Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) “shaking” to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85–95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable “undifferentiated MSC-pool” property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues.

中文翻译：

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在体外生成具有增强多能性的骨髓源性间充质基质/干细胞球体的振荡培养方法

间充质基质/干细胞 (MSC) 通常会扩展为贴壁单层，在重复传代后很容易失去其增殖和多向细胞潜能。浮动培养系统可用于生成 MSC 球体，有望克服与传统贴壁培养相关的限制，同时促进无支架细胞移植。然而，长期培养后球体的表型特征尚不清楚。此外，再生疗法需要新的培养系统来维持其未分化状态。在这项研究中，我们建立了一种新的培养方法，采用三维（3D）“摇动”来使用骨髓来源的 MSC 生成 MSC 球体。小鼠或人类 MSC 的浮动 3D 培养物在 1 个月内摇动 (85–95 rpm) 后形成球体。这些球状体保持了它们的成骨、成脂和成软骨分化能力。贴壁培养的小鼠和人 MSC 的成脂分化能力在几代后丧失，通过振荡培养显着恢复。值得注意的是，人类 MSC 球体表现出可再生的“未分化 MSC 池”特性，其中未分化的 MSC 从重复接种在塑料培养皿上的球体中生长。这些数据表明，摇动培养方法可以维持和恢复单层扩增后丧失的多能性，从而显示出作为间充质组织再生疗法的潜力。几段后丢失，通过摇动培养显着恢复。值得注意的是，人类 MSC 球体表现出可再生的“未分化 MSC 池”特性，其中未分化的 MSC 从重复接种在塑料培养皿上的球体中生长。这些数据表明，摇动培养方法可以维持和恢复单层扩增后丧失的多能性，从而显示出作为间充质组织再生疗法的潜力。几段后丢失，通过摇动培养显着恢复。值得注意的是，人类 MSC 球体表现出可再生的“未分化 MSC 池”特性，其中未分化的 MSC 从重复接种在塑料培养皿上的球体中生长。这些数据表明，摇动培养方法可以维持和恢复单层扩增后丧失的多能性，从而显示出作为间充质组织再生疗法的潜力。