Reid Gregory, Cerino Giulia, Melly Ludovic, Fusco Deborah, Zhang Chunyan, Reuthebuch Oliver, Milan Giulia, Marsano Anna
Cardiac Surgery and Engineering Group, Department of Biomedicine, University of Basel and University Hospital of Basel, 4031, Basel, Switzerland.
Department of Cardiac Surgery, University Hospital of Basel, 4031, Basel, Switzerland.
Stem Cell Res Ther. 2025 May 1;16(1):220. doi: 10.1186/s13287-025-04286-6.
The rapid formation and long-term maintenance of functional vascular networks are crucial for the success of regenerative therapies. The stromal vascular fraction (SVF) from human adipose tissue is a readily available, heterogeneous cell source containing myeloid lineage cells, mesenchymal stromal cells, endothelial cells and their precursors, and pericytes, which are important for vascular support. Previous studies showed that seeding SVF cells under perfusion and pre-culturing them on three-dimensional (3D) collagen sponges enhances the vascular cell component in vitro while accelerating vascularization and improving human cell engraftment in vivo compared to static pre-culture. However, generating a perfusion-cultured SVF patch over a 5-day period is both costly and challenging for clinical translation. To overcome these limitations, this study explores a no-pre-culture strategy by comparing perfusion-based seeding with static cell loading on 3D sponges. The hypothesis is that perfusion-based seeding enhances in vivo cell engraftment and angiogenic potential by loading different SVF cell subpopulations onto 3D scaffolds during the seeding process.
SVF-cells are seeded onto collagen scaffold using two approaches: a closed system perfusion bioreactor for 18 h or static loading onto the sponge surface. The in vitro cell distribution and baseline cytokine profiles were evaluated. Subsequently, human cell engraftment and differentiation were assessed in vivo using a nude rat subcutaneous implantation model. Analyses included the survival of transplanted human cells, the functionality and maturation of newly formed blood vessels within the SVF-patch.
Perfusion seeding significantly reduced the number of myeloid cells and achieved uniform spatial distribution across the construct. Vascular endothelial growth factor release was significantly increased following perfusion culture, whereas pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1β were decreased. In the short term, perfusion culture enhanced uniform vascularization and SVF cell engraftment in vivo. However, the long-term differences between the perfusion-seeded and static-seeded groups diminished.
Eliminating the need for prolonged pre-culture offers a feasible and cost-effective strategy for advancing regenerative cell-based therapies by reducing pre-culture times while preserving therapeutic efficacy. Perfusion-based seeding offers significant short-term benefits, including enhanced vascularization and cell engraftment, though long-term differences compared to static seeding are minimal. Further investigation is needed to evaluate its potential in a diseased ischemic heart model.
功能性血管网络的快速形成和长期维持对于再生治疗的成功至关重要。人脂肪组织的基质血管成分（SVF）是一种易于获取的异质性细胞来源，包含髓系细胞、间充质基质细胞、内皮细胞及其前体，以及对血管支持很重要的周细胞。先前的研究表明，与静态预培养相比，在灌注条件下接种SVF细胞并在三维（3D）胶原海绵上预培养可增强体外血管细胞成分，同时加速血管生成并改善体内人细胞植入。然而，在5天内生成灌注培养的SVF贴片对于临床转化来说既昂贵又具有挑战性。为了克服这些限制，本研究通过比较基于灌注的接种与在3D海绵上的静态细胞加载来探索一种无预培养策略。假设是基于灌注的接种通过在接种过程中将不同的SVF细胞亚群加载到3D支架上，增强体内细胞植入和血管生成潜力。
使用两种方法将SVF细胞接种到胶原支架上：在封闭系统灌注生物反应器中接种18小时或静态加载到海绵表面。评估体外细胞分布和基线细胞因子谱。随后，使用裸鼠皮下植入模型在体内评估人细胞植入和分化。分析包括移植人细胞的存活、SVF贴片中新生血管的功能和成熟情况。
灌注接种显著减少了髓系细胞数量，并在构建体中实现了均匀的空间分布。灌注培养后血管内皮生长因子释放显著增加，而肿瘤坏死因子-α和白细胞介素-1β等促炎细胞因子减少。短期内，灌注培养增强了体内均匀的血管生成和SVF细胞植入。然而，灌注接种组和静态接种组之间的长期差异减小。
通过减少预培养时间同时保持治疗效果，消除长时间预培养的需求为推进基于细胞的再生治疗提供了一种可行且具有成本效益的策略。基于灌注的接种具有显著的短期益处，包括增强血管生成和细胞植入，尽管与静态接种相比长期差异很小。需要进一步研究以评估其在患病缺血性心脏模型中的潜力。
