[Literature Review] Key Technologies in Artificial Meat Development—The Impact of Serum-Free Medium Components

“ clean meat As a growing global population seeks alternative sources of dietary protein, the successful commercialization of this product will require the development of efficient and cost-effective serum-free cell culture media. It is well known that serum-free media elicit different cellular growth behaviors compared to serum-based media; however, comprehensive data on the impact of serum-free culture on the nutritional requirements of meat-related cell types have yet to be reported. Edward N. O’Neill Waiting for someone at Essential 8 Cultured in serum-free medium and in conventional serum-containing medium C2C12 The cells and culture medium components were analyzed.

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Preparation of cells before the experiment:

Cell: C2C12 Cell;

Culture medium: 40% of the DMEM 、40% of the Ham’s F10 、20% of the FBS And the other one 2.5ng/mL Recombinant human basic fibroblast growth factor;

Culture method: Maintain the cells in tissue culture medium ( TC on polystyrene culture dishes, with 5000 a cell /cm² Get vaccinated and use Try-pLE Express At reaching approximately 75% Passage upon confluence. Count using the trypan blue method via a hemocytometer;

Culture conditions: 37°C and 5%CO₂。

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Experimental method:

To put TC For handling polystyrene six-well plates Matrigel Coating, use 0.15mg/mL The protein solution is diluted and routinely spread thinly.将 P10 of the C2C12 Cells are resuspended in serum-free medium. Essential 8 or PCGM in the culture medium, and with 10⁵ a cell / Well inoculation, with a total volume per well 3mL Culture medium. Shortly thereafter, will... 2 μL Hoechst 33342 The concentrated stock solution of the nuclear stain is added to each well, resulting in a final concentration in each well of: 0.2 μg/mL After cell seeding, the... 6 The孔板 remains undisturbed in the cell culture incubator. At the observation time points, use ImageXpress Pico The high-content screening microscopy system imaged the synergistic response groups from three replicate wells in each of the two medium groups.

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Detection method:

1. Glucose, lactic acid: HP 1100 High-performance liquid chromatography (HPLC) HPLC ) System and refractive index ( RI ) Used in conjunction with detectors Aminex HPX-87H Column.

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2. Amino acids: Use REBEL Analyzer ( 908 Devices ) Measure the amino acid concentrations in fresh and spent culture media. Use 0.20 μm The sample is prepared using a sterile filter to remove cellular debris, and then... REBEL Dilute with a diluent. Using microfluidic capillary electrophoresis and high-pressure mass spectrometry ( CE-HPMS ) Each sample is analyzed in triplicate. The analytes are automatically identified based on migration time and mass.

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3. FGF2 : User FGF2 enzyme-linked immunosorbent assay (ELISA) ELISA ) Kit. Use SpectraMax iD3 Multi-mode flatbed reader in 450nm Read at the location 96 The final colorimetric absorbance in the well determination plate.

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Experimental results:

1. Cell density: By monitoring the culture process every 24 Record cell density per well once per hour (Figure). 1 ), it was determined that, compared to traditional serum-containing culture media, serum-free E8 The culture medium supports a similar level of cell growth. E8 Cell count on day 3 The sky increases exponentially and reaches its maximum value more rapidly (higher than PCGM cell density in the culture medium at any given time), but then began to decline slightly, and subsequently at day... 6 The number of cells significantly decreased (possibly due to increased cell death and shedding). PCGM Cell density on day 2 The growth rate of the index is coming to an end, but it continues to increase gradually until the ... 7 Heaven.

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Figure 1 Changes in cell density

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2. Glucose and Lactic Acid Concentrations: Data (Figure) 2A and 2B ) indicates that glucose utilization was very similar between the two culture groups until day 🛑. 4 About a day, although PCGM Cells in the group continue to grow, E8 The number of cells in the group has decreased, but... PCGM Its use is slowing down, but... E8 Its use is still ongoing. Although... PCGM The initial lactate concentration in the former was significantly higher than that in the latter. E8 However, during the seven-day culture period, the relative rates of lactate accumulation were similar in the two media.

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3.FGF2 The concentration: E8 The extra in the middle FGF2 Used as a means to replace FBS The main components of the function. Data (figure) 2C ) indicates that in the cultured 3 or 4 About a day, in both culture media. FGF2 The concentration began to decline significantly, which roughly coincided with the time when the corresponding cell density ceased to exhibit exponential growth. Although in cultures grown in both types of medium, FGF2 Concentration at approximately 5 or 6 The time point reached its minimum measurable value, but thereafter cell density was maintained for at least several days. Overall, the relative... FGF2 There is no significant difference in consumption patterns.

