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Efficiently Analyze the Cell Growth Characteristics of an Autologous Chondrocyte Culture


– An application of the confluency checker software to cell processing –

Efficiently Analyze the Cell Growth Characteristics of an Autologous Chondrocyte Culture

Background 

Two factors that are important for autologous chondrocyte implantation and the development of new regenerative medicines are a stable cell culture process and efficient analysis of cell growth characteristics. Both factors have challenges associated with them. Achieving a stable culture process requires accurate, repeatable cell confluency information to  predict the best time for cell passage. The analysis of cell growth characteristics is challenging because users want to acquire accurate data without having to grow large quantities of cells. Olympus introduced the CKX-CCSW software to improve the cell culture process and help solve these problems.

CKX-CCSW Confluency Checker Software

Combined with a microscope and digital camera, the software enables the confluency of live cells to be measured. A fast, simple cell counting procedure along with highly accurate and reproducible data enable consistent, predictable cell culture under constant conditions. Furthermore, the number and density of the culture can be measured without the need to disrupt the culture, minimizing the probability of contamination. Important cell growth data, such as the doubling time (DT) and population doubling level (PDL), are obtained  efficiently and nondestructively without disturbing the cells.

The CKX41microscope, DP27 digital camera, and CKX-CCSW software.
The CKX41microscope, DP27 digital camera, and CKX-CCSW software.

DT and PDL are calculated from the growth curve

Effect of CKX-CCSW Confluency Checker Software

1.Overcoming the challenge of stabilizing the culture process of autologous chondrocytes
The timing of cell passage normally depends on visual assessment, and this can lead to human error and variation of the passage timing. The CKX-CCSW software provides accurate confluency information of cells growing in culture vessels, so users can pass cells at the suitable time.

2.Overcoming the challenge of efficiently acquiring cell growth characteristic analysis data
The CKX-CCSW confluency checker software provides accurate, repeatable cell growth data, so users can time cell passage and use fewer stocks in the culture process.

Appropriate Number of Cells for Passage

Fewer stocks needed when using the CCSW

 Verification of Accuracy

To evaluate the performance of the CKX-CCSW software, the number of cells counted by the software were compared to the number of cells calculated from a fluorescent stained image. Focusing the microscope is an important factor for acquiring an accurate growth curve, and the software’s focusing indicator produced accurate results. The software’s  accuracy was estimated to be 84–111%, confirming that the software could create a reliable cell growth curve.

  • Phase contrast image
    Phase contrast image
  • Fluorescent stained nuclei image
    Fluorescent stained nuclei image

Accuracy Compared to Fluorescent Imaging (n=3)

By using CKX-CCSW it is possible to draw the growth curve equivalent to counting accuracy obtained by fluorescence observation in a low density to high density cell culture state despite non-stained cells. 

Conclusion

The combination of the CKX41 microscope, DP27 digital microscope camera, and CKX-CCSW confluency checker software enable users to accurately estimate the proper timing for cell passage while minimizing human error. The software makes it easy to acquire accurate, repeatable cell growth data without changing the workflow in the cell culture room.

This research was partly granted by Japan Agency for Medical Research and Development (AMED) and Ministry of Economy, Trade and Industry in their program entitled “Development of Basic Evaluation Technology for Industrialization of Regenerative Medicine”. Sample preparation and image data are courtesy of OLYMPUS-RMS CORP.

Products used for this application

融合检测器

CKX-CCSW

  • 无染色培养细胞及融合的测量
  • 降低污染风险
  • 操作快捷简便
  • 利用定量分析提高细胞培养过程的质量
紧凑型符合人体工学设计的细胞培养解决方案

CKX53

  • 预对中相衬
  • 反转对比 (IVC)技术获得清晰的三维观察效果
  • 带3孔位滑块的荧光
显微数码专用相机

DP27

  • 500万像素彩色CCD
  • 存档的文件具有高的像素分辨率,且同时具有高质量色彩和高保真度
  • 用于明场观察最佳的对比度和彩色模式

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