Towards the development of robust and meaningful potency assays for MSCs

FS Abukar1, IB Wall1

1 University College London (UCL), Department of Biochemical Engineering, Torrington Place, London, WC1E 7JE

In the UK alone, there are 146,000 people that have a Myocardial Infarction (MI) every year. Multiple clinical trials have explored MSCs for therapeutic application for ischemic cardiac injury. Currently there are no potency assays in the clinical setting that give a reliable measurement of MSCs mode of action, other than simple surface marker expression, which does not predict function. For this reason, there is a need to develop in vitro assays that will be predictive of functional activity of the MSC product. To be predictive, the assays need to be performed using parameters that more closely mimic the environment in the body, because measuring cell attributes in ambient conditions does not inform on how the cells will behave in vivo. Vascular assays can be used as platforms to study the hypothesis that MSCs functionally support blood vessels as a key mode of action in ischemic injured tissues. Vascular assays have traditionally lacked robustness and reproducibility, particularly since biological materials are highly variable. Our work focuses on developing robust assays with increased reproducibility. To achieve this, one aim of the project is to use Design of Experiments (DOE) to systematically optimize key parameters that affect the tubule-formation capacity of human endothelial cells, with an aim of making the assays more standardized and lowering assay costs in the process. In the current work, we found that tubule network formation could be controlled using lower quantities of an optimized matrix hence increasing the number of assays achievable from a single batch of substrate. Previous literature has indicated that efficiency of tubule forming capacity decreases drastically after endothelial cells reach passage 10. Therefore further work was conducted to investigate the impact of passage number on tubule formation capacity on the optimized matrix. Automated cell
counting methods were used and were validated using manual counts. Here we show that higher passages than the ones presented in the literature, are still able to form networks when using optimized matrices, therefore increasing the possibility for higher throughput release assay development. Ultimately the optimized methods will enable MSCs to become a real therapy and replace more costly and less reliable tests currently in the market.

Fig. 1: 3D response graphs comparing branch point counts for two different types of matrix, passage number and matrix volume.

REFERENCES: 1 D. Donovan, N.J. Brown, E.T. Bishop, C.E. Lewis (2001) Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo in Angiogenesis, 4: 113-121.1.