Scaling Up Stem Cell Culture

Tissue culture-treated polystrene cultureware has long been the gold standard for culturing adherent cells for research. There are good reasons for using 2D polystyrene culture as it is a cheap, easy-to-use material where cell proliferation and behavior are clearly visible.

However, for certain types of cell culture – stem cells in particular – there are major limitations to the use of conventional flasks and plates. Different stem cell types do not attach well to standard tissue culture-treated polystyrene and require feeder cells or specialized coatings for attachment and growth. We have discussed the need for coating surfaces for stem cell culture in an earlier article.

The scalability of 2D culture is another important limitation. Many stem cell applications, including for clinical use, require cell numbers far higher than those needed for in vitro studies. However, increasing the scale of a 2D culture project requires significant increases in labor, consumables and incubator space – all of which lead to higher costs. So, what are the better alternatives for scaling up stem cell culture?

From well plate to bioreactor
Bioreactors are often the most suitable alternative to 2D culture when needing high cell numbers as they are available in a wide range of sizes, so scaling up (or down) only requires minimal modifications to a protocol.

A key benefit of culturing in bioreactors is the excellent control over culture parameters. Stem cells are among the most sensitive cells in the body when it comes to environmental conditions, so even small fluctuations can have a detrimental effect on cell quality. Critical parameters for successful stem cell culture that can be tightly controlled in bioreactors are:

• pH. In static 2D culture, the pH of culture medium changes over time. Bioreactor culture systems have the ability to regulate pH by adding acid or base to the medium, or by changing the CO₂ level.
• Oxygen tension. During culture, stem cells may require different levels of dissolved oxygen, mimicking normoxic and hypoxic conditions in vivo. By altering the oxygen concentration in the headspace, bioreactors exert precise control over oxygen levels.
• Shear stress. Fluid flow inside bioreactors influences the behavior of stem cells, due to shear stresses on their cell membranes. Both the speed and shape of the impeller(s) directly determine the strength of fluid flow that cells experience.

Bioreactors can measure key parameters in real-time, which provides better process control – as well as valuable information for reporting and quality control. Furthermore, culturing stem cells in bioreactors often takes up less lab space than large amounts of 2D cultureware and saves time in replacing culture media. Single-use bioreactors also facilitate production of cells to GMP standards.

One type of bioreactor that is commonly used in stem cell culture is a stirred-tank reactor. It consists of a vessel that holds the culture medium, one or more impellers that create gentle, consistent fluid flow, and various tools that measure and control culture parameters. These tools include sensors for monitoring pH, temperature and dissolved oxygen as well as pumps to bring in acid or base, devices for temperature control (e.g. a heating blanket), and tubes or spargers to bring in gasses such as O₂ and CO₂. This setup, combined with specialized software, enables good control over culture parameters as well as straightforward scale-up of stem cell culture.

Surface or no surface?
Stem cells, like many other cells, depend for their survival on being anchored to a matrix. It is therefore important to ensure that stem cells have a suitable 3D environment for proliferating and for retaining their phenotype when cultured in a bioreactor. There are two methods of providing this environment in a stirred-tank bioreactor – microcarriers and cell aggregates – and different types of stem cell favor one method over another.

Microcarriers are polymer particles (typically 100–300 μm in diameter), which provide a high surface area for cell attachment in a small volume. They are well suited for growing mesenchymal stem cells (MSCs) – multipotent cells that give rise to bone, cartilage and muscle cells.

When seeded on microcarriers in low concentrations (e.g. 10 cells per particle), MSCs will proliferate and spread across the surface of the particle forming a monolayer. Under appropriate culture conditions MSCs will retain the ability to differentiate into multiple cell types and can be harvested using standard techniques^1,2.

Culture of pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), on microcarriers is more challenging. These cells require highly specific surface properties in order to attach, proliferate and retain their phenotype. As a result, coating of microcarriers with Matrigel or extracellular matrix proteins might be required^3,4.

An alternative to the use of microcarriers for growing pluripotent stem cells in bioreactors is to form cell-only aggregates. When a suspension of single cells is added to a stirred-tank bioreactor, they can, under the right conditions, spontaneously clump together into small aggregates. These aggregates have been shown to grow to over 100 μm and achieve a fourfold increase in cell number by day 7 without premature differentiation, providing a useful alternative to microcarrier-based bioreactor culture^5–8.

Conclusion
Many applications of stem cell culture require cell numbers that are not easily achievable with conventional 2D culture. These applications require a scalable culture process with reliable parameters for consistent culture.

Stirred-tank bioreactors, represent a scalable option that offers precise control over parameters to which stem cells have a high sensitivity. Growing stem cells in a bioreactor, either as cell-only aggregates or attached to microcarriers, is a proven, commonly used method for efficient stem cell expansion in large-scale applications.

References
[1] https://www.eppendorf.com/appnote305/
[2] Moloudi R et al. Inertial-Based Filtration Method for Removal of Microcarriers from Mesenchymal Stem Cell Suspensions. Nature Scientific Reports 2018;8(12481): 1–10.
[3] Lam ALT et al. Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures. Stem Cells and Development 2014;23 (14): 1688–1703.
[4] Chen AKL et al. Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Research 2011;7(2): 97–111.
[5] https://www.eppendorf.com/product-media/doc/en/339060/Fermentors-Bioreactors_Application-Note_292_DASbox_BioBLU-03_Scalable-Expansion-Human-Pluripotent-Stem-Cells-Eppendorf-BioBLU-03-Single-Bioreactors.pdf
[6] Singh H et al. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. Stem Cell Research 2010;4: 165–179.
[7] Villa-Diaz LG et al. The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells 2013;31(1): 1–7.
[8] Steiner D et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nature Biotechnology 2010; 28(4): 361–366.