Pathak Juhi, Singh Shiva Pratap, Pathak Manisha, Goel Anjana, Soni Yogesh Kumar, Singh Manoj Kumar
Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, Mathura, 281 122, Uttar Pradesh, India.
Department of Biotechnology, GLA University, Mathura, 281, 406, Uttar Pradesh, India.
Mol Biol Rep. 2025 Jan 12;52(1):116. doi: 10.1007/s11033-025-10227-6.
Extracellular matrix (ECM) proteins play a crucial role in regulating the biological properties of adherent cells. For cryopreserved fibroblasts, a favourable ECM environment can help restore their natural morphology and function more rapidly, minimizing post-thaw stress responses.
This study explored the functional responses of cryopreserved enriched caprine adult dermal fibroblast (cadFibroblast) cells to structural [collagen-IV and rat tail collagen (RTC)] and adhesion ECM proteins (laminin, fibronectin, and vitronectin) under in vitro culture conditions. The cryopreserved cadFibroblasts were evaluated for cell morphology, confluence, viability, cell adhesion, colony-forming units, proliferation, population doubling time migration (scratch wound healing assay), and quantitative real-time PCR for gene expression analyses when cultivated on surfaces coated with ECM proteins. A significantly (p < 0.05) higher cellular responses regarding cell adhesion ability, proliferation, CFUs, migration rate and differential gene expression of cell adhesion (β integrin, β tubulin, and E-cadherin) associated marker genes were observed with adhesion ECM proteins (laminin, fibronectin, and vitronectin) than with structural ECM proteins (collagen-IV and RTC). RT-PCR analyses revealed ECM-dependent differential template expression in cultured post-thawed cadFibroblasts.
Overall, while comparing different types of ECM proteins, adhesion ECM proteins (laminin, fibronectin, and vitronectin) provide a relatively better niche that supports adhesion, growth, proliferation, and migration of post-thawed cadFibroblasts compared with structural ECM proteins (collagen-IV and RTC) and the culture without ECM. The outcomes of the present study are crucial for replicating physiological conditions in experimental models for future research involving cryopreserved dermal fibroblasts or any other cell type, which can lead to future developments in tissue engineering and regenerative medicine.
细胞外基质（ECM）蛋白在调节贴壁细胞的生物学特性方面起着至关重要的作用。对于冷冻保存的成纤维细胞，良好的ECM环境有助于更快地恢复其天然形态和功能，将解冻后的应激反应降至最低。
本研究探讨了在体外培养条件下，冷冻保存的富集山羊成年真皮成纤维细胞（cadFibroblast）对结构型ECM蛋白（IV型胶原蛋白和大鼠尾胶原蛋白）和黏附型ECM蛋白（层粘连蛋白、纤连蛋白和玻连蛋白）的功能反应。当在涂有ECM蛋白的表面培养时，对冷冻保存的cadFibroblasts进行细胞形态、汇合度、活力、细胞黏附、集落形成单位、增殖、群体倍增时间迁移（划痕伤口愈合试验）以及用于基因表达分析的定量实时PCR评估。与结构型ECM蛋白（IV型胶原蛋白和大鼠尾胶原蛋白）相比，黏附型ECM蛋白（层粘连蛋白、纤连蛋白和玻连蛋白）在细胞黏附能力、增殖、集落形成单位、迁移率以及细胞黏附相关标记基因（β整合素、β微管蛋白和E-钙黏蛋白）的差异基因表达方面表现出显著（p < 0.05）更高的细胞反应。RT-PCR分析揭示了冻融后cadFibroblasts培养物中ECM依赖性差异模板表达。
总体而言，在比较不同类型的ECM蛋白时，与结构型ECM蛋白（IV型胶原蛋白和大鼠尾胶原蛋白）以及无ECM培养相比，黏附型ECM蛋白（层粘连蛋白、纤连蛋白和玻连蛋白）提供了一个相对更好的微环境，支持解冻后cadFibroblasts的黏附、生长、增殖和迁移。本研究结果对于在涉及冷冻保存的真皮成纤维细胞或任何其他细胞类型的未来研究的实验模型中复制生理条件至关重要，这可能会引领组织工程和再生医学的未来发展。
