始数据的值和 FDR 来看，只有 11 个与长链非编码 RNA 相关的基因、12 个与黄体化相关的基因和 3 个与卵泡成熟相关的基因分别在 FC、PH 和 HD 培养系统中专门表达。定量实时 PCR 验证和下一代测序数据具有 90% 以上的相关性。对独家表达的 SDEG 的生物信息学分析表明，新鲜吸出的 GCs 是来自小卵泡（直径 <8 mm）的 GCs 的真正代表，PH 下的 GC 球体保持线粒体功能，在 HD 系统中培养 6 天的那些模拟炎症排卵所需的环境。所以，