Who would have thought that something as minuscule as a cell could be so valuable? But they are – cell suspensions are liquid cultures in which individual cells are freely suspended in a nutrient-rich liquid medium. Cell suspensions can be of plant, animal, or microorganisms. They have several uses, such as to produce valuable products like phytochemicals, proteins, enzymes, and antibodies in industries.
In this study, plant cell suspensions are studied. In order to produce more phytochemicals and other valuable products, biomass productivity must be taken into account. Biomass productivity determines how fast new organic material (biomass) gets produced by the cells.
Plant cell suspensions are made by placing a clump of cells (inoculum) into a liquid growth medium, and shaking on a shaker. Shake flasks are usually used for this purpose on a laboratory level. For producing useful products at a larger scale, the cells are usually transferred to a bioreactor in a step wise volume increase: lab scale, pilot scale and to industrial scale.
But the problem arises when transferring the plant cell suspension from shake flasks to the bioreactor, even at lab scale. There is a dip in the biomass productivity. In fact, the biomass productivity was unchanged when increasing the volume in shake flasks, but it dipped when transferred to the bioreactor, even at lab scale. This could be because of the change in hydrodynamics (fluid motion and its interaction with the surrounding forces) during transfer. Generally the design of the bioreactor is chosen by hit and trial methods to match the shake flask biomass productivity at the bioreactor. But plant cells have a growth time of 10 to 20 days and it is labor intensive and time consuming to choose the design with this approach. In this study, the authors Ms. Vidya Muthulakshmi Manickavasagam, Prof. Nirav Bhatt, and Prof. Smita Srivastava from the Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology (IIT) Madras, Chennai, India (Prof. Nirav Bhatt is also affiliated with the Department of Data Science and Artificial Intelligence, Wadhwani School of Data Science and Artificial Intelligence, Indian Institute of Technology (IIT) Madras, Chennai, India), and Prof. Kameswararao Anupindi from the Department of Mechanical Engineering, Indian Institute of Technology (IIT) Madras, Chennai, India, have used Computational fluid dynamics (CFD) to choose the impeller (the rotating part of a machine to move a fluid by rotation) design for the bioreactor, for the model system, Viola odorata (sweet violet/wood violet – this is a small flowering plant known for its fragrant purple flowers and medicinal properties).
A two-phase CFD model was used to characterise the non-Newtonian fluid dynamics of the plant cell suspension in a stirred tank reactor using different impeller designs such as, a setric, Rushton, and marine impeller. The simulations were performed adopting an Euler-Euler approach, which is a mathematical model used to describe the two-phase flow. The developed CFD model demonstrated that setric impeller was a suitable choice for V. odorata cell cultivation among the three impellers. The setric impeller offers good mixing at the bioreactor bottom while causing less damage to the cells. This is particularly important because plant cells tend to settle down and hence it is important to have good mixing at the bottom to prevent settling. They can also be easily damaged by the forces acting in the fluid because of the impeller rotation and so setric impeller was identified to be suitable among the three impellers.
Also, studying the hydrodynamics experienced by a plant cell suspension in a shake flask can help in smooth translation to the bioreactor level. This can be achieved by studying parameters like volumetric mass transfer coefficient, energy dissipation rate and shear rate.
Unfortunately, quantifying these parameters is not straightforward by direct experimental measurement. CFD can be used to study these parameters as well.
To understand which parameter is favourable for plant cells in shake flasks, even when the volume is increased, CFD model with the fluid properties of V. odorata cell suspension in shake flasks of volume ranging from 250 to 3000 mL were developed and the fluid flow was simulated. The shake flask parameters such as velocity, energy dissipation rate, mass transfer rate, and shear rate were then quantified to study the effect of flask volume on mixing, mass transfer, and shear environment in conditions suitable for cultivation of plant cell suspensions.
It was found that minimising the velocity gradients in bioreactors could help achieve shake flask biomass productivity. This was in line with the finding that setric impeller is suitable, as setric impeller had lower velocity gradients than the other two impellers and could match shake flask biomass productivity. Further, it was found that maintaining a constant shear environment could serve as a suitable scale-up parameter for V. odorata cell cultivation in bioreactors. This finding can be useful for scaling up the bioreactors from lab scale to pilot and industrial scale.
Thus, employing Computational fluid dynamic modeling, the authors were able to rationally design bioreactors for V. odorata and this approach can be extended for rational selection of other design and operational parameters in the bioreactors and scale -up, for V. odorata and potentially other plant cells.
Dr. Ashok Kumar Srivastava, a retired Professor from the Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, New Delhi, India, acknowledged the importance of the findings of this study with the following appreciative comments: “Plants are excellent source of bio-active compounds, however they feature slow growth, thereby necessitating scale up of bioreactor cultivation of plant cells. CFD model was used to understand the hydrodynamic culture behavior during shake flask cultivation for V.odorata cells wherein it emerged that “maintenance of constant shear” is the most suitable criterion for scale up of the plant cell cultivation. These novel conclusions are extremely important for successful scale up of plant cell cultivation from shake flask to bioreactor for mass scale production of medicinal compounds in society.
The contents are adequately described by the authors in simple and attractive manner. I would like to highly recommend its inclusion as Tech Talk article.”
Prof. Nathalie Giglioli-Guivarc’h, Professor and Director at the Biomolecules and Plant Biotechnologies Laboratory, The University of Tours, France, also commented at length on the work done by the authors, giving her appreciative comments as follows: “Mastering the scaling up of plant cell culture volumes is crucial for establishing an industrial-scale bioproduction process. This scaling up is a particularly challenging aspect to manage. The article by Manickavasagram et al. (Biotechnology Journal 2025; 20: e70086), proposes the use of computational fluid dynamics (CFD) modeling to study the impact of culture volume on the hydrodynamics of plant cell suspensions grown in flasks of increasing size. The application of these mathematical models of cell cultures is still underdeveloped, and is becoming a growing field, in which progress is made through a multidisciplinary approach, thanks to effective collaboration between biologists and researchers in the field of applied mathematics.
The biological model studied here has no known mathematical properties, and the studies are conducted on the system of differential equations rely on numerical simulations. Thus, based on results from cell growth experiments and the identification of the basal part of the flask, which contains the cell suspension, as the computational domain, the authors developed a CFD model for the shake flask geometries. This model uses differential equations to represent the evolution of several variables, including the two-phase fluid flow, the effect of orbital agitation on the liquid phase geometry, the height of the liquid phase relative to the gas phase in the flask, and the KLa coefficient. In conclusion, variations in KLa do not represent a good criterion, unlike the shear effect of the culture. These data allow for the proposal of technical improvements in the design of more efficient bioreactors for scaling up plant cell culture.”
Article by Akshay Anantharaman
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