IxCell Biotechnology Co., China (People's Republic)
Abstract: Mesenchymal stem cells (MSCs) have emerged as a promising tool in regenerative medicine and cell therapy due to their ability to differentiate into various cell types and their immunomodulatory properties. However, the traditional use of serum-containing media in MSC culture has raised concerns regarding variability, contamination, and ethical issues associated with animal-derived components. Serum-free culture medium (SFM) offers a viable alternative that addresses these challenges. In this study, we developed a novel cGMP-grade medium for large-scale MSC expansion.
DOE (Design of Experiments) methods were adapted, and multiple rounds of optimization were performed. All the components were cGMP grade and commercially available. Cell growth was investigated by calculating doubling time. MSC surface markers were measured using flowcytometry. The capability of MSC differentiation was assessed using standard protocol.
The medium developed in this study incorporates chemicals and human recombinant proteins, such as bFGF and other growth factors, and is free of undefined animal origin components. MSCs can be expanded up to 20 passages. The cell doubling time ranges from 15.5 hours in passage 5 to 24.4 hours in passage 16 and the cells stopped growing after passage 20. High purity of MSCs was achieved and evidenced by cell surface marker staining. The percentage of cells positive for CD73, CD90, and CD105 but negative for CD31, CD34, CD45, and HLA-DR was 99%. The medium was used to culture MSCs in multi-layer cell factory and generated consistency production with very little batch-to-batch variability. No significant difference was observed among MSCs from different donors in terms of identity，purity and the capability to be differentiated into adipocyte, chondrocyte, and osteocyte lineages.
We have systematically developed a serum-free culture medium for MSC expansion. The MSC manufacturing processes are more consistent and scalable by using this medium. This serum-free culture medium is a significant advancement in MSC research and application, offering enhanced safety, consistency, and efficiency. Its use is expected to facilitate the translation of MSC-based therapies into clinical practice, providing new treatment options for a wide range of medical conditions.
