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

Summary

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.

Abstract

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.

Introduction

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 approaches^2,3,4,5. 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 HCC^3,4. 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 organoids^6,7,8. 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 passaging^6,8. In the subsequent sections, a comprehensive account of the operational intricacies and pertinent factors involved in the construction of HCC organoids will be presented.

Protocol

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.

1. Tissue dissociation
   1. Preparation of supplies and materials
      1. Collect 1-2 cm³ of HCC tissue from patients who have not received any previous local or systemic treatment before the operation. After surgical resection, keep the tissue at 4 °C in tissue preservation solution (Table 1) until processing.
         NOTE: High-quality fresh tissue samples are essential for the successful establishment of organoids. It is important to process samples promptly, ideally within 1-4 h after surgical resection to maintain tissue viability. Perform all subsequent procedures in an aseptic environment using sterile materials and reagents.
      2. Preheat ultra-low attachment surface cell culture plates (24 wells) for 1 h in an incubator at 37 °C. Thaw the frozen basement membrane extract (BME) overnight at 4 °C until just before use.
         NOTE: To ensure complete thawing of the BME, it must be placed on ice for at least 3 h. Complete melting of the BME is essential for successful organoid culture in the subsequent steps.
      3. Before use, ensure that the digestion solution (Table 1) is warmed to a temperature of 37 °C.
         NOTE: Prepare the digestion solution freshly in an aseptic environment and utilize it promptly.
   2. Organizational processing flow
      1. In a laminar flow cabinet, cut the tumor tissue into small pieces (0.5-1 mm³) using surgical scissors on a Petri dish. Transfer the clipped tissue to a 15 mL conical tube and add 5-10 mL of basal medium (Table 1).
         NOTE: The basal medium (Table 1) can be stored at 4 °C for up to 1 month.
      2. Use a barotropic pipette to wash away as much blood as possible and let it stand for 1-2 min to remove some of the supernatant, including any blood cells and floating fat clots. Repeat this procedure twice.
      3. Centrifuge the mixture at 300 × g for 5 min at room temperature, and carefully remove the supernatant. Add 5 mL of the prewarmed digestion solution (Table 1) to the trimmed tissue.
      4. Rotate the tube at 37 °C for digestion.
      5. After 30 min of initial digestion, look for small clusters of cells under the microscope. Check every 5-10 min to avoid overdigestion.
         NOTE: The time required for tissue digestion will depend on the size and type of tissue. Tissue digestion should not exceed 90 min. Overdigested tissue will appear under the microscope as a large sheet of individual cells. The appropriate level of digestion is indicated by the fact that most of these are cell clusters and have a grape-like appearance.
      6. Stop digestion by adding cold basal medium (Table 1) and filter into a new 50 mL tube with a 100 µm cell filter. Add cold basal medium to a volume of 50 mL.
      7. Centrifuge the cells at 2 × g for 10 min at 8 °C. Carefully remove the supernatant and resuspend the pellet by adding a further 50 mL of cold basal medium (Table 1). Repeat this step twice.
         NOTE: Low-speed centrifugation is used to allow small cell clusters to settle down, while blood cells and cell debris are still suspended in the supernatant; the process is repeated several times to obtain a relatively pure tissue cell mass.
2. Organoid plating
   1. After the second wash, remove as much of the supernatant as possible and resuspend the desired number of cells (1,000-5,000 cells per well of a 24-well plate) in the appropriate BME for plating.
      NOTE: Always keep the BME at 4 °C before use.
   2. Add BME and suspend small cell clusters in Advanced DMEM/F-12 by adding BME and gently pipetting up and down until the cell aggregates are completely suspended. Control the concentration of the BME between 30% and 50%.
   3. Seed 50 µL BME droplets mixed with cell clusters in the center of 24-well culture plates.
      NOTE: As far as possible, do not let the droplets spread to the sidewall of the well plate. The sidewall of the low-adsorption plate is not in a low-adsorption state, and contact between the droplets and the sidewall will lead to adhesion, which is not conducive to the subsequent incubation.
