Various methods used for quantifying senescence of MSCs.
Top: our label-free autofluorescence method. Bottom left: fluorescence flow cytometry detection of β-galactosidase activity. Bottom right: flow cytometry forward scatter measurements for MSC size determination.
Science
Human mesenchymal stromal cells (MSCs) have demonstrated consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation of preclinical results to actual healthcare outcomes has been challenging, partly due to the inability to assess quality of the cells during the biomanufacturing process. One such quality parameter is cellular senescence. It indicates the aging process inside the cell whether due to the donor who provided the cells, or due to inefficient or inconsistent cell culture processes. Senescent cells are typically not as efficacious.
Current methods for characterising MSC senescence are often destructive and rely on addition of biochemical labels whose own quality require validation. In contrast, our method is label-free and utilises MSCs’ own autofluorescence (natural emission of light by cellular organelles) without killing the cells. By minimising manipulation of the cells when measuring them, our method has the potential to provide real-time assessment and thereby exert finer quality control of cell therapy to improve their performance.
Societal Impact
Cell therapy holds tremendous therapeutic promise because they can repair tissues and perform complex functions that traditional drugs cannot. However, if the manufacturing operations are inconsistent and inefficient, cell therapy will continue to be very expensive without a guarantee for their quality. MSC therapy is one such example, despite demonstrating strong performance in a wide range of animal models. The quality of MSC therapy is hugely dependent on the age of the donor who donated the MSCs. It is also dependent on the duration of cell culture, because the cells continue to age outside the body.
We have developed a technology to monitor this age-related quality of MSCs. Our innovation keeps the cells alive during monitoring to improve accuracy of measurement. It also avoids the use of additional reagents, which reduces the supply burden and avoids the need for quality control of the additional reagents. We hope our technology will enable real-time monitoring and simplify operations to allow for more automation and therefore cost efficiencies.
Technical Summary
In this study, we compared and evaluated various methods in quantifying MSC senescence. Current methods include flow cytometry based on cell size and fluorescently labelled β-galactosidase activity. We developed a method that measures autofluorescence from endogenous fluorophores including lipopigments.
We benchmarked our method and conventional methods against the current standard in β-galactosidase analysis. Both methods demonstrated statistically significant differences between early-stage and senescent MSCs. These results suggest that our label-free autofluorescence method could be effective and easily adopted by the cell manufacturing and therapy industry for real-time monitoring.
Reference
Zhai, W., Tan, J., Russell, T., Chen, S., McGonagle, D., Naing, M.W., Yong, D., Jones, E. Multi pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label free methods. Scientific Reports. https://doi.org/10.1038/s41598-020-79831-9. 2021. 11:1054