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Tissue Engineering

Tissue Engineering - Poster Session B

Poster L19 - Evaluating the impact of the microenvironment on the stromal cell phenotype using decellularized lymph node sections

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Lymph nodes (LN) are an essential part of the immune system, helping fight off infections and preventing tumor growth in our bodies. We are particularly interested in the lymph node paracortex zone, rich in T-cells and fibroblastic reticular cells (FRCs), in which antigen-presenting cells present peptide antigens to the T cells to activate or regulate them. Furthermore, in this LN compartment, FRCs regulate transport of antigens and other cytokines and build the LN Extracellular Matrix (ECM). However, it is still unknown whether the LN microenvironment is responsible for regulating FRC phenotype or whether FRCs are responsible for shaping the environment in which they develop. Our hypothesis is that the LN ECM determines their phenotype in the LN. To test our hypothesis we developed a novel in vitro platform for FRC culture using decellularized LN sections. Here, we aimed to establish a protocol that would allow for decellularization while fully preserving the ECM components of the T cell zone of the LN amenable for FRC seeding.

Materials and

Methods:  LNs were embedded in 6% agarose and sliced using a vibrotome at a thickness of 100 um. The decellularization was conducted at various concentrations of SDS detergent, ranging from 0.1% to 2.5% followed by 1% Triton-X treatment in whole LNs or just in the LN sections that specifically contained T cells. To select these LN sections before decellularization, confocal microscopy imaging was performed after staining using CD3, B220, alpha-smooth muscle actin, and DAPI antibodies (Figure 1). The decellularization protocol was optimized via varying the concentrations of SDS (Figure 3A), the comparison of homogenized vs. non-homogenized tissue (Figure 3B), and the effectiveness of slicing LN sections (Figure 3D). The success of the chosen protocol was confirmed through PicoGreen Assay as well as DAPI staining to validate DNA removal. Then, the sections were sterilized with ethanol and an antibiotic mix, and the FRC cells were seeded on top of the sections at the densities of 10,000 and 20,000 cells/ml and cultured in sterile conditions for 24 hours. Then, FRCs were harvested via enzymatic digestion and the LN sections were stained for confocal imaging to confirm the success of the seeding procedure. We are currently assessing the GAG content on the sections as well as phenotyping harvested FRCs by flow cytometry.

Results, Conclusions, and Discussions: We found that the optimal SDS concentrations were of 0.1% for LN sections and 0.2% for the whole LNs followed by decellularization via 1% Triton-X. Higher concentration of detergent disintegrated the tissue without a significant improvement in removal of cells. We also saw that this optimized protocol allowed for near complete decellularization with the lowest signal being 0.006222 ug/ml DNA and no signal from the DAPI channel in decellularized sections, while maintaining the ECM (Figure 4). Only the LN sections with areas of T cells surrounded with alpha-smooth muscle actin FRCs were chosen. Our sterilization technique with ethanol was effective and did not produce the growth of any bacteria, and thus can be safely used. After successful sterilization and FRC seeding, the confocal imaging showed significant DAPI signal in the FRC seeded sections (Figure 5) and our enzymatic digestion protocol for FRC harvesting after 24 hours culture in the LN sections yielded ~30,000 cells/ml. The microenvironment of the LNs likely shapes the phenotype of its stromal cells, and the ECM structure facilitates the FRC function. FRC seeding of decellularized and sterilized LN sections allowed for a suitable environment for FRC attachment and viability and did not promote cell proliferation. Ongoing experiments are assessing the phenotype of the seeded FRC cells to test whether the LN-like environment promotes LN-like FRC phenotype, as well as quantifying the amount of GAG remaining after the decellularization procedures to confirm that the decellularization process preserves the LN ECM components.

Acknowledgements (Optional):