Home › Cell Biology › A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants
Cell Biology JoVE (Open Access) Citable · DOI

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

DOI: 10.3791/58672-v
What you'll learn
  • ✓ Prepare OP9/DLL1 feeder cells for iPSC co-culture differentiation
  • ✓ Differentiate iPSC into immature T cells in vitro
  • ✓ Generate tumor antigen-specific thymic emigrants using 3D organ culture
  • ✓ Characterize and validate iTE populations for immunotherapy applications
Protocol

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to naïve T cells with the capacity for proliferation, memory formation, and tumor suppression.

Difficulty
advanced
Total time
~4–6 weeks (iPSC differentiation ~3 weeks, 3D thymic culture ~1–2 weeks, analysis ongoing)
Model organism
Mouse iPSC-derived cells; OP9/DLL1 stromal support
Biosafety
BSL-1

Steps

1
Prepare OP9/DLL1 stromal cells for co-culture

Culture and prepare OP9/DLL1 feeder cells expressing Delta-like ligand 1 to support T cell differentiation. Plate cells at appropriate density for subsequent iPSC co-culture.

▶ 00:57
2
Differentiate iPSC into immature T cells

Co-culture induced pluripotent stem cells with OP9/DLL1 cells in vitro to generate immature T cell precursors. Culture conditions support hematopoietic and T cell lineage commitment.

▶ 02:08
3
Generate iTE in three-dimensional thymic culture

Transfer immature T cells into a 3D thymic organ culture system to promote positive and negative selection, generating tumor antigen-specific thymic emigrants with naïve-like properties.

▶ 05:00
4
Analyze and validate iTE populations

Characterize generated iTE by flow cytometry and functional assays to confirm naïve T cell phenotype, tumor antigen specificity, and capacity for proliferation and memory formation.

▶ 08:00
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