Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, Institute for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University, Chongqing, China.
College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, China.
Biotechnol J. 2024 Feb;19(2):e2300427. doi: 10.1002/biot.202300427.
Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210 rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60 rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1% ± 3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.
三维（3D）细胞培养因其能够更准确地模拟体内细胞环境而在生物医学研究中受到广泛关注。使用悬滴法形成 3D 细胞球状体已成为生成均一大小球状体的一种具有成本效益的关键方法。本研究旨在验证一次性实验室用品——尖端再填充晶片（TrW）在促进 3D 细胞培养方面的潜力。TrW 可通过将溶液吸到晶片的孔中来轻松生成悬滴。TrW 的表面润湿性和厚度确保了悬滴的机械稳定性。含有 60µL 溶液的悬滴即使在以 210rpm 进行轨道摇晃时也能牢固地附着在 TrW 上。出色的抗机械振动能力使得可以使用惯性聚焦来促进球状体的形成。通过活/死细胞染色、定量聚合酶链反应（qPCR）分析和细胞骨架染色证明了这一点，结果表明以 60rpm 进行水平轨道旋转 15 分钟可促进细胞聚集，并最终导致 3D 球状体的形成。三个 TrW 中每个包含 60 个悬滴，球状体的收获率为 96.1%±3.5%。除了生成单培养 3D 球状体外，基于 TrW 的悬滴平台还能够形成多细胞球状体，并按需对球状体进行配对和融合。TrW 是一种一次性用品，不需要任何制造或表面改性程序，进一步增强了其在常规生物实验室中的应用潜力。
