领先的Cas蛋白、LAMP检测技术、原料酶提供商

构建3D细胞结构的好方法

Claire Wilhelm

2017-09-12

《Nature Communication》杂志2017年9月12日在线发表了法国巴黎狄德罗大学Claire Wilhelm研究员的一篇研究论文，研究报告了一种可以形成心脏细胞的胚胎干细胞(ESC)磁驱动三维(3D)聚合物。这一成果被视为显著区别于传统技术的、可构建细胞3D组织结构的全新方法。同时报告了胚胎干细胞结构的3D磁组装以及针对干细胞分化的远程力学刺激。

再生医学是指利用生物学及工程学方法，创造出具备正常结构和功能的组织和器官，替代人体失去的或功能受损的组织器官。通过单个细胞创造3D组织结构并刺激它们形成特定细胞类型是再生医学的一个重要目标。大量研究表明力学因素可以影响干细胞分化，但是许多都是集中在2D结构上。

胚胎干细胞具有体外培养无限增殖、自我更新和多向分化的特性——这种从早期胚胎或原始性腺中分离出来的细胞，无论在体外还是体内环境，都能被诱导分化为机体几乎所有的细胞类型。根据此前已有的大量研究，科学家发现力学因素可以影响干细胞分化，但是到目前为止，许多研究只是集中在2D结构上。

研究发现，将氧化铁纳米颗粒融入胚胎干细胞中，即可以制成此类结构。这些细胞一旦磁化后，就能通过磁场对它们进行远程操控以形成3D聚合物，再通过力学刺激分化成心脏细胞。

研究团队的实验表明，在胚胎干细胞中加入磁性氧化铁粒子，并不会影响其分化成不同类型细胞的活力和能力。

该过程使研究人员能够在无任何生物化学触发物的情况下，研究胚胎干细胞的分化情况，被认为代表了一种有别于传统技术的、制造细胞3D组织结构的新方法。

Figure 1

Schematic illustrating the different steps involved in the magnetic stretcher. a Nanoparticles incorporation in ESCs, b EBs formation from magnetized ESCs driven by a magnetic microtip, and c EBs magnetic stimulation in situ, in the 3D geometry, and without the need for a supporting matrix

Figure 2Optimization of embryonic stem cell (ESC) magnetic labeling. a Magnetic labeling of ESCs at different extracellular iron concentrations (for a fixed incubation time of 30 min) and during different incubation periods (for a fixed iron concentration of [Fe] = 2 mM). b Perls’ Prussian blue staining of ESCs after labeling with different concentrations of extracellular iron (between 0.5 mM and 2 mM), and a fixed incubation time of 30 min. Scale bar: 250 µm. c Transmission electron micrograph of ESC after labeling for 30 min at [Fe] = 2 mM (successive zooms of framed areas). Scale bar: 5 µm. Nanoparticles are all located inside the lysosomes. d Cell viability testing using Alamar Blue detection of cell metabolic activity. Cell viability was calculated relative to the control (unlabeled cells in complete medium) and was measured 2 h after different incubation periods (in RPMI) with different extracellular iron concentrations and incubation times. e Expression of pluripotency genes Oct4, Nanog and Sox2 measured by real-time PCR. The gene expression level was calculated with respect to RPLP0 mRNA and expressed as compared to control (unlabeled cells, cultured in complete medium with LIF, = 1 ± SEM). A positive control was added in which the LIF has been removed during 5 days before analysis (culture in complete medium without LIF). One can note that only one condition led to a significant upregulation (Oct4—incubation at 2 mM for 30 min). However the gene was upregulated <1.5-fold (1.3-fold exactly). Besides, higher doses (2 h incubation at 2 and 5 mM) provide the same Oct4 expression as the control. f Expression of several genes characteristic of the different embryonic layers in hanging drop EB formation conditions with 1000 unlabeled (control, blue bars) or labeled cells (magnetic, red bars), 5 days (open bars) and 7 days (solid bars) after initiation of differentiation. All values were calculated with respect to RPLP0 mRNA and normalized by the expression value of the same gene measured at day 0. Two-sample t-test was used to compare the control group with the magnetic group, for same gene and same day; *p < 0.05; **p < 0.01; ***p < 0.001. All error bars represent the SEM