Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model.
Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model.
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DOI：
10.1089/ten.tea.2021.0217
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发表时间：
2022-06
影响因子：
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通讯作者：
A. Detwiler;K. Polkoff;Lewis S. Gaffney;D. Freytes;J. Piedrahita
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文献类型：
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作者：
A. Detwiler;K. Polkoff;Lewis S. Gaffney;D. Freytes;J. Piedrahita
目前由人真皮成纤维细胞和水凝胶支架组成的基于细胞水凝胶的皮肤移植物倾向于使全层皮肤伤口的收缩最小化并支持皮肤再生。然而，所用的真皮成纤维细胞来源之间没有比较。使用成人或新生儿包皮真皮成纤维细胞的产品通常在体外扩增并在多次传代后使用，而没有清楚地了解该初始生产步骤对细胞行为的质量和再现性的影响。基于二维组织培养扩增对细胞增殖和基因表达的已知影响，我们假设供体年龄和培养时间的差异可能会影响成纤维细胞填充的胶原基质中的细胞特性和收缩行为。使用猪皮肤作为基于其在结构和伤口愈合特性方面与人类皮肤相似的模型，我们分离了三种不同供体年龄的猪真皮成纤维细胞用于2D增殖测定和3D细胞填充胶原基质收缩性测定。在2D细胞培养中，从第1代到第6代，所有年龄组之间的倍增时间保持相对一致。在收缩力试验中，在体外第1代，胎儿和新生儿组收缩更快，产生的收缩力大于成人组。然而，在培养五代后，不同年龄之间的收缩力没有差异。这些结果显示了水凝胶支架中的细胞反应如何基于供体年龄和体外培养时间而不同，并表明生物工程皮肤产品的细胞组分的一致性可能有利于一致、可靠的皮肤移植物和移植物体内模型的生物制造。使用生物工程皮肤移植物的未来研究和治疗应考虑结果如何根据供体年龄和接种前培养时间而变化。
Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. 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 influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in 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 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding.