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Article | Open Access | Online First

Generation of patient-derived glioblastoma organoids: a comparative study of enzymatic digestion and mechanical fragmentation methods

Jian Zhang1, Jiping Liu1, Yanghua Shi1, Lanyang Li1, Chen Wang1, Mingjie Rong1, Bangbao Tao2, Hong Tan3, Wei Deng4, Chunhui Cai1( ), Xinxin Han1,5( )
Shanghai Lisheng Biotech, Shanghai 200092, China
Department of Neurosurgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200092, China
Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai, China
LongHua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
Organ Regeneration X Lab, LiSheng East China Institute of Biotechnology, Peking University, Nantong 226299, China
Show Author Information

Highlights

• Successfully generated four patient-derived organoids.

• RNA-seq was used to monitor gene expression changes at various stages of organoid growth.

• Both propagation methods successfully maintain the typical characteristics and immune microenvironment of glioblastoma (GBM) organoids.

• The GBM organoids prepared by mechanical fragmentation retained vascular architecture.

Graphical Abstract

In this study, we employed enzymatic digestion and mechanical fragmentation to generate Glioblastoma (GBM) organoids.Utilizing photography, RNA sequencing (RNA-seq), and histological staining, we meticulously documented and compared the morphological and molecular features of the organoids derived from both methods.Our findings underscore the preservation of GBM’s key characteristics, including its unique tissue architecture and gene expression patterns.

Abstract

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.

Keywords

glioblastoma (GBM)organoidspatient-derived modelsRNA sequencingheterogeneity

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Cell Organoid
DOI: 10.26599/CO.2024.9410004
Cite this article:
Zhang J, Liu J, Shi Y, et al. Generation of patient-derived glioblastoma organoids: a comparative study of enzymatic digestion and mechanical fragmentation methods. Cell Organoid, 2024, https://doi.org/10.26599/CO.2024.9410004

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Received: 02 February 2024
Revised: 29 April 2024
Accepted: 12 May 2024
Published: 08 November 2024
© The Author(s) 2024. Published by Tsinghua University Press

The articles published in this open access journal are distributed under the termsof the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, distribution andreproduction in any medium, provided the original work is properly cited.
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