Optimizing media composition for patient-derived organoids in ovarian cancer: a narrative review

Introduction

Background

Ovarian cancer (OC) is the eighth most commonly diagnosed cancer in women and the fifth leading cause of cancer-related death among women globally (1,2). In 2025, an estimated 3,100 Canadian women will be diagnosed with OC and 2,000 will die from it (2). The majority of patients are diagnosed at an advanced stage (3). High-grade serous ovarian carcinoma (HGSOC), the most prevalent subtype, accounts for approximately 70% of OC cases (4), is characterized by ubiquitous TP53 mutations (5), frequent homologous recombination deficiency (HRD) including BRCA1/2 mutations, and pronounced inter- and intratumoral heterogeneity (6). Despite the known heterogeneity, current treatment paradigm with combination of surgery and platinum-based chemotherapy in the upfront setting has not changed in the past decades.

Recent advances, including poly (ADP-ribose) polymerase (PARP) inhibitors, folate receptor antibody-drug conjugates, have expanded treatment options for patients. While these therapies have primarily been studied in the recurrent setting, ongoing trials are now evaluating their efficacy in the frontline setting. However, despite these advances, individualizing therapy selection using biomarkers remains an imperfect science. Current biomarker strategies often fail to predict response reliably across heterogeneous patient populations, leading to suboptimal treatment outcomes. This limitation underscores the critical need for predictive ex vivo models, such as patient-derived organoids (PDOs), that can more accurately assess individual tumor responses and enable truly personalized therapy selection beyond static biomarker profiles (7-9).

Rationale and knowledge gap

PDOs have revolutionized the study of cancer by enabling the ex vivo culture of 3D structures that retain key genetic, morphological, and functional characteristics of the patient’s tumor. These models are particularly valuable in translational research as they offer platforms for personalized drug testing, biomarker discovery, and insights into individual-specific tumor biology (7-9). In solid cancers, such as colorectal and pancreatic cancer, successful growth of PDOs using standardized protocols has been achieved in up to 70% (10-12). In contrast, the establishment of PDOs from OC has low reported success rates (~20%) and lack standardized protocols (13-16).

For example, Kopper et al. [2019] reported successful derivation of 23 organoids lines from 56 organoids samples (41%) (17), while Hill et al. [2018] achieved a 26% success rate for HGSOC PDOs capable of drug screening (18). Reasons for low success rates include poor tissue quality (often necrotic or fibrotic), inconsistent sample processing, and significant heterogeneity in the criteria used to define PDOs “success”, whether based on initial expansion, passaging potential, or therapeutic applicability (15,19). Compounding these difficulties is the lack of standardized culture media for OC PDOs generation (15,19). Most protocols borrow media compositions originally optimized for gastrointestinal cancers, incorporating components such as epidermal growth factor (EGF), R-spondin 1, Noggin, Wnt3a, and pathway inhibitors like A83-01 and Y-27632 (17,19). However, it remains unclear which of these components are essential or beneficial for OC specifically. For example, while bone morphogenetic proteins (BMPs) pathway inhibition via Noggin is a staple of colorectal PDOs protocols (20), its utility in OC models is inconsistently applied (21-23). Similarly, the inclusion of β-estradiol, a hormone relevant to estrogen receptor-positive tumors, is variably used across OC studies despite limited evidence for its necessity in HGSOC (14,17,19,24). Overall, a more nuanced approach to translating media design from other solid tumors to HGSOC PDOs is needed.

In addition to culture media composition, upstream factors such as tissue acquisition, handling, and preservation play a critical role in determining PDO establishment success and reproducibility. Ovarian tissue cryopreservation (OTC) is an established clinical strategy for fertility preservation, with robust evidence demonstrating restoration of endocrine function and successful reproductivity. While primarily developed for clinical use, OTC also provides a valuable framework for standardized tissue preservation and biobanking, which is directly relevant to translational research applications such as PDO generation (25).

Other cryopreservation techniques, including conventional slow freezing and vitrification, differ in their impact on tissue architecture and cellular viability. Studies have shown that these approaches can influence stromal integrity, angiogenic potential, and the secretion of key factors such as vascular endothelial growth factor (VEGF) (26,27), as well as metabolic recovery following thawing. These biological differences may affect the survival and proliferative capacity of tumor cells in vitro, thereby influencing PDO derivation efficiency.

Despite these considerations, variability in cryopreservation protocols, including differences in ischemia time, cryoprotectant exposure, and storage conditions, is rarely accounted for in PDO studies. As a result, heterogeneity in tissue preservation may represent an underrecognized source of variability in PDO success rates, alongside differences in media composition. Integrating standardized biobanking and cryopreservation practices into PDO workflows may therefore be essential to improving reproducibility across studies.

Given the current limitations, including low success rates, variable definitions of success, and inconsistent media formulations, there is a demonstrated need to systematically compare published protocols, identify key media components, and assess the rationale behind their use to identify opportunities to improve reproducibility and efficiency. Furthermore, investigating whether media formulations used for other TP53-mutant cancers such as pancreatic cancer or glioblastoma (GBM) may offer transferable strategies that could guide future optimization.

