Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

Abstract

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 µm to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffinembedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 µm depth) by confocal microscopy.

Future Match Making: When Pediatric Oncology Meets Organoid Technology

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Fusion-negative rhabdomyosarcoma 3D organoids to predict effective drug combinations: A proof-of-concept on cell death inducers

“…In brief, RMS_Os were fixed and processed as described before. 78 Immunohistochemistry (IHC) was performed on an automated immunostainer (Ventana discoveryXT, Roche) using rabbit Omni map DAB kit. Organoids’ slides were stained with HPS (Hematoxylin Phloxine Saffron), or the following antibodies: anti-Desmin (1/50, Dako, cat.…”

Fusion-negative Rhabdomyosarcoma 3D-organoids as an innovative model to predict resistance to cell death inducers

“…FNRMS-derived organoids were fixed and processed as described before 80 . In brief, immunohistochemistry (IHC) was performed on an automated immunostainer (Ventana discoveryXT, Roche) using rabbit Omni map DAB kit.…”

“…no. M7240) were applied 30 min at RT, as described previously 80 . Sequential immunofluorescence was performed using OPAL 520 (Survivin, green), OPAL 690 (Ki-67, red), and cells were counterstained with DAPI.…”