Name: Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture
Uploaded: 2023-08-18T14:50:00
Description: Watch this Scientific Journal Video about Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture at JoVE.com

hepatocellular carcinoma tissue dissociation for organoid culture

تحسين تكوين عضوي HCC لتحديد الهدف وتطوير الأدوية

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

تسليط الضوء على المؤلف: التحقيق في التسبب في سرطان الكبد باستخدام المواد العضوية المشتقة من المريض 

تطوير وتحسين نموذج عضوي مشتق من سرطان الخلايا الكبدية البشرية لتحديد الهدف المحتمل واكتشاف الأدوية

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

Watch this Scientific Journal Video about Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture at JoVE.com

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture  

تفكك أنسجة سرطان الخلايا الكبدية للثقافة العضوية

لبدء تفكك الأنسجة ، اجمع سنتيمترا إلى سنتيمترين مكعبين من سرطان الخلايا الكبدية أو أنسجة HTC من المرضى غير المعالجين. ثم ضع المنديل في محلول حفظ الأنسجة واحتفظ به عند أربع درجات مئوية حتى المعالجة. الآن ، سخن ألواح زراعة الخلايا السطحية ذات التعلق المنخفض للغاية لمدة ساعة واحدة في حاضنة محددة عند 37 درجة مئوية.قم بإذابة مستخلص الغشاء القاعدي طوال الليل عند أربع درجات مئوية. باستخدام مقص جراحي ، قم بتقطيع أنسجة الورم إلى قطع صغيرة على طبق بتري داخل غطاء التدفق الصفحي. انقل هذه القطع إلى أنبوب مخروطي حجمه 15 ملليلتر وأضف خمسة إلى 10 ملليلترات من الوسط القاعدي.باستخدام ماصة باروتروبيك ، اغسل أكبر قدر ممكن من الدم. اترك الخليط يستقر لمدة دقيقة إلى دقيقتين قبل إزالة بعض المواد الطافية ، بما في ذلك أي خلايا دم وجلطات دهنية عائمة. بعد ذلك ، قم بطرد الخليط عند 300 غرام لمدة خمس دقائق في درجة حرارة الغرفة.ثم استخرج بعناية المادة الطافية وأضف خمسة ملليلتر من محلول الهضم قبل التسخين إلى الأنسجة المشذبة. ضع الأنبوب عند 37 درجة مئوية في دوار للحصول على الهضم الأمثل. بعد 30 دقيقة من الهضم الأولي ، استخدم المجهر للبحث عن مجموعات الخلايا الصغيرة.لإيقاف الهضم ، أضف خمسة ملليلتر من الوسط القاعدي البارد إلى الأنبوب. ثم استخدم مرشحا خلويا حجمه 100 ميكرومتر للغربلة في أنبوب جديد سعة 50 ملليلتر. املأ الأنبوب بوسط قاعدي بارد للوصول إلى حجم إجمالي قدره 50 ملليلتر.بعد ذلك ، قم بطرد الخلايا عند اثنين G لمدة 10 دقائق عند ثماني درجات مئوية. بمجرد الانتهاء ، قم بإزالة السائل الطافي بعناية. أعد تعليق الحبيبات في 50 ملليلتر من الوسط القاعدي البارد للحصول على حبيبات الخلية للطلاء العضوي.

Research

JoVE Journal

Cancer Research

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture

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

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...