Objective

This review aims to (I) summarize and compare media compositions used in OC PDOs studies; (II) highlight key differences and potential rationales for media component selection; (III) draw comparative insights from other cancer types with frequent TP53 mutations; and (IV) propose recommendations for standardizing OC PDOs culture conditions. We present this article in accordance with the Narrative Review reporting checklist (available at https://cco.amegroups.com/article/view/10.21037/cco-2026-1-0013/rc).

Methods

This review was conducted to map the available evidence on OC PDOs (Table 1). A structured search was performed in PubMed using the keywords (“ovarian cancer” OR “ovarian carcinoma” OR “OC”) AND (“Organoids”[MeSH] OR “patient-derived organoid*” OR “PDO” OR “PDOs” OR “tumor organoid*” OR “cancer organoid*” OR “organoid culture”). This initial search identified 188 articles. Abstracts were independently reviewed by K.H., M.C., and G.L. to ensure they met the predefined inclusion criteria and full texts were reviewed by all authors. Disagreements were resolved through discussion and consensus. Articles not meeting these criteria were excluded for the following primary reasons: review articles; use of commercial PDO kits; lack of detailed media/culture information; not focused on PDO establishment; non-English publications. Full-text articles were then reviewed by all authors. The final selected articles met the following criteria: (I) the study was a primary research article (no reviews); (II) the authors developed the PDOs and culture media independently; (III) no commercial PDO kits were used; (IV) detailed information on media composition and culture conditions was provided; and (V) the studies were published no earlier than Hill et al. [2018], which was the first paper to address this topic directly (18). In total, 10 articles were included.

Further, we conducted our own preliminary results and developed OC PDOs. All patients provided written informed consent for the collection, storage, and research use of tumor material and associated clinical data, with the option to withdraw consent at any time. Biobanking and research protocols were approved by the Institutional Review Board of the McGill University Health Centre (Montreal, Quebec, Canada; IRB #A08-M79-13B). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell pellets were resuspended in Matrigel^TM (80 µL per dome), with the number of domes adjusted according to pellet size, and plated in 24-well plates. Plates were incubated at 37 °C with 5% CO₂ for 10 minutes to allow Matrigel polymerization. Following polymerization, 600 µL of either base medium derived from Hill et al. [2018] (18), M1 or M2 culture medium, as previously described by Senkowski et al. [2023] (19), was added to each well, and each sample was cultured in both media conditions in parallel. Primocin was added to all cultures to prevent microbial contamination.

Culture medium was refreshed every 3–4 days, and PDO growth and morphology were monitored regularly using inverted light microscopy. Representative images were acquired using the Thermo EVOS XL Inverted Imaging Microscope.

Results

Summary of recent and relevant OC PDO studies

Table 2 presents a summary of the most recent and relevant studies involving OC PDOs.

PDO sources and derivation

In total, 289 OC PDOs were attempted, of which 32 reached the author-defined threshold of success (11.2%). The median number of PDOs developed was 34 [range from 13 (24) to 57 (31); interquartile range (IQR), 16–49.5]. Across the studies, the median patient cohort size was 27. Most studies included HGSOC, with some also incorporating low grade serous ovarian carcinoma (LGSOC), mucinous (MOC), clear cell (OCCC), endometrioid (EOC), and carcinosarcoma (OCS) subtypes. Tumor samples for PDOs derivation were obtained from solid tumors (64.4%), ascites (17.1%), and pleural effusion (18.5%).

The ability to passage PDOs varied considerably: Hill et al. [2018] achieved 6 to 30 passages (18), Kopper et al. [2019] reported 5 to 32 passages (17), Hoffman et al. [2020] established lines lasting up to 26 passages (14), and Senkowski et al. [2023] derived PDOs for up to 20 passages (19). Studies varied in their inclusion of BRCA mutation status. Hill et al. [2018] (18) and Kopper et al. [2019] identified BRCA1/2 mutations and tested sensitivity to DNA repair inhibitors (17). Thorel et al. [2025] (29), Senkowski et al. [2023] (19), and Hoffman et al. [2020] confirmed tumor identity in PDOs via TP53 mutation concordance (14).

Definition of success

Definitions of successful PDOs derivation among the articles differed. Hoffman et al. [2020] defined success as stable long-term culture beyond one year (14). Maenhoudt et al. [2020] distinguished between short-term success (≤4 passages) and long-term success (>4 passages, ≥1 year) (24) while Thorel et al. [2025] defined long-term success as growth beyond 8 passages (29). Senkowski et al. [2023] used a four-point validation: ≥10 passages, absence of growth arrest, expansion of tumor cells, and retention of TP53 mutations (19). Hill et al. [2018] (18) and Chen et al. [2024] (30) did not define success but reported PDO formation from multiple patient samples. Other studies, such as Bi et al. [2021] (28), Yoshimura et al. [2025], and Farsinejad et al. [2025], did not formally define success (31,32). For our results, we define short-term success as beyond three passages and long-term success as beyond 10 passages.

