Generating suspension-adapted human mesenchymal stromal cells (S-hMSCs) for the scalable manufacture of extracellular vesicles

Abstract

Human mesenchymal stromal cells (hMSCs) are a naturally adherent cell type and one of the most studied cellular agents used in cell therapy over the last 20 years. Their mechanism of action has been primarily associated with paracrine signalling, which has contributed to an increase in the number of studies focused on hMSC-related extracellular vesicles (EVs). In this study, we demonstrate for the first time that human telomerase reverse transcriptase (hTERT) immortalised hMSCs can be adapted to suspension culture, eliminating the need for microcarriers or other matrixes to support cell growth. This novel cell line, named suspension hMSCs (S-hMSCs), has a doubling time of approximately 55 hours, with a growth rate of 0.423 d?1. Regarding its immunophenotype characteristics, S-hMSCs retained close to 90% of CD73 and CD105 expression levels, with the CD90 receptor being downregulated during the adherent to suspension adaptation process. An RNA sequencing analysis showed an upregulation of the transcripts coding for CD44, CD46 and CD47 compared to the expression levels in AT-hMSCs and hTERT-hMSCs. The cell line herein established was able to generate EVs using a chemically defined medium formulation with these nanoparticles averaging 150 nm in size and displaying the markers CD63, CD81, and TSG101, while not expressing the negative marker calnexin. This body of evidence, combined with the visual confirmation of EV presence using transmission electron microscopy, demonstrates the EV-producing capabilities of the novel S-hMSCs. This cell line provides a platform for process development, drug discovery and translational studies in the EV field.