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Abstract Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross‐linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein‐1, macrophage colony‐stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN‐γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN‐γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN‐γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN‐γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN‐γ.
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Serial Passaging Affects Stromal Cell Mechanosensitivity on Hyaluronic Acid Hydrogels
Abstract There is a tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work, a methacrylated hyaluronic acid hydrogel platform is leveraged to study stromal cell mechanotransduction. Hydrogels are first formed through thiol‐Michael addition to model normal soft tissue (e.g., lung) stiffness (E ≈ 1 kPa). Secondary cross‐linking via radical photopolymerization of unconsumed methacrylates allows matching of early‐ (E ≈ 6 kPa) and late‐stage fibrotic tissue (E ≈ 50 kPa). Early passage (P1) human bone marrow mesenchymal stromal cells (hMSCs) display increased spreading, myocardin‐related transcription factor‐A (MRTF‐A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF‐A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models.
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- Award ID(s):
- 2046592
- PAR ID:
- 10491130
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Macromolecular Bioscience
- Volume:
- 24
- Issue:
- 1
- ISSN:
- 1616-5187
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/mabi.202300110