Reported derivation efficiencies across studies

Reported derivation efficiencies varied widely depending on tissue source, histologic subtype, and study-specific definitions of success. Across studies using solid tumor samples from epithelial OC, derivation efficiencies ranged from 13.8% (27) to 65% (17). In contrast, Hill et al. [2018] reported a 100% derivation rate for PDOs established from pleural effusion samples (18).

When focusing specifically on solid HGSOC samples, Kopper et al. [2019] (17) and Maenhoudt et al. [2020] (24) reported derivation efficiencies of 55% and 36%, respectively, with corresponding long-term culture efficiencies of 61% and 63%. These findings underscore the strong influence of tissue source and histologic subtype on reported PDO establishment rates.

Media composition

Currently, there is no standardized protocol or consensus regarding optimal conditions for culturing PDOs in OC. In order to address this, we compiled a comparative analysis of the culture media components used in the most widely cited OC PDOs studies (Tables 3,4), highlighting both the most commonly used ingredients and those shared across multiple protocols. We compared media compositions across 10 foundational studies that established PDO models for OC, with a focus on HGSOC and other subtypes (Tables 3,4). While a core set of components was common to most protocols, concentrations and supplemental factors varied considerably.

Basal media and core additives

All studies used Advanced DMEM/F12 as the basal medium, supplemented with GlutaMAX and B27. Penicillin/streptomycin or Primocin was added in each study to prevent microbial contamination. Antioxidants such as N-acetylcysteine were consistently included to enhance cell viability by reducing reactive oxygen species (ROS).

Signal pathway inhibitors and growth promoters

A83-01, a TGF-β receptor inhibitor that maintains stemness and inhibits epithelial-to-mesenchymal transition (EMT), was included in all protocols (0.25–0.5 µM), except for Chen et al. [2024] (30), who omitted it. Nicotinamide, a PARP and sirtuin inhibitor that enhances PDOs expansion and suppresses differentiation, was universally present, though concentrations varied widely from 1 mM (14) to 10 mM (18,29,32). ROCK inhibitor Y-27632, used to prevent anoikis and improve survival during passaging, was included by most, with doses typically around 10 µM.

Wnt and BMP pathway modulation

Medium developed by Hill et al. [2018] was enriched with Wnt pathway activators including R-spondin, Noggin, fibroblast growth factors (FGF), and EGF (18). Subsequently, Kopper et al. [2019] expanded this formulation by adding hydrocortisone, forskolin, β-estradiol, HGF, and Heregulin β1, while maintaining Wnt3a activation (17). Noggin and R-spondin1 (RSPO1), essential for Wnt/BMP pathway regulation, were commonly used. Noggin was added at ~100 ng/mL in most studies (except where unspecified), while RSPO1 concentrations ranged from 10% conditioned medium (17) to 1 µg/mL (19). Some variability was observed in RSPO1 levels and the source (conditioned media vs. recombinant protein), which may influence signaling potency.

Hormonal and epithelial growth support

β-estradiol was included in five studies, with concentrations around 10–100 nM, potentially to maintain ovarian lineage or epithelial characteristics. EGF was universally included at concentrations from 5–50 ng/mL. FGF10, another epithelial growth factor, was variably added {e.g., 10 ng/mL in Hill et al. [2018] (18), up to 100 ng/mL in Bi et al. [2021] (28) and Yoshimura et al. [2025] (31)}, indicating less consensus on its necessity.

Buffering and stability components

HEPES was used as a buffering agent (1%–10 mM) in most protocols to maintain pH stability in culture. B27 supplement was included in nearly all protocols, providing essential vitamins, antioxidants, and lipids necessary for PDOs development.

Differences in media composition

The study by Hill et al. [2018] served as the baseline reference for OC PDOs culture, providing the initial medium formulation from which subsequent protocols evolved (18). Besides the core components, medium developed by Hill et al. [2018] (18) was enriched with Wnt pathway activators including R-spondin1, Noggin, FGF, and EGF. Subsequently, Kopper et al. [2019] expanded this formulation by adding hydrocortisone, forskolin, β-estradiol, HGF, and Heregulin β1, while maintaining Wnt3a activation (17).

However, using the media composition composed by Kopper et al. [2019] (17), Hoffman et al. [2020] reported that HGSOC PDOs failed to grow in this medium, necessitating several modifications (14). Notably, the authors share that the only essential paracrine growth factor for HGSOC cultures was EGF. In contrast to healthy PDOs derived from tissues like intestine, fallopian tube, stomach, and liver, which require exogenous Wnt activation, none of the HGSOC PDOs lines could be maintained in media containing the Wnt agonist Wnt3a. Moreover, continuous BMP inhibition via Noggin proved detrimental, removal of Noggin was necessary for successful culture in 13 out of 15 HGSOC samples, suggesting a fundamentally different role for BMP and Wnt signaling in the regulation of stemness in malignant versus normal ovarian tissues.

Maenhoudt et al. [2020] further refined OC PDOs culture conditions, identifying Neuregulin-1 as a potent enhancer of PDOs development and growth (24). They also introduced N2 supplement, N-acetylcysteine, and IGF1, achieving a 44% derivation efficiency across 12 PDOs lines. Building on this, Bi et al. [2021] reported a 75% efficiency by excluding p38i, IGF1, and HGF from their formulation (28).

