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

Introduction

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.

Methods

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 CellTracker^TM 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.

Results

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

 
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.

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

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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