biostudies-literatureFu JJNational Natural Science Foundation of ChinaNatural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission187-199https://www.ebi.ac.uk/biostudies/studies/S-EPMC8010094biostudies-literatureUnknown14(2)A fast and low-cost fabrication process of flexible hanging drop chips for 3D spheroid cultures was proposed by cutting and bonding Parafilm^®, a cohesive thermoplastic. The Parafilm^® Hanging Drop Chip (PHDC) was assembled by two-layer of Parafilm^® sheet with different sizes of holes. The hole on the upper layer of the Parafilm^® is smaller than the hole on the bottom layer. The impact of hole size and sample volume on hanging drop formation and 3D spheroid formations in the hanging drop were investigated. The results showed that 20 <i>µ</i>L solution on PHDC with a 3 mm hole could form stabile drop and facilitate spheroid formation. The initial cell number determinates the size of the formed spheroids. Exchanging liquid from the upper hole of the Cellular and molecular bioengineeringCutting and Bonding Parafilm^® to Fast Prototyping Flexible Hanging Drop Chips for 3D Spheroid Cultures.PMC80100943187275311702047cstc2019jcyj-msxmX0211Fu JJLi YYu LLi CMLv XHHe XWang LXfalseCutting and Bonding Parafilm^® to Fast Prototyping Flexible Hanging Drop Chips for 3D Spheroid Cultures.A fast and low-cost fabrication process of flexible hanging drop chips for 3D spheroid cultures was proposed by cutting and bonding Parafilm^®, a cohesive thermoplastic. The Parafilm^® Hanging Drop Chip (PHDC) was assembled by two-layer of Parafilm^® sheet with different sizes of holes. The hole on the upper layer of the Parafilm^® is smaller than the hole on the bottom layer. The impact of hole size and sample volume on hanging drop formation and 3D spheroid formations in the hanging drop were investigated. The results showed that 20 <i>µ</i>L solution on PHDC with a 3 mm hole could form stabile drop and facilitate spheroid formation. The initial cell number determinates the size of the formed spheroids. Exchanging liquid from the upper hole of the 2021-01-01T00:00:00Z2021 Apr2025-04-04T18:41:52.836Z2025-04-04T18:41:52.836ZS-EPMC80100943386849910.1007/s12195-020-00660-x