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Kino-oka, Masahiro

Yamamoto, Riku

YUNHAO, LIAO

English

Engineering Conferences International

INVESTIGATE THE STABILITY OF SEEDING PROCESS OF MESENCHYMAL STEM CELL ON MICROCARRIERS BY CONSIDERING HETEROGENEITY   LIAO YUNHAO, Department of Biotechnology, Graduate School of Engineering, Osaka University liao_yunhao@bio.eng.osaka-u.ac.jp Riku Yamamoto, Department of Biotechnology, Graduate School of Engineering, Osaka University  Masahiro Kino-oka, Department of Biotechnology, Graduate School of Engineering, Osaka University and Research Base for Cell Manufacturability, Graduate School of Engineering, Osaka University  Key Words: mesenchymal stem cell, microcarrier, cell attachment, seeding process, stability.  Regenerative medicine based on mesenchymal stem cells (MSCs) has become one of the important directions to be explored for future cell therapy 1. Such cells have a multi-lineage differentiation ability, can modulate immune responses, and can also promote tissue regeneration through the secretome 1. At the present time, one of the major issues facing the use of MSCs in the clinic is that the number of cells obtained from the tissues is much less than the number of cells needed for treatment 2, and therefore, an in vitro expansion process is necessary. Microcarriers (MCs), which are a class of small beads with diameters ranging from 100 to 200 μm, could be used as a substrate for cell adhesion. MC-based suspension cultures have been recognized as a potential tool for cost-effective large-scale cell expansion process due to providing a larger surface area per unit volume compared to planar cultures 2.  Cell attachment on microcarriers is initial and necessary during the expansion process. Excessive cell attachment will result in a large amount of wasted inoculum as well as a low number of cells obtained by further expansion. In addition to the overall amount of adherence of cells, the effect of the seeding process is reflected in variations of the number of cells adhering on individual MCs which creates spatial heterogeneity in cell density in the same bioreactor. Such cell densities could act as signals from the local microenvironment and have an impact on the differentiation potential of MSC through mechanotransduction 3. Consequently, this would lead to heterogeneity in the cell population, which would negatively affect the stability of the expansion process. In order to understand this variation of cell numbers on individual MCs, 3-dimensional images of MCs and MSCs labelled by fluorescence with nuclei were analyzed for direct counting of the number of cells on a single MC. This experiment used MC concentration, CMC (cm2/mL) as a process parameter, controlled cell seeding density of 6000 cells/cm2 and a static strategy by no mixing after seeding, with three replications per group.   As can be seen in the Figure 1, comparing to the high CMC condition, the mean value is lower which demonstrate fewer overall numbers of attached cells in this condition. The distribution of cell per MCs fluctuated more in the low CMC condition than in the high CMC condition while it showed significant difference in same condition. However, the high CMC condition showed a higher heterogeneity indicated by the larger box size. Our research provided a method to analysis the stability of seeding process, and the result suggest that the MC concentration is a factor to cause the heterogeneity of seeding. Therefore, we suggest that the stability of seeding process should be considered while developing the MC expansion process.   [1] Andrzejewska, et al. (2019). Concise review: mesenchymal stem cells: from roots to boost. Stem cells, 37(7), 855-864. [2] Barekzai, J et al. (2020). Bioprocess development for human mesenchymal stem cell therapy products. New Adv. Ferment. Processes, 1-25. [3] Raman, N. et al. (2022). Mechanotransduction in mesenchymal stem cells (MSCs) differentiation: a review. International Journal of Molecular Sciences, 23(9), 4580. Figure 1. bee swarm and box plot of cell per MCs in lower and higher CMC condition. (Blue triangles indicate the mean value; significant difference was observed with a p-value less than 0.0001 after u-test.) 

Investigate the stability of seeding process of mesenchymal stem cell on microcarriers by considering heterogeneity

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1060&context=cell_gene_therapies_viii&type=additional

Investigate the stability of seeding process of mesenchymal stem cell on microcarriers by considering heterogeneity

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Engineering Conferences International