Bioreactor for Cultivated Meat Production

Bioreactor Instrumentation and Control

Given the sensitivity of animal cells, monitoring and controlling production process parameters is a mandatory task requiring meticulous attention. Monitoring must be systematically organized and should provide clear, unambiguous results. This data is crucial for assessing the physiological and morphological status of cells and identifying cell growth stages, which are essential for successful culture. Consequently, collecting this culture data aids in process optimization and facilitates scale-up, establishing a vital link between data acquisition and process efficiency.

Therefore, to ensure bioreactor efficiency, appropriate instrumentation must be employed, complemented by rapid and accurate data analysis. Furthermore, it is preferable that all operations for monitoring and control be automated to eliminate any manual intervention. This combination is essential for effectively guiding biotransformation processes to peak performance, avoiding contamination, or process variations.

Ensuring optimal cell growth requires monitoring multiple parameters. These include specific cellular parameters such as morphology, density, viability, and size; physical and mechanical parameters like agitation, shear stress, gas partial pressure, temperature, and density; and physicochemical parameters, including medium pH—a factor influenced by the combined concentrations of nutrients, salts, and metabolites within the medium.

To meet the need for consistent and reliable monitoring, three sensor approaches are employed: online sensors, characterized by sterilizable probes immersed in the culture medium or scaffold matrix; non-contact sensors, positioned externally to the bioreactor and utilizing techniques such as spectrophotometry or ultrasonics for monitoring; and indirect sensing, encompassing methods like parameter correlation and offline analysis, where samples are collected and evaluated in a laboratory setting.

Physical and Mechanical Parameters

Physical and mechanical parameters are critical factors in cell growth, as they directly influence the cellular environment. For instance, agitation and shear stress affect cell morphology and viability, while factors such as slight deviations in temperature and gas partial pressure impact metabolic rates and overall culture process efficiency.

The ideal internal temperature of a bioreactor depends on the cells being cultured. Typically, for optimal performance, sensors should be capable of reading temperatures within the 10°C–40°C range, with a maximum reading deviation of 0.5°C. As bioreactor volume increases, implementing additional sensor points is recommended to achieve uniform readings. For this purpose, online resistance temperature detectors (RTDs) are employed for cell temperature monitoring due to their high precision and rapid response times. However, thermistors (non-contact sensors) and thermocouples are more cost-effective and may be suitable for single-use technologies (SUT). Their sensitivity, however, can be lower depending on the specific model.

Since cells rely on oxygen to meet their metabolic demands, the effective supply of oxygen-rich air is critical. To determine the partial pressure of oxygen, it can be closely correlated with dissolved oxygen (DO), the variable commonly used for monitoring. Additionally, monitoring dissolved carbon dioxide is equally important, as it is one of the primary metabolites directly influencing the pH of both the extracellular and intracellular environments. Dissolved ammonia should also be monitored, as it exhibits potent inhibitory effects even at low concentrations. Moreover, it can arise both as a metabolic byproduct and through non-enzymatic degradation of glutamine, a key amino acid present in culture media. Generally, two primary sensor types are employed for dissolved gas monitoring. First are online electrochemical sensors, which utilize various electrodes. These sensors are fast, capable of rapidly monitoring different molecules, and highly sensitive. Second, online optical sensors, though more expensive and slightly slower than electrochemical sensors, are gaining popularity. They stand out for their high resistance to interference and low maintenance requirements. Beyond these online methods, offline analysis is also an option. Alternatively, spectrometers or analyzers specifically designed for the target parameter can be coupled to the reactor.

The mechanical state of cells is a critical factor in monitoring and controlling processes within bioreactors, with its importance varying depending on the specific reactor and growth medium used. This is particularly crucial when cells are susceptible to shear stress, as shear stress can affect their morphology and viability. Agitation is a key factor in many reactors and is typically a fixed variable optimized during scale-up. Therefore, it requires strict control to ensure cell integrity and function. For instance, in air-lift reactors or bubble columns, aeration rate and bubble size must be controlled to maintain optimal conditions. In stirred tank reactors (STRs), attention must be paid not only to aeration but also to agitation rate, with particular focus on impeller design and efficiency. Additionally, continuous monitoring of cell morphology is necessary to assess the impact of these mechanical factors. Further details regarding cell morphology monitoring will be discussed subsequently.

Physicochemical Parameters

The pH of the culture medium provides valuable information about process conditions. Analysis in conjunction with other variables aids in better understanding cellular metabolic behavior. Similar to dissolved gases, pH is monitored using electrochemical and optical sensors.

