A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels
A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels
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DOI：
10.1089/ten.tec.2013.0051
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发表时间：
2014-03-01
影响因子：
3
通讯作者：
Putnam, Andrew J.
中科院分区：
文献类型：
--
作者：
Carrion, Bita;Janson, Isaac A.;Putnam, Andrew J.
间充质干细胞（MSC）显示多能特性，使其成为潜在治疗应用的理想选择。MSC通常在组织培养塑料上培养为单层，但越来越多的证据表明它们可能随着时间的推移在体外失去其多能性，并最终不再保留与体内驻留MSC的任何相似性。三维（3D）培养系统，更紧密地概括了MSC和其他细胞类型的生理环境，越来越多地探索其支持和维持细胞表型的能力。在我们自己的大部分工作中，我们利用了纤维蛋白，一种天然的蛋白质基材料，在伤口愈合过程中充当临时细胞外基质。已证明纤维素可用于许多组织工程应用，并已在临床上用作止血材料。其由凝血酶介导的纤维蛋白原改变驱动的快速自组装使纤维蛋白成为有吸引力的3D基质，细胞可以在其中粘附，扩散，增殖并进行复杂的形态发生程序。然而，存在对简单的成本有效的方法的显著需求，以安全地取回包封在纤维蛋白水凝胶内的细胞，以进行额外的分析或将细胞用于治疗。在这里，我们提出了一个安全有效的协议，从3D纤维蛋白凝胶分离间充质干细胞。我们成功提取方法的关键成分是纳豆激酶，它是枯草杆菌蛋白酶家族的一种丝氨酸蛋白酶，具有很强的纤溶活性。我们的数据表明，使用纳豆激酶从3D纤维蛋白凝胶中回收的MSC不仅是可行的，而且保留了其增殖和多谱系潜力。经证明，对于MSC，该方法可以容易地适用于从3D纤维蛋白凝胶构建体中检索任何其他细胞类型，用于各种应用，包括扩增、生物测定和体内植入。
Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.