On the Horizon: The integration of adipocytes into 3D cultured meat

Fat is a key component of a flavorful and juicy piece of meat. In a study done by Kerry, taste takes precedence: 73% of consumers surveyed named it as a must have feature for alternative protein. Healthy fats are also an accepted and sought after component of a comprehensive diet, after years of “low-fat” products. Most species-specific flavors are attributed to the profile of fatty acids in the meat and also lipophilic, or fat-soluble, components1. Only around 20% of the fat found in meat is located within muscle cells themselves while the other 80% comes from intramuscular adipocytes, or fat cells2. As a recent Forbes article wrote, “hungry investors should focus their appetite on the juiciest bit: cultured fat.” The juicy sizzle of a hamburger or steak that hits the grill is what’s lacking in many plant based options. It’s what entices the consumer in smell, taste, sound, and texture. Giving a consumer a palatable, complete experience that is the same as or better than a traditional cut of meat is imperative for the industry’s success. For cell cultured meat, which is real meat, fat is a must have feature. The challenge will be culturing muscle and fat together, as this co-culturing process has challenges such as cost as well as chemistry hurdles in the lab. Although many companies in the space are actively involved in R&D to generate cultivated fat, only a few have debuted products containing co-cultured muscle and fat cells. Most notably, Aleph Farms, with a total of $14.4 M in funding according to Crunchbase, announced the successful development of a 3D bioprinted steak containing both muscle and fat cells this past February3. Other companies developing cultivated fat include Hoxton Farms, Mission Barns, and Meatable, among others. As the cultured meat industry moves towards creating whole 3D cuts of meat, incorporating fat will be increasingly important for delivering realistic marbling, taste, and texture.

Advantages of Cultured Fat

There are several reasons related to product quality that make cultivated fat development worthwhile, and companies are choosing to invest considerable amounts to achieve these integrated products. The taste and textural improvement of cultured meat is one of the largest advantages. Fat is extremely important for delivering essential flavor components. Fats from vegetable sources that are used in plant-based meat typically do not contain components or lipid profiles that match conventional meat12. For this reason, fat derived from animal adipocytes would be the ideal way to convey a similar taste profile to conventional meat. Additionally, plant-based fats typically have lower melting temperatures and thus meat containing these fats would behave differently during the cooking process, and ultimately have an impact on juiciness and texture12.

Another more infrequently discussed advantage of cultivated fat is the potential for minimizing the intake of environmental pollutants which are found in the fat of conventional meat. These pollutants are particularly dangerous because of their ability to bioaccumulate in both animal and human fat tissue13. Cultivation of fat would allow for more control over input and the ability to reduce or completely eliminate these toxins from cultured meat. Mission Barns, which has $28.2 M in funding according to Crunchbase, highlights this capability on their website, comparing conventional meat which may contain heavy metals and toxins to their products which contain none. As certain of these toxins have been linked to cancer, endocrine dysfunction, and reproductive and developmental defects, removing dietary sources of these persistent environmental toxins would be of great benefit to human health14. These safety and health advantages over conventional meat could be marketed to consumers to persuade either a transition from conventional to cultured meat, or an incorporation into their current diet.

Brief Background on Adipocytes (fat cells) and Differentiation

Adipocytes, or fat cells, play key roles in energy storage in the form of lipid droplets and are important mediators of endocrine signaling and communication with other tissues to regulate metabolic processes. In animals, adipocytes are found throughout the body, surrounding most organs and tissue types including muscle. White adipocytes originate from stem cells which can be differentiated to pre-adipocyte cells and then further to mature adipocytes which are capable of storing lipids.

In an academic or biomedical research setting, pre-adipocytes, or pre-fat cells, are induced to become mature adipocytes through treatment with insulin, steroids, and antidiabetic drugs like rosiglitazone. These chemicals would not be appropriate for cultured meat and food related processes. Alternative and food-compatible methods of adipocyte differentiation do exist, however. Treatment of bovine adipogenic precursor cells (basically baby fat cells) with fatty acids from vegetable oils has been shown to lead to differentiation and maturation of bovine adipocytes (fat cells in cows)4.

Co-culture Strategies and Potential Complications

Culturing cells in large batches has been a feature solely of the biomedical industry until fairly recently. For medical purposes, adipocyte tissue engineering has been used for soft tissue regeneration. Often, growing fat in an environment that is conducive to culturing fat and muscle cells together is time consuming and costly. When it comes to the incorporation of fat cells into muscle tissue for cultivated meat products, companies have two methodological choices: (1) culture muscle and fat separately and then combine, or (2) co-culture muscle and fat precursor cells together and utilize a differentiation media which works for both cell types. Both approaches maintain their own set of difficulties.

If the goal is to create a whole cut of meat, it is important that an investment is made into a scaffold, or framework for the fat to grow on, that can support growing muscle and fat together. One challenge that will need to be confronted when choosing a scaffold is that fat and muscle cells need different levels of environmental stiffness during the growth process. Fat cells are typically quite fragile and prone to floating, two properties that make them difficult to work with. Finding a company or scientist that is familiar with these finicky properties is imperative to the success of culturing fat. These processes may require diligent practices to delicately manipulate the two cell types, muscle and fat. Recent advancements in the field of adipose, or fat, tissue engineering have explored the possibility of bioprinting mature fat cells or fat cell clusters 6,7. With the right bioink to encapsulate the cells and proper printing conditions, fat cells remained viable during and following the bioprinting process. These publications illustrate the potential for utilizing bioprinting to combine mature fat cells with muscle cells in a patterned manner. Bioprinting is a promising way to skirt the challenge of integrating muscle and fat.

