Patient-Derived Three-Dimensional Tumor Organoid Model for In Vitro Chimeric Antigen Receptor T Cell Screening

Linjun Liang; Yue Shi; Minzhu Ji; Yang Xu; Ru Zhang; Yong Cui; Liang Li

doi:10.3791/69113

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Method Article

Patient-Derived Three-Dimensional Tumor Organoid Model for In Vitro Chimeric Antigen Receptor T Cell Screening

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DOI:

10.3791/69113

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December 30th, 2025

Linjun Liang¹ , Yue Shi² , Minzhu Ji³ , Yang Xu⁴ , Ru Zhang⁵^,⁶^,⁷ , Yong Cui¹ , Liang Li²

¹Department of Oncology, Shenzhen Qianhai Shekou Free Trade Zone Hospital, ²Department of Pharmacology, Joint Laboratory of Guangdong-Hong Kong Universities for Vascular Homeostasis and Diseases, School of Medicine, Southern University of Science and Technology, ³Department of Digestive Disease, Shenzhen Qianhai Shekou Free Trade Zone Hospital, ⁴Department of Human Cell Biology and Genetics, School of Medicine, Southern University of Science and Technology, ⁵Department of Surgical Endoscopy, Shenzhen People's Hospital (The First Affiliated Hospital, Southern University of Science and Technology; The Second Clinical Medical College, Jinan University), ⁶Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong, ⁷Institute of Digestive Disease, State Key Laboratory of Digestive Disease, Faculty of Medicine, The Chinese University of Hong Kong

Summary

This protocol establishes an in vitro co-culture model using patient-derived tumor organoids and chimeric antigen receptor T (CAR-T) cells to evaluate the specific cytotoxic activity of CAR-T cells against solid tumor cells.

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has achieved exciting clinical efficacy in hematological malignancies, but CAR-T cell therapy for solid tumors still requires further development. Patient-derived tumor organoids are in vitro disease models that retain patient heterogeneity and have been used to test the efficacy and safety of chemotherapy and targeted drugs. This method describes an in vitro efficacy testing model of co-culturing tumor organoids with CAR-T cells. Colorectal cancer samples from patients are constructed into tumor organoids with a three-dimensional (3D) structure in a basement membrane matrix. The tumor organoids can grow stably and be passaged continuously. Mature tumor organoids are separated from the matrix by washing and centrifugation, and CAR-T cells are added at different effector-to-target (E:T) ratios to form an immune-organoid co-culture system. After 6-24 h, the morphology and intercellular structures of the organoids are observed by bright-field imaging. This experiment further performs dead cell staining in the co-culture system, which can reflect the viability of the organoids and evaluate the cytotoxic effect of CAR-T cells on tumor organoids. This experiment can effectively observe the interaction between CAR-T cells and 3D tumor organoids, and the results can be used to assess the tumor-killing activity of CAR-T cells.

Introduction

Chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable clinical success in treating hematological malignancies¹. However, its application in solid tumors remains limited due to several major obstacles, including tumor heterogeneity and antigen loss, physical and chemical barriers within the tumor mass, and the immunosuppressive tumor microenvironment²^,³. These challenges contribute to the limited efficacy of CAR-T cell therapy in solid tumor settings.

In vitro evaluation of CAR-T cell efficacy is a critical step during preclinical deve....