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Updated: Nov 30, 2025
Isolation of Murine Lymph Node Stromal Cells
Published on: August 19, 2014
Tissue-Engineered Stromal Reticula to Study Lymph Node Fibroblastic Reticular Cells in Type I Diabetes
Freddy Gonzalez Badillo1,2, Flavia Zisi Tegou1,2, Riccardo Masina2
1Department of Biomedical Engineering, University of Miami, 1251 Memorial Dr, Coral Gables, FL 33146 USA.
Introduction:
Fibroblastic reticular cells (FRCs) support and remodel the lymph node (LN), express and present self-antigens to T cells to promote tolerance. In Type 1 diabetes (T1D), decrease in FRC frequency and in their expression of T1D-related self-antigens may hinder tolerogenic engagement of autoreactive T cells. FRC reticular organization in LNs is critical for adaptive immunity. Thus, we engineered LN-like FRC reticula to determine if FRC reticular properties were altered in T1D and to study engagement of autoreactive T cells in vitro.
Methods:
We characterized FRC networks in pancreatic and skin-draining LNs of 4- and 12-week old non-obese diabetic (NOD) and diabetes resistant NOR mice by immunofluorescence. Murine FRCs isolated from NOR, NOD or human pancreatic LNs were cultured in collagen sponges for up to 21 days before immunofluorescence and flow cytometry analysis. NOD FRCs expressing T1D antigens were co-cultured with CellTrace-labeled specific T cells in 2D or in scaffolds. T cell engagement was quantified by CD25 upregulation, CellTrace dilution and by T cell tracking.
Results:
FRC networks in both 4- and 12-week old NOD LNs displayed larger reticular pores than NOR controls. NOD FRCs had delayed scaffold remodeling compared to NOR FRCs. Expression of the gp38 FRC marker in NOD FRCs was lower than in NOR but improved in 3D. FRC reticula expressing T1D antigens promoted higher engagement of specific T cells than 2D.
Conclusion:
We engineered LN-like FRC reticula that recapitulate FRC organization and phenotype of T1D LNs for studying tolerogenic autoreactive T cell engagement in T1D.
