Cultured meat production: what we know, what we don’t know and what we should know

Abstract Certain companies in the rapidly expanding cultured meat space claim that cultured meat is exactly the same in taste, flavour and nutrition as traditional meat, but without the need for animal slaughter, and bringing benefits for human health and the environment. It is important to point out that cultured meat is still in the early phases of development and, in many cases, the claims are based on assumptions rather than facts. In this work, we outline several knowledge gaps, mainly based on technical issues, with repercussions related to the possible benefits as well as economic and regulatory challenges. Highlights: Cultured meat made entirely of cultured cells does not yet exist Cultured meat production still faces several hurdles including the omission of animal-derived components and economic viability A lack of transparent production methods limits the evaluation of product characteristics and environmental impact


Introduction
Though cultured meat companies have emerged only in the last 10 years, the underlying science dates back about 4 decades and was developed within the medical field, mostly in academic labs.The aim of the underlying tissue engineering research was (and is) to create 3-dimensional cell-based constructs which resemble a tissue as it occurs in the body.Applications include (damaged) tissue replacement, drug screening and disease modelling.As for skeletal muscle tissue, interestingly, initial developments were based on cultured chicken myotubes (Vandenburgh and Kaufman 1979;Vandenburgh and Kaufman 1980;Vandenburgh et al. 1988) and then later moved to human myotubes towards clinical applications (Gholobova et al. 2020).Despite this time span, still, many issues remain to be solved to recreate large (exceeding 1 mm thickness) pieces of tissue, some of which also apply to the cultured meat field.The challenges for the cultured meat field have been summarised in (Thorrez and Vandenburgh 2019) and currently, these remain.

Definitions of cultured meat
Cultured meat is a term that covers several types of products.In a broad sense, it is a hybrid product that in part contains animal cells.Other ingredients could be derived from plants, algae, bacteria, or fungi to formulate the bulk of the product.In addition, synthetic ingredients may also be added, such as colourants, binders, flavourings, to enhance the techno-sensorial properties of the product.The final product is typically shaped to resemble ground meat, used for manufacturing sausages, hamburgers and nuggets.The most well-known example of such a product is the cultured meat hamburger that was presented in 2013 by Mark Post, which was based on thousands of cultured tissue strips (Kupferschmidt 2013).These strips contained either animal-derived collagen or fibrin mixed with bovine cells.It is unknown whether the cells were differentiated to myotubes and if so, to what extent.Since these strips were white, beetroot juice and saffron were added for colour.Breadcrumbs and a binder were added to provide some texture, and flavour was enhanced by caramel and cooking in butter (Kupferschmidt 2013).
CONTACT Lieven Thorrez lieven.thorrez@kuleuven.be Another example of a hybrid cultured meat product is Good Meat chicken nuggets, which was the first cellbased product authorised for sale and can be boughtpending reservation-in a butchery and a few restaurants in Singapore.The nuggets contain immortalised chicken fibroblasts, that are not able to form myotubes and probably rely on other plant-based components to mimic the fibrous texture.
One might argue that cultured meat should be indistinguishable from meat.This would imply that the tissue structure is grown in vitro with a similar result as what occurs in vivo.A steak is then typically used as a reference, representing complexly organised myofibers with deposits of intramuscular fat surrounded by extracellular matrix.In 2018, Aleph Farms claimed to produce the first cultured steak, which was based on textured soy protein, making it a hybrid product discussed above (Ben-Arye et al. 2020).To understand how cultured meat differs from meat, it is important to refer to the development and structure of skeletal muscle.During embryonic development, cells fuse to form myotubes, typically containing hundreds of nuclei.In vitro, this fusion can be induced after the cell expansion, and the fusion process takes a few days.Nevertheless, the resulting myotubes in vitro are composed of tens of nuclei at best (Okamoto et al. 2022;Terrie et al. 2022), not hundreds (Cramer et al. 2020).The next step in muscle development is maturation of the myotubes.For muscle to exert their contractile function, large amounts of contractile proteins are being synthesised which are subsequently organised in a contractile apparatus with a striated appearance.During this maturation process, the highly organised contractile apparatus takes up most of the cell volume and is pushing the nuclei to the periphery, and the myotube transitions to a myofiber.The difference with fused bovine myotubes in vitro can easily be appreciated by comparing the amount of contractile proteins.Microscopic images reveal fused bovine myotubes that typically are very thin with central nuclei (Ben-Arye et al. 2020;Kolkmann et al. 2022).Simultaneously, surrounding cells produce an extracellular matrix, which is of key importance to the organisation and mechanical characteristics of the muscle tissue.This maturation process takes a much longer time, starting during foetal development and continuing postnatally.Depending on the species, muscle development to its mature state takes months or even years.At present, in vitro-formed myotubes lack maturation (Yoshimoto et al. 2020).To reach better maturation, protocols and devices must be further developed which allow the stimulation of the tissue (Terrie et al. 2022).In any case, further tissue development beyond cell fusion will add time to the production process and will inevitably result in higher production costs.Therefore, to this date, cultured steak remains fully hypothetical.Despite the lack of evidence, mainstream media have uncritically echoed the claims of cultured meat companies.

