琼脂糖离心下降测定:一种嵌入类器官进行组织学分析的技术

癌细胞的3D类器官培养概括了人类癌症的许多病理生理特征。因此，这些体外培养物构成了癌症研究的绝佳模型。

为了处理这些类器官，首先培养在合适的基底膜基质中生长的预成型3D类器官。加入所需的细胞回收培养基并孵育。该培养基促进基质凝胶的解聚，将 3D 类器官释放到溶液中。

心该混合物以沉淀类器官。含有基质组分的回收培养基保留在上清液中。弃去上清液。接下来，将多聚甲醛溶液添加到类器官沉淀中。该化学物质进入类器官细胞并与氨基酸结合，引起蛋白质交联和样品固定。此步骤对于任何下游组织学应用都至关重要。

类器官沉淀以分离和去除含多聚甲醛的上清液。将温热的融化琼脂糖加入类器官中。使用针头将沉淀的类器官分离并分散到液体琼脂糖中。让琼脂糖凝固并包埋类器官。使用含有类器官的琼脂糖塞进行下游组织学测定。

为了处理用于组织学的类器官，请从孔中取出现有培养基，注意不要吸出基底膜穹顶。加入等体积的细胞回收溶液，并将板在4摄氏度下孵育60分钟。

孵育后，使用移液器移开基底膜圆顶并用移液器吸头将其压碎。将解离的圆顶和细胞回收溶液收集到 1.5 毫升管中。将试管在 300 x g 和 4 摄氏度下离心 5 分钟，然后取出上清液并将其放在一边。

保存所有上清液，直到确认类器官存在的最后一步。加入所需体积的冷PBS，轻轻上下移液，以机械移出沉淀，而不会破坏类器官。重复离心并除去上清液。

室温下将沉淀固定在匹配体积的 4% PFA 中 60 分钟。固定后，重复离心步骤和PBS洗涤。然后，缓慢加入 200 微升温热的 2% 琼脂糖，并立即但轻轻地使用连接到 1 毫升注射器的 25 号针头将细胞沉淀从管壁上分离，而不会破坏它。

对于琼脂糖旋降法，使用 25 号针头添加琼脂糖后立即将细胞沉淀从管壁上分离至关重要。

脂糖完全凝固后，用连接到 1 毫升注射器的 25 号针头将其从管子上分离，并将其转移到新的 1.5 毫升管中。用 70% 乙醇填充管，并继续进行组织脱水和石蜡包埋的常规方案。

• Spin-down - A process to separate substances of different densities by centrifugation.
• Organoid paraffin embedding - A technique to preserve biological tissues in paraffin wax.
• Cell recovery solution - A solution used to dissociate cells from a matrix for further analysis.
• Agar embedding - A method to preserve tissue samples in an agarose gel block.
• Agarose embedding protocol - A detailed procedure for encasing biological samples in agarose.

• Introduce Spin-down – A method utilized to separate different-density particles by centrifugal force (e.g., spin-down).
• Key Concepts – Overview of tissue sample preservation techniques such as paraffin and agar embedding (e.g., organoid paraffin embedding).
• Underlying Mechanisms – Description of the procedure to detach and process organoids (e.g., cell recovery solution).
• Connect to Experiment – Explanation of the importance of proper organoid and agarose handling in the experiment.

• What is the spin-down technique and how is it applied in the procedure?
• How does organoid paraffin embedding contribute to tissue preservation?
• What role does cell recovery solution play during the dissociation of organoids?

• Practical Applications – Spin-down and embedding techniques in biological and medical research (e.g., spin-down).
• Industry Impact – Areas like pathology and histology benefit from these methods (e.g., organoid paraffin embedding).
• Societal Importance – Advancement in disease diagnosis and treatment (e.g., cell recovery solution).
• Link to Scientific Advancements – Development of improved tissue preservation and analysis methods.