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Biomicrofluidics / Volume 5 / Issue 4 / REGULAR ARTICLES

Biomicrofluidics 5, 044117 (2011); http://dx.doi.org/10.1063/1.3669371 (13 pages)

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

Huaying Chen1,2, Jingjing Li1, Han Zhang1, Musen Li2, Gary Rosengarten3, and Robert E. Nordon1

1Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia
2School of Materials Science and Engineering, Shandong University, Ji’nan, China
3School of Mechanical and Manufacturing Engineering, University of New South Wales, Sydney, Australia

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(Received 26 April 2011; accepted 19 November 2011; published online 15 December 2011)

Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 μl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis.

© 2011 American Institute of Physics

Article Outline

  1. INTRODUCTION
  2. MATERIALS AND METHODS
    1. Static cell culture experiments
    2. Media degradation study
    3. Biocompatibility of tubing
    4. Microscope incubator
    5. Microwell perfusion system
    6. Microwell perfusion culture
    7. Image and statistical analysis
  3. RESULTS AND DISCUSSION
    1. Biocompatibility of the perfusion system
    2. Microwell array perfusion culture
    3. Clonal throughput and analysis of cell cycle time heterogeneity
  4. CONCLUSION

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KEYWORDS and PACS

Keywords

biological techniques, cellular biophysics, probability, stochastic processes

PACS

ARTICLE DATA

Digital Object Identifier
http://dx.doi.org/10.1063/1.3669371

PUBLICATION DATA

ISSN

1932-1058 (online)

Publisher

$abstract.society.value is a Member of CrossRef American Institute of Physics

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