Microwell perfusion array for high-throughput, long-term imaging of clonal growth

Area of Science:

• Cell Biology
• Biotechnology
• Microfluidics

Background:

• Single-cell fate mapping via time-lapse microscopy is crucial for understanding cell differentiation.
• Stochastic branching processes in cell division require extensive clonal division histories for accurate measurement.
• Current methods face limitations in imaging throughput for large-scale clonal analysis.

Purpose of the Study:

• To investigate condensing cell imaging information into a small region to maximize live cell imaging throughput.
• To develop a high-throughput system for clonal analysis of non-adherent cells using continuous live cell tracking.
• To assess the biocompatibility and optimize protocols for long-term cell culture in a microwell perfusion array.

Main Methods:

• Utilized a microwell perfusion array (16 μl volume, 600 microwells) for high-throughput clonal analysis.
• Examined biocompatibility of various cell culture components including buffer systems, tubing, substrates, and media.
• Implemented an intermittent perfusion protocol for long-term time-lapse imaging of KG1a cells.

Main Results:

• Successfully cultured and tracked 1500 clones simultaneously for 6 days with 3-minute scanning intervals at 100× magnification.
• Demonstrated the feasibility of high-throughput clonal analysis for non-adherent cells.
• Identified advantages including continuous long-term tracking, increased imaging throughput, and enhanced microenvironment control.

Conclusions:

• Perfusion microwell arrays offer significant advantages for continuous, high-throughput live cell tracking.
• These devices facilitate detailed analysis of cell lineage development and probabilistic life events like mitosis.
• The developed system advances the capacity for studying complex cellular processes at the single-cell level.