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Mock meat- an expansion to tissue cell culture

This blog provides an in-depth insight into cell-based meat production. It reviews the overall art of bioprocess technologies in animal cell culture, and tissue engineering- cell lines, culture media, scaffolding, and bioreactors in clean/mock meat production.

Introduction- A protein-rich diet is essential for good health. But the reality is that in the future protein-based food systems will not fulfill the demands on a global scale as conventional meat products or traditional meat products are the only source of meat consumption. Meat consumption contributes to 35% of the total protein requirement of the Indian population. According to FAO, the meat demands increase up to 70% globally and by 2050, the protein requirements of almost 9.8 billion people need to be fulfilled sustainably and economically without causing food security and production issues.

  • Conventional meat- Animal slaughter poses threats to human health, the environment, and welfare. Slaughtering brings the risk of animal diseases like swine & avian influenza, salmonella, campylobacter, epidemics, and antibiotic misuse to the ecosystem. Herds are responsible for environmental destruction -14.5% of GHG emissions, 30% of terrains, and 8% of global freshwater have already contributed to depletion. Furthermore, rearing animals poses significant public health threats from antibiotic resistance and zoonotic disease epidemics as well as acute consumer risk in the form of foodborne illness from fecal contamination introduced during slaughter and rendering.

  • This increase in deforestation, ocean dead zones, and water and air pollution have increased the concerns about growing numbers of farm animals and the consequences of keeping them and their treatments. It is advised to improve conditions to omit ethical and health issues.

  • What is clean/ cultured meat? The term “cultured meat”, also described as in vitro, synthetic, or lab-grown meat refers to meat produced in a bioreactor with tissue engineering technology. Compared to conventional meat, cultured meat is secured in terms of financial concerns, animal welfare ethics, resource shortages, and public health issues. Cultured meat (CM) not only satisfies the demands on a global scale but also at the added nutritional value to the diet. These products have a similar taste and texture as conventional animal meat with nutritional attributes.

  • In recent years, it is practical to use skeletal muscle tissue engineering, stem cells, cell co-cultures, and tissue culture methods for in vitro meat culturing. Scientists use advances in tissue engineering; they obtain muscle tissue from a section of living samples with cell cultures. Therefore, it is important to develop efficient, safe, and large-scale production of cultured meat to reduce the production costs effectively.

Cells resources- The main challenge in tissue culture for cultured meat is to obtain starter cells to conduct effective proliferation and differentiation. Generally, stem cells are good at producing a population. Stem cell research is an established niche in animal science, crucial for clean meat research. Stem cells are a population of progenitor cells, which can differentiate into specialized tissues and increase their number by proliferation.

Several stem cell types have been identified, and cell sources applied to tissue engineering.

  • Bovine embryonic stem cells (ESC), could potentially differentiate into all cell types required to recapitulate skeletal muscle development.

  • Mesenchymal stem cells (MSC) are another promising candidate, due to their abundance and roles during muscle development, and their ability to differentiate into myocytes, adipocytes, fibroblasts, and ECs.

  • Induced pluripotent stem cells (iPSC) can also be considered a cell source for clean meat production. iPSC is simple to produce and is extensively studied, making them valuable for lowering the cost and research needed to develop stable stem cell lines.

  • Satellite cells (SCs) are adult stem cells that can be found in skeletal muscle tissues and participate in muscle repair.

These cells are first expanded and then induced for differentiation into muscle cells. Once they are converted into muscle cells, they are further grown in bioreactors to increase their number.

Serum-free media- Generally for animal cell culture serum-free media are used widely. They contain attachment factors, micronutrients, trace elements, growth factors, hormones, and protective elements for fast growth. It provides high yield via growth-promoting factors for a variety of mammalian cells. A serum-free medium usually consists of basal medium and medium supplements. The basal medium generally comprises amino acids, vitamins, glucose, and inorganic salts, which are essential factors in cell growth and metabolism.

The only drawback of using serum-free media is that they bring high contamination risks to the batch. Otherwise, it is a low-cost and safe medium. These mediums can facilitate large-scale production, promote easy handling in tissue engineering, and downstream processes, and scale up cultured meat.

Important considerations for bioreactor design- The following are considered key factors to be considered in the design of an upstream bioprocess for mammalian cells.

