- 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
. 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 techniques1,2.
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 required3,4.
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 culture5–8. 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.