More recently, Senkowski et al. [2023] compared two media formulations (M1 and M2) across 17 HGSOC PDOs samples, reaching a derivation efficiency of 53% (19). The key differences between M1 and M2 were the inclusion of Heregulin β1, forskolin, EGF, and hydrocortisone, though the two media variations enhanced PDOs growth. Their study also found that Noggin increased cellular attachment while reducing PDOs formation, and that R-spondin 1, nicotinamide, and prostaglandin E2 had no significant effects. Importantly, they were able to sustain PDOs growth for at least 10 passages, with no observed growth arrest and successful expansion of cancer cells.

Standardization between studies

Despite substantial heterogeneity in culture conditions and experimental aims, OC PDOs have been successfully applied in drug testing and functional assays, demonstrating their value as preclinical models. However, across the literature, the level of detail used to report PDO generation, derivation efficiency, success criteria, and histotype-specific context remains inconsistent, which complicates cross-study interpretation and limits reproducibility at the level of reporting rather than protocol design (66).

For example, Yoshimura et al. [2025] successfully generated 57 OC PDOs (31). Their culture medium differed significantly from previous protocols [e.g., Senkowski et al., 2023 (19)], being enriched with Wnt3A agonist, R-spondin1, and Noggin. They performed high-throughput drug screening using nine OCCC-derived PDOs, identifying proteasome inhibitors as potential therapeutic candidates. They further validated this vulnerability by CRISPR-Cas9-mediated knockout of the ARG2 gene. Although the media composition is included, explicit definitions of successful organoid establishment, expansion rate, and passage thresholds were not specified, making it difficult to contextualize their reported success relative to other studies. Similar gaps in reporting are observed across multiple studies, where PDO formation is described without clear documentation of expansion capacity, duration of culture, or criteria used to classify cultures as successful.

Similar limitations in reporting expansion rate, passage number, and success definitions were observed in studies by Farsinejad et al. [2025] (32) and Thorel et al. [2025] (29), despite robust mechanistic findings. Although these reporting differences do not detract from the biological conclusions drawn within individual experimental frameworks, the absence of standardized reporting of expansion thresholds and success definitions complicates contextual interpretation across studies, even when media composition and experimental focus are similar.

Our experience

In total, 23 tissue-derived PDOs were established from 23 OC patients representing diverse histologic and molecular subtypes who underwent either primary or interval debulking surgery. The cohort included eight EOC, one OCCC, 12 HGSOC, two adult granulosa cell tumors of the ovary (AGCT), and one OCS (Table 5). All HGSOC cases were diagnosed at advanced stage; among these, eight demonstrated aberrant p53 overexpression, two showed a p53-null pattern, and p53 status was unavailable for two cases.

As an initial step in media optimization, a base culture medium {adapted from Hill et al.’s [2018] media (18)} containing ADDF+++, R-spondin, Noggin, SB202190, A83-01, EGF, FGF10, prostaglandin E2, N-acetyl-L-cysteine, nicotinamide, B27 supplement, Y-27632, basic FGF, NRG1, and Normocin was used. Under these conditions, none of the 14 tissue-derived samples expanded beyond passage three. Based on this lack of expansion, two media formulations established by Senkowski et al. [2023], media M1 and M2, were subsequently used to better support OC PDO growth (19).

Following optimization to the M1 and M2 media formulations, successful PDO derivation, defined as expansion beyond the third passage, was achieved in two out of ten samples. Both models that demonstrated this short-term expansion were derived from endometrioid ovarian carcinomas from patients with stage IA disease (Patients #14 and #17; Table 5). One of these PDOs (PDO ID OC-15) achieved long-term expansion, reaching passage 20. In parallel with improved passaging capacity, overall organoid size and confluency increased following media optimization (Figure 1). No appreciable differences in growth behavior were observed between the M1 and M2 conditions (Figure 2).

Figure 1 Comparison of organoid confluency and size in different media. Representative brightfield images of organoid growth in medium M0 (stage IC endometrioid ovarian carcinoma) and in media M1 and M2 (stage IA endometrioid ovarian carcinoma). All images were captured at passage one, with M0 captured at ×4 magnification and M1 and M2 at ×10 magnification. Increased confluency and larger organoid size were observed in M1 and M2 compared to M0, indicating enhanced growth with media optimization.

Figure 2 Comparison of organoid growth in media M1 and M2 across patient cases. Representative brightfield images of organoids derived from four different patient tissue samples, cultured in media M1 and M2. Images were captured at ×10 magnification. Organoid size and confluency appear comparable between M1 and M2, with no substantial differences in growth observed.

Discussion

Our review highlights that despite the increasing interest in modeling OC biology, PDOs development varies considerably in their protocols, definition of success and derivation outcomes. These differences stem from the diversity in study designs, tissue sources, and intended downstream use of PDOs.

