Mesenchymal stem cells (MSCs) hold immense promise in regenerative medicine due to their multilineage differentiation potential. However, large-scale expansion of these therapeutic cells can be hampered by a significant challenge: cell clumping. While some degree of clumping may be beneficial for cell-cell interactions, which can play a role in certain cellular functions like differentiation, excessive clumping during cell passaging can negatively impact cell growth, viability, and ultimately, their therapeutic efficacy.

This blog post explores the detrimental effects of cell clumping in MSC cultures and delves into established strategies to mitigate this phenomenon.

Understanding the Causes of Cell Clumping

Excessive cell clumping can be a challenge during passaging of MSC cultures. Here are some key reasons why MSCs clump:

• High Cell Density: As cells proliferate, they move closer together, which increases the likelihood of physical contact. This proximity facilitates the formation of cell clumps. High cell densities can lead to rapid depletion of nutrients and accumulation of metabolic waste products in the culture medium. These suboptimal conditions can stress cells, causing them to adhere more readily to each other as a survival mechanism.
• Over-digestion: Excessive treatment with enzymes like trypsin, used for detaching adherent cells, can damage cell membranes. This damage can make cells more prone to stress and apoptosis and increase the likelihood of cells sticking to one another, leading to clumping.
• Environmental Stress: Mechanical stress during handling, repeated temperature changes, or other stresses can injure cells, leading to increased cell-cell adhesion as a stress response.
• Cell-Cell Adhesion: MSCs express molecules, such as cadherin and integrin, on their surface that promote adhesion to other cells. This inherent “stickiness” allows them to clump together.
• Extracellular Matrix (ECM) Production: MSCs secrete components of the ECM, a network that provides support and influences cell behaviour. The secreted ECM can act as a scaffold, bringing cells closer together and facilitating aggregation.

The Pitfalls of Clumping

While some cell-cell contact is beneficial, excessive aggregation creates a microenvironment detrimental to MSC health and therapeutic potential. These clusters limit the even distribution of vital nutrients and oxygen, particularly to cells within the core. This uneven distribution has several consequences, as we’ll explore in the following pitfalls of clumping:

• Uneven Nutrient and Gas Diffusion: Large clumps create a barrier to the even distribution of essential elements like nutrients and oxygen. Cells in the center of the clump have limited access to these factors, hindering their growth and metabolic activity. This can lead to stunted proliferation as cells deprived of nutrients and oxygen will struggle to divide and replicate, ultimately reducing the overall yield of viable MSCs. Additionally, limited access to oxygen can push cells towards anaerobic respiration, a less efficient process that produces less energy and potentially harmful byproducts.
• Increased Cell Death: The lack of nutrients and oxygen within the clump can eventually lead to cell death. Dead and dying cells within the clump release debris, further promoting aggregation and creating a vicious cycle. This significantly reduces the overall viability of the MSC population.
• Heterogeneity in the Cell Population: Clumps often contain a mix of cells in different stages of growth and differentiation. This heterogeneity can be problematic for downstream applications that require a uniform cell population with consistent characteristics. For example, some cells within a clump may be more differentiated and less desirable for therapeutic purposes.
• Challenges in Downstream Analysis and Applications: Techniques like flow cytometry, which analyse individual cells, become unreliable when dealing with clumps. Clumps can clog filters or microfluidic devices used in cell sorting and other downstream applications.
• Altered Cell-Cell Interactions: While some cell-cell contact is beneficial for MSCs, excessive aggregation can lead to abnormal cell-cell interactions. This can potentially disrupt important signalling pathways and influence the differentiation potential of the cells.
• Vascular Obstruction Risks: Clumping creates a significant challenge for downstream applications, particularly intravascular delivery (injection into blood vessels). Large clumps can obstruct blood flow, potentially leading to serious complications like stroke or organ damage. This underscores the need for strategies that not only minimise clump formation during culture but also ensure proper dispersion before injection.

Combating the Clumping Conundrum:

Researchers have developed several strategies to promote even distribution and prevent excessive aggregation during MSC expansion:

• Optimise Cell Density: Maintaining an optimal cell density is crucial. Low seeding densities (e.g. 5,000-6,000 cells/cm²) can reduce the likelihood of cell-to-cell contact, thereby minimising clumping. Regular monitoring and subculturing of cells before they reach high confluency can help maintain an optimal density. Do not let cells grow to 100% confluency before subculturing, as over-confluence leads to the formation of tight cell sheet which clumps together after trypsinisation. Subculture cells when they have reached approximately 80% confluence.
• Use of Enzymatic and Non-Enzymatic Dissociation Methods: Proper dissociation of cells during passaging is critical. Enzymatic methods using trypsin or collagenase can effectively dissociate cells, but care must be taken to avoid over-trypsinisation, which can damage cells. Using chelating agent, such as EDTA, can block cell surface adhesion molecules, or a gentler enzyme such as TryPLE can be helpful. Non-enzymatic methods, such as cell scrapers or dissociation buffers, can also be used to gently separate cells without compromising their viability.
• Optimise Culture Media:  The composition of the culture media plays a vital role in cell behaviour. Media supplemented with appropriate growth factors can support cell growth while minimising clumping. Additionally, adjusting the pH and osmolarity of the media to optimal levels can enhance cell viability and reduce aggregation.
• Gentle Handling and Regular Monitoring: Gentle handling of cells during passaging and media changes is essential. Using wide-bore pipettes and minimising physical agitation and avoid excessive pipetting or vortexing can prevent the formation of cell clumps. Perform a shorter spinning time at a lower speed to prevent mechanical stress e.g. 3-5 min at 200-300g. Regular monitoring of cell cultures under the microscope allows for early detection of clumping, enabling timely interventions.
• Coating Culture Surfaces: Coating culture surfaces with ECM proteins such as fibronectin, laminin, or collagen can promote better cell adhesion to the substrate rather than to each other. This approach not only prevents clumping but also supports cell growth and differentiation.
• Use of Bioreactors: For large-scale cultures, bioreactors provide a controlled environment that can minimise cell clumping. Stirred-tank bioreactors, for instance, maintain cells in suspension and ensure even distribution of nutrients and oxygen, reducing the likelihood of clump formation.
• Storage Conditions and Procedures: The storage solution, duration of time that cells remain in suspension, and the freeze-thawing procedure can all significantly influence cell viability and the propensity for clumping before transplantation. Ensuring optimal storage conditions and minimising the time cells spend in suspension can help maintain cell integrity and reduce aggregation.
• Use of Fresh Cells: Whenever possible, use fresh cells for transplantation. Freshly cultured cells typically exhibit higher viability and less tendency to clump compared to cells that have been stored or subjected to freeze-thaw cycles. This can improve the efficiency and safety of cell-based therapies.

By implementing these multifaceted strategies, researchers can effectively minimise cell clumping in MSC cultures. This not only enhances cell health, yield, and therapeutic potential, but also ensures a more uniform cell population for more reliable experimental results and successful clinical applications. As the field of regenerative medicine continues to advance, overcoming the hurdle of cell clumping will be pivotal in unlocking the full therapeutic potential of MSCs.

References

Cui LL, Kinnunen T, Boltze J, Nystedt J, Jolkkonen J. Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study. Stem Cells Int. 2016;2016:1764938. doi: 10.1155/2016/1764938.

Ge J, Guo L, Wang S, et al. The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. Stem Cell Rev Rep. 2014;10(2):295-303. doi:10.1007/s12015-013-9492-x