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Quantifying Confluency of iPS Cell Colonies after Derivation


Induced pluripotent stem (iPS) cells continue to be at the frontier of regenerative medicine and drug discovery research due to their potential applications. The workflow of sustaining, growing, and preparing iPS cells for research purposes involves many processes. Improving these procedures to increase efficiency and mitigate cell death is essential to the advancement of all stem cell-related research. Although relatively new, stem cell research relies heavily on operator experience and skill, meaning human error plays a large role in a colony’s efficacy. Denoting the confluency of iPS cells is a valuable tool for optimizing colonies, differentiation and obtaining reproducible results.

Example of iPS Cell Research

Example of iPS Cell Research

Solution

Figure 1. Image of iPS cell colonies (left) and the result of confluency measurements (right)
Figure 1. Image of iPS cell colonies (left) and the result of confluency measurements (right)


Obtaining multiple images with the Olympus CKX53 tissue culture microscope during the iPS cells culture maintenance process enables the CKX-CCSW software to provide accurate confluency data. Three, six, and ten images were taken  at different points in a 6-well plate across six days. These images were imported into the CKX-CCSW software, where cells and background were first denoted (figure 1).

From there, the cells and background were shown across the entire image in blue and green, and the confluency was calculated for all images. Average confluency and standard deviations were calculated on days one, three, and six when three, six, and ten images were used. Despite the significant change  in the number of images used, figure 2 shows that the results from even a small number of images can produce accurate growth curves.

Figure 2. Record of the changes in colonies, confluency of iPS cells using the CKX-CCSW software
Figure 2. Record of the changes in colonies, confluency of iPS cells using the CKX-CCSW software
 

Figure 3. Growth curve of colonies confluence of iPS cells (colonies’ 3 images) Device and Software used: OLYMPUS CKX53 microscope, Camera DP22, CKX-CCSW software
Objective lens: PLN2X (NA 0.06, WD 5.8 mm) Observation method: Brightfield 
Container: 6-well plate 
Cell: Feeder-free iPS cells
Courtesy iPS PORTAL Inc.

Figure 3. Growth curve of colonies confluence of iPS cells (colonies’ 3 images)

Conclusion

When used in combination with the CKX53 microscope, the Olympus CKX-CCSW software enables you to measure and record colonies, confluence efficiently without scraping colonies of iPS cells from a culture vessel. Using the maintenance culture process of iPS cells as an example, this application note demonstrates that you can obtain data without variations between operators in many maintenance culture processes after deriving iPS cells using a 100 mm dish and a 24-well plate*. It also supports a highly reproducible cell culture process by recording and storing quantitative data.

Products used for this application

Confluency Checker

CKX-CCSW

  • Measurement for Non-stain Cultured Cells and Confluence
  • Less Risk for Contamination
  • Quick and Easy Operation
  • Improve Cell Culture Process Quality with Quantitative Analysis
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CKX53

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