Research design informs media protocols and sample selection

Short-term PDO models were emphasized in Chen et al. [2024] (30), Hill et al. [2018] (18), and Farsinejad et al. [2025] (32), where cultures were maintained primarily for ex vivo drug testing or functional assays. While these models had limited passage potential, they were highly relevant for correlating immediate drug response with clinical outcomes. For example, Chen et al. [2024] demonstrated that PDO sensitivity to chemotherapeutics paralleled patient responses, offering strong translational utility despite limited scalability (30). Similarly, Hill et al. [2018] used modified culture conditions to explore responses to PARP and ATR inhibitors in HRD-stratified PDOs, linking in vitro sensitivity to underlying homologous recombination status (18).

Long-term PDO cultures, such as those established by Kopper et al. [2019] (17), Maenhoudt et al. [2020] (24), and Thorel et al. [2025] (29) are particularly valuable for biobanking, molecular tracking, and therapeutic modeling. These models retain genomic and phenotypic fidelity to the original tumor over time. In contrast, short-term models [Chen et al., 2024 (30); Hill et al., 2018 (18)] are best suited for rapid drug sensitivity profiling and may better align with real-time clinical decision-making workflows.

The inclusion of BRCA status and treatment history in some studies also highlights the potential for PDOs to stratify therapeutic response. For example, Hill et al. [2018] used PDOs to validate sensitivity to olaparib in BRCA-mutated samples (18). Similarly, Thorel et al. [2025] correlated PDO drug responses with patient treatment outcomes (29).

Subtype-specific differences were also reflected in media choices. Yoshimura et al. [2025] developed PDOs from CCC and adapted media composition accordingly, incorporating FGF10 and A83-01, and established a high-throughput drug screening platform (31). Bi et al. [2021] explored PDO culture from multiple gynecologic malignancies (including OC), demonstrating successful propagation from both fresh and cryopreserved tissues using a standard set of PDOs-supporting factors (28). This study highlighted the feasibility of biobanking and the robustness of their culture system across tumor types.

The source of tumor tissue also influenced success rates. Hill et al. [2018] reported 100% efficiency using pleural effusion samples (18), while studies using ascites or solid tumor tissue, such as Thorel et al. [2025] reported lower efficiency (13.8%) (29). This suggests that cell viability and tumor cell density in liquid samples may confer a growth advantage. Interestingly, while most studies focused on PDOs derived from solid tumor tissue, few investigated ascites-derived models or microenvironmental mimicry. Media compositions were often optimized for epithelial cell growth but lacked stromal or immune components, a limitation acknowledged by several groups. Furthermore, definitions of PDO “success” varied—ranging from initial sphere formation to sustained passaging and functional validation—underscoring the need for standardized metrics across studies.

Balancing Wnt and growth factor signaling in OC PDO media composition

The same problem of limited concordance between studies is evident in the variable media components used for OC PDO’s culture. For instance, Senkowski et al. [2023] (19) and Hoffman et al. [2020] (14) demonstrated that low-Wnt or Wnt3a-free conditions were optimal for the expansion and stability of HGSOC PDOs, as high Wnt signaling could promote undesired differentiation or limit long-term propagation. In contrast, Kopper et al. [2019] (17) and Maenhoudt et al. [2020] (24) included more extensive supplementation, such as p38 inhibitors (SB202190), PGE2, and FGF10, enabling long-term cultures (>10 passages) and higher fidelity to the original tumor’s morphology and mutational landscape. Thorel et al. [2025] further validated the stability of such long-term PDOs, showing that they could be maintained for over a year while preserving clinical treatment response patterns (29).

Evidently, Noggin, R-spondin1, and Wnt3A are highly debated components due to their potent but variable influence on Wnt signaling, pathway essential for cell maintenance and differentiation. While these factors are known to promote PDOs growth in certain epithelial tissues (67), their effects in OC are inconsistent and histotype-dependent (17,18). For instance, R-Spo1 and Wnt3A synergistically enhance canonical Wnt signaling, which may support long-term expansion of PDOs, but in some OC subtypes such as HGSOC excessive Wnt activation has been associated with loss of tumorigenic potential or induced differentiation rather than self-renewal (17). Noggin, a BMP pathway inhibitor, further complicates the balance by preventing differentiation signals, yet its necessity varies across protocols and histotypes (18). The lack of consensus stems from the heterogeneity of OC, where each histological subtype shows distinct molecular and signaling dependencies (68,69). As a result, while these components are indispensable in some systems (67), their inclusion in OC media remains controversial, often requiring empirical optimization and raising questions about standardization and cross-study comparability.

Our own experience in developing OC PDOs aligns with findings by Senkowski et al. [2023] (19) and Maenhoudt et al. [2020] (24) where low-Wnt conditions and the inclusion of TGF-β and ROCK inhibitors supported robust PDOs formation. However, we also observed morphological heterogeneity and differential growth responses depending on the tumor source and inclusion of ascitic fluid, suggesting that even subtle media variations can yield significant effects on PDOs viability and phenotype. These nuances emphasize the importance of tailoring media composition not only to the cancer subtype but also to the source material and intended downstream applications.

