Decellularized lymph node sections with preserved extracellular matrix for stromal cell culture

TL;DR

Researchers developed a method to create thin, cell-free scaffolds from lymph nodes to study how the extracellular matrix influences immune cell behavior.

Contribution

A new protocol for decellularizing lymph node slices that preserves ECM and supports cell culture and imaging.

Findings

Decellularized lymph node slices retained collagen and GAG levels similar to native tissue.

The slices supported long-term fibroblastic reticular cell culture and co-culture with T cells.

The method enabled high-resolution imaging and revealed altered protein expression in cultured cells.

Abstract

The lymph node (LN) extracellular matrix (ECM) is produced by stromal cells like fibroblastic reticular cells (FRCs) and supports adaptive immunity by guiding immune cell interactions. Disruption of this ECM in cancer and chronic inflammation has been shown to promote disease progression. While interactions between cells and the LN ECM are critical for immunity, they remain difficult to study due to limitations in current models and reliance on animal studies. To address this, LNs could be decellularized to generate cell-free scaffolds that are subsequently reseeded with cells to study how the native LN microenvironment influences cellular behavior. Existing whole-organ decellularization methods preserve ECM features but yield dense scaffolds that restrict uniform cell seeding, limit nutrient diffusion, and hinder imaging analyses. Here, we present a protocol that combines vibratome sectioning (200-μm slices) with detergent decellularization (0.1% SDS and 1% Triton-X) to generate thin LN slices from mouse and human tissues. Decellularized LNs had comparable collagen and GAG concentrations to native tissue, and immunofluorescence staining showed the presence of other ECM proteins. Decellularized sections sustained 21-day FRC culture, enabled FRC-T cell co-culture, and supported high-resolution imaging and flow cytometric analyses, revealing altered gp38 and PDGFRα expression in FRCs relative to 2D culture.

The online version contains supplementary material available at 10.1038/s41598-025-28782-0.

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