High content analysis reveals distinct morphological profiles of human MSCs under different culture conditions

Background & Aim The number of clinical trials employing Mesenchymal Stem Cells (MSC) for the treatment of human diseases has grown substantially in the last decade. In this context new characterisation tools for cell and media manufacturing process are required. With this in mind, we sought to develop an automated high-content imaging assay to characterize the morphological profile of MSC in response to different media formulations. Methods, Results & Conclusion High content analysis of high resolution images indicated that MSC growing in absence of serum displayed distinct morphological features compared to SC MSC. Specifically, in the absence of serum cells showed a smaller and more circular morphology, with less protrusions compared to SC MSC, which displayed an elongated shape, with numerous cellular protrusions. These phenotypic differences were expressed for instance by a significant higher circularity index and lower form factor by SF MSC, which measure respectively the overall roundness and spreading of a cell. Both automated workflows were able to successfully cluster the two experimental groups based on several morphological descriptors, allowing the generation of a morphological profile. Of note, the assay produced similar morphological profiles using MSC from two donors. The development of serum-free (SF) and chemically defined media (CDM) for the clinical production of human MSCs is a high priority within the cell therapy community. Small molecules as media supplements are an attractive tool as they are generally easily available, chemically defined and tunable, and chemical media design presents an attractive opportunity for improving MSC manufacturing. In this context, we are developing a novel analytical technology aimed at designing chemically defined media (CDM) tailored to the MSC production process. For this purpose, MSC Morphology Profile will be used as one of the read out assays to screen small molecules, with the goal of identifying molecules that sustain proliferation of MSC while retaining their potency. Taken together, we report the development of a high-content assay to readily capture and analyse the morphological profile of MSC grown in different culture conditions and quantitatively showed that serum influences MSC morphology. Importantly, the assay may represents a straightforward strategy to benchmark MSC preparation and media formulations during production processes for therapeutic application.