Table of Contents
- Introduction
- Shear stress
- Factors that affect shear forces in the bioreactor
- How to reduce shear stress in your process
- Conclusion
- Sources
Introduction
The natural environment of the cells requires fluids for all the biological and chemical processes to take place. Similarly, it is key to recreate a fluid environment in the bioreactor so the bioprocess can run efficiently.
However, cells that are used in bioprocesses are exposed to different forces and stress factors that they would not be exposed to in their natural environment. One of these factors, especially predominant in culturing mammalian and insect cells, is shear.
In this blog post, mainly factors which influence the occurrence of shear forces will be discussed, as well as how to implement this knowledge in bioreactor design to have an efficient bioprocess and reduce the shear stress.
Shear Stress
Shear is a force that acts tangentially to the surfaces over which it is applied. Shear stress is developed due to the presence of velocity gradients within the fluid flow.
In other words, slower fluid flow will reduce the speed of an adjacent layer of fluid in the same flow, whilst a faster layer of flow will have an accelerating effect on the slower layer.
When different parts of the fluid move relative to each other that exerts forces on the cells caught in the middle. Thus, shear stress can be looked at as a relation between agitation speed and cell death.
Occurrence of the shear forces exerted on the cells in the bioreactor can be found/expected in the following examples:
- Collisions between cells and reactor parts like the stirrer blade,
- Collisions between microcarriers,
- Interactions with gas bubbles, as for instance the surface tension exerted on a cell when it wants to spread over the bubble surface,
- Forces generated at the formation of the bubble,
- The movement of the bubble through the liquid from the sparger to the top,
- Bursting bubbles on the surface,
- Foaming - forces present in the foam,
- Pumping of cells.
When shear forces exerted on the cells are too large the consequence is cell death. This is undesirable as it leads to decrease in viable cells and release of cell contents (proteases, unprocessed product, host cell proteins, DNA,..). Consequently, the product quality and yield decrease. It can cause interference with measuring equipment and control.
The goal is to limit cell death as much as possible. To do so, some of the following factors have to be considered in bioreactor design.
Factors that affect shear forces in the bioreactor
Cell size and mobility
Why are mammalian and insect cells so sensitive? First of all, they lack a cell wall, because they come from a multicellular organism where there is no need for one. Second, they are relatively large and their cell size influences the occurrence and size of forces exerted on them.
Table 1. Different cell types and their susceptibilities to damage in the bioreactor environment.
|
Cell type |
Size |
Shear sensitivity |
|
Microbial cells |
1-10 µm |
Low |
|
Plant cells |
100 µm |
Moderate/high |
|
Plant cell aggregates |
Up to 1 – 2 cm |
High |
|
Animal cells |
20 µm |
High |
|
Animal cells on microcarriers |
80-200 µm |
Very high |
Small cells in a shear field experience only a minor velocity difference. Whereas bigger cells experience much greater velocity differences and thus larger forces in the same shear field.
Third, cells may be immobilised on microcarriers, which influences cell mobility. The velocity difference experienced is larger since there is a larger difference between the centre and the border of the carrier.
Through rotation the cell can lessen the shear forces exerted on itself by turning away from the shear and through the flattening of the cell. However, the immobilisation makes it more rigid and more shear sensitive because it cannot adjust to the forces.
Figure 1. A small cell in a shear field feels only a minor velocity difference. A larger cell in the same shear field feels a much greater velocity difference and thus experiences larger forces.
Agitation
Fluids in a bioreactor are always in motion due to applying agitation with the goal to achieve efficient mixing. Agitation is needed to get homogenous conditions in the bioreactor and prevent settling. In addition, sufficient mixing is needed to supply oxygen through mass transfer (sklicuj se na blog OTR).
Fluids are in constant motion, they either flow through pipes or over solid parts of the bioreactor. Therefore, this causes velocity variations of the fluid, depending on the position of the flow path. The fluid flow in the context of velocity variations is represented by streamlines, which show the net effect of the fluid motion.
However, the net motion of the fluid represented by streamlines does not always show us the whole picture. Streamlines can represent continuous flow, while when you look closely smaller particles in the fluid can actually be moving in an erratic way.
