Multi-omics uncovers potential of microcarrier culture for improving therapeutic quality of mesenchymal stem cells

From Left: Dr Alan Lam, Dr Alison Lee, Dr Zhou Lihan, Dr Andy Tan, Dr Steve Oh

Science

Mesenchymal stem cells (MSCs) are stem cells that can become fat, bone, and cartilage tissues. Researchers and companies are developing MSCs as a therapy to regenerate damaged tissues in the body. Critically, MSCs need to be attached to a surface to grow. At small scales, MSCs can be grown in flasks, which provide a flat 2D surface. At large scales, a lot more surface area must be provided in the same volume to save costs. This is achieved using microcarriers—small beads with spherical 3D surfaces on which MSCs can attach and grow. To the MSCs, this necessary transition drastically changes their working environment. In this study, we systematically studied the expression of genes, miRNAs, and cytokines by a human MSC line grown on flat 2D surfaces in flasks versus spherical 3D surfaces on microcarriers, in collaboration with MiRXES, a Singapore-headquartered biotechnology company. Our scientists confirmed that the change from 2D to 3D culture is beneficial to the MSCs, and 3D culture should be encouraged to maintain or even improve the therapeutic characteristics of MSCs.

Societal Impact

MSCs represent a type of cell therapy. Unlike traditional biopharmaceuticals, cell therapy is a living product and therefore highly sensitive to its processing conditions. During scale-up, MSC therapy necessarily transitions from 2D to 3D culture. While this change is beneficial by enabling economy of scale, it casts doubt on the quality of the MSC product. By uncovering that scale-up improves the beneficial features of MSCs on a molecular basis, our study shows that 3D culture should be encouraged to maintain or even improve the therapeutic characteristics of MSCs.

Technical Summary

In this study, we systematically studied the transcriptome, miRNome and cytokine expression of human Wharton’s Jelly-derived MSCs grown in monolayer (2D) versus microcarrier-based (3D) cultures. Cells at different growth phases were harvested for microarray and intracellular miRNA profiling by MiRXES’s mSMRT-qPCR platform. At the same time, conditioned media were collected for cytokine measurement by multiplex immunoassay and extracellular miRNA profiling by mSMRT-qPCR platform. Gene set enrichment analysis, miRNA-gene network analysis, and pathway enrichment analysis were employed to investigate differences in the phenotypic and genotypic characteristics of hMSCs between both cultures. Our results identified two distinct patterns of transcriptomes, miRNomes, and secretomes between the cultures. Regulatory pathways upregulated in microcarrier culture compared to monolayer culture include osteogenic differentiation capacity, angiogenesis, immunomodulation, cell migration, wound healing and, interestingly, cardiac repair. Moreover, cell harvest at the appropriate growth phase is also critical to maximize therapeutic qualities of the MSCs. In conclusion, this study demonstrated that microcarrier-based bioprocessing may improve post-expansion cellular properties and product quality for MSC therapies.

Figure 1. Integrative analyses of genes, miRNAs and cytokines of hMSCs in microcarrier vs. monolayer processes

References

A.T. Lam, A.P. Lee, P. Jayaraman, K.Y. Tan, D. Raghothaman, H.L. Lim, H. Cheng, L. Zhou, A.H. Tan, S. Reuveny, and S. Oh, Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures. Stem cell research. 53(2021): p. 102272.