   4. Allow the plates with the added droplets to solidify at 37 °C for 20 min. At the end of the incubation, add 500 µL of prewarmed medium (Table 1) to each well and incubate in a cell incubator at 37 °C.
3. Organoid culture
   1. Refresh the culture medium (Table 1) once every 2-3 days. At the beginning of the culture, culture the HCC organoids in HCC isolation medium (Table 1) for 2 weeks.
   2. After 2 weeks of incubation with HCC isolation medium, replace the medium with HCC organoid expansion medium (Table 1) (stored at 4 °C for up to 2 weeks).
   3. Refresh the culture medium once every 3 days.
   4. After 7-10 days of culturing with HCC expansion medium when the organoid has reached the appropriate density or size, initiate experimental interventions or passage or lyophilize the organoids as needed.
4. Organoid passaging
   1. Preparation of supplies and materials
      1. Preheat ultra-low attachment surface cell culture plates (24 wells) for 1 h in an incubator at 37 °C. Thaw frozen BME overnight at 4 °C until just before use.
      2. Prewarm organoid harvest solution and trypsin substitute at 37 °C for 30 min.
   2. passaging process
      1. After removing the culture medium from the well plate, transfer the organoid suspension to a 15 mL tube.
      2. Add organoid harvesting solution according to the amount of BME (500 µL of organoid harvesting solution per 50 µL of BME) by scraping and pipetting up and down using a 1,000 µL pipette gun.
         NOTE: Avoid air bubbles throughout the process to prevent loss of organoids as the presence of air bubbles indicates higher pipetting pressure.
      3. Incubate for 30 min at room temperature.
      4. Gently aspirate the BME with a 1,000 µL pipette and observe that the BME is completely dissolved. Observe every 10 min until a clear organoid cell cluster is observed, then centrifuge at 400 × g for 5 min at room temperature and remove as much of the supernatant as possible.
         NOTE: At this stage, the organoid can be frozen and preserved.
      5. For enzymatic digestion of organoids, add 1-5 mL of the trypsin substitute (according to the number of organoids) and incubate at 37 °C for 2 min. Observe under the microscope the degree of enzymatic digestion to determine whether the organoids dissociate into small clusters of 2-10 cells; if not, continue the enzymatic digestion.
         NOTE: Overdigestion will result in the lysis of organoid cell clusters into single cells, decreasing their viability and prolonging subsequent culture time.
      6. Add the appropriate volume of cold basal medium (Table 1) to stop the digestion.
      7. Centrifuge the organoids at 400 × g for 5 min at 8 °C; remove the supernatant as much as possible.
      8. Resuspend the desired number of organoids (1,000-5,000 organoids per well of a 24-well plate) in the appropriate matrix for plating. Follow the same steps as in section 1.2.
         NOTE: The plating density should be optimized according to the growth rate and size of the organoids.
      9. Passage the organoids every 10 days at a ratio of 1:3 or 1:4 depending on the density of organoid growth.
5. Organoid cryopreservation and resuscitation
   1. Cryopreservation of organoids
      1. Prepare lyophilization tubes, each tube confluent with two wells of a 24-well plate containing organoids.
      2. Follow steps 1.4.2.1 to 1.4.2.4 for organoid passaging to obtain organoids without BME, and gently resuspend the organoids by adding 500 µL/well of organoid lyophilization solution to a 24-well plate.
         ​NOTE: Depending on the growth status and size of the organoids, it is recommended to cryopreserve the organoids in culture up to the third generation.
      3. Transfer the suspension into lyophilization tubes and place it in a gradient-cooled box at -80 °C. After gradient cooling at -80 °C for at least 24 h, transfer the tubes to liquid nitrogen for long-term storage.
   2. Resuscitation of organoids
      1. Incubate lyophilized tubes in a 37 °C water bath and stop incubation when only a small block of ice remains.
      2. Centrifuge at 400 × g for 5 min at 8 °C to remove the supernatant completely.
      3. Follow sections 1.2 and 1.3 for subsequent operations.

Representative Results

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.

Discussion

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 cultivation^6,8,9. 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 models^10,11.

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 challenges^11,12,13.

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 therapy^4,5. 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 organoids^6,9. 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.

Materials

Name	Company	Catalog Number	Comments
[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