When the flow is slower the representation by streamlines is more accurate and is therefore called laminar flow. However, in fast flowing fluids the particles often cross streamlines and form local areas of swirling fluid called eddies. Such fluid behaviour is characteristic for turbulent flow.
In order to achieve homogeneous conditions by effective mixing the flow must be turbulent. Transition from a laminar to turbulent flow depends on velocity, viscosity and density of the fluid, as well as the geometry of the bioreactor. The fluid flow is characterized in a parameter called Reynolds number.
In a cylindrical vessel stirred by a central rotating stirrer, turbine or propeller the Reynolds number is calculated by this formula:
Where Re is the Reynolds number, Ni is rotational stirrer speed (rps), ρ is density of the fluid (kg.m-3), µ is the fluid viscosity (kg.m-1.s-1), Di is the diameter of impeller (m).
Most stirred tank systems have a laminar flow at Re ≤ 10 and a fully turbulent flow at Re ≥ 104. Consequently, there is a relatively large transition region of the flow regime of 10 ≤ Re ≤ 104.
To accelerate diffusion and achieve turbulent flow, the agitation rate can be increased. However, high agitation rates are accompanied by shear forces caused by eddy formation. This is explained by the eddy length model. In a turbulent bioreactor (Re>1000) large eddies are formed which break up into smaller ones, while transferring their energy to smaller ones.
Eddies larger than the cells engulf the cells or in other words cells just float along with the eddy. However, if the eddies are equal or smaller than the cell diameter, they can interact with the cell and exert a large force on the cell, causing damage to it and eventually killing it.
Figure 2. Visual representation of the effect of eddy sizes on the cells in the fluid environment.
This is the most general theory used to correlate agitation to cell death.
To prevent shear damage, eddy size should be bigger than the cell diameter. When designing a bioprocess, checking if eddy sizes become equal or smaller than cells at any of the values of N (rotational stirrer speed), is crucial. Eddy size can be calculated with this formula:
Where, λk is the eddy size (m), η is dynamic viscosity (N.s.m-2), ρ is density of medium (kg.m-3), εT is the energy dissipation (m2.s-3), Np is the power number, Di is the diameter of impeller (m).
Viscosity of the liquid
As mentioned previously, to achieve effective mixing the flow must be turbulent, which is indicated by the Reynolds number (Re). As can be seen from the formula for calculating Re, turbulence is inversely proportional to viscosity. Meaning, that non turbulent flow and poor mixing are most likely to occur with highly viscous fluids.
When the viscosity of the liquid increases mixing time consequently increases significantly, which makes it harder to achieve homogenous conditions. In addition, achieving turbulence is laboured and stagnant zones are formed in the bioreactor.
This can be solved, in theory, by increasing the power input, however, that is impractical in practice since it increases power costs.
If we have a fixed power input and viscosity is low, the eddy size will be small and mixing will be efficient. In contrast, more viscous liquid will result in bigger eddies and laboured mixing.
Throughout the bioprocess, the viscosity of the media changes. This change is caused by increasing cell size and concentration, as well as substrate and product concentrations, especially when the product is a polymer.
Aeration through sparging
As explained, in mixed bioreactors cells are exposed to a complex fluid environment caused by turbulence. Constantly supplying oxygen into the cell culture is crucial for the success of the bioprocess. Hence, efficient sparging methods are important to ensure cells have enough oxygen for growth and productivity, with low shear stress and reduced foaming tendency.
To achieve high mass transfer performance, efficient gas utilization and low-pressure drop, in bioreactors sparging of air is usually utilized. However, sparging or aeration can cause additional damage to the shear-sensitive cells and further complicate the environment.
Kunas and Papoutsakis performed experiments in 1990, where they increased the agitation rate in a reactor with and a reactor without a headspace. In a reactor without a headspace increasing rpm to 800 caused no damage to the cells, while with a headspace cell death started to occur at 200 rpm already. It appeared that the bursting of bubbles and vortex formation was responsible for cell death.
Cells grown in the absence of sparging can tolerate very high agitation rates without significant cell damage.
Figure 3. Bubble rapture at the air-liquid interface.
