One Size Fits All? Not for MSC Bioreactors
The manufacture of clinically-relevant mesenchymal stem cells (MSCs) is not without its challenges. MSCs are inherently heterogeneous cell populations; thus, it is challenging to produce consistent populations of cells that reliably retain their stemness, regenerative potential, and immunomodulatory properties. Another issue in generating batches of therapeutic MSCs is in efficiently scaling-up production. Increasing the scale is costly in terms of space, media, and labor requirements. Finally, from a practical standpoint, MSCs are adherent cells that benefit from growing in 3D cultures instead of simple 2D monolayer culture (1,2).
Bioreactors provide a solution to many of the challenges associated with the manufacture of large batches of clinical-grade cells. There are various bioreactor platforms and set-ups that support the growth of MSCs, each with their own advantages.
Types of Bioreactors
Stirred tank bioreactors
The traditional set-up that most people will picture when they think of a bioreactor is the stirred tank bioreactor. Stirred tank bioreactors are vessels that contain a motorized propellor that maintains the flow of the liquid inside.
Stirred tank bioreactors provide a larger surface area relative to volume compared to traditional monolayer culture. This feature reduces the amount of both space and growth medium required, both of which represent significant expenses in cell manufacture. Therefore, stirred tank bioreactors represent a cost-effective solution for large-scale production of MSCs (3,4). Importantly, since MSCs are adherent cells, selecting an appropriate growth matrix is essential for robust culture in stirred tank bioreactors.
MSCs are sensitive to shear forces (5). One problem with stirred tank bioreactors is the mechanical agitation provided by the propellors, which can impact the phenotype of the cell population. Therefore, rocking bioreactors are often selected as a gentler alternative. Rocking bioreactors have been shown to support the growth of clinically-relevant numbers of MSCs. In fact, rocking bioreactors enable cell populations to grow more quickly than cells in traditional culture, making them a potentially more efficient solution. These cells are phenotypically similar to flask-grown MSCs (6).
Vertical-wheel suspension bioreactors
Vertical-wheel suspension bioreactors are another gentler alternative to stirred tank bioreactors. A large vertical wheel-shaped propellor located at the bottom of the tank provides the agitation in these vessels. This innovation provides uniform mixing and a low shear environment and has been shown to produce a higher yield of proliferative and less apoptotic MSCs (7).
Hollow-fiber bioreactors are 3D culturing systems that contain thousands of hollow cylindrical fibers - semi-permeable tubes bundled to form networks. Cells are seeded into the spaces in the center of the fibers, where they are allowed to expand. Hollow fiber bioreactors are very small but facilitate the growth of high cell densities. They also use significantly less medium and growth factors than traditional cell culture methods.
Hollow fibers have been effectively used for large-scale exome production from MSCs, successfully attaining 7.5 x higher yields than traditional cultures (4,8). The therapeutic characteristics of MSCs from both bone marrow and umbilical cord tissue are also not altered, supporting their use in clinical manufacture (9).
The efficient expansion of cells will be an essential aspect of developing clinical-grade advanced therapies. Innovations that reduce costs, improve safety, and enhance reproducibility will be crucial as we move into a new era of regenerative medicine. A range of bioreactor technologies and platforms should continue to be explored for their ability to offer feasible manufacturing solutions in generating MSC therapies.
1. Jossen, V., van den Bos, C., Eibl, R. & Eibl, D. Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges. Applied Microbiology and Biotechnology vol. 102 3981–3994 (2018) doi: 10.1007/s00253-018-8912-x
2. Levy, O. et al. Shattering barriers toward clinically meaningful MSC therapies. Science advances vol. 6, no 30 (2020) doi: 10.1126/sciadv.aba6884
3. Lam, A. T. L. et al. Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors. Cytotherapy 19, 419–432 (2017) doi: 10.1016/j.jcyt.2016.11.009
4. Yan, X. et al. Dispersible and Dissolvable Porous Microcarrier Tablets Enable Efficient Large-Scale Human Mesenchymal Stem Cell Expansion. Tissue Eng. - Part C Methods (2020) doi:10.1089/ten.tec.2020.0039.
5. Jossen, V. et al. Mass Production of Mesenchymal Stem Cells — Impact of Bioreactor Design and Flow Conditions on Proliferation and Differentiation. in Cells and Biomaterials in Regenerative Medicine (InTech, 2014). doi:10.5772/59385.
6. Das, R. et al. Preparing for cell culture scale-out: establishing parity of bioreactor- and flask-expanded mesenchymal stromal cell cultures. J. Transl. Med. (2019) doi:10.1186/s12967-019-1989-x.
7. Lembong, J. et al. Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System. Bioengineering 7, 73 (2020) doi: 10.3390/bioengineering7030073.
8. Yan, I. K., Shukla, N., Borrelli, D. A. & Patel, T. Use of a, hollow fiber bioreactor to collect extracellular vesicles from cells in culture. in Methods in Molecular Biology (2018). doi:10.1007/978-1-4939-7652-2_4.
9. Mennan, C., Garcia, J., Roberts, S., Hulme, C. & Wright, K. A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells. Stem Cell Res. Ther. 10, (2019). doi: 10.1186/s13287-019-1202-4.