In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates
In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates
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DOI：
10.1007/s00441-014-1939-0
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发表时间：
2014-11-01
影响因子：
3.6
通讯作者：
Leung, Kai P.
中科院分区：
文献类型：
--
作者：
Rettinger, Christina L.;Fourcaudot, Andrea B.;Leung, Kai P.
间充质干细胞（Mesenchymal stem cells，MSCs）具有自我更新和向沿着多种细胞谱系分化的能力，在多种治疗中具有潜在的应用前景。这些细胞通常在组织培养塑料上以单层培养，但在体外可能随着时间的推移而失去其细胞特异性。人们越来越关注通过三维（3D）技术培养贴壁细胞，以重现3D体内条件。我们描述了一种新的方法，用于产生和培养兔骨髓间充质干细胞作为无支架的三维细胞聚集体，通过使用微图案化的威尔斯通过强制聚集技术。通过活/死染色和5-乙炔基-2 '-脱氧尿苷（EdU）掺入来评估MSC聚集体的活力和增殖能力。酶联免疫吸附试验和基于抗体的多重蛋白质测定被用来量化释放的生长因子和趋化因子。通过定量实时聚合酶链反应评价了相对于单层生长的MSC的作为3D聚集体的MSC的基因表达谱。兔MSC能够形成致密的细胞聚集体，并在3D培养中保持活力长达7天。我们还证明了在3D条件下培养的MSC中与血管生成和伤口愈合相关的基因和蛋白质表达增强。体外试管形成和划痕试验显示，创伤后，响应于3D条件培养基，上级新血管形成和更大的细胞恢复和迁移。我们的数据进一步表明，脂肪来源的干细胞聚集体在加速伤口愈合和减少瘢痕形成方面比真皮成纤维细胞或骨髓来源的MSC具有更大的潜力。
Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.