Evaluation of cell-culture condition in CV8000’s internal stage incubator

Introduction

Live-cell imaging is an extremely efficient technique that allows the collection of cellular timelapse information in a single image acquisition. The technique requires the preparation of devices that maintain an optimum environment (temperature, CO₂ concentration, humidity control) for preserving cells in their normal condition. This technical note shows the evaluation results of the internal stage incubator of the CellVoyager CV8000 high-content analysis system. The evaluation consisted of the investigation of cell proliferation variation within well plates, and comparison against a conventional CO₂ incubator.

Experimental procedure

Fig 1(a). Timelapse movie: A1 well (72hrs)	Fig 1(b). Timelapse movie: B2 well (72hrs)
Fig 1(c). Timelapse movie: C3 well (72hrs)	Fig 1(d). Timelapse movie: D6 well (72hrs)

1. HeLa cells stably expressing Azami-Green (green fluorescent protein) were seeded (500 cells/well) in 250uL of medium in 96-well plates (Greiner #655896), and incubated for 24hrs. (Two plates were prepared.)
2. One plate was incubated for 72 hours in a conventional CO₂ incubator. Imaging was performed at 0hrs and 72hrs using the CV8000.
   The other plate underwent live cell imaging using the CV8000. Whole well imaging was performed at 3hr intervals for 72hrs with a 4x objective lens.
3. Analysis was performed using the high-content analysis software CellPathfinder.
   The total area covered with cells in each well was calculated to evaluate the proliferation of cells. The variation of proliferation rate across wells was investigated. A comparison with the conventional CO₂ incubator was also made.

Fig 1(e). Line graphs of cell proliferation for each well

Vertical axis: total area (total area of cells in that well) Horizontal axis: time (0-72hrs)
Cell proliferation deteriorated in the plate’s corner wells; however, it continued at a good rate in the other wells.
Time-lapse movie under the similar condition: comparison of each wells:  Play

Fig 2. Variation of cell proliferation rates within the well plate (n=3)

(A) Total area ratio for each well (total area at 72hrs / total area at 0hrs)
96 well average: 7.7
96 well coefficient of variation (standard deviation/average): 8.5×10^-2
36 well coefficient of variation (perimeter wells): 1.1×10^-1
60 well coefficient of variation (except perimeter wells): 5.1×10^-2

(B) Change in total area ratio over time (error bars show standard deviation)
After 24hrs, there was no variation in the total area ratio across groups. Even after 72hrs, perimeter wells (except corner wells) showed similar results to the center wells.

Fig 3. Proliferation ratio comparison with a conventional CO₂ incubator (n=3)

Comparison of the cell proliferation ratio 72hrs after seeding, for each well, between a regular CO₂ incubator and the CV8000
CV8000 total area ratio / CO₂ incubator total area ratio×100
(The nearer the value to 100, the more similar cell proliferation was between the CV8000 and the CO₂ incubator.)
96 well average: 90
36 perimeter well average: 81
60 well average (except perimeter wells): 96

Discussion

The results showed there was little variation in cell proliferation across wells (except perimeter wells) in the CV8000’s internal incubator, and its performance was comparable to that of a conventional CO₂ incubator. It is generally understood that cell proliferation rates deteriorate on the perimeter of well plates due to culture medium evaporation. In this evaluation, however, a large variation in proliferation rates between the perimeter wells (except corner wells) and center wells was not observed. These results indicate that the CV8000’s internal incubator possesses sufficient performance for live-cell imaging.