ovarian cancer organoid passaging and cryopreservation for long-term organoid cultivation

卵巣がんオルガノイドバイオバンキングの標準化

Clinical management of patients with epithelial ovarian cancer remains challenging due to its heterogeneous clinical presentation at advanced stages and high recurrence rates1. Improving our understanding of ovarian cancer development and biological behavior requires research approaches that address the patient-specific variability during the course of the disease, treatment response, and histopathological as well as molecular features2.
Biobanking, characterized by the systematic collection and long-term preservation of tumor samples derived from ovarian cancer patients along with their clinical information offers the preservation of a large patient cohort in different disease stages, including tumor samples from primary debulking surgeries, after neoadjuvant chemotherapy and from recurrent disease. It holds valuable potential for advancing cancer research serving as a resource of promising prognostic biomarkers and therapeutic targets3. However, conventional biobanking methods, such as formalin fixation and freezing, are not amenable to conducting functional studies on the original tumor samples due to the loss of viability and the disruption of the native three-dimensional tissue architecture4,5.
Studies of molecular mechanisms, in oncology and beyond, crucially depend on the use of appropriate experimental models that faithfully reflect the biology of the disease and maintain in vitro properties of the tissue observed in vivo. Patient-derived organoids, based on the preservation of the renewal potential, reproduce in the lab the original structure and function of the epithelium and allow testing in a patient-specific context. Therefore, they have emerged as highly promising tools for cancer research and personalized medicine, bridging the gap between clinical diversity and laboratory research6,7,8,9. Tailored therapeutic strategies based on individual drug responses of organoid lines and testing of the functional relevance of molecular profiles, can potentially be directly applied to patient care10,11. The possibility of long-term cultivation including patient-specific characteristics and the collection of relevant prospective clinical data over time holds great promise to identify novel prognostic and predictive factors involved in disease progression and resistance mechanisms3,9.
Nonetheless, building a biobank that includes organoids from different tumor samples requires a combination of strict adherence to complex methodology and setting up protocols for easy maintenance12. Process standardization ensures that the biobank can be established and maintained efficiently by trained staff even at high turnover, while at the same time adhering to the highest quality standards13. Several studies reported the successful generation of stable ovarian cancer organoid lines corresponding to the mutational and phenotypical profile of the original tumor with varying efficiency rates. Still, routine bio banking remains challenging in practice, particularly for long-term stable growth of lines, which is a prerequisite for large-scale expansion or successful genomic editing.
In particular, the issue of expandability remains vaguely defined in the field as organoids that show slow and limited growth potential are occasionally counted as established lines. As initially demonstrated by Hoffmann et al., a study whose principal findings provided the basis for this further developed protocol, optimal handling of ovarian cancer tissue requires a unique strategy to accommodate heterogeneity14. Phenotypic characterization of the organoids obtained by this method and close similarity with parental tumor tissue were confirmed by panel DNA sequencing and transcriptomics analysis of mature cultures (4-10 months of cultivation) demonstrating the stability of the model8,9,12,14.
In contrast to the paracrine environment that regulates the homeostasis in the healthy fallopian tubes, the epithelial layer, which likely yields high-grade serous ovarian cancer (HGSOC), cancer regeneration potential, and organoid formation capacity, is less dependent on exogenous Wnt supplementation. Moreover, active Bone Morphogenetic Protein (BMP) signaling, characterized by the absence of Noggin in organoid medium, proved to be beneficial for the establishment of long-term cultures from ovarian cancer solid tissue deposits14,15. During systematic biobanking of solid deposits of ovarian cancer, we have confirmed these findings and set up the pipeline, with details outlined in this protocol that ensures sustained long-term expansion in the majority of cases. We find that parallel testing of different media compositions and seeding modalities when working with primary isolates are essential to improve the establishment of long-term stable organoid lines and to increase yields enabling robust propagation and expansion to multi-well formats required for downstream experiments16.
Furthermore, the purity and quality of the samples collected during surgery are of crucial importance for the translational potential of ovarian cancer organoids in basic research and molecular diagnostics. The complexity of the clinical presentation of HGSOC requires close cooperation between the surgeons, oncologists, and the scientists in the lab to ensure that relevant material is correctly identified, transport conditions are kept constant, and organoid lines are generated with high efficiency representing the most important characteristics of the disease of each patient. This protocol provides a standardized but adaptable framework to capture the full potential of ovarian cancer organoids, considering the heterogeneity that characterizes ovarian cancer16,17. Notably, this protocol enables reliable biobanking of the broad spectrum of ovarian cancer clinical presentation, including different histological types (high-grade and low-grade ovarian cancer, LGSOC), different deposits from the same patients who exhibit differences in stemness regulation, tissues from surgeries in post neoadjuvant setting, biopsy material, and samples from surgeries in the recurrent phase of disease progression.

