We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.
Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent 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' 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.
Human-biopsied tissues were obtained from the respective patient at the Affiliated Cancer Hospital and Institute of Guangzhou Medical University, and informed consent was obtained from the patients. See the Table of Materials for details about all materials, reagents, and instruments used in this protocol.
1. Establishing patient-derived HCC organoids from surgical samples
NOTE: The establishment of HCC organoids encompasses various stages, namely tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation. The process of tissue dissociation requires a duration of 2 h, while the seeding of organoids onto a plate takes approximately 40 min. Following this, the initial generation of HCC organoids undergoes a culture period of 10-14 days using HCC isolation medium. Once a satisfactory density is achieved, organoid passages are conducted, which requires 1 h. Subsequent cultures of the organoids are then maintained using HCC expansion medium for 7-10 days, which may vary depending on the growth rate and condition of the organoids.
Upon implementing the aforementioned procedure, the emergence of HCC organoid spheroids is typically observable within a span of 3 days (Figure 1). Figure 1A,B show the established HCC organoid, which promptly develops compact spheroids characterized by rounded edges and permeable cytosol on the initial day of establishment. During the growth of HCC organoids, the use of different concentrations of BME had different effects on the growth rate of the organoids. We cultured two patient-derived HCC organoids in 10%, 30%, 50%, and 100% of BME for 12 days in HCC expansion medium (Table 1) and found that the BME was intact at 30% and 50% and the organoids were close in size and had the largest diameter at these BME concentrations. At 10% BME, the BME was the most fragmented, with a narrow space for organoid growth and the smallest diameter. At 100% BME, the BME was the most intact, but the diameter of the organoids was in the middle. Therefore, it is recommended to control the concentration of BME at 30-50% in HCC organoid culture (Figure 2). The proliferating HCC organoid can be effectively propagated in accordance with the prescribed protocol, attaining a size exceeding 500 µm in each culture after three generations of culture (Figure 3). Subsequent experiments, including drug intervention and immunohistochemical staining, can be conducted upon achieving this size. Utilizing this culturing strategy, we achieved substantial growth of HCC organoids, reaching sizes exceeding 1,000 µm within a 20-day culture period (Figure 4). Immunohistochemical staining of both HCC organoids and paired tumor tissues revealed similarities in the expression of marker genes (Figure 5).
Figure 1: HCC organoids derived from patients' surgical tissues. (A,B) Representative images of robust HCC organoid cultures on the first day of plating. (C,D) The HCC organoid cultures after 5 days of cultivation. Scale bars = 500 µm (A,C), 250 µm (B,D). Abbreviation: HCC = hepatocellular carcinoma. Please click here to view a larger version of this figure.
Figure 2: To investigate the growth of HCC organoid models at different BME concentrations. HCC organoids A and B were cultured at the same planting density for 12 days in 10%, 30%, 50%, and 100% BME using HCC expansion medium (Table 1). Scale bars = 1,000 µm (A), 250 µm (B). Please click here to view a larger version of this figure.
Figure 3: HCC organoids in long-term amplification. (A,B) Representative images of HCC organoids on the tenth day of passage 8. Scale bar = 500 µm (A), 250 µm (B). Abbreviation: HCC = hepatocellular carcinoma. Please click here to view a larger version of this figure.
Figure 4: Maximum size of HCC organoids. Representative images of HCC organoids in a 20-day culture period in passage 8 . Scale bar = 250 µm. Please click here to view a larger version of this figure.
Figure 5: Histopathological characteristics of HCC organoids and paired tumor tissues. The expression of gene markers for HCC organoids and paired tumor tissues, HCC marker AFP, differentiated hepatocyte markers HNF4A and ALB, ductal markers SOX9 and EPCAM, and bile marker KRT19 were detected by immunohistochemistry. Scale bar = 100 µm. Abbreviations: AFP = Alpha Fetoprotein; HNF4A = Hepatocyte Nuclear Factor 4 Alpha; ALB = albumin; SOX9 = SRY-Box Transcription Factor 9; EPCAM = epithelial cell adhesion molecule; KRT19 = Keratin 19. Please click here to view a larger version of this figure.
Table 1: Composition of media and solutions. Please click here to download this Table.
One notable benefit of patient-derived organoid models lies in their capacity to faithfully replicate the biological characteristics of tumors, encompassing tissue structure and genomic landscape. These models demonstrate a remarkable level of accuracy and effectively mirror the heterogeneity and progression of tumors, even over extended periods of cultivation6,8,9. Through the utilization of this refined organoid culture protocol, we have effectively established patient-derived HCC organoid models, facilitating sustained growth in vitro, as well as the ability to cryopreserve and subsequently resuscitate organoids as required for experimental purposes. In comparison to preceding protocols, these organoids exhibit enhanced growth rates and are capable of attaining greater dimensions in culture. Subsequently, we subjected the well-established organoids to antibody-coupled drugs derived from prescreened potential oncogenic targets, leading to notable therapeutic outcomes that align with in vivo PDX animal models10,11.
The objective of this study was to improve on the shortcomings of previous HCC organoid establishment protocols, resulting in the formation of larger-sized HCC organoids under stable passaging and culture conditions. We optimized the concentration of BME to improve the growth rate of organoids while reducing the cost of the experiment; added an important factor, CHIR99021, to the original culture formula, which can promote the self-renewal of stem cells in organoids and enhance the proliferation of organoid; improved the removal method of BME, from the original mechanical separation method to the use of a special harvesting solution, to increase the amount of organoid obtained and improve the efficiency; supplemented the operation steps of organoid cryopreservation and resuscitation, constructed the whole process of HCC organoid culture. With this organoid culture protocol, it is possible to achieve an average organoid size of 800 µm in a single 20-day culture period, with a few robust HCC organoids growing to over 1,000 µm. The ultimate goal is to enhance the precision of treatment strategies for HCC and establish a dependable model and platform for drug development. These efforts will contribute to the improvement in HCC patient survival rates and the provision of novel solutions to combat drug resistance challenges11,12,13.
