Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid growth and high heterogeneity, posing challenges for fundamental research and personalized drug screening due to the lack of suitable models. GBM organoids serve as an innovative research tool, providing a valuable model for studying the biological characteristics of GBM. In this study, we successfully generated 4 GBM organoids and employed enzymatic digestion and mechanical fragmentation techniques for subsequent cultivation. Through continuous observation, pathological assessment, and RNA sequencing (RNA-seq), we observed that all the organoids generated through both methods demonstrated good growth characteristics. The organoids derived from mechanical fragmentation not only achieved a two-dimensional (2D) area of ~ 1.5 mm² but also exhibited distinct vascular structures. The organoids derived from enzymatic digestion achieved a 2D area of approximately 0.8 mm². Furthermore, RNA-seq analysis has revealed that organoids cultured using two distinct methods exhibit a heterogeneous cellular composition, comprising a total of 20 cell types (endothelial, immune cells ...). Our studies show that both methods successfully maintained the essential characteristics of GBM, encompassing its distinctive tissue structure and gene expression patterns. Each method exhibits its own attributes, contributing to the understanding of GBM organoids.

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal malignancy associated with significant morbidity, necessitating the urgent development of an effective chemotherapeutic assay for ICC patients. In this study, we have successfully established an advanced culture method for ICC organoids that can be utilized with both single-cell assembly and tissue fragmentation initiation techniques. These ICC organoids maintain the morphological characteristics, including mutation profiles and frequency (46.9% in organoid and 48.5% in tumor tissue) of IDH1 genes, and 1733 high-frequent overlapped mutated genes (94.2%). Additionally, ICC biomarkers such as CK7 and CK19 also maintain a similar pattern compared with the original tissue. Furthermore, RNA-seq analysis reveals upregulation of immune-related genes in single-cell assembly organoids. The significantly changed genes including IL9R (4.4-fold), IL2RB (3.2-fold), CCR4 (3.5-fold), TESPA1 (4.4-fold), ZAP70 (4.3-fold) and CD6 (4.3-fold) in log scale. These evidence both indicating the presence of viable and active immune cells. Overall, our findings present an advanced and user-friendly culture approach for generating ICC organoids adaptable to diverse experimental objectives.

Ovarian cancer, a common gynecologic tumor, is associated with a high mortality, due to challenges in early detection within the reproductive system. According to our previous research, cultivating patient-specific organoids from mechanically sheared tissues can be utilized for drug response evaluation but has limitations for high-throughput screening efficiency due to their inconsistent size. In this research, we focused on organoids developed from single-cell suspensions to address the critical requirement for uniformity in organoid size. By the day 3 of culture, single-cell suspensions rapidly and spontaneously aggregated into spherical structures with a more consistent size. Notably, the organoids of sample OVA-37 were ten times larger after 8 days of culture. Transcriptomic analysis was used to compare the two organoid culture techniques, demonstrating that the variations between different organoid culture methods were minimal, with higher variability observed among patients. Gene set enrichment analysis (GSEA) revealed only minor discrepancies in specific pathways, such as TGF-β and tight junctions. Furthermore, treatment with carboplatin in a 96-well plate setup resulted in reproducible drug responses, as evidenced by coefficients of variation lower than 40%. This finding suggests that single-cell suspension-cultured organoids can be employed for reproducible high-throughput drug screening. This approach holds potential for personalized drug screening in ovarian cancer and may contribute to the development of novel therapeutic strategies.