著者スポットライト:卵巣がんにおける個別化医療の推進 

卵巣がんオルガノイドのバイオバンキング戦略:組織学的サブタイプと疾患病期にわたる患者間の不均一性への対処

The best clinical management in cutting-edge translational research for patients with advanced ovarian cancer has highest priority at our institution. With the biobanking of patient-derived organoids, we have achieved major progress towards individually-tailored management. This enables us to evaluate patient-specific characteristics and to identify predictive biomarkers.We have made significant advances in recent years in improving radical multivisceral surgery, chemotherapy, anti-angiogenic therapy, and also PARP inhibitor therapy in patients with advanced ovarian cancer and improved survival rates even with that. Now, the next big step would be to better predict which patient would benefit from which specific treatment. Currently, clinical treatment decisions are based on general histopathological features and on information on homologous recommendation deficiency or tested in paraffin-embedded tumor tissue.Biologic models that evaluate specific responses to chemotherapy and to targeted treatments have not yet been implemented in clinical routine. We have identified key characteristics of ovarian cancer tissue, specifically the culture requirements necessary to sustain its renewal potential in vitro, and use this knowledge to achieve robust long-term growth of organoid lines and establish biobank from samples at various stages of the disease. Our day-to-day experience in biobanking of ovarian cancer organoids effectively shows a remarkable heterogeneity in the biology of this malignancy, which could pave the way to new classification or provide the basis for new therapeutic solutions targeting STEMI's potential.

Watch this Scientific Journal Video about Ovarian Cancer Organoid Passaging and Cryopreservation for Long-Term Organoid Cultivation at JoVE.com

卵巣癌オルガノイド継代と凍結保存による長期オルガノイド培養

まず、24ウェルまたは48ウェルプレートで培養した卵巣がんオルガノイドを採取します。氷冷した基礎培地を添加し、プレートの底部をピペットで操作またはこすり、BME-2マトリックス液滴を破砕します。次に、細胞懸濁液を15ミリリットルのチューブに移し、氷上に保管します。1ミリリットルの氷冷基礎培地を加えます。均一に膨張させるために2〜3つのテクニカルリプリケートウェルを組み合わせた後、チューブを300Gで5分間遠心分離し、懸濁液を光に対してチェックして、BME-2ゲルがないことを確認します。オルガノイドの凍結保存には、前述のように細胞口蓋を調製し、細胞口蓋を1ミリリットルの氷冷凍結保存培地に再懸濁します。細胞懸濁液を標識済みの1.8ミリリットル極低温チューブに移します。

ovarian-cancer-organoid-passaging-cryopreservation-for-long-term

Research

JoVE Journal

Cancer Research

Ovarian Cancer Organoid Passaging and Cryopreservation for Long-Term Organoid Cultivation

263 ビュー.  University Hospital, Ludwig-Maximilian-University (LMU) Munich. まず、24ウェルまたは48ウェルプレートで培養した卵巣がんオルガノイドを採取します。氷冷した基礎培地を添加し、プレートの底部をピペットで操作またはこすり、BME-2マトリックス液滴を破砕します。次に、細胞懸濁液を15ミリリットルのチューブに移し、氷上に保管します。1ミリリットルの氷冷基礎培地を加えます。均一に膨張させるために2〜3つのテクニカ?...

ビデオ: 卵巣癌オルガノイド継代と凍結保存による長期オルガノイド培養

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...