3D time lapse imaging and quantitative analysis of the active migration of human vascular endothelial cells into a multilayered cell sheet


Time lapse confocal imaging has been an essential method to investigate the 3D dynamic behaviors of cells in tissue cultures. For long-term live cell imaging, it is critical to reduce phototoxic damage to the cells caused by repeated laser scanning. Yokogawa CSU (confocal scanner unit) is a confocal unit using a microlens-enhanced dual Nipkow disk confocal optical system, which has been shown to be less harmful to living cells compared to conventional single beam scanning devices. The CQ1 is an all-in-one confocal quantitative imaging cytometer based on the CSU. Here we report the 3D time lapse live cell imaging in a multilayered cell sheet using CQ1.


  1. Five-layered myoblast cell sheets were constructed from human skeletal muscle myoblasts (HSMM) and human skeletal muscle fibroblasts (HSMF) .
  2. HSMMs and HSMFs were labeled with CellTrackerTM Orange
  3. Human umbilical vein endothelial cells (HUVEC) expressing GFP (GFP-HUVEC) were overlaid by the cell sheet and co-cultured.
  4. Time lapse imaging (67 hours, 30 min interval, 40x objective lens , 49 fields) was performed by CQ1 equipped with an internal incubation chamber to regulate culture environment.



1.Dynamic migration and network formation of GFP-HUVECs captured by 3D time lapse imaging

Time lapse images of the cell sheet. 
Time lapse movie Play

Fig. 1-1. Time lapse images of the cell sheet.
Images were reconstructed of the field indicated by the yellow frame in the large field stitched image in Method fig.2.

Migration of the GFP-HUVECs into the cell sheet.

Fig. 1-2. Migration of the GFP-HUVECs into the cell sheet.
Single slice images showing the migration of HUVECs into upper layers. (Rows, from top to bottom) Single slice images of layers 3, 2, 1 and corresponding Y-Z plane images of the cell sheet. (Columns, from left to right) Images acquired at 0, 17, 34 and 51 hr incubation. The image filed is the same as fig. 1-1.

2.Quantification of the migration of GFP-HUVECs into the five-layered cell sheet

Temporal change of the distribution GFP-HUVECs in the cell sheet.

Fig. 2. Temporal change of the distribution GFP-HUVECs in the cell sheet.
GFP fluorescence intensity in each layer was indicated as the ratio against the total GFP intensity in the cell sheet.

Summary & Discussions

  • GFP-HUVECs dynamically migrated upward into the five-layered cell sheet constructed from HSMMs and HSMFs.
  • The GFP-HUVECs formed a reticulate network in the horizontal plane in the middle layers.
  • Long-term 3D time lapse imaging by CQ1 revealed a dynamic process of the active migration and the formation of the cellular network in the multilayered cell sheet.
  • CQ1 would be a powerful research tool in tissue engineering as well as regenerative medicine and drug screening.

Data provided by Dr. Nagamori, Osaka Institute of Technology
Reference: Nagamori E. et al., Biomaterials, 34, 662-668. (2013)

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