As mentioned, cellular metabolism correlates with changes in medium pH. These metabolic pathways respond differently to glucose and amino acid concentrations and consumption, generating byproducts of cell growth that increase concentrations of inhibitory components like lactic acid, ammonia, and carbon dioxide. Controlling glucose and amino acid supply has been shown to help maintain a balanced metabolic environment, slow metabolite accumulation, and enhance cell growth and productivity.

Monitoring reagent and product concentrations during bioprocesses is critical for understanding and controlling pH and cell growth, thereby further optimizing the process. In addition to electrochemical sensors, various coupled instrumentation techniques are employed for this purpose. For instance, Raman spectroscopy enables online monitoring of lactate, glucose, and up to 20 amino acids. UV-Vis spectroscopy can detect metabolites and nutrients at different wavelengths, while mass spectrometry can identify volatile organic compounds. Offline analysis is also possible using these and other methods, such as liquid chromatography and gas chromatography.

While some of these methods may be costly and challenging to implement at scale, they can reduce expenses by controlling feedstock input into the culture medium, thereby minimizing waste. Balancing cost and efficiency is crucial for optimizing bioprocesses.

Cell Parameters

In the production of cell-based meat, where cells are the product, precise monitoring of cell condition is critical. Tracking cell viability and physiological state ensures product quality and safety. For in-depth morphological analysis, microscopic examination of reactor samples is essential, providing insights into cell structure and growth patterns. However, manual procedures for assessing cell count, viability, density, and size can be time-consuming and prone to human error. To overcome these limitations, technologies such as flow cytometry and cell counters have been developed. These advanced methods automate the analysis process, reducing time and bias.

Furthermore, automated technologies integrated with reactors can efficiently perform routine assessments. For instance, Raman spectroscopy effectively monitors cell density and concentration, providing real-time data critical for process optimization. Dielectric spectroscopy sensors are used to evaluate biomass viability, offering key insights into cell condition. Moreover, Doppler ultrasound technology, renowned for its non-invasive approach, has been proposed for measuring cell concentration and viability.

Five Challenges and Future Developments in Bioreactor Design and Scale-Up

Although cultured meat represents a promising sustainable and environmentally friendly alternative to conventional meat, its production process still faces several challenges in achieving appropriate industrialization and commercialization. For instance, the cost of media used for two process steps: proliferation and differentiation. To properly cultivate eukaryotic cells, media must be nutrient-rich to meet the energy and cofactor requirements for cell growth. Compared to media used for microbial cultures, eukaryotic cell media require more components, particularly amino acids, vitamins, and trace elements. Some commercial media formulations provide all necessary nutrients. However, they are often animal-based (using bovine or equine serum), which contradicts the slaughter-free nature of cultured meat, and these components are costly. Additionally, specific factors must be used at each process step to induce cell proliferation or differentiation. These specific components can be obtained through recombinant technologies. Finally, nutrient-rich media are more susceptible to contamination. Antibiotics can be added to prevent contamination; however, regulations typically strictly prohibit the misuse of these compounds due to the potential for developing resistant bacteria, and adding another ingredient to the medium increases its cost. Another challenge regarding contamination is the proper sterilization of equipment, primarily bioreactors. This process must be well-executed to ensure contamination risks are minimized. In-line cleaning (CiP) and in-line sterilization (SiP) strategies are commonly adopted as they are automated systems requiring no manual intervention. However, these systems consume significant amounts of water and energy. An alternative approach involves using wave bioreactors or single-use STRs, as the bags are already sterile and can be discarded at the end of the process. Nevertheless, environmental concerns arise with these systems due to increased plastic waste generation.

The production of lab-grown meat typically involves cultivating muscle cells in bioreactors. However, meat is a complex structure composed of muscle, blood, fat, and bone cells. Despite recent scientific efforts, co-cultivating different cell types in an efficient manner has not yet been achieved. Consequently, the products obtained are usually ground meat or processed items such as hamburgers, chicken nuggets, and meatballs. Currently, it is not possible to produce “steak” or “steak-like” products, and substantial research remains necessary. Nevertheless, the advantage of cultured meat lies in the ability to adjust or enhance nutritional value—including protein content—by utilizing certain safe additives. Furthermore, it is possible to genetically engineer the cells, though this raises discussions about the biosafety of genetically modified organisms. Since the cells used to produce cultured meat rely on adhesion, a solid substrate is typically required for proper growth during two production steps. This necessitates the use of scaffolds—biopolymers that serve as the matrix for cells to attach to during the process. These materials must strictly comply with minimum food safety standards and be biocompatible or biodegradable to remain in the product or be easily separated before commercialization. Industrial-scale production of synthetic meat scaffolds remains uncommon, naturally increasing process costs through these biomaterials. Furthermore, distinct scaffolds are often required for each step (proliferation and differentiation), further inflating final costs. Thus, scaffolds must be carefully selected to be compatible with applied bioreactors, cost-effective, scalable in production capacity, and derived from safe, non-animal sources.