Hypotheses on differences between studies

Variability across studies likely reflects a combination of biological, technical, and clinical factors. For example, HGSOC often exhibits distinct growth characteristics and media requirements compared with endometrioid or CCC (17,31), potentially influencing PDOs initiation efficiency.

Molecularly, HGSOC is defined by near-universal TP53 mutations and frequent alterations in homologous recombination repair genes, including BRCA1 and BRCA2 (70). In contrast, other histologic subtypes are enriched for distinct oncogenic drivers, such as activating mutations in the MAPK pathway (e.g., KRAS, BRAF) in LGSOC (71), alterations in Wnt/β-catenin signaling (e.g., CTNNB1) in endometrioid carcinoma (72-74) and frequent ARID1A and PIK3CA mutations in CCC (75,76).

Importantly, substantial molecular heterogeneity exists not only between OC subtypes but also within individual histotypes, with considerable variation in mutational profiles, pathway activation, and genomic instability across patients. This intra- and intertumoral heterogeneity likely contributes to variable PDO establishment and expansion outcomes and underscores the need for flexible, context-aware media optimization strategies rather than uniform culture conditions.

Tissue quality is another critical factor. Tumors obtained from metastatic or peritoneal sites may exhibit greater necrosis, inflammatory infiltration, or stromal overgrowth than those collected from primary lesions, reducing the proportion of viable epithelial tumor cells available for culture (77,78).

Moreover, prior patient treatment exposure can have a profound impact on PDOs establishment. Tumors collected after chemotherapy or targeted therapy may contain subpopulations of drug-resistant cells with altered growth requirements or may be composed primarily of therapy-induced senescent or apoptotic cells, thereby lowering the probability of successful culture (77).

Our results compared to others

Our findings demonstrate that successful PDO establishment and maintenance using our current culture conditions are strongly influenced by tumor histologic subtype and media composition. While key media modifications showed success in low-grade endometrioid carcinoma samples, our protocol was not successful in any other subtypes, suggesting the importance of tailoring media composition based on the tumor subtype.

Consistent with prior reports, our data underscore the critical role of media formulation in supporting PDO expansion. The base medium (M0), inspired by Hill et al. [2018] (18), was insufficient to sustain organoid growth beyond early passages in most cases, whereas growth factor–enriched formulations (M1 and M2) improved short-term expansion. Despite these improvements, HGSOC samples in our cohort consistently failed to achieve long-term expansion, even under optimized conditions. Notably, this limitation persisted despite the use of a media formulation identical to that reported by Senkowski et al. [2023], who demonstrated successful long-term expansion of PDOs derived from HGSOC (19). In contrast, the only PDOs in our study that achieved sustained expansion were derived from endometrioid carcinomas, highlighting a clear subtype-specific divergence in growth requirements. Together, these findings suggest that media formulations optimized for HGSOC may not be directly transferable across OC subtypes and, conversely, that distinct molecular contexts, such as those present in endometrioid tumors, may confer differential responsiveness to current culture conditions.

While M1 and M2 showed comparable short-term growth performance, subtle differences in passaging potential suggest a nuanced influence of each medium component. Similar to Senkowski et al. [2023] (19), we found that some PDOs could sustain multiple passages under optimized conditions. Furthermore, they demonstrated that organoids cultured under these refined conditions closely mirrored the genetic and histological characteristics of the original tumors. While genomic fidelity was not assessed in our study, the subtype-specific growth patterns and morphological heterogeneity observed in culture align with prior reports, suggesting that optimized media conditions can preserve key tumor-intrinsic features.

Importantly, our findings confirm and expand upon the work of Senkowski et al. [2023] (19) by applying similar media optimization strategies across a broader range of OC histologies, including endometrioid ovarian carcinoma as well as less common subtypes such as clear cell carcinoma and carcinosarcoma. Collectively, these observations reinforce the necessity for context- and subtype-specific media development and support ongoing efforts to establish more robust and broadly applicable organoid platforms across the spectrum of ovarian malignancies.

Cross-cancer insights

As powerful tools for investigating drug response and treatment efficacy, PDOs have been successfully used to model a wide range of cancer types, enabling cross-cancer comparisons. This allows OC PDOs to be analyzed in relation to models from other tumor types, such as pancreatic (79), colorectal (77), and GBM (80), offering valuable insights into shared vulnerabilities, signaling dependencies, and media optimization strategies across TP53-dominant malignancies.

TP53 mutations are present in approximately 50–75% of human pancreatic ductal adenocarcinomas (PDAC), typically arising after an initiating oncogenic event involving KRAS activation. These mutations often lead to the expression of a stable but dysfunctional p53 protein, which contributes to genomic instability and tumor progression (81). This mutational sequence and TP53 dominance make PDAC biologically comparable to HGSOC, where TP53 alterations are also a hallmark feature.