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4. Amino acid concentration: (Figure 2E ) PCGM and E8 Most of the initial amounts of each amino acid between them ( D0 ) The concentrations are similar and consistent with their respective basal medium formulations. The differences may be due to... PCGM middle FBS caused by the unknown amino acid profile of the ingredients. For any given medium, many amino acid concentrations did not significantly decrease over time, suggesting that these amino acids were either not utilized or not incorporated into cellular metabolic cycles. As for some amino acids, E8 The culture medium group appears to consume the same as... PCGM faster than the cells in the middle / More complete 。 For example, with PCGM Compared to the concentrations of glutamine and glycine in the medium, on day ... of culture, 4–5 Heaven, E8 The consumption of glutamine and glycine in the middle group increased significantly. By the ... 3 About a day, E8 Serine is almost completely depleted, marking the point at which exponential growth ceases. Beyond this, with the exception of a few amino acids such as arginine, the overall utilization trend for most amino acids is similar.

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Figure 2 Glucose, lactic acid, FGF2 Changes in amino acid concentration

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Experimental conclusion:

The results show that, C2C12 Myoblast cell line in E8 The performance in [this medium] is at least as good as that in serum-containing culture media (Figure [number]). 1 ). C2C12 It is an immortalized muscle cell line that differs from adult stem cells in important physiological aspects; adult stem cells may differ from... Clean Meat More relevant. Therefore, in order to achieve better processing efficiency, Clean Meat The industry may need to develop its own immortalized cell lines, such as... C2C12 However, from different species and lineages, Clean Meat There are significant differences in the medium requirements among the relevant cell types, making it necessary to explore and optimize the medium composition for each cell type individually.

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The experiment examined the kinetics of glucose and lactate concentrations (Figure: 2A and B ) demonstrates the intriguing effects of culture medium type on cellular metabolic behavior. The glucose utilization and lactate accumulation in serum-containing and serum-free media are essentially similar, suggesting that there is no significant difference in metabolic activity among cells cultured in different media, and indicating that serum-free media may not directly affect glucose metabolism. However, the complex interactions among many additional growth factors, cytokines, and other signaling molecules present in serum also warrant further investigation.

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Amino acid kinetic analysis (Figure) 2E ) indicates that many amino acids are not significantly consumed by cells grown in either serum-containing or serum-free culture media; therefore, they may not need to be included in the optimized formulation. Clean Meat The culture medium either does not need to contain such a large amount of amino acids—or at least does not necessarily have to. However, the experimental results also revealed intriguing differences in amino acid metabolism between cells grown in the two types of culture media. The observed differences in glutamine utilization suggest that... E8 The cells in the culture medium are experiencing a higher rate of glutamine catabolism until glutamine is depleted by day... 4 will be completely depleted in about a day; E8 Aspartic acid and alanine from the central metabolism (metabolic products of glutamine degradation) until the ... 4 The low consumption rate of the medium supports this view. The biochemical differences in amino acid metabolism between serum-containing and serum-free culture media are intriguing and warrant further investigation.

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Although the overall between the two media FGF2 There was no significant difference in trends. With PCGM In comparison, in serum-free E8 Cells grown in the culture medium utilize more... FGF2 This may be due to the lack of other growth-promoting factors commonly found in serum, resulting in... FGF2 As a SFM Grown in China “ Substrate ” More important than in other situations. E8 In the culture medium FGF2 The more, E8 The higher the total number of cells obtained. However, does this degree of increase determine... Clean Meat The need for high levels of expensive growth factors in culture media remains an unresolved issue.

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Characterizing most Clean Meat Relatively little work has been done on the metabolic requirements of specific cell types; consequently, many of the more detailed metabolic assumptions made about these cell types stem from our understanding of how they behaved in earlier studies, which typically employed serum-containing culture media. These assumptions may hinder the overall ease and success of transitioning cells to fully serum-free and chemically defined culture media. It is essential to optimally tailor serum-free culture media for various cell types, achieving maximum efficiency at the lowest possible cost. Clean Meat Bioprocessing can require significant time and effort. Perhaps, it’s possible to create... Clean Meat cell lines, thereby achieving greater versatility, which could be instrumental in research. Clean Meat Cell / A promising goal for experimentalists in the field of process engineering.

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Original text: https://doi.org/10.1016/j.fufo.2023.100226

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