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How to achieve monoclonality?

Maria Milla

When isolating single cells for cell line development or other downstream analysis, monoclonality is a central issue. Upon placing a cell into a new container, we must make sure that the targeted cell is the only one that is being deposited therefore when cell growth is detected, we can be sure that the population is monoclonal.

Using our systems for sorting, only the target cell is held to the cantilever by applying a smooth negative pressure and, therefore, monoclonality is given to a certain degree. During the picking phase of sorting, the FluidFM micropipette is immersed in the well/dish on which the cell to be picked is found, and which it typically contains a suspension of cells floating in the liquid (A). When the probe is retracted for transfer, the droplet around the cantilever can contain some of these floating cells (B). When the probe is then immersed in the target container, these additional cells can end up growing in the same surface as the initially selected cell.  

To avoid this effect and make sure that each well/dish contains only the desired cell, it is recommended to do a so called “monoclonality wash”. With this, the probe with the picked-up cell will be immersed several times in different containers filled with fresh cell media before being transferred to the final target dish/well (C-F). 

This way, any cells retained in the droplet will simply end up in one of the washing containers, ensuring a single cell in the final destination and, thus, a monoclonal expanded culture (F). The recommended number of washings is 3. In case the target cell gets lost during the process, the holding pressure should be slightly more negative.

According to our experiments with Jurkat cells, this washing protocol increases monoclonality on the final wells at least 70%.