Cell and Gene Therapy Catapult Cell Confluency Tool

Simply upload your cell culture image and the cell confluency tool will automatically segment the image into areas covered by cells from the background to determine cell culture confluency. The confluency tool provides fast and repeatable measurements enabling the operator to make rapid key in-process decisions about the cell culture.

Repeatable visual estimations can be hard to achieve even by an experienced scientist and variability in estimates between operators will affect the process and output consistency. Automated cell confluency measurement removes inter- and intra-operator variability and provides accurate data for successful cell culture-dependent processes. Furthermore, automating the assessment of cell confluency allows the operator to make rapid key in-process decisions about the cell culture.

• Automated, fast and repeatable measurements
• Cross imaging platform compatible
• Ability to review and download processed images
• Free and easy to use

A texture segmentation method determines which pixels contain cells and which are background. Confluency is the number of pixels containing cells divided by the total number of pixels in the image multiplied by 100. This measurement and an image with the cell boundaries marked in red are available for online review and download by the operator. Cell boundaries may be incorrectly assigned at high cell confluency. Always check the processed images to ensure that the cell boundaries marked in red align with the edges of the cells. If the default method has failed, try the high confluency method.

While the default method should work for most images, the high confluency method has been specifically developed for images of cell cultures at high confluency. We recommend using the high confluency method if the default method has not correctly segmented the cells from the background.

This tool has been optimised for use with pluripotent stem cells (hESC’s and iPSC’s). It should work with other cell types, but please let us know how it goes as it will help us to develop the tool.

This tool will work with *.png and *.tif/.tiff images.

The tool performs best with high-quality images:

• Phase-contrast
• Even illumination across the image
• Image in focus
• Adequate exposure

Issues which may affect the image quality and therefore the confluency assessment, include:

• Debris, bubbles, scratches, etc
• Uneven illumination across the image
• Overexposure
• Low resolution
• Condensation on the cell culture vessel

While we have done our best to develop a tool that is robust to these issues, the tool will be less accurate with poor quality images.

The tool assesses cell culture images magnified in the range x4 to x10. The tool can be used outside of this range, but the confluency estimate may be less accurate if the area contained within the image only represents a small area of the cell culture surface (high magnification). In such a scenario, we recommend taking multiple images for assessment to increase the reliability of the measurement. Any feedback on your experience with this tool, including issues with particular images, cell types, or imaging systems, is highly appreciated. This information will be used to further develop the tool.

The maximum number of images that can be uploaded in a batch is 30 and the maximum file size per image is 20mb. If you need to upload more images than this per batch, please get in touch, or an alternative is to split images into smaller batches.

You can use the tool as many times as you like, always for free.

If the image contains a white block scale bar it will be removed from the analysis and will not contribute to cell confluency.

The tool should work with any phase-contrast image. It has been tested on several different imaging platforms and given accurate results. Please let us know which imaging system you use to help us further develop the tool.

The number of images and their size, as well as the speed of the network connection, will affect the time required for the analysis. If you encounter problems while uploading your images, please verify your network connection and reload the page.

You should always review the cell confluency results before downloading the output file. Once the image processing is done, go through your images and verify that the tool has appropriately segmented cells from the background during the analysis. If this is not the case, click on ‘’switch method’’ to select the high confluency method. If both methods fail to give satisfactory results, you can input your own confluency estimations by clicking on “add override”. Manually input data will be highlighted in the output file, allowing you to keep track of any changes made to the analysis.

The images submitted are stored anonymously in the cloud to aid in the further development of the tool. These images are only accessible by the Cell and Gene Therapy Catapult and will only be used for this purpose.

The Cell and Gene Therapy Catapult is a non-profit organisation set up to facilitate the growth of the cell & gene therapy sector within the UK. We are mandated to identify and solve common industry issues to accelerate the commercial and clinical delivery of cell & gene therapies. One of the biggest barriers inhibiting the development and manufacture of pluripotent stem cell (PSC)-derived therapies is process robustness and control.

We identified that objective assessment of confluency was a key bottleneck for technology transfer and process success in the PSC field. Cell confluency is used to identify the point at which key stages of these processes; cell passage and differentiation, should be initiated. In providing a free tool to automatically assess confluency, we hope that therapy developers can gather this important data at an early stage in their pipeline, increasing process consistency and facilitating industrialisation and manufacturing.

Images uploaded on this tool are kept for image processing algorithm development purposes only. Data are anonymous and will never be shared with third parties.

For more information, please contact the Cell and Gene Therapy Catapult at the following email address: cellconfluency@ct.catapult.org.uk

An internal server error could occur if you have a slow internet connection. If this occurs please upload images in smaller batches or contact us at cellconfluency@ct.catapult.org.uk