Establishment and identification of organoid derived from patients with gastric cancer based on suspension culture_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LUO Wei , SU Yuanhui , YU Cheng , CHAI Changpeng , TANG Huan , MIAO Long , WANG Zhengfeng ,  XU Hao

The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou 730000, P. R. China;

Corresponding author：

XU Hao, Email: amoyxu@126.com

Keywords：

gastric cancer; organoid; suspension culture; identification

DOI：

10.7507/1007-9424.202401079

Video：

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Objective To explore the feasibility of organoid culture derived from the patients with gastric cancer by suspension culture. Methods The fresh gastric cancer tissues of the 3 patients with gastric cancer were selected, which were digested with mixed enzymes and then made into cell suspensions, and were inoculated into ultra-low attachment plates to culture organoid by suspension. When the organoid growth was dense, the passage and freezing were carried out. The formation process of organoid was observed under the inverted microscope (IM). Further the consistency between the organoid and primary gastric cancer tissue was evaluated by hematoxylin-eosin (HE) and immunohistochemical (IHC) staining. Results In this study, an organoid that could be passaged and frozen was successfully established in one patient. The results under the IM showed that the organoid was initially spherical in shape (cultured on day 5), then gradually became short rod-shaped on day 10, showed a branching like change on day 15, and formed irregular glandular tubular structures on day 20. The structures between the organoids and primary gastric cancer tissues were highly similar by HE staining. The IHC staining results showed that the expressions of low molecular weight cytokeratin (CK-LMW), p53, and Ki67 in the organoid and its corresponding primary gastric cancer tissue were basically the same. That was, the CK-LMW and p53 expressions were positive in the organoid and primary gastric cancer tissue, and the Ki67 was highly expressed (with a positive rate of approximately 70%). Conclusion Based on the preliminary research results of this study, it suggests that the suspension culture can be used to establish organoid derived from patients with gastric cancer, which is in accordance with primary tumor tissue at the tissue and cellular levels.

Citation： LUO Wei, SU Yuanhui, YU Cheng, CHAI Changpeng, TANG Huan, MIAO Long, WANG Zhengfeng, XU Hao. Establishment and identification of organoid derived from patients with gastric cancer based on suspension culture. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2024, 31(8): 924-927. doi: 10.7507/1007-9424.202401079 Copy

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8.	Vlachogiannis G, Hedayat S, Vatsiou A, et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science, 2018, 359(6378): 920-926.
9.	Nanki K, Toshimitsu K, Takano A, et al. Divergent routes toward Wnt and R-spondin niche independency during human gastric carcinogenesis. Cell, 2018, 174(4): 856-869.
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12.	Li G, Ma S, Wu Q, et al. Establishment of gastric signet ring cell carcinoma organoid for the therapeutic drug testing. Cell Death Discov, 2022, 8(1): 6. doi: 10.1038/s41420-021-00803-7.
13.	Choi W, Kim YH, Woo SM, et al. Establishment of patient-derived organoids using ascitic or pleural fluid from cancer patients. Cancer Res Treat, 2023, 55(4): 1077-1086.
14.	Gan Z, Qin X, Liu H, et al. Recent advances in defined hydrogels in organoid research. Bioact Mater, 2023, 28: 386-401.
15.	Kozlowski MT, Zook HN, Chigumba DN, et al. A Matrigel-free method for culture of pancreatic endocrine-like cells in defined protein-based hydrogels. Front Bioeng Biotechnol, 2023, 11: 1144209. doi: 10.3389/fbioe.2023.1144209.
16.	Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel. Commun Biol, 2021, 4(1): 1387. doi: 10.1038/s42003-021-02910-8.
17.	Broguiere N, Isenmann L, Hirt C, et al. Growth of epithelial organoids in a defined hydrogel. Adv Mater, 2018, 30(43): e1801621. doi: 10.1002/adma.201801621.
18.	Capeling MM, Huang S, Childs CJ, et al. Suspension culture promotes serosal mesothelial development in human intestinal organoids. Cell Rep, 2022, 38(7): 110379. doi: 10.1016/j.celrep.2022.110379.
19.	Kumar SV, Er PX, Lawlor KT, et al. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells. Development, 2019, 146(5): dev172361. doi: 10.1242/dev.172361.

1. Malvezzi M, Bonifazi M, Bertuccio P, et al. An age-period-cohort analysis of gastric cancer mortality from 1950 to 2007 in Europe. Ann Epidemiol, 2010, 20(12): 898-905.
2. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018, 68(6): 394-424.
3. Ferlay J, Colombet M, Soerjomataram I, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer, 2019, 144(8): 1941-1953.
4. Hunt RH, Camilleri M, Crowe SE, et al. The stomach in health and disease. Gut, 2015, 64(10): 1650-1668.
5. Huch M, Koo BK. Modeling mouse and human development using organoid cultures. Development, 2015, 142(18): 3113-3125.
6. Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol, 2014, 15(10): 647-664.
7. Seidlitz T, Merker SR, Rothe A, et al. Human gastric cancer modelling using organoids. Gut, 2019, 68(2): 207-217.
8. Vlachogiannis G, Hedayat S, Vatsiou A, et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science, 2018, 359(6378): 920-926.
9. Nanki K, Toshimitsu K, Takano A, et al. Divergent routes toward Wnt and R-spondin niche independency during human gastric carcinogenesis. Cell, 2018, 174(4): 856-869.
10. Yan HHN, Siu HC, Law S, et al. A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening. Cell Stem Cell, 2018, 23(6): 882-897.
11. Drost J, Clevers H. Organoids in cancer research. Nat Rev Cancer, 2018, 18(7): 407-418.
12. Li G, Ma S, Wu Q, et al. Establishment of gastric signet ring cell carcinoma organoid for the therapeutic drug testing. Cell Death Discov, 2022, 8(1): 6. doi: 10.1038/s41420-021-00803-7.
13. Choi W, Kim YH, Woo SM, et al. Establishment of patient-derived organoids using ascitic or pleural fluid from cancer patients. Cancer Res Treat, 2023, 55(4): 1077-1086.
14. Gan Z, Qin X, Liu H, et al. Recent advances in defined hydrogels in organoid research. Bioact Mater, 2023, 28: 386-401.
15. Kozlowski MT, Zook HN, Chigumba DN, et al. A Matrigel-free method for culture of pancreatic endocrine-like cells in defined protein-based hydrogels. Front Bioeng Biotechnol, 2023, 11: 1144209. doi: 10.3389/fbioe.2023.1144209.
16. Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel. Commun Biol, 2021, 4(1): 1387. doi: 10.1038/s42003-021-02910-8.
17. Broguiere N, Isenmann L, Hirt C, et al. Growth of epithelial organoids in a defined hydrogel. Adv Mater, 2018, 30(43): e1801621. doi: 10.1002/adma.201801621.
18. Capeling MM, Huang S, Childs CJ, et al. Suspension culture promotes serosal mesothelial development in human intestinal organoids. Cell Rep, 2022, 38(7): 110379. doi: 10.1016/j.celrep.2022.110379.
19. Kumar SV, Er PX, Lawlor KT, et al. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells. Development, 2019, 146(5): dev172361. doi: 10.1242/dev.172361.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Establishment and identification of organoid derived from patients with gastric cancer based on suspension culture

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