Y24 - Extracellular vesicles (EVs) and silk fibroin (SF) can enhance the therapeutic efficacy of multiple cell populations

Introduction: : Cell therapy involves the substitution of damaged or dead cells with healthy substitutions. This involves direct replacement with mature differentiated cells, or the use of stem/progenitor cells to repopulate injured tissues. Cell delivery has proved challenging, as cells may be lost upon direct injection into the bloodstream, or unable to integrate into the disease milieu. Silk fibroin (SF) is an FDA-approved, biodegradable material with low immunogenicity that can be processed and orientated into a variety of different structures such as tubular vessels or hydrogels. Extracellular vesicles (EVs) are a class of novel therapeutic secretions that contain a variety of proteins and micro RNAs, that promote angiogenesis, repair, and regulate inflammation. EVs can be soak-loaded into SF before use to further enhance the regenerative potential of the material and enhance integration upon delivery. Enrichment of SF with EVs represents a novel bioengineering platform for the enhancement of a variety of cell therapeutics. This study investigated the use of SF+EVs as a bioengineered solution to augment multiple cell types for regenerative medicine applications.

Materials and

Methods: : : EVs were purchased/characterized from RoosterBio, Inc. Mature human endothelial cells (ECs), endothelial colony forming cells (ECFCs), fibroblasts (HDFs) and smooth muscle cells (SMCs) were treated with EVs to assess effects on viability/proliferation using AlamarBlue. Cells were grown on either tissue culture plastic (TCP) or silk films. Proliferation was assessed at either 24/72h (films) or 48/96h (TCP) post-treatment. Growth media was used as a positive control. Adhesion to films was assessed by seeding cells on EV pre-loaded films. Cells were fixed/stained for DAPI and phalloidin to assess the number of cells adhered, and cytoplasmic area respectively. Proliferation was analyzed using two-way ANOVA and adhesion was analyzed using one-way ANOVA. All data is presented as fold change to corresponding vehicle control.

Results, Conclusions, and Discussions:: EVs were internalized by all cell types. EVs enhanced the proliferative potential of all cells on TCP after 96h (P= < 0.0005), with ECFCs showing enhanced proliferation after 48h (P= < 0.0005). On films, EVs enhanced the proliferative potential of all cell types after 24 and 72h post-seeding (P= < 0.0001). For adhesion, all cells showed increased cell adherence and cytoplasmic area (P= < 0.0005).
We conclude that SF can be enhanced with EVs to promote the adhesion and proliferation of multiple cell types. A combination of SF with EVs further enhanced this potential, serving as an attractive platform for cells to adhere to, and enhance subsequent downstream proliferation. Future work will investigate how EVs further enhance the in vitro bioactivity of these therapeutic populations with regards to wound closure and migration, as well as elucidating the genetic mechanisms which may be responsible for these enhancements.

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