Mello Eleonora, Sorrentino Stefano, Bucciarelli Alessio, Cordelli Ermanno, De Luca Elisa, Nygaard Haakon, Wendt Stefan, Rainer Alberto, Gigli Giuseppe, Moroni Lorenzo, Polini Alessandro, Mozetic Pamela
Institute of Nanotechnology (NANOTEC), National Research Council, c/o Campus EcoTekne, via Monteroni, 73100, Lecce, Italy.
Department of Mathematics and Physics "Ennio De Giorgi", University of Salento, via Arnesano, 73100, Lecce, Italy.
Stem Cell Res Ther. 2025 Jul 31;16(1):417. doi: 10.1186/s13287-025-04547-4.
Neuronal spheroids represent an easy and versatile solution to model neuronal tissue in vitro. Conventional approaches to generate spheroids lack accurate size control, scalability, and customizability. This is even more exacerbated in case of pluripotent stem cell (PSC) derived spheroids, which remain challenging to standardize. Microwell devices address these limitations, providing an optimal balance between accessibility and scalability. With the aim of optimizing culture conditions, we parametrically investigated the role of microwell geometry on the formation and maturation of iPSC-derived motor neuron precursor (MNP) spheroids.
We developed a customizable mold device using Digital Light Processing (DLP) 3D printing to fabricate agarose microwell arrays with distinct aspect ratios for culturing hiPSC-derived MNP spheroids with high reproducibility. We generated nine different pyramidal microwell array geometries for culturing size-controlled spheroids in the 40-140 μm diameter range. We then evaluated the differential expression of genes related to cell proliferation and motor-neuron differentiation as function of microwell geometry and spheroid size.
Our results indicate that spheroid size is significantly influenced by the microwell geometry, reliably due to cell partitioning at the seeding stage. Expression of proliferation and differentiation markers, such as motor neuron and pancreas homeobox 1 (MNX1) and Islet-1 (ISL1) transcription factors, is also dependent on microwell geometry and spheroid morphological descriptors.
Our approach enables the scalable production of size-controlled MNP spheroids and underscores the effect of geometrical confinement on regulating motor neuron differentiation.
神经元球体是一种在体外模拟神经元组织的简便且通用的方法。传统的生成球体的方法缺乏精确的尺寸控制、可扩展性和可定制性。在多能干细胞（PSC）衍生的球体中，这种情况更加严重，其标准化仍然具有挑战性。微孔装置解决了这些限制，在可及性和可扩展性之间提供了最佳平衡。为了优化培养条件，我们参数化地研究了微孔几何形状对诱导多能干细胞（iPSC）衍生的运动神经元前体（MNP）球体形成和成熟的作用。
我们使用数字光处理（DLP）3D打印技术开发了一种可定制的模具装置，以制造具有不同纵横比的琼脂糖微孔阵列，用于以高重现性培养hiPSC衍生的MNP球体。我们生成了九种不同的金字塔形微孔阵列几何形状，用于培养直径在40 - 140μm范围内的尺寸可控的球体。然后，我们评估了与细胞增殖和运动神经元分化相关的基因的差异表达，作为微孔几何形状和球体大小的函数。
我们的结果表明，球体大小受到微孔几何形状的显著影响，这可能是由于接种阶段的细胞分区所致。增殖和分化标志物的表达，如运动神经元和胰腺同源框1（MNX1）以及胰岛-1（ISL1）转录因子，也取决于微孔几何形状和球体形态描述符。
我们的方法能够可扩展地生产尺寸可控的MNP球体，并强调了几何限制对调节运动神经元分化的影响。
