Considerations on cultivation system-related shear forces and the impact on cell quality
Shear forces—mechanical forces that occur when layers of fluid move relative to each other—play a pivotal role in cell culture systems, especially for adherent cells like mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs). Understanding and controlling these forces are essential in cell and gene therapy applications to ensure optimal cell growth, differentiation, and viability.
The cause of shear forces and shear stress impact on cell behavior in culture
In cell culture, shear forces arise from fluid movement within bioreactors or culture vessels. These forces can influence cell behavior in several ways, while the quality and functionality of the cultured cells are paramount:
- Cell attachment and morphology: Adherent cells require attachment to a substrate for proper growth and function. Excessive shear forces can disrupt cell attachment, leading to altered cell morphology or detachment (1).
- Proliferation and differentiation: Shear forces can modulate signaling pathways that govern cell proliferation and differentiation. For MSCs and iPSCs, controlled shear stress has been shown to direct lineage specification, affecting the efficiency of differentiation protocols (2).
- Viability: High shear forces can cause physical damage to cells, reducing viability, a critical consideration in therapeutic applications where cell integrity is crucial (3).
- Gene expression: Shear stress can alter gene expression profiles, affecting the therapeutic potential of cells (3).
Ultimately, the modulation of these cellular responses by shear forces has significant implications for the success of Cell and Gene Therapy (CGT) applications. Therapeutic efficacy depends not only on the biological potency of the cells but also on the consistency and scalability of the manufacturing process. Understanding and controlling shear stress in cell culture is thus essential to optimize both patient outcomes and production efficiency.
Cell culture systems differences
Bioreactors and cell culture systems are engineered systems designed to provide environments for cell cultures, with shear forces varying based on design:
Fixed Bed Bioreactors: These systems immobilize cells on stationary substrates, minimizing shear forces and providing a stable environment conducive to the growth of shear-sensitive cells like MSCs and iPSCs. However, cell harvest efficiency can be reduced because cells are sterically hindered from exiting the bed after detachment, making retrieval more challenging compared to other bioreactor designs.
Fluidized Bed Bioreactors: In these systems, cells often grow on microcarriers—small beads that provide a surface for adherent cells to attach while being suspended by upward fluid flow. This dynamic environment enhances nutrient distribution but also introduces shear forces that must be carefully managed to prevent cell damage (3). Porous microcarriers can offer internal surfaces that shield cells from excessive shear, potentially improving cell protection and yield. However, cell harvest efficiency can be reduced in porous microcarriers because cells are sterically hindered from exiting the bed after detachment, making their recovery more difficult.
Roller Bottles: Roller bottles are traditional culture vessels rotated to provide a dynamic environment for cell growth. The gentle rolling motion generates low shear forces, making roller bottles a traditional option for the cultivation of adherent cells. This approach is particularly used for MSC and iPSC cultures due to their sensitivity to shear stress (4,5). Additionally, roller bottles often have limitations in scaling up and providing uniform nutrient and oxygen distribution, which can compromise the quality and yield of cells for cell and gene therapy applications. Compared to more advanced systems like the CellScrew®, roller bottles may fall short in surface area efficiency and the consistency of environmental conditions. In contrast, low shear mixed systems like the CellScrew® enable more uniform environmental conditions by promoting better fluid dynamics, ultimately improving cell viability and productivity.
Multi-stack culture vessels: These rely on diffusive matter transport and offer an alternative with greater surface area compared to roller bottles but often face challenges in maintaining consistent nutrient and oxygen gradients throughout the stacks. Shear forces in these systems are negligible.
Each system presents a trade-off between maintaining optimal cell health through controlled shear and providing scalable culture conditions. While traditional and static systems (e.g., multi-stacks, roller bottles) offer gentler environments for sensitive cells, they are often limited in scale and consistency. Conversely, more dynamic systems may enhance scalability and nutrient distribution at the expense of exposing cells to greater shear stress.
With an innovative design that combines the best of both worlds—the scalability of 3D systems and the environmental advantages of low-shear environments—next-generation cultivation vessels, like the CellScrew®, can support both excellent cell health and efficient manufacturing scalability.
Shear forces are a critical factor in the design and operation of cell culture systems, especially for adherent cells, including MSCs, and iPSCs used in Cell and Gene Therapy. Understanding the interplay between shear forces and cell behavior enables the optimization of culture conditions, leading to improved cell yields and therapeutic outcomes. To drive progress in CGT manufacturing, researchers, engineers, and clinicians must prioritize culture systems that balance cell integrity with scalable, efficient production.
References
(1) Paddillaya N, Mishra A, Kondaiah P, Pullarkat P, Menon GI, Gundiah N. Biophysics of Cell-Substrate Interactions Under Shear. Front Cell Dev Biol. 2019;7:251. doi: 10.3389/fcell.2019.00251. Available from: https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2019.00251/full
(2) Limraksasin P, Nattasit P, Manokawinchoke J, et al. Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells. Sci Rep. 2022;12:19021. Available from: https://doi.org/10.1038/s41598-022-21479-8. doi: 10.1038/s41598-022-21479-8. Available from: https://www.nature.com/articles/s41598-022-21479-8#citeas
(3) Mand M, Hahn O, Meyer J, Peters K, Seitz H. Investigation of the Effect of High Shear Stress on Mesenchymal Stem Cells Using a Rotational Rheometer in a Small-Angle Cone–Plate Configuration. Bioengineering. 2024;11(10):1011. Available from: https://doi.org/10.3390/bioengineering11101011. doi: 10.3390/bioengineering11101011.
(4) Hassan MNFB, Yazid MD, Yunus MHM, Chowdhury SR, Lokanathan Y, Idrus RBH, et al. Large-Scale Expansion of Human Mesenchymal Stem Cells. Stem Cells Int. 2020;2020:9529465. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC7378617/
(5) Dhahri W, Sadikov Valdman T, Wilkinson D, Pereira E, Ceylan E, Andharia N, et al. In Vitro Matured Human Pluripotent Stem Cell-Derived Cardiomyocytes Form Grafts With Enhanced Structure and Function in Injured Hearts. Circulation. 2022;145(18):1412-1426. Available from: https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.053563
