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Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model

Amber Detwiler, Kathryn Polkoff, Lewis Gaffney, Donald Freytes, Jorge Piedrahita
doi: https://doi.org/10.1101/2021.11.29.469875
Amber Detwiler
1Comparative Medicine Institute, North Carolina State University, Raleigh, NC
2Joint Department of Biomedical Engineering, North Carolina State/ University of North Carolina-Chapel Hill, Raleigh NC
3Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
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Kathryn Polkoff
1Comparative Medicine Institute, North Carolina State University, Raleigh, NC
3Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
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Lewis Gaffney
1Comparative Medicine Institute, North Carolina State University, Raleigh, NC
2Joint Department of Biomedical Engineering, North Carolina State/ University of North Carolina-Chapel Hill, Raleigh NC
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Donald Freytes
1Comparative Medicine Institute, North Carolina State University, Raleigh, NC
2Joint Department of Biomedical Engineering, North Carolina State/ University of North Carolina-Chapel Hill, Raleigh NC
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  • For correspondence: dofreyte@ncsu.edu
Jorge Piedrahita
1Comparative Medicine Institute, North Carolina State University, Raleigh, NC
3Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
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ABSTRACT

Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding.

IMPACT STATEMENT Little is known about the impact of donor age and time in culture on the contraction of the 3D hydrogel-based graft. These results show how cellular phenotypes differ based on donor age and time in culture, which could account for important inconsistencies in biomanufacturing of skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. Future in vivo studies could help determine the best donor age and time in culture for improved wound healing outcomes or more reproducible in vitro testing constructs.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* Contact Authors: Donald O. Freytes, Ph.D., Joint Department of Biomedical Engineering, North Carolina State University, University of North Carolina-Chapel Hill, 4208D Engineering Building III, Campus Box 7115, Raleigh, NC 27695, dofreyte{at}ncsu.edu, dfreytes{at}unc.edu, Office: 919-513-7933

  • ↵** Contact Authors: Jorge Piedrahita, Ph.D., Comparative Medicine Institute, Biomedical Partnership Center 20001, Campus Box 8401, Raleigh, NC 27695, japiedra{at}ncsu.edu, Office: 919-515-7407

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 30, 2021.
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Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model
Amber Detwiler, Kathryn Polkoff, Lewis Gaffney, Donald Freytes, Jorge Piedrahita
bioRxiv 2021.11.29.469875; doi: https://doi.org/10.1101/2021.11.29.469875
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Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model
Amber Detwiler, Kathryn Polkoff, Lewis Gaffney, Donald Freytes, Jorge Piedrahita
bioRxiv 2021.11.29.469875; doi: https://doi.org/10.1101/2021.11.29.469875

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