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High throughput generating stable spheroids with tip-refill wafer.

Xiaoyan Yang1, Rong Pan1, Ke Ning1

  • 1Key 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.

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Summary
This summary is machine-generated.

A novel tip-refill wafer (TrW) enables efficient 3D spheroid formation using hanging drops. This cost-effective method is robust against mechanical stress, facilitating spheroid generation for biomedical research.

Keywords:
3D cell cultureco-culturehanging droptip-refill wafertumor spheroid

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Area of Science:

  • Biomedical Engineering
  • Cell Biology
  • Biotechnology

Background:

  • Three-dimensional (3D) cell cultures offer a more accurate in vivo cellular environment compared to traditional 2D cultures.
  • Hanging drop technology is a key method for generating uniform 3D cell spheroids cost-effectively.
  • Developing accessible and efficient platforms for 3D cell culture is crucial for advancing biomedical research.

Purpose of the Study:

  • To evaluate the utility of a disposable tip-refill wafer (TrW) as a platform for generating 3D cell spheroids via hanging drops.
  • To assess the mechanical stability and spheroid formation efficiency of the TrW-based hanging drop system.
  • To explore the potential of the TrW platform for both mono-culture and multicellular spheroid formation.

Main Methods:

  • Utilized a tip-refill wafer (TrW) for the facile generation of 60-µL hanging drops.
  • Assessed hanging drop stability under mechanical stress, including orbital shaking at 210 rpm.
  • Employed inertial focusing via horizontal orbiting (60 rpm for 15 min) to promote spheroid formation.
  • Validated spheroid quality using live/dead cell staining, qPCR, and cytoskeleton staining.

Main Results:

  • Hanging drops on the TrW demonstrated exceptional mechanical stability, resisting detachment during vigorous shaking.
  • Horizontal orbiting effectively induced cell aggregation and the formation of 3D spheroids.
  • High spheroid harvest rates (96.1% ± 3.5%) were achieved across multiple TrWs.
  • The platform successfully facilitated the formation of mono-culture and multicellular spheroids, including spheroid pairing and fusion.

Conclusions:

  • The tip-refill wafer (TrW) provides a simple, robust, and cost-effective platform for 3D cell spheroid formation using hanging drops.
  • The TrW's mechanical stability and compatibility with inertial focusing streamline spheroid generation for research applications.
  • This disposable, ready-to-use system enhances the accessibility of 3D cell culture techniques in standard biological laboratories.