近端培养系统:一种研究细胞间旁分泌信号传导的体外培养技术

在旁分泌信号传导中，细胞产生并分泌信号分子，诱导邻近细胞的反应。要模拟旁分泌信号传导，首先在适当的培养基中制备卵巢癌细胞和间皮细胞的单细胞悬液。

接下来，在无菌培养皿中取一个倒置的多孔膜插入物。将癌细胞悬液移液到插入膜的底面上以形成液体圆顶。孵育以使细胞沉淀并附着在膜上。

置插入物以排出介质。将插入物转移到含有生长培养基的新鲜培养皿中，以滋养粘附在膜底面的癌细胞。现在，将间皮细胞悬液添加到插入孔中。孵育以使细胞粘附在膜的另一侧，拒绝两种细胞类型之间的任何直接接触。

培养过程中，两种细胞类型都会分泌信号因子。这些分子通过多孔膜向邻近细胞扩散。它们与特定的细胞受体结合，并在两种细胞类型中诱导复杂的信号级联反应。

首先将卵巢癌细胞与1毫升0.25%胰蛋白酶从80%汇合混合物中分离40至60秒，然后用6毫升完全生长培养基中和反应。通过离心收集细胞，并将沉淀重悬于 5 毫升新鲜完全生长培养基中。

计数后，将细胞稀释至每毫升完全生长培养基浓度100,000个卵巢癌细胞，并根据实验条件将三个倒置的0.4微米孔组织培养处理的聚碳酸酯膜细胞培养插入物放入适当大小的无菌组织培养皿中。根据实验条件标记插入物，小心地将癌细胞以同心圆移液到每个插入物的底部，从膜的中间开始向外移动，形成一个800微升的圆顶。

细胞接种到插入物的底面时要格外小心，以免含有培养基的细胞的圆顶在孵育过程中从插入物的边缘流出。

所有插入物都接种完毕后，盖上培养皿并小心地将插入物放入细胞培养箱中。约4小时后，用2毫升0.25%胰蛋白酶分离HPMC一至两分钟，然后用12毫升完全生长培养基中和反应。

通过离心收集HPMC，并将沉淀重悬于10毫升完全生长培养基中进行计数。将细胞稀释至每毫升完全生长培养基 300,000 HPMC，并将 2.5 毫升新鲜完全生长培养基添加到 6 孔培养板的每个孔中。

使用无菌镊子，将每个插入物的右侧朝上放入 6 孔板的每个孔中，以便将下部卵巢癌细胞附着表面浸入培养基中。然后，将 1.5 毫升 HPMC 接种到每个插入物的孔中，并将板放入细胞培养箱中 72 小时。

• Paracrine Signaling - Cellular communication through secreted molecules affecting neighboring cells.
• Mesothelial Surface - The membrane where the mesothelial cells adhere and function.
• Culture System - Set-up where cells are grown under controlled conditions, often in vitro.
• Corning 10-013-CV - Specific type of culture dish used for cell adhesion and growth experiments.
• In Vitro Cell Culture Techniques - Methods used to cultivate cells in controlled environments outside of the living organism.

• Introduce Paracrine Signaling – Paracrine signaling is the process by which cells communicate and influence the behavior of neighboring cells via secreted molecules (e.g., paracrine).
• Key Concepts - We can simulate this biological process in an in vitro cell culture system using human mesothelial cells and cancer cells (e.g., culture system).
• Underlying Mechanisms - Both cell types secret signaling factors which bind to receptors and trigger complex responses (e.g., mesothelial surface).
• Connect to Experiment - To study this, cells are cultivated on either side of a porous membrane in a culture dish (e.g., corning 10-013-CV).

• What is paracrine signaling and how does it work in an in vitro culture system?
• How are mesothelial and cancer cells cultured for this study?
• What are the key concepts and mechanisms underlying paracrine signaling?

• Practical Applications - Understanding paracrine signaling could improve diagnosis and treatment of diseases like cancer (e.g., in vitro cell culture techniques).
• Industry Impact - The knowledge acquired is beneficial for the medical and pharmaceutical sectors (e.g., culture system).
• Societal Importance - The broader implications extend to improving patient outcomes and quality of care (e.g., mesothelial surface).
• Link to Scientific Advancements - The use of in vitro cultivation techniques continues to advance our understanding of cell communication (e.g., corning 10-013-CV).