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首页> 外文期刊>RSC Advances >Ultrahigh-throughput generation and characterization of cellular aggregates in laser-ablated microwells of poly(dimethylsiloxane)
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Ultrahigh-throughput generation and characterization of cellular aggregates in laser-ablated microwells of poly(dimethylsiloxane)

机译：聚二甲基硅氧烷激光烧蚀微孔中细胞聚集体的超高通量生成和表征

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Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complicated to manufacture, or lack the surface packing densities that would significantly improve MCA production. To address these concerns, we custom-modified a commercial laser cutter to provide complete control over laser ablation and directly generate microwells in a poly(dimethylsiloxane) (PDMS) substrate. We achieved ultra rapid microwell production speeds (>50 000 microwells per h) at high areal packing densities (1800 microwells per cm(2)) and over large surface areas for cell culture (60 cm2). Variation of the PDMS substrate distance from the laser focal plane during ablation allowed for the generation of microwells with a variety of sizes, contours, and aspect ratios. Casting of high-fidelity microneedle masters in polyurethane allowed for non-ablative microwell reproduction through replica molding. MCAs of human bone marrow derived mesenchymal stem cells (hMSCs), murine 344SQ metastatic adenocarcinoma cells, and human C4-2 prostate cancer cells were generated in our system with high uniformity within 24 hours, and computer vision software aided in the ultra-high-throughput analysis of harvested aggregates. Moreover, MCAs maintained invasive capabilities in 3D migration assays. In particular, 344SQ MCAs demonstrated epithelial lumen formation on Matrigel, and underwent EMT and invasion in the presence of TGF-beta. We expect this technique to find broad utility in the generation and cultivation of cancer cell aggregates, primary cell aggregates, and embryoid bodies.
机译：细胞的聚集体，也称为多细胞聚集体（MCA），已经在癌症生物学，再生医学和发育生物学领域中用作微型组织。但是，小的MCA（每个聚集体少于100个单元）仍然难以以高均匀度进行大量生产。强制聚集到微孔中为形成一致的聚集体提供了一种有希望的解决方案，但是微孔的商业来源价格昂贵，制造复杂或缺少可以显着提高MCA产量的表面堆积密度。为了解决这些问题，我们定制修改了商用激光切割机，以提供对激光烧蚀的完全控制，并直接在聚二甲基硅氧烷（PDMS）基板上生成微孔。我们在高面积填充密度（每平方厘米1800微孔（2））和较大的细胞培养表面积（60 cm2）上实现了超快速的微孔生产速度（每小时> 50,000微孔）。烧蚀过程中PDMS衬底与激光焦平面的距离变化允许生成具有各种尺寸，轮廓和长宽比的微孔。聚氨酯浇铸高保真微针母盘，可以通过复制模制进行非烧蚀微孔复制。我们的系统在24小时内以高度均匀性生成了人类骨髓源性间充质干细胞（hMSCs），鼠类344SQ转移性腺癌细胞和人C4-2前列腺癌细胞的MCA，并且计算机视觉软件辅助了超高收获的骨料的通量分析。此外，MCA在3D迁移分析中保持了侵入性功能。特别地，344SQ MCA表现出在基质胶上的上皮内腔形成，并且在存在TGF-β的情况下经历了EMT和侵袭。我们期望该技术在癌细胞聚集体，原代细胞聚集体和胚状体的产生和培养中找到广泛的用途。

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