Designing the future of cultivated meat with cost-effective stem cell media and RNA delivery

This article is based on a poster originally authored by Jonathan Faherty, Alex Rimmer, Samuel East, and Catriona Jamieson.

Cellular agriculture has the potential to revolutionize environmentally friendly food production, but its high raw material costs currently impede its financial viability.

Stem cell culture media is one area with significant potential for improvement because it is often expensive and prone to containing components that render it unsuitable for food production.

Uncommon is a pioneering biotechnology company based in the UK working to develop an inexpensive, animal-free culture media designed to produce cultivated meat as an alternative to conventionally farmed meat (Figure 1).

This article describes Uncommon’s use of automated liquid handling and Design of Experiments (DoE) methodology to drive the development of low-cost, animal-free media, as well as optimizing cost-effective RNA delivery.

Figure 1. Uncommon’s process to produce cultivated meat at scale. Considerations need to be taken at every step to reduce the cost impact of the final product. Image Credit: SPT Labtech

Development of 2D piPSC growth media

The team at Uncommon performed parallel investigations into the impact of four media growth factor supplements (A, B, C, and D) at three different concentrations. JMP and Synthace software generated unique trial formulations (Figure 3) and specific instructions for the dragonfly discovery liquid dispenser.

A series of two-dimensional cultures in 384-well plates were inoculated with 300 µL media. These were incubated, with feedstocks replenished using apricot S3.

Cultures were stained with DAPI before being imaged, and the cell count per well was measured and fed back into JMP to enable further optimization of the growth media.

Figure 2. Iterative process used to optimize growth media for 2D iPSC cultures. Image Credit: SPT Labtech

Figure 3. Design space visualization of the conditions that were tested in 2D cell culture. Replicates of the central formulation were included to investigate inherent biological variation. Image Credit: SPT Labtech

Initial data highlighted that Factor-A strongly promoted 2D cell growth, while conversely, Factor-D impeded 2D cell growth (Figure 4). The workflow was repeated using a broader range of Factor-D concentrations to investigate further.

Data from these further investigations (Figure 5) confirmed that Growth Factor-D was required for cell growth, but a critical threshold exists whereby further supplementation impedes cell growth.

This result was determined to be consistent across Factor-D's rHuman and rPorcine homologs, demonstrating the team’s capacity for rapidly formulating optimized iPSC growth media and its ability to proactively respond to an ever-changing regulatory landscape.

Figure 4. Initial data showed that Factor-A strongly promoted 2D cell growth, while Factor-D impeded 2D cell growth. Image Credit: SPT Labtech

Figure 5. Follow-up experiments uncovered that Growth Factor-D is required for cell growth but only up until a specific concentration. This was demonstrated in parallel using both rHuman and rPorcine homologs. Image Credit: SPT Labtech

Optimizing growth media for three-dimensional cell culture

3D assays more closely replicate bioreactor conditions, where cells will ultimately be grown in suspension. These assays enable the team to screen media at a much smaller scale, saving resources and costs.

The team also investigated the four Growth Factors’ (A, B, C, D) relative contributions to total 3D biomass to help further reduce costs.

These additional experiments followed a similar workflow (Figure 6), leveraging a combination of DoE and dragonfly discovery to generate unique formulations and dispense media into 48-well plates.

Total biomass was measured using tile scan imaging and AI image analysis following 2-4 days of incubation.

Figure 6. Iterative process used to optimize growth media for small-scale 3D cell cultures. Image Credit: SPT Labtech

Initial data showed that the most significant contributions came from Growth Factor-A and Growth Factor-B, and the interaction between these two supplements. By plotting this data together in a heat map (Figure 7A), it was determined that both are required for a viable 3D culture, but neither growth factor was sufficient on its own.

This workflow was repeated using a total of 10 different concentrations of both Growth Factor-A and Growth Factor-B (Figure 7B), enabling identification of the minimum concentrations required to achieve peak biomass production.

Since these experiments took place, findings have been incorporated into large-scale bioreactors (3 L and 50 L) to achieve successful and cost-effective production at scale.

Figure 7. Heat map showing the effect of Growth Factor-A and Growth Factor-B on 3D cell culture growth using 3 (A) and 10 (B) different concentrations of each. Image Credit: SPT Labtech

Conclusion

The powerful combination of DoE and automated liquid handling discussed in this article enables hundreds of high-throughput experiments, allowing researchers to simultaneously investigate the effect of multiple factors, as well as the interaction between different factors.

Uncommon has been successful in leveraging this approach to optimize media supplement formulation for 2D and 3D iPSC cell cultures.

The resulting media is 1,000x cheaper compared to commercial media's cost.

This approach has also been employed to optimize the composition of the RNA delivery system to differentiate iPSCs into fat and muscle cells, resulting in further improved workflow cost-effectiveness.

Acknowledgments

Produced from materials originally authored by Jonathan Faherty from SPT Labtech; and Alex Rimmer, Samuel East, and Catriona Jamieson from Uncommon.

About SPT Labtech

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