Another historical difficulty of a muscle and fat cell co-culture approach is the ability of one cell type to influence the behavior of the other. Research shows that the growth rate and gene expression profiles of myoblasts (muscle precursor cells) are altered when cultured in the presence of fat cells. This is likely due to the secretion of specific proteins acting as signaling molecules that are sensed by the other cell type and influence their function. These have been known to promote gene expression that codes for fat cells in premature muscle cells, and alter cell metabolism. One study found that co-culture of mature fat cells and muscle cells from mice leads to the domination of the culture by the fat cells, and reduces the ability of the muscle cells to grow and multiply in a proper way9. Such results suggest possible obstacles in achieving efficient muscle differentiation and maturation with this approach, although the effects of these secreted signaling factors could potentially be minimized by frequent cycling and filtering of media. The challenge here will be growth media costs, as a company may not have the bandwidth to constantly cycle their media.

At this stage both of the proposed strategies appear to be viable options and are employed by various early stage cultivated meat companies. Ivy Farm Technologies, using the first tactic, grows each cell type separately before combining to create the final product10. On the other hand, Meatable, which has $62.9 M in funding according to Crunchbase, has developed stem cells that can be grown together to form both muscle and fat.11 As scaffold technologies and media formulations improve it will be interesting to see what new methods for co-culture arise.

Concluding Remarks

As the cultured meat industry grows and companies approach the point of commercial release of products, fat sources that contribute the flavors and texture of conventional meat fat will be necessary to draw new consumers to their products. The integration of fat cells with muscle cells will be even more important in recreating realistic whole cuts of meat. With innovations in tissue engineering on the rise, many of the large and immediate challenges of 3D cultured meat are being tackled, including vascularization (making sure nutrients can move around the tissue) and scaffolding, allowing for more focus on developing these downstream incorporation strategies. The implementation of novel co-culture tactics and technology will be required to overcome the discussed historical difficulties associated with these approaches; however the result will presumably be well worth the investment in innovation.

References

1. Walker EL, Hudson MD. Encyclopedia of Meat Sciences.; 2014.

2. Ben-Arye T, Levenberg S. Tissue Engineering for Clean Meat Production. Front Sustain Food Syst. 2019;3:46. doi:10.3389/fsufs.2019.00046

3. Bandoim L. World’s First 3D Bioprinted And Cultivated Ribeye Steak Is Revealed. Published February 12, 2021. Accessed June 4, 2021. https://www.forbes.com/sites/lanabandoim/2021/02/12/worlds-first-3d-bioprinted-and-cultivated-ribeye-steak-is-revealed/?sh=5ef53c764781

4. Mehta F, Theunissen R, Post MJ. Adipogenesis from bovine precursors. In: Methods in Molecular Biology. Vol 1889. Humana Press Inc.; 2019:111–125. doi:10.1007/978–1–4939–8897–6_8

5. Post MJ, Levenberg S, Kaplan DL, et al. Scientific, sustainability and regulatory challenges of cultured meat. Nat Food 2020 17. 2020;1(7):403–415. doi:10.1038/s43016–020–0112-z

6. Louis F, Piantino M, Liu H, et al. Bioprinted Vascularized Mature Adipose Tissue with Collagen Microfibers for Soft Tissue Regeneration. Cyborg Bionic Syst. 2021;2021:1–15. doi:10.34133/2021/1412542

7. Colle J, Blondeel P, De Bruyne A, et al. Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering. J Mater Sci Mater Med. 2020;31(4):1–15. doi:10.1007/s10856–020–06374-w

8. Kuppusamy P, Kim D, Soundharrajan I, Hwang I, Choi KC. Adipose and muscle cell co-culture system: A novel in vitro tool to mimic the in vivo cellular environment. Biology (Basel). 2021;10(1):1–12. doi:10.3390/biology10010006

9. Seo K, Suzuki T, Kobayashi K, Nishimura T. Adipocytes suppress differentiation of muscle cells in a co-culture system. Anim Sci J. 2019;90(3):423–434. doi:10.1111/asj.13145

10. Morrison O. UK start-up predicts lab-grown sausages on shelves by 2023 after technology ‘breakthrough.’ Published May 19, 2021. Accessed June 4, 2021. https://www.foodnavigator.com/Article/2021/05/19/UK-start-up-predicts-lab-grown-sausages-on-shelves-by-2023-after-technology-breakthrough

11. Ellis J. Meatable banks $47m from DSM, others for its cellular pork & beef. Published March 25, 2021. Accessed June 4, 2021. https://agfundernews.com/meatable-banks-47m-from-dsm-others-to-realize-the-pluri-potential-of-its-cellular-pork-beef.html

12. Southey F. Cultivating animal fat for plant-based meat: ‘Nobody wants a burger that tastes of coconut.’ Published March 4, 2021. Accessed June 22, 2021. https://www.foodnavigator.com/Article/2021/03/04/Cultivating-animal-fat-for-plant-based-meat-Nobody-wants-a-burger-that-tastes-of-coconut

13. Guo W, Pan B, Sakkiah S, et al. Persistent organic pollutants in food: Contamination sources, health effects and detection methods. Int J Environ Res Public Health. 2019;16(22). doi:10.3390/ijerph16224361

14. Kogevinas M. Human health effects of dioxins: Cancer, reproductive and endocrine system effects. Hum Reprod Update. 2001;7(3):331–339. doi:10.1093/humupd/7.3.331

Written by Anna Goddi, Associate & Thea Burke, Strategist for Helikon Consulting

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Scientific consulting in alternative proteins — plant-based proteins, cellular agriculture, and cell-based meat

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Helikon Consulting

Helikon Consulting

Scientific consulting in alternative proteins — plant-based proteins, cellular agriculture, and cell-based meat

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