Towards animal-free components
Considerable efforts have been made in the development of serum-free cell culture media.Typically, growth media for mammalian cells contain 10-30% serum, mostly foetal bovine serum (FBS).Efforts to create alternative media for cultured meat production so far have met limited success.Different formulations available on the market, including media and supplements, that are primarily designed for pluripotent stem cell culture, have been compared for bovine myoblasts (Kolkmann et al. 2020).Although some of these showed shortterm cell growth, the cells could not proliferate as fast as in a serum-containing medium.More recently, two groups were able to develop serum-free media formulations which allowed cell growth over 15-25 population doublings, however with limited retention of their fusion capacity (Stout et al. 2022;Kolkmann et al. 2022).Additionally, serum-free cultures usually require the simultaneous use of special matrices for cell attachment, such as recombinant vitronectin or laminin.Besides recombinant proteins, other non-typical cell culture supplements from various sources have been proposed such as algal extracts (Okamoto et al. 2022) and plant protein isolates (Stout et al. 2023).The proposed supplements are rarely validated beyond short-term cell growth.A thorough characterisation of cells grown in the presence of these supplements is still needed.

Economic challenges in cultured meat production
The economic viability of cultured meat is to a large extent dependent on the amount and price of media and growth substrates that are needed.A main limitation of developed media and matrix proteins is their high cost.As the market for recombinant proteins is growing, existing and new companies are now competing in the cellular agriculture field, which may bring down the price (Singh et al. 2022).When referring to upscaling processes, sometimes the term "brewing" is used.However, mammalian cell culture is very different from brewing processes involving yeast.Most importantly, the doubling time of yeast is about 1.5 h and that of bacteria is <1 h, whereas the doubling time of animal cells is in the range of 24 h, which makes animal culture extremely vulnerable to microbial contamination.This vulnerability imposes additional costs associated with maintaining high levels of sterility in every step of the culture process.Moreover, as discussed above, mammalian cells require much more to grow than just water and sugar.Long culture periods in combination with expensive medium composition make it difficult to reduce the cost of the production process.The animal body can be seen as a very complex bioreactor that grows and maintains >10 kg mass per litre of blood.In comparison, the yield from stir-tank bioreactors is currently in the range of 2-50 g/L (Humbird 2021).To obtain 1 kg of cellular material, the final volume will thus be >10 litre and likely a multitude of that as at least parts of the medium need to be replaced regularly.It is clear that media costs would need to drop below a fraction of a euro for cultured meat to come at par with the cost of meat.Current media prices at the research scale are still at least a factor 1000 too high.Other concerns regarding upscaling, such as manufacturing facilities have also been raised (Wood et al. 2023).In an extensive techno-economic analysis, it was concluded that the price is unlikely to drop below 25$/kg of wet cell mass (Humbird 2021).

Taste, texture and nutritional value of cultured meat
When perhaps technological barriers and economic issues can be resolved, the next question is whether the texture, taste and nutritional value of meat can be approximated.Still not much is known regarding these characteristics (Fraeye et al. 2020).The primary reason for that is the lack of products available to test, compounded by the lack of transparency in existing products and the definition of cultured meat.First cultured meat prototypes are mainly hybrid products containing a mixture of plant and cell-based ingredients, but no requirements exist on what should be the percentage of cultured cells.Can a plant-based product containing a few percent of cultured cells represent cultured meat in nutritional profile?Conventional meat is a good source of highly bioavailable amino acids, is rich in specific minerals (e.g.heme-iron, zinc) and vitamins (especially B-vitamins, e.g.B12, and vitamin D), and contains long-chain polyunsaturated fatty acids and bioactive compounds such as creatine and carnosine.Plant-based alternatives have other nutritional benefits but cannot be considered equivalent in terms of nutritional composition.Diets exclusively consisting of plant-sourced foods may be deficient in several critical nutrients and may require supplementation.It is therefore interesting to know whether cultured cells would fill these nutritional gaps or will have to be fortified as well.Recently, it has been shown that the amino acid profile and taste of cultured bovine and avian satellite cells do not yet match that of meat (Joo et al. 2022).Cultured satellite cell mass, of course, is still quite far from a final product.The addition of fat cells, for example, is shown to contribute both to the aroma and texture (Dohmen et al. 2022).Moreover, post-mortem processes determine the transition from muscle to meat (Fraeye et al. 2020).The formation of the actomyosin complex is determining properties such as tenderisation and flavour development.The intracellular content of cultured cells may be very different from adult muscle fibres as already discussed above.Therefore, it is not clear to what extent meat maturation processes would be applicable for cultured meat production.The textural properties of cultured meat in the form of sausage were found to be similar to traditional meat (Paredes et al. 2022), however, the described cultured meat was a black box.Therefore, until cultured meat is defined more strictly, it is impossible to tell what nutritional, textural and taste properties can be attributed.