Bioreactors in the game- Among the reasons that large-scale production has yet to be seen are the difficulties associated with reactors and the process scaling up of cultured meat. In the biotech industry, there is an inverse relationship between a product's market size and its selling price. As of today, most products derived from mammalian cell cultures are in the high-value, low-volume territory, e.g., therapeutics and pharmaceuticals. So, here we have listed a few common bioreactors used in cell culture production on a large scale.

1. Bubble column reactor- single-use reactors are becoming easily popular. Bubble columns (including airlift reactors) show great potential and have low shear stress while operating as no agitation provision is there in the reactor. However, the local eddy energy dissipation associated with bursting bubbles is a major source of cell damage. Many scientists lost interest in bubble columns for this application.

2. Stirred tank reactor- STRs are preferred bioreactors over bubble columns where the cells are either freely suspended or attached to suspended microcarriers. As it is a continuous operation, 4-5 reactors should work weekly to yield smoother supply, causing high operational costs.

  • Microcarriers must be used in conjunction with a stirred tank or bubble column bioreactor for suspended cultures. The use of microcarriers in animal cell cultivation has long been established, and no major technical obstacles are expected to its application in cultured meat production. Ideally, if the microcarrier is biodegradable and/or edible, it can be integrated into the final product, eliminating a downstream separation step. General materials used in microcarriers are usually cross-linked pectin and polypeptides. It is easy to harvest cells from microcarriers with the same viability and physical importance. If intensive agitation is used in STRs, it is expected that microcarriers handle shear forces well without much destruction.

3. Perfusion tank reactor- The popularity of perfusion reactors with 3D scaffolding in tissue engineering has convinced several researchers that it is the best technique for cultured meat production. The flow of medium is laminar with minimum back mixing in this reactor. The backbone of the perfusion reactor is the scaffold.

  • Scaffolds- Tissues are difficult to grow in media without mechanical support, a solid surface is required for attachment and maximizing their surface. Scaffolds are macroporous sponge-like substances that provide mechanical support to the tissues. These scaffolds are fabricated in such architectural dimensions- 2D and 3D and molecular composition that they could be beneficial for clean meat production. If they have a striated texture that helps in resemblance to the structure of muscle cells, it gives more definition to cell growth. Scaffold material is defined by pore size distribution, porosity, and pore interconnectivity, and they are often generated from edible materials, such as proteins, polysaccharides, and native ECM. Other edible and inedible polymers like collagen, cellulose, and synthetic polymers can also be used as base materials in the preparation of scaffolds. The scaffold of higher porosity is meant to give improved nutrient flow ECM deposition, cell viability, cell adhesion, and cell proliferation.

Perfusion reactors meet-

  • Stable operation

  • Easy scale-up

  • Versatile- can be used for any cell type and any culture conditions

  • Lower investment cost on reactor

  • High-density culture

  • Product harvesting continuously

  • High productivity

  • Dilution rate not limited

  • Separation of viable and dead cells is possible

Based on the typical specific oxygen uptake of an animal cell, it is required to build a reactor that maintains mass transfer at certain dissolved oxygen in the batch. Additional factors like airflow and pressure applied through the bottom do not cause any damage to the cells. As animal cells need both oxygen and carbon dioxide for their growth, coarse sparger should provide a fast transfer rate of air. The fact that cells must be 100% retained back with the same viability, cross-flow filtration or tangential flow filtration method would be required for the recycling procedure. The following image depicts how reactor design impacts the operational parameters of the batch.

The potential success of cultured meat is for both industries and academia as we can see in the above image. This shows India's diversification in terms of company specialization, research and development, and business creation opportunities.

Applications and conclusion- It has been indicated that cultured meat involves approximately 78%–96% lower greenhouse gas emissions, 99% lower land use, and 82%–96% lower water use. hence it is procured that the overall energy balance will tip in favor of cultured meat when indirect costs and environmental benefits are considered.

There are online monitoring systems to analyze the quality of cultured meat and provide food safety guarantee technology with a low chance of bacterial contamination. The quality of cultured meat can be optimized through a controlled culture system and post-processing such as the composition proportion, nutrient content, taste, and flavor. Cultured meat will use safe and moderate concentrations of preservatives such as sodium benzoate to protect the growing meat from microbes so that no harm is given in any way.







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