Since the development of PDAC PDOs models by Boj et al. [2015] (82), the field has advanced significantly. Their study reported an PDOs establishment efficiency of 75–83%, highlighting the robustness of the model. Importantly, they found that the inclusion of Noggin, prostaglandin E2 (PGE2), EGF, Wnt3A, nicotinamide, R-spondin1, and A83-01 significantly improved PDOs viability and propagation (83), allowing the cultures to be maintained for up to 20 passages. These PDOs could also be successfully cryopreserved and recovered, enhancing their utility for long-term biobanking and functional studies. Such findings emphasize the importance of optimizing signaling environments in TP53-driven cancers and support PDAC PDOs as a stable and scalable preclinical model.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide (84), following breast and lung cancers, and remains the second leading cause of cancer-related mortality globally, surpassed only by lung cancer (2). CRC progression is closely associated with the dysregulation of several key signaling pathways, particularly WNT/β-catenin, TP53, and TGF-β/Smad signaling (85). PDOs models have been instrumental in studying these molecular mechanisms and in advancing personalized approaches to CRC therapy (86).

In a foundational study, Sato et al. [2011] demonstrated that a combination of Wnt3A, R-spondin1, Noggin, EGF, prostaglandin E2 (PGE2), nicotinamide, and the TGF-β inhibitor A83-01 was essential for the long-term expansion and maintenance of human CRC PDOs (67). Among these, nicotinamide was shown to be particularly critical for sustained culture longevity. Further optimization efforts have revealed that factors such as Wnt3A, SB202190 (a p38 MAPK inhibitor), and even oxygen concentration significantly impact CRC PDOs proliferation and viability. Under optimized conditions, PDOs generation efficiencies have approached 100%, underscoring the remarkable responsiveness of CRC cells to niche-derived signaling inputs and the value of finely tuned culture systems in modeling this disease (78,87).

GBMs are the most aggressive and lethal form of primary brain tumors, characterized by extensive intratumoral heterogeneity, infiltrative growth, and profound resistance to standard therapies. These features contribute to the poor prognosis associated with GBM, with median survival rarely exceeding 15 months despite surgery, radiation, and chemotherapy (88). PDOs models have emerged as a promising approach to better understand GBM biology and to explore therapeutic responses in a patient-specific manner (89).

Interestingly, in contrast to the complex and growth factor-enriched media formulations required for PDOs derived from epithelial cancers such as ovarian, pancreatic, or colorectal tumors, GBM PDOs (GBO) culture relies on a relatively simpler, serum-free neural medium. As described by Jacob et al. [2020], GBO medium consists of a 1:1 mixture of DMEM/F12 and Neurobasal, supplemented with GlutaMAX, non-essential amino acids (NEAAs), penicillin-streptomycin, N2 supplement, B27 supplement without vitamin A, 2-mercaptoethanol, and human insulin. This composition supports the growth of GBM cells in a manner that preserves their native architecture and heterogeneity, without requiring exogenous growth factors such as EGF or FGF2. The streamlined nature of this medium reflects the neural origin of GBM and its intrinsic capacity for self-renewal, while also facilitating the maintenance of key histopathological and molecular features seen in patient tumors (90).

Other factors that could play a factor in PDOs success

Beyond TP53 status, homologous recombination (HR) capacity [whether homologous recombination deficient (HRD) or proficient (HRP)] may also contribute to variability in PDOs establishment and maintenance. HRD tumors, often driven by germline or somatic mutations in BRCA1/2 or other HR pathway genes, exhibit profound genomic instability, which not only shapes therapeutic vulnerabilities but may also influence ex vivo growth dynamics (91,92). For instance, HRD cells tend to accumulate unrepaired DNA damage, potentially limiting long-term culture stability, whereas HRP tumors may display more robust proliferative capacity but reduced chemosensitivity in pharmacological assays (93). Moreover, the metabolic adaptations linked to HR deficiency, such as increased reliance on specific nucleotide biosynthesis pathways or oxidative stress responses, could translate into distinct nutrient or signaling requirements in culture media (94). These observations suggest that considering HRD versus HRP status when tailoring PDOs protocols may improve both derivation efficiency and predictive power for therapeutic response. Importantly, although many cancers with dysfunctional TP53, such as OC PDOs, utilize media formulations that share core elements—such as EGF, B27, N2, and R-spondin1—the loss of TP53 alone does not appear to be the primary determinant of media needs or success. This indicates that while TP53 mutations are central to tumor initiation and progression, they do not uniformly dictate the exogenous signaling inputs required for PDOs maintenance and long-term propagation in vitro (8). For instance, TP53-mutant CRC PDOs depend heavily on canonical Wnt signaling, requiring components like Wnt3A and R-spondin1 to sustain growth and structure (67). In contrast, pancreatic cancer PDOs, which often harbor both TP53 and KRAS mutations, grow efficiently in Wnt-free media, instead relying on FGF10 or FGF2, alongside other epithelial-supporting factors (82). Likewise, GBO, despite frequently harboring TP53 alterations, are maintained in neural culture media that completely lack Wnt or R-spondin supplementation, reflecting their neuroectodermal lineage and distinct developmental context (90).

In addition to intrinsic tumor biology and culture conditions mentioned previously, variability in tissue acquisition, handling, and preservation may represent an underrecognized source of heterogeneity in PDO establishment success. While most studies focus on media composition, upstream factors such as tissue transport time, cold ischemia duration, cryopreservation protocols, cryoprotectant exposure, and storage conditions can significantly impact cell viability and subsequent organoid formation (25). Integrating standardized biobanking and cryopreservation practices into PDO workflows may therefore be essential to improving reproducibility across studies.