Regulatory frameworks for cell-based foods authorisation
Worldwide, a number of countries are putting regulatory procedures forward for cell-based food authorisation.Some countries, such as Israel, Brazil and Qatar, announced that they started working on these procedures, while others have them already outlined.Till now, worldwide only two products have been authorised for sale: chicken nuggets from Good Meat (US and Singapore) and cultivated chicken from Upside Foods in the US (announced on the companies' websites).In the European Union, the framework is the regulation 2015/2283 on novel foods, in which it is specified that novel foods must undergo a safety evaluation by the European Food Safety Authority (European Parliament and Council 2015).If genetic modification methods are used, the EU has additional regulations that may apply.A problem for regulatory agencies, academics and ultimately all consumers, is the unavailability of protocols and product information in the public domain.Despite the sentiment to move towards open science, developed methods in the cultured meat field are largely kept secret.Production processes should be known in order to gauge the possible dangers.This may reveal potential sources of chemical or biological contaminants, such as trace products, leachables from the used equipment, etc.Though the legislative framework was already put in place in several countries, the applications are somewhat lagging behind.This indicates that the technology and economics of cultured meat have not yet reached the level required for sufficient production.

Environmental impact of cultured meat
The environmental impact of cultured meat is based on many assumptions, as still, no protocols are available that fully describe an existing cultured meat product (Rodr ıguez Escobar et al. 2021).Though initial life cycle assessment (LCA) studies suggested reductions in energy, water and land use (Tuomisto and Teixeira de Mattos 2011), later studies gradually attenuated those benefits.A recent analysis (Tuomisto et al. 2022) was based on the use of Chinese Hamster Ovary (CHO) cells.Nor the species, nor the tissue type seemed very much of interest for consumption, and they do not have the capacity to fuse and form myotubes.In addition, the CHO cells (which are already immortalised cells) had been mutagenized to optimise growth.

The use of immortalised cell lines
The use of immortalised cells, which can divide indefinitely, might be necessary to obtain sufficient cell numbers.However, it has given rise to controversy whether or not these may pose risks to inducing cancer following consumption.Several barriers are present, making such claim very unlikely.First, it should be pointed out that although immortalised cells are able to divide, they do not necessarily have other characteristics such as growth in a hypoxic environment.Tumour cells could only grow in a xenogeneic environment in the absence of an active immune system.Furthermore, cells would need to pass the epithelial barrier of the upper gastrointestinal system, before being destroyed in the acidic environment of the stomach.Heat treatment of cultured meat products at the end of the production phase also alleviates any hypothetical concern related to living cells.However, the use of immortalised cells may lead to other concerns, for example, the risk for allergic reactions.The Food and Agriculture Organisation points out that some cell lines, in particular shellfish, maybe a source of potential new allergens (FAO & WHO 2023).

Future perspectives
There are now worldwide >150 companies active in the field of cultured meat which together have raised $2.8 billion since 2016 (GFI 2022).Claims about cultured meat being similar in taste to traditional meat, but better for animals and the environment, certainly attract investment.However, there is very little publicly available evidence that these claims hold true at present.Overcoming the existing challenges will require a number of technological developments, which are difficult to follow and verify in a highly competitive and secretive environment of R&D start-ups.
Academic research has also been gaining interest in the 10 years that passed since the demonstration of the first cultured meat hamburger.Some scientific progress has been made regarding the development of serum-free media and characterising the expansion of animal cell lines.In the next decade, many issues regarding creating tissue structure, determination of nutritional content, characterisation of potential risks and calculation of environmental impact need to be tackled further.To independently investigate all the open questions, it is important that next to investments in companies, academic research is funded.