It is well-known that eukaryotic cells required for cultured meat production are generally more sensitive to process conditions than microorganisms. Consequently, scaling up and achieving high cell densities can pose challenges. Process control over fundamental conditions—such as temperature, pH, dissolved oxygen, carbon source consumption, and cell growth—is critical. Some bioreactor systems (e.g., STR) can be easily controlled through well-established strategies. However, other bioreactor types may require enhanced strategies due to control difficulties, particularly those with solid substrates holding fixed cells (e.g., hollow fibers and fixed beds). Cells are also shear-sensitive, necessitating low agitation rates or specific mixing strategies to preserve cell integrity. Nevertheless, inadequate mixing leads to poor homogenization, creating gradients within the bioreactor. Low aeration rates cause poor mass transfer and may even lead to excessive CO₂ accumulation within the bioreactor (a product of cellular respiration). Elevated CO₂ levels subsequently lower the medium's pH. These consequences highlight the complexity of controlling eukaryotic cell culture conditions. Scaling up certain bioreactors remains challenging, as models and relationships have yet to be fully established and developed. Generally, the geometric relationships within bioreactor models between two scales (laboratory and pilot, or pilot and industrial) must remain consistent to ensure accurate process reproducibility during scale-up. Furthermore, certain key process parameters—primarily related to agitation and aeration (thus guaranteeing minimum conditions for cell growth)—can be maintained across different scales. Bench-scale and pilot-scale bioreactors serve as models and testing grounds for varying process parameter conditions, paving the way for subsequent industrial applications.

Since scaling up certain models used in artificial meat production has not yet been established, alternative strategies can be employed to achieve the desired final product concentration. This involves scaling up and/or parallelization strategies, where multiple smaller bioreactors are utilized instead of a single large bioreactor. This approach reduces the impact of shear stress during scaling up and minimizes the need to develop scaffolds compatible with large bioreactors. However, this represents a more labor-intensive approach compared to a single large bioreactor. Another strategy facilitating scale-up is computational fluid dynamics (CFD), which serves as a valuable tool for simulating bioreactor conditions and determining optimal cell growth parameters. CFD enables analysis of fluid behavior, characteristics, and interactions, aiding in parameter prediction and providing valuable insights into biological processes through simulation. This facilitates further optimization and scale-up.

The fabrication of bioreactors for cultured meat production requires customization, as each process step demands specific conditions. Therefore, open dialogue with bioreactor manufacturers is essential to identify the optimal design for the proposed process. A disruptive innovation involves using 3D printing as a step in cultured meat production, constructing meat products by extruding bioink layer by layer. Typically, cell expansion steps are still required before printing; however, bioreactors for differentiation are not necessarily needed. Theoretically, the final product is a structured 3D object, unlike the unstructured meat obtained without bioprinting. Post-processing may alter certain characteristics of the final product, making its adjustment crucial before commercialization. Although it is a disruptive technology with significant potential for cultured meat production, it remains an emerging and costly technique, necessitating thorough research and understanding before implementation.

Conclusions and Future Prospects

Cultured meat technology is undergoing significant advancement, necessitating major progress in developing new and/or improved bioreactor models to cultivate cells correctly and efficiently. The unique characteristics of various mammalian cells employed for this purpose demand research and development into novel equipment and conditions, including medium composition, agitation, mass transfer, structure, and the composition of microcarriers serving as fundamental supports. Nevertheless, conventional bioreactor types remain viable for cultured meat production with certain modifications. For instance, STR and air-lift bioreactors remain feasible and effective options for culturing these cells under appropriate control and instrumentation. Other bioreactor options, including hollow-fibre, fixed-bed, and single-use bioreactors, also warrant consideration. Consequently, the design and manufacture of bioreactors for cultured meat production necessitate thorough discussion between researchers and manufacturers to identify effective solutions.