Mode of 3D Cell Culture Systems

Different modes of cell culture systems are used in MSC cultures as well as in bacterial cultures. The most commonly used modes are batch, semi-batch, continuous and perfusion [66]. Cell density, nutrients and metabolites concentrations, and culture system parameters can be demonstrated with mathematical formula with some kinetic equations, such as mass

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balances and Monod equation. The modes of cell culture systems are presented below (Figure 2.6.).

Figure 2.3. Schematic showing different cell culture modes (modified from [66]).

Batch systems include the mode of operation without any nutrient addition after cell inoculation. The working volume of the reactor is constant throughout the culture. On the one hand, as the cells proliferate, the nutrients present in the medium turn into metabolic residues at the end of cell culture. In the batch production process, all nutrients are introduced at the beginning of the culture, while oxygen, which plays a crucial role in intracellular metabolic reactions, is continuously fed from the sparger. In addition, base solutions such as sodium bicarbonate, sodium hydroxide and CO2 gas are added externally for pH control. Due to its simplicity, the batch culture system is widely used. Generally, the cells use in large-scale studies are cultured in fixed flasks, mixed flasks or small and medium-sized bioreactors. Since the number of cells in the end of cell culture is generally lower than other operating systems, it can be said that the fed-batch system is inefficient. This is mainly due to the fact that nutrients cannot be added above certain limits initially. Because the medium inhibitions are described as a result of changes in osmotic pressure [67]. In addition, during batch production,

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the medium of the cells is continuously changing. Again, the most crucial disadvantage of these operating systems is the inhibition effect of metabolites increasing towards the end of culture.

Half batch systems: This production method differs from batch production in that the nutrients consumed by the cell during culture are added to the culture from the outside. In the half-batch production process, the initial working volume is much lower than the final volume.

During culture, the final volume is reached by adding medium or concentrated nutrients. The most important feature of the half-batch process is that the number of cells and product concentration is much higher than the batch system. It is possible to keep the culture period on average 7-10 days. In this way, the final cell number has been reported to reach up to 10 ×10⁶ cells/mL [68]. In half-batch cultures, the accumulation of toxic metabolites negatively affects cell proliferation, viability and product formation, thus reducing productivity. Metabolites affecting most of the specified parameters are lactate and ammonia [69]. However, it is possible to reduce the formation of toxic metabolites by providing optimal feeding strategies.

The most crucial characteristic of half-batch production is the need for a more complex operation than fed-batch culture, but on the other hand cell and product yields are significantly enhanced. Furthermore, compared to perfusion systems, due to its considerably short culture period makes validation relatively easy. Because of these properties, many biotechnology companies use this production method [70].

Continuous systems: The continuous operating system is based on the introduction of the fresh medium into the reactor at a continuous flow rate, on the one hand, the continuous removal of the homogeneous cell suspension from the reactor at a rate equal to the feed rate, thereby keeping the reactor volume constant. The continuous production process allows a well-defined description of the steady-state between nutrient concentrations in the bioreactor and different rates of biological reactions. Therefore, this operating method is a powerful tool for cell characterization [71]. The most critical limiting parameter in continuous cultures is the gradual decrease in the cell number. This is because the cell uptake rate at the reactor is

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equal to or greater than the cell growth rate. The maximum specific growth rates in mammalian cells are between (μmax), 0.02–0.05 h^-1 (0.50-1 day^-1), thus cell uptakes usually not exceed 2x10⁶ cells mL^-1 in studies.

Perfusion systems: In the perfusion operating system, which similar to the continuous operating system, the cells are immobilized in the reactor. However, the medium is circulated in the system at a specific flow rate. This system is the most complex but also the most efficient operating system. The use of spin filters that hold cells in bioreactors in perfusion cultures is well-known. This operating system has been widely used in laboratory and industrial production [70]. The most significant limitation of the continuous cell culture system, the low productivity caused by cells leaving the bioreactor, is eliminated. Mass equivalents of the nutrient and product are previously applied for continuous cell culture.

Thus, the perfusion system can be applied in almost all reactor types. Also, heterogeneous bioreactors often work in perfusion mode. Homogeneous reactors can also be perfused if suitable separation devices such as spin filters are used. Although the maximum number of cells to be reached with the perfusion system in homogeneous reactors is reported as 10⁷-10⁸ cells/mL, when heterogeneous reactors are used, the cell number can reach much higher values, such as 10⁹ cells/mL. Although the product concentrations reported in this production method vary, the most common range is 100-500 mg/L.