Human mesenchymal stem cells (hMSCs), also known as marrow stromal cells, are a self-renewing population of adherent, multipotent progenitor cells with the capacity to differentiate into several cell lineages (1). In defined in vitro assays, hMSCs have been shown to readily differentiate into lineage-specific cells that form bone, cartilage, fat, tendon, and muscle tissues (1, 2). Mesenchymal stem cells also provide support and maintenance for the other major stem cell population in the bone marrow, the hematopoietic stem cells (2).
Human mesenchymal stem cells hold great promise as therapeutic agents because of their differentiation ability (thus their potential to replace damaged tissue) and for their immunomodulatory properties. A large number of clinical trials are underway that are using hMSCs in a variety of indications, including bone/cartilage disease, cancer, heart disease, gastrointestinal disease, diabetes, autoimmunity, and neurodegenerative diseases; hMSCs are also being used in drug discovery as a replacement for primary cells and animal models for initial toxicity and effector function screening of new compounds (3, 4). However, a key challenge remains for both drug discovery and clinical applications: obtaining a sufficient number of cells at reasonable cost (5).
The large-scale, industrialized production of hMSCs is necessary to advance these cells into clinical trials and to deliver the large quantities needed for drug discovery screening and lead optimization. Bridging the gap between basic research and large-scale manufacturing of stem cells for clinical trials requires the expansion of well-characterized cells produced under tightly controlled, consistent, reproducible culture conditions that adhere to cGMP standards. cGMP stem-cell culture systems require well-defined, optimized processes that support stem-cell expansion and differentiation to ensure consistent cell populations with uniform properties and predictable behaviors. Additionally, vessels used for expansion must allow rapid analysis of small volumes containing the actual cells to confirm that the expansion and harvest methods are yielding the expected cell populations. Because stem cells are the product, the sample size must be small enough to ensure that valuable product is not wasted.Human mesenchymal stem cells have historically been isolated based on the ability of these cells to form adherent cell layers in culture and the concomitant lack of adherence of other cells in the bone marrow stroma, such as hematopoietic stem cells, adipocytes, and macrophages (1). However, the multilayer flatbed culture (2D) methods currently used for stem-cell expansion are cumbersome, time-consuming, do not allow for constant monitoring of cell characteristics throughout the expansion process, and they introduce a high degree of variability. These limitations make this method suboptimal for the manufacturing of clinical-grade hMSCs. Furthermore, the culture protocols for multilayer vessels require high labor cost, which in turn results in high cost of goods overall. Thus, the development of culture conditions that can be monitored and that produce high numbers of stem cells at low cost is warranted.
One possible solution for overcoming the limitations of 2D multilayer flatbed culture methods is the use of stirred tank bioreactors in which the stem cells are grown on a microcarrier scaffold for suspension (3D). In these 3D cultures, cell samples and medium can be analyzed throughout the expansion process and the growth process can be tightly controlled (e.g., oxygen, pH, glucose, glutamine, lactate, and ammonia). This article desribes the utility of EMD Millipore's 3-L single-use, bench-scale bioreactor (Mobius CellReady bioreactor, EMD Millipore, Billerica, Mass.) for the expansion of hMSCs.