hepatocellular carcinoma organoid culture and digestion to study liver cancer pathogenesis

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Hepatocellular carcinoma (HCC), a prevalent and extensively diverse tumor1, has garnered considerable attention within the medical community. The presence of lineage plasticity and substantial heterogeneity in HCC suggests that tumor cells originating from various patients and even distinct lesions within the same patient may manifest dissimilar molecular and phenotypic traits, thereby presenting formidable obstacles in the advancement of innovative therapeutic approaches2,3,4,5. Consequently, there is an imperative need for enhanced comprehension of the biological attributes and mechanisms of drug resistance in HCC to inform the formulation of more efficacious treatment strategies.
In recent decades, researchers have dedicated their efforts to the development of in vitro models for the purpose of studying HCC3,4. Despite some advancements, limitations persist. These models encompass a range of techniques, such as the utilization of cell lines, primary cells, and patient-derived xenografts (PDX). Cell lines serve as in vitro models for long-term culture of tumor cells obtained from HCC patients, offering the benefits of convenience and facile expansion. Primary cell models involve direct isolation and culture of primary tumor cells from patient tumor tissues, thereby providing a representation of biological characteristics that closely resemble those of the patients themselves. PDX models entail the transplantation of patient tumor tissues into mice, with the aim of more faithfully simulating tumor growth and response. These models have been instrumental in HCC research, yet they possess certain limitations, including the heterogeneity of cell lines and the inability to fully replicate in vivo conditions. Furthermore, prolonged in vitro cultivation may result in the deterioration of the cells&#39; original characteristics and functionalities, posing challenges in accurately representing the biological properties of HCC. Additionally, the utilization of PDX models is both time-consuming and costly3.
To address these limitations and more accurately replicate the physiological attributes of HCC, the utilization of organoid technology has been introduced as a promising research platform capable of surpassing previous constraints. Organoids, which are three-dimensional cell models cultured in vitro, have the ability to replicate the structure and functionality of actual organs. However, in the context of HCC, there exist certain challenges in establishing organoid models. These challenges include insufficiently detailed descriptions of HCC organoid construction procedures, a lack of comprehensive protocols for the entire process of HCC organoid construction, and the typically small size of cultured organoids6,7,8. In light of the typically limited dimensions of cultured organoids, we endeavored to tackle these challenges through the development of a comprehensive protocol encompassing the entirety of HCC organoid construction6. This protocol encompasses tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation. By optimizing the procedural steps and refining the composition of the culture medium, we have successfully established HCC organoid models capable of sustained growth and long-term passaging6,8. In the subsequent sections, a comprehensive account of the operational intricacies and pertinent factors involved in the construction of HCC organoids will be presented.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Our lab aims to investigate the molecular pathogenesis of liver cancer and identify novel therapeutic targets for drug development. We would like to answer how cancer initiates, metastasis, and progress to drug tolerance utilizing novel disease models such as patient derived organoids. The current experimental challenges are in two aspects.First, there is a lack of complete set of protocols for constructing hepatocellular carcinoma organoids for the whole process. Second, the size of cultured HCC organoids is too small for subsequent experimental processing. In this protocol, we provide the details of the optimized organoids culture and subsequent transfer and freezing procedures, as well as the precautions to be taken during all the procedures.We optimized the composition of the organoid medium for organoids with close to the original tissue characteristics. We may monitor the tumor evolution using the patient-derived organoids and try to identify novel molecular and therapeutic targets for HCC treatment.

Watch this Scientific Journal Video about Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis at JoVE.com

Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis  

To perform organoid plating, first, remove the supernatant from the centrifuge and washed hepatocellular carcinoma cells. Re-suspend the cells in the 50 microliters of basement membrane extract, or BME, per well, for plating. Next, suspend the cells in advanced DMEM/F12.Then gently pipette the cell aggregates until an even suspension is seen. Add BME to the suspension, ensuring that the BME concentration is between 30 and 50%Now seed 50 microliters of BME droplets with cell clusters in the center of a 24 well plate. Allow the droplets to solidify at 37 degrees Celsius for 20 minutes.Add 500 microliters of prewarmed medium to each well and incubate in a cell incubator at 37 degrees Celsius. Refresh the culture medium every two to three days. After two weeks, replace the isolation medium with the organoid expansion medium.After seven to 10 days of culturing, once the organoids have reached the appropriate density, process the cultures as needed. To passage the organoids, first preheat ultra low attachment surface cell culture plates in an incubator. Next, thaw the frozen BME overnight at four degrees Celsius until right before usage.Prewarm the organoid harvest solution and trypsin substitute at 37 degrees Celsius for 30 minutes. Following the preparation stage, remove the culture medium from the organoid culture plate. Then transfer the organoid suspension into a 15 milliliter tube.Now add organoid harvesting solution in proportion to the amount of BME. To mix the suspension, use a 1000 microliter pipette gun to scrape and pipette up and down. After incubating the tube at room temperature for 30 minutes, use a one milliliter pipette to carefully aspirate the BME.Ensure that the BME is fully dissolved. Monitor the extract every 10 minutes until a clear organoid cell cluster is visible. Then centrifuge at room temperature at 400 g for five minutes.Remove as much of the supernatant as feasible. To start enzymatic digestion of the hepatocellular carcinoma organoids, add one to five milliliters of the prewarmed trypsin substitute to the cultured organoid pellet. Incubate the suspension at 37 degrees Celsius for two minutes.Use a microscope to verify if organoids dissociate into small clusters of two to 10 cells. To stop the digestion, add an appropriate volume of cold basal medium. Then centrifuge the organoids at 400 g for five minutes at eight degrees Celsius.Carefully remove as much supernatant as possible. Re-suspend the desired number of organoids in the proper matrix for plating. Every 10 days passage the organoids, depending on the density of organoid growth.HCC organoids spheroids were observed within three days of culture. Compact spheroids with rounded edges and permeable cytosol were seen on the initial day of establishment. Organoids were similarly sized and had the largest diameter when cultured in 30 to 50%BME.The BME was most fragmented at 10%resulting in the smallest organoids. The BME was most intact at 100%but resulted in organoids with intermediate diameter. The proliferating HCC organoid attained a size exceeding 500 micrometers in each culture after three generations.Substantially sized HCC organoids of higher than 1000 micrometers were obtained within a 20 day culture period.

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Research

JoVE Journal

Cancer Research

167 次观看.  Affiliated Cancer Hospital and Institute of Guangzhou Medical University. 要进行类器官接种，首先，从离心机中取出上清液并洗涤肝细胞癌细胞。将细胞重新悬浮在每孔 50 微升基底膜提取物 （BME） 中进行铺板。接下来，将单元格悬浮在高级 DMEM/F12 中。然后轻轻移液管细胞聚集体，直到看到均匀的悬浮液。将BME添加到悬浮液中，确保BME浓度在30%至50%之间，现在在24孔板的中心接种50微升BME液滴和细胞簇。让液滴在 37 摄氏度下凝固 20 分钟。