In this study, layer‐by‐layer coatings composed of heparin and collagen are proposed as an extracellular mimetic environment on nerve guide conduits (NGC) to modulate the behavior of Schwann cells (hSCs). The authors evaluated the stability, degradation over time, and bioactivity of six bilayers of heparin/collagen layer‐by‐layer coatings, denoted as (HEP/COL)6. The stability study reveals that (HEP/COL)6is stable after incubating the coatings in cell media for up to 21 days. The impact of (HEP/COL)6on hSCs viability, protein expression, and migration is evaluated. These assays show that hSCs cultured in (HEP/COL)6have enhanced protein expression and migration. This condition increases the expression of neurotrophic and immunomodulatory factors up to 1.5‐fold compared to controls, and hSCs migrated 1.34 times faster than in the uncoated surfaces. Finally, (HEP/COL)6is also applied to a commercial collagen‐based NGC, NeuraGen, and hSC viability and adhesion are studied after 6 days of culture. The morphology of NeuraGen is not altered by the presence of (HEP/COL)6and a nearly 170% increase of the cell viability is observed in the condition where NeuraGen is used with (HEP/COL)6. Additionally, cell adhesion on the coated samples is successfully demonstrated. This work demonstrates the reparative enhancing potential of extracellular mimetic coatings.
- Award ID(s):
- 2051582
- NSF-PAR ID:
- 10314356
- Date Published:
- Journal Name:
- Cells Tissues Organs
- ISSN:
- 1422-6405
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Mesenchymal stem cells (MSCs) secrete a diversity of factors with broad therapeutic potential, yet current culture methods limit potency outcomes. In this study, we used topographical cues on polystyrene films to investigate their impact on the secretory profile and potency of bone marrow-derived MSCs (hBM-MSCs). hBM-MSCs from four donors were cultured on topographic substrates depicting defined roughness, curvature, grooves and various levels of wettability.
Methods The topographical PS-based array was developed using razor printing, polishing and plasma treatment methods. hBM-MSCs from four donors were purchased from RoosterBio and used in co-culture with peripheral blood mononuclear cells (PBMCs) from Cell Applications Inc. in an immunopotency assay to measure immunosuppressive capacity. Cells were cultured on low serum (2%) for 24–48 h prior to analysis. Image-based analysis was used for cell quantification and morphology assessment. Metabolic activity of BM-hMSCs was measured as the mitochondrial oxygen consumption rate using an extracellular flux analyzer. Conditioned media samples of BM-hMSCs were used to quantify secreted factors, and the data were analyzed using R statistics. Enriched bioprocesses were identify using the Gene Ontology tool
enrichGO from theclusterprofiler. One-way and two-way ANOVAs were carried out to identify significant changes between the conditions. Results were deemed statistically significant for combinedP < 0.05 for at least three independent experiments.Results Cell viability was not significantly affected in the topographical substrates, and cell elongation was enhanced at least twofold in microgrooves and surfaces with a low contact angle. Increased cell elongation correlated with a metabolic shift from oxidative phosphorylation to a glycolytic state which is indicative of a high-energy state. Differential protein expression and gene ontology analyses identified bioprocesses enriched across donors associated with immune modulation and tissue regeneration. The growth of peripheral blood mononuclear cells (PBMCs) was suppressed in hBM-MSCs co-cultures, confirming enhanced immunosuppressive potency. YAP/TAZ levels were found to be reduced on these topographies confirming a mechanosensing effect on cells and suggesting a potential role in the immunomodulatory function of hMSCs.
Conclusions This work demonstrates the potential of topographical cues as a culture strategy to improve the secretory capacity and enrich for an immunomodulatory phenotype in hBM-MSCs.
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2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. 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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1159/000521609
