(M1030-11-72) Guided Assembly of Lymph Node Stromal Cells Using a Biomaterial Formulation

Purpose: Cell-based immunotherapies have gained remarkable interest following their promising therapeutic outcomes. However, the primary challenge in cell therapy is mimicking the in vivo microenvironment to preserve their biological functions. Fibroblastic reticular cells (FRCs) are recognized for actively steering immune responses. FRCs form a three-dimensional (3D) reticular network while maintaining cell-to-cell contacts in lymph nodes critical for their optimal functions. They maintain an anti-inflammatory milieu by regulating immune cells which makes them a promising cell therapy for hyperinflammatory and autoimmune conditions. The anti-inflammatory therapeutic potential of FRCs is highlighted by literature reports showing the suppression of inflammation in a murine sepsis model [1]. However, the localized delivery of FRCs in a guided 3D organization necessary for optimal functions remains unexplored. To this end, we developed a biotinylated self-assembling peptide (EAKbt) to promote FRCs into reticular cell clusters to achieve a 3D delivery. We hypothesize that avidin-mediated cross-linking of EAKbt forms a supramolecular assembly that promotes FRC cluster formation. We further investigate the cell cluster characteristics for the developed biomaterial formulation.

Methods: A self-assembling peptide AEAEAKAKAEAEAKAK biotinylated at the N-terminal (EAKbt) was used as the biomaterial platform for delivering human lymphatic fibroblasts. The biomaterial scaffolds were formulated by cross-linking EAKbt with varying concentrations of avidin to yield the final avidin: biotin molar ratio of 1:30 (high), 1:150 (medium), and 1:750 (low). The peptidic assembly was characterized using scanning electron microscopy (SEM). Human lymphatic fibroblasts were seeded and cultured with the peptidic assembly and brightfield images were captured at pre-determined times. The 3D nature of the clusters was determined by analyzing the morphological attributes of the clusters through image analysis using ImageJ software. Cell viability of the delivered cells was confirmed using live-dead staining.

Results: We demonstrate the formation of avidin-mediated cross-linked assemblies using the biotinylated EAK peptide. SEM analysis shows a denser peptidic assembly of EAKbt+avidin compared to EAKbt itself (Fig. 1) indicative of scaffold formation. The peptide assembly drives the formation of reticular clusters of human lymphatic fibroblasts. The cluster formation was robust and the 3D features of the clusters were proportional to avidin concentration (Fig. 2A). Higher solidity values indicate non-attaching cells, thereby supporting 3D organization of delivered cells (Fig. 2C). The clusters were retained over time as a function of avidin concentration (Fig. 2B). The cells maintained their viability as observed from the live-dead staining. 3D structure of cells was observed in EAKbt+avidin, while cells formed 2D multilayers in the absence of avidin (Fig. 3). Taken together, EAKbt-avidin formulation shows the potential to deliver FRCs in a 3D organization mimicking the in vivo microenvironment favorable for cell therapy.

Conclusion: The preliminary data supports our hypothesis that avidin-mediated cross-linking of EAKbt fosters the formation of 3D clusters of human lymphatic fibroblasts. The observed effects were robust with the delivered cells retaining their cluster forms and viability. Additional studies to investigate their effect to orchestrate an anti-inflammatory immune response will provide further insights into the therapeutic potential of this biomaterial formulation.

References: 1. Fletcher, A.L., et al., Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis. Science translational medicine, 2014. 6(249): p. 249ra109-249ra109.

Fig.1 Scanning electron microscopy (SEM) imaging of EAKbt-avidin formulation. SEM images of (A) EAKbt and (B) EAKbt+avidin showing denser peptidic assembly in the latter.

Fig. 2 Formation and characterization of reticular clusters of human lymphatic fibroblasts. (A) Representative brightfield microscopic images showing cell cluster formation in the presence of EAKbt-avidin as a function of avidin concentration; (B) Effect of EAKbt+Avidin on retaining clusters cultured till day 7; (C) Image analysis of clusters (day 7) showing higher solidity in high avidin group representing uniform outer morphology (absence of adhering (2D), elongated cells) indicative of 3D organization; **** represents p < 0.0001 (One-way ANOVA with multiple comparisons).

Fig. 3 Live-dead staining of cell clusters. Fluorescent microscopy images of live-dead staining for cells depicting live (green) and dead (red) in the biomaterial formulation.