Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, USA.
Joint Department of Biomedical Engineering, North Carolina State University/University of North Carolina-Chapel Hill, Raleigh, North Carolina, USA.
Tissue Eng Part A. 2022 Oct;28(19-20):833-844. doi: 10.1089/ten.tea.2021.0217. Epub 2022 Aug 4.
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 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 . 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 , 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 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. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and 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. The information from this study can be extrapolated to future studies using human dermal fibroblasts in an model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible testing constructs.
目前，由人真皮成纤维细胞和水凝胶支架组成的基于细胞的水凝胶皮肤移植物倾向于最大限度地减少全层皮肤伤口的收缩，并支持皮肤再生。然而，用于真皮成纤维细胞的来源之间没有比较。使用成人或新生儿包皮真皮成纤维细胞的产品通常在经过多次传代后进行扩展和使用，而不清楚该初始生产步骤对细胞行为的质量和可重复性的影响。基于二维组织培养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富含成纤维细胞的胶原基质中的细胞特性和收缩行为。使用猪皮作为模型，基于其在结构和伤口愈合特性上与人皮的相似性，我们分离了来自三个不同供体年龄的猪真皮成纤维细胞，用于二维增殖测定和三维细胞填充胶原基质收缩测定。在 2D 细胞培养中，从第 1 代到第 6 代，所有年龄组的倍增时间相对保持一致。在收缩测定中，胎儿和新生儿组在第 1 代时比成年组收缩更快，产生的收缩力更大。然而，在培养 5 个传代后，年龄之间的收缩力没有差异。这些结果表明，水凝胶支架中的细胞反应如何根据供体年龄和培养时间的不同而不同，并表明生物工程皮肤产品中细胞成分的一致性可能有益于生物制造一致、可靠的皮肤移植物和移植物模型。在播种前，使用生物工程皮肤移植物的未来研究和治疗应考虑根据供体年龄和培养时间的不同，结果可能会有所不同。 影响声明 关于供体细胞年龄和培养时间对细胞水凝胶基皮肤移植物收缩的影响知之甚少。这些结果表明，猪成纤维细胞的细胞表型如何根据供体年龄和培养时间的不同而不同。在解决生物工程皮肤移植物生物制造中的重要不一致性时，这些信息很有帮助。这些发现与使用生物工程皮肤移植物模型的研究和治疗相关，并且可以在生物工程皮肤构建体的生产过程中提高可重复性和一致性。本研究的信息可以推广到未来在模型中使用人真皮成纤维细胞的研究，以帮助确定最佳供体年龄和培养时间，以获得最佳的伤口愈合结果或更可重复的测试构建体。