However, it is imperative to consider multiple aspects of the discourse. Primarily, a significant obstacle arises from the diminished efficacy of establishing HCC organoids, particularly for patients in patients with intermediate- to advanced-stage HCC who have undergone diverse therapeutic interventions, including transcatheter arterial chemoembolization (TACE), chemotherapy, and targeted therapy4,5. Frequently, the tumor activity of excised HCC specimens is compromised, leading to a decreased rate of success in generating organoids. Meanwhile, in the previous establishment of HCC organoids by other teams, it was found that there is a correlation between the proportion of proliferating cells in the tumor, the grade of differentiation of the tumor, and the establishment of organoids6,9. Hence, the achievement of a high success rate in the development of HCC organoids is intricately tied to the treatments employed prior to the patient's surgery, as well as the need for adjunctive judgments of the pathologist after the tumor tissues have been obtained. Additionally, while organoids are purported to replicate the heterogeneity of HCC, discerning whether an organoid in culture originates from a solitary cell clone or encompasses a blend of cells with distinct genetic profiles poses a challenge. Consequently, further inquiries are imperative to augment our comprehension of the clonal dynamics and genetic diversity within HCC organoids. Furthermore, it is imperative to address the inherent limitations of current plate-based organoid culture techniques. One notable constraint is the restricted growth potential of organoids. The culture of HCC organoids using this protocol, it is possible to achieve diameters of more than 1,000 µm. However, in the later stages of culture, blackened necrosis often occurs in the core region of the organoid, due to insufficient nutrient and oxygen supply, which limits the development potential of the organoids. Therefore, there is an urgent need to devise strategies aimed at enhancing nutrient delivery and oxygenation within the organoid culture system.
In summary, patient-derived organoid models present notable benefits in accurately reproducing the biological attributes of HCC. Nevertheless, there are still obstacles to be overcome regarding the efficacy of organoid establishment, clonal dynamics, and the limitations inherent in organoid culture techniques. By addressing these challenges, the usefulness of HCC organoid models for comprehending the disease and advancing drug development endeavors can be further augmented.
The authors have nothing to disclose.
This research was supported by the National Natural Science Foundation of China (82122048; 82003773; 82203380) and Guangdong Basic and Applied Basic Research Foundation (2023A1515011416).
[Leu15]-gastrin I human | Merck | G9145 | |
1.5 mL Microtubes | Merck | AXYMCT150LC | |
A8301 (TGFβ inhibitor) | Tocris Bioscience | 2939 | |
B27 Supplement (503), minus vitamin A | Thermo Fisher Scientific | 12587010 | |
B-27 Supplement (503), serum-free | Thermo Fisher Scientific | 17504044 | |
BMP7 | Peprotech | 120-03P | |
Cell strainer size 100 μm | Merck | CLS352360 | |
CHIR99021 | Merck | SML1046 | |
Collagenase D | Merck | 11088858001 | |
Corning Costar Ultra-Low | Merck | CLS3473 | |
Costar 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Individually Wrapped, Sterile | Corning | 3473 | |
Costar 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Individually Wrapped, Sterile | Corning | 3471 | |
Cultrex Organoid Harvesting Solution | R&D SYSTEMS | 3700-100-01 | Organoid harvesting solution |
Cultrex Reduced Growth Factor BME, Type 2 PathClear (BME) | Merck | 3533-005-02 | |
DAPT | Merck | D5942 | |
Dexamethasone | Merck | D4902 | |
DMSO | Merck | C6164 | |
DNaseI | Merck | DN25 | |
Dulbecco's Modified Eagle Medium/Ham's F-12 | Thermo Fisher Scientific | 12634028 | Advanced DMEM/F-12 |
Earle’s balanced salt solution (EBSS) | Thermo Fisher Scientific | 24010043 | |
Forceps | N/A | N/A | |
Forskolin | Tocris Bioscience | 1099 | |
GlutaMAX supplement | Thermo Fisher Scientific | 35050061 | |
HEPES, 1 M | Thermo Fisher Scientific | 15630080 | |
Leica DM6 B Fluorescence Motorized Microscope | Leica | N/A | |
N2 supplement (1003) | Thermo Fisher Scientific | 17502048 | |
N-acetylcysteine | Merck | A0737-5MG | |
Nicotinamide | Merck | N0636 | |
Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes | Thermo Fisher Scientific | 339651 | |
Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes | Thermo Fisher Scientific | 339653 | |
Penicillin/streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
Recombinant human EGF | Peprotech | AF-100-15 | |
Recombinant human FGF10 | Peprotech | 100-26 | |
Recombinant human FGF19 | Peprotech | 100-32 | |
Recombinant human HGF | Peprotech | 100-39 | |
Recombinant human Noggin | Peprotech | 120-10C | |
Rho kinase inhibitor Y-27632 dihydrochloride | Merck | Y0503 | |
R-spodin1-conditioned medium | (Broutier et al.) | N/A | Secretion of cell lines |
Surgical scissors | N/A | N/A | |
Surgical specimen of tumor removed from HCC patients | Affiliated Cancer Hospital and Institute of Guangzhou Medical University | N/A | |
TNFα | Peprotech | 315-01A | |
TrypLE Express Enzyme (1x), no phenol red | Thermo Fisher Scientific | 12604013 | Trypsin substitute |
Wnt-3a-conditioned medium | (Broutier et al.) | N/A | Secretion of cell lines |