Establishment of Patient-Derived Organoid Model from Hydrothorax and Ascites

Abstract

Organoids are miniaturized organs cultured in vitro, exhibiting self-renewal and self-organization capabilities. In comparison to traditional 2D cell cultures, organoids can reproduce the spatial structure and functionality resembling corresponding tissues or organs, providing highly physiologically relevant in vitro systems. Consequently, they can be utilized for simulating disease occurrence, testing the tumor drug sensitivity and co-culturing with other cells. This study aims to establish a Patient-Derived Organoid (PDO) model using hydrothorax and ascites samples. To ensure cell viability, it is essential to store the collected malignant fluid at 4°C. Upon arrival at the laboratory, the fluid should be divided into centrifuge tubes for subsequent centrifugation, allowing the collection of cell precipitates. These cell precipitates need to be washed multiple times with DPBS until the supernatant appears clear. If the pellet contains red blood cells, they should be lysed, followed by cell counting. Depending on the desired seeding density, cells should be resuspended in Matrigel and plated as domes in a culture plate. The plate should then be inverted and placed in a 37°C incubator for 30 minutes to allow the Matrigel to solidify. Once solidified, complete culture medium should be added, and the culture plate should be placed in a 37°C incubator for further incubation. Organoid growth can be monitored using microscopy to assess their development and progression. Once the organoids have reached a stable growth state, immunohistochemistry can be performed to identify specific cell types or markers within the organoids. Furthermore, the drug responsiveness of the organoids can be evaluated through cell viability assays, enabling researchers to determine the effects of different drugs or organoid viability and function.