Microfluidic Generation of Gradient Hydrogels to Modulate Hematopoietic Stem Cell Culture Environment
Microfluidic Generation of Gradient Hydrogels to Modulate Hematopoietic Stem Cell Culture Environment
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DOI：
10.1002/adhm.201300263
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发表时间：
2014-03-01
影响因子：
10
通讯作者：
Harley, Brendan A. C.
中科院分区：
文献类型：
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作者：
Mahadik, Bhushan P.;Wheeler, Tobias D.;Harley, Brendan A. C.
骨髓提供影响造血干细胞（HSC）行为的空间和时间可变信号。虽然多个生物分子信号和骨髓细胞群体已被提出作为HSC命运的关键调节因子，但需要新的工具来探测它们的重要性和作用机制。在这里，描述了一种基于微流体混合平台的新方法，以创建小体积的3D水凝胶构建体，其包含受HSC小生境启发的细胞和基质成分的重叠模式。该方法用于以不依赖于水凝胶密度和细胞/颗粒尺寸的方式产生含有荧光微球、MC 3 T3-E1成骨细胞、原代鼠造血干细胞和祖细胞（HSPC）及其组合的相反梯度的水凝胶。描述了三种不同的分析方法以在多个尺度下表征这些水凝胶的局部性质：1）整个构建体荧光分析; 2）构建体内单个细胞的多光子成像; 3）从培养后的水凝胶中检索离散的子区域。这里报告的方法允许在单个光学半透明的3D生物材料中创建细胞和材料线索的稳定梯度，以实现一系列关于微环境信号如何影响细胞命运的研究。
The bone marrow provides spatially and temporally variable signals that impact the behavior of hematopoietic stem cells (HSCs). While multiple biomolecular signals and bone marrow cell populations have been proposed as key regulators of HSC fate, new tools are required to probe their importance and mechanisms of action. Here, a novel method based on a microfluidic mixing platform to create small volume, 3D hydrogel constructs containing overlapping patterns of cell and matrix constituents inspired by the HSC niche is described. This approach is used to generate hydrogels containing opposing gradients of fluorescent microspheres, MC3T3-E1 osteoblasts, primary murine hematopoietic stem and progenitor cells (HSPCs), and combinations thereof in a manner independent of hydrogel density and cell/particle size. Three different analytical methods are described to characterize local properties of these hydrogels at multiple scales: 1) whole construct fluorescent analysis; 2) multi-photon imaging of individual cells within the construct; 3) retrieval of discrete sub-regions from the hydrogel post-culture. The approach reported here allows the creation of stable gradients of cell and material cues within a single, optically translucent 3D biomaterial to enable a range of investigations regarding how microenvironmental signals impact cell fate.