OTC provides a relevant clinical framework demonstrating that ovarian tissue can be successfully frozen, stored at ultra-low temperatures, and later thawed while retaining biological viability, including the capacity to restore endocrine function (95). Although primarily developed for fertility preservation, this approach highlights the feasibility of preserving complex ovarian tissue architecture and cellular function over time, which is directly relevant to translational applications such as PDO generation.

Importantly, different cryopreservation techniques, including slow freezing and vitrification, can differentially affect tissue integrity (96). Slow freezing may introduce ice crystal formation that can damage cellular and stromal components, whereas vitrification minimizes ice formation by inducing a glass-like state, potentially preserving tissue architecture more effectively (26). This aligns with observations that cryopreservation reduces metabolic activity and is associated with decreased secretion of VEGF, which may impact tissue viability and subsequent organoid formation (27). However, variability in protocols including differences in cooling rates, cryoprotectant composition, and handling procedures can lead to inconsistent outcomes in terms of cellular viability, angiogenic signaling, and metabolic recovery following thawing.

Collectively, these observations suggest that heterogeneity in tissue preservation represents a potential confounding factor in PDO success rates across studies. Without accounting for these upstream variables, differences attributed to media composition alone may be incomplete. Standardizing tissue handling and cryopreservation protocols alongside culture conditions may therefore be critical to improving the consistency, scalability, and translational relevance of OC PDO models.

Conclusions

Given the treatment resistance associated with recurrent OC, there is a critical need for robust, personalized preclinical models to inform therapy. While prior studies have demonstrated the feasibility of PDO generation in these cancers, further work is needed to improve success rates, expand the diversity of tissue sources, and characterize morphological and functional variability across PDOs. The variability in media composition and culture parameters across studies likely contributes to differences in derivation efficiency and passage potential. Importantly, few studies addressed microenvironmental components or immune contexture, pointing to future opportunities for co-culture systems or PDOs-on-chip integrations.

To address these discrepancies and promote more consistent reporting, the adoption of standardized terminology and success benchmarks for PDOs-based studies is essential. First, establishment efficiency should refer to the percentage of patient samples that give rise to viable PDOs within a defined early window. Expansion capacity can be defined as the ability of an PDOs culture to be passaged at least five times while maintaining consistent morphology, proliferation, and cell viability. Cryopreservation recovery should involve the successful thawing and regrowth of PDOs with high viability (e.g., ≥80%) and comparable growth kinetics to fresh cultures. In addition, functional success should be evaluated along two axes: drug testing capability, defined by the PDOs’ ability to yield reproducible pharmacological data (e.g., consistent dose-response curves across replicates), and molecular fidelity, assessed by the retention of key genetic alterations from the parental tumor, such as TP53 or KRAS mutations. Finally, long-term culture stability should be determined by the maintenance of structural integrity, proliferation, and phenotypic consistency over the passages without evidence of drift or selection bias.

Standardizing success criteria, potentially incorporating culture duration, molecular fidelity, and functional performance, will be critical for reproducibility and cross-study comparison. Until then, defining success should be aligned with the intended purpose of the PDO system: short-term therapeutic screening vs. long-term modeling and preservation. Overall, success rates remain low (22–36%), particularly for long-term cultures, underscoring the challenges that persist in this field. Standardization of media components, implementation of quality control measures such as frozen section assessment, and consensus on success definitions are urgently needed to advance reproducibility. Looking forward, future work should not only draw from lessons learned in other TP53-mutated cancers but also consider the unique molecular and biological features of OC subtypes to develop optimized, clinically meaningful PDO models.

Collectively, this comparative analysis underscores that while core components of PDO media are broadly conserved, optimization for OC subtypes, particularly HGSOC, requires careful modulation of Wnt and BMP signaling, mechanical stress inhibitors, and potentially subtype-specific growth factors. The diversity in reported media compositions and outcomes reflects both the complexity of OC biology and the evolving nature of PDOs technology. Future efforts toward harmonization and biomarker-driven customization may enable more reproducible and clinically translatable models.

Acknowledgments

We thank Céline Domecq for assistance with maintaining PDO cultures. We thank Drs. Anne-Marie Mes-Masson of Centre hospitalier de l’Université de Montréal, Trevor Shepherd of University of Western London Ontario, and Nikolina Radulovich of Unity Health Network for assistance in optimizing PDO development protocol and media compositions.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://cco.amegroups.com/article/view/10.21037/cco-2026-1-0013/rc

Peer Review File: Available at https://cco.amegroups.com/article/view/10.21037/cco-2026-1-0013/prf

Funding: This work was supported by the Canada Graduate Research Scholarship—Master’s Award from Canadian Institutes of Health Research.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://cco.amegroups.com/article/view/10.21037/cco-2026-1-0013/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the McGill University Health Centre (Montreal, Quebec, Canada; IRB #A08-M79-13B). All patients provided written informed consent for the collection, storage, and research use of tumor material and clinical data.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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