Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants
Published: July 31, 2023
Abstract
Source: Vizcardo, R., et al. A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants. J. Vis. Exp. (2019).
This video demonstrates a protocol for generating functional antigen-specific T cells from iPSC-derived immature T cells using a three-dimensional thymic culture system. Co-culturing the iPSC-derived immature T cells with endogenous lymphocyte-depleted mice fetal thymic lobes resulted in successful maturation of the T cells to form CD8αβ+ MHC class I+ iPSC-derived thymic emigrants (iTEs).
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. 3D Thymic Organ Culture to Generate iTE Harvest mouse fetal thymic lobes and deploy endogenous lymphocytes by deoxyguanosine (dGUO) treatment. On day 7 of dGUO treatment, take four new 10 cm dishes and fill each with 20 mL of complete media (Roswell Park Memorial Institute Media 1640 [RPMI 1640] + 10% FBS + 1x…
Representative Results
Figure 1: Immunohistochemistry of thymic lobes seeded with iPSC-derived immature T cells. Top: H&E staining of a thymic lobe with and without seeding of iPSC-derived immature T cells. From second top to bottom: confocal images of the sectioned lobes stained with DAPI (nucleus), CD3 (T cell), and merge. Scale bars = 100 µm. <p class="jove_content" fo:keep-together.wit…
Divulgaciones
The authors have nothing to disclose.
Materials
| 2-deoxyguanosine | Sigma-Aldrich | 312693-72-4 | |
| 2-Mercaptoethanol (1,000x) | Thermo Fisher Scientific | 21985-023 | |
| FBS | Gemini | 100-500 | |
| Flt-3 ligand | R&D Systems | 427-FL | |
| Interleukin-7 | R&D Systems | 407-ML | |
| MEM Non-Essential Amino Acids Solution | Gibco | 11140050 | |
| MEM powder | Gibco | 61100061 | |
| Sodium Pyruvate | Thermo Fisher Scientific | 11360-070 | |
| Stem Cell Factor (SCF) | R&D Systems | 455-MC | |
| Penicillin/streptomycin | Thermo Fisher Scientific | 15140-122 | |
| Phosphate-buffered saline pH 7.4 (1x) | Thermo Fisher Scientific | 10010-023 | |
| RPMI 1640 | Gibco | 11875093 | |
| 10 cm dish | Corning, Inc | 353003 | |
| Perfecta3D Hanging Drop Plate | Sigma-Aldrich | HDP1096 | |
| Forceps | DUMONT | 0108-5PO | |
| OP9/N-DLL1 | Riken Bioresource center | Cat# RCB2927; RRID:CVCL_B220 | |
| GlutaMAX (100x) | Thermo Fisher Scientific | 35050-061 |
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Citar este artículo
Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants. J. Vis. Exp. (Pending Publication), e21533, doi: (2023).