Bursting of the bubbles happens at the surface of the medium when they disengage from the culture. When that happens intense hydrodynamic stress and high levels of energy dissipation are generated. These effects together can cause severe damage to the cells, located in the so-called killing volume.
The killing volume of a bursting bubble consists of the bubble film cap and the liquid layer surrounding the bubble.
Cells in the culture, typically animal cells, attach to the rising bubbles and are retained and concentrated in the thin layer of the film cap, where the bubble ruptures.
In the liquid of the bursting bubble, shear forces are generated of high magnitudes. Shear forces combined with high liquid velocities of the rapidly receding film cause cell death.
As an example, experiments with insect cells were performed to measure cell death in each 3.5 mm bubble that ruptured at the surface. The measured concentration of the killed cells was about 103 cells from an initial suspension of 106 cells/ ml. A rate at which, most of the cells would succumb to cell death in only a few hours.
To reduce these effects there are several alternative approaches for supplying oxygen to the culture.
The first approach is to enhance pressure in the bioreactor in order to enhance oxygen solubility. However, this is a very costly process.
The second option is bubble-free aeration, where permeable materials are used for the diffusion of oxygen. Permeable materials pose a challenge in themselves as they might limit oxygen diffusion through the membrane.
The third possibility is micro-sparging or aeration with microbubbles. Microbubbles are small bubbles with a diameter of less than 100 µm, that have lower coalescence and bursting tendency, leading to lower shear stress exerted on the cultivated cells. In addition, microbubbles have high mass transfer capacity, which leads to lower gassing rates needed to supply sufficient oxygen and reduced foaming tendency.
While the bubbles travel through the liquid medium they dissolve completely in the surrounding medium, before reaching the top of the bioreactor. This in turn reduces the need for antifoaming agents.
Figure 4. Comparison of the fate of a macroscopic bubble and a microbubble in the culture medium of a bioreactor.
How to reduce shear stress in your process
To overcome the challenges posed by shear forces on the cells, all the previously mentioned factors should be considered in the bioreactor design process.
Some of the possible solutions that could be tested and applied in bioreactor design are:
- Proper impeller choice and placement,
- Addition of antifoaming agents,
- Application of protective additives, with surface-active properties, in suspension culture,
- Micro-sparging,..
Conclusion
In bioreactors, fluid properties and bioreactor geometry play a key role in determining effective mixing. When culturing, especially insect, plant or mammalian cells, their sensitivity must be taken into account while still providing sufficient agitation for mixing, gas dispersion, as well as mass and heat transfer. Therefore, we should find the volume and agitation rate at which oxygen supply is sufficient and shear forces are kept minimal.
To conclude, mixing and fluid flow together with the effects of shear, have a significant influence on system productivity and the success of a bioprocess.
Sources
- Stem Cells Culture Bioreactor Fluid Flow, Shear Stress and Microcarriers Dispersion Analysis Using Computational Fluid Dynamics: http://www.biotechrep.ir/article_69217_d41d8f11ebd299013d747fa36e9f3321.pdf
- Mass transfer and shear in an airlift bioreactor: Using a mathematical model to improve reactor design and performance (https://www.sciencedirect.com/science/article/pii/S0009250911000583)
- Introduction to CFD (https://www.sciencedirect.com/science/article/pii/B9780128015674000015)
- Bioprocess Engineering Principles (Second Edition) - Mass Transfer (https://www.sciencedirect.com/science/article/pii/B9780122208515000101)
- Macintyre F. 1972. Flow patterns in breaking bubbles. J Geophys Res 77: 5211–5228.
- Papoutsakis ET. 1991a. Fluid-mechanical damage of animal cells in bioreactors. Trends Biotechnol 9:427–437. Papoutsakis ET. 1991.
- Media additives for protecting freely suspended animal cells against agitation and aeration damage. Trends Biotechnology 9:316–324.
- J.D. Michaels, J.E. Nowak, A.K. Mallik, K. Koczo, D.T. Wasan, E.T. Papoutsakis, Interfacial properties of cell culture media with cell-protecting additives, Biotechnol. Bioeng. 47 (1995) 420_430.
- Mechanisms of animal cell damage associated with gas bubbles and cell protection by medium additives (https://www.sciencedirect.com/science/article/abs/pii/0168165695001337)
