Bioprocess development for production of 3D tissues to underpin creation of engineered meat

我们养活自己的方式正在成为一个日益严重的问题，许多发展中国家人口和收入的增长正在推动全球肉类需求的增长。畜牧业约占人为温室气体排放总量的14.5%，占用70%的耕地和27%的淡水用于维持牲畜和饲料生产1。传统的肉类生产及其在延续气候危机中的作用是一个明显的问题，目前正在影响气候，并将变得更糟。培养肉代表了这个问题的解决方案，通过使用实验室技术而不是牲畜饲养和屠宰从小细胞样品中生产实验室生长的动物组织。这项技术比畜牧业有许多好处，大大减少了土地和淡水需求以及温室气体排放。此外，在受控条件下生产的肉类可以不含可能感染消费者的细菌和病毒。这是在没有过度使用抗生素的情况下实现的，而过度使用抗生素会使抗生素耐药性疾病的增加持续下去。受控的生产条件还支持可定制的营养成分，这些营养成分可以富含所需的营养素，以消除缺乏，并降低某些化合物的水平，如胆固醇，这些化合物可能是有害的。2013年，汉堡的销售额达到2000万欧元，最近的一份好食品研究所报告描绘了一个不断增长的行业，仅在2020年就有3.66亿美元的投资。随着Good Meat公司在新加坡的一家餐馆销售养殖鸡肉，为消费者带来商业上可行的大规模养殖肉类的竞赛正在进行中。尽管GFI报告令人鼓舞地声明，商业上可行的大规模养殖肉类不需要更多的基本技术突破，但仍需要进行大量研究，以解决日益负担得起的生物化学和工程挑战，扩大生产规模，模仿屠宰肉的质地和营养成分。该项目旨在通过工程水凝胶胶囊生产培养肉组织，一种三维支架，细胞可以在其上生长成三维网络。这些包封的细胞可以在生物反应器中培养，不同细胞类型的共培养可以帮助复制肉组织的复杂性。为了模仿牲畜肉，培育的肉将需要一个由肌肉，脂肪和结缔细胞组成的复杂结构，共同提供肉的味道和营养价值。因此，牛间充质干细胞（bMSCs）将用于该项目，因为它们不仅易于生长，易于分离，而且还具有分化为肌肉和脂肪细胞的能力，这两者对于复杂的仿肉组织的共培养都是必需的。这些bMSCs将在其上生长的水凝胶胶囊将需要适合一系列参数。它们需要可食用并提供适合粘附细胞生长的3D结构，这些特征将由水凝胶生物化学组成定义，为了彻底研究理想的生长条件，将探索一系列水凝胶和胶囊尺寸。胶囊大小可以通过微调生产参数来改变，例如水凝胶注射速率和剪切力，并且可以使用膜挤出技术来选择特定的大小范围。该项目旨在定义一种优化的工艺，通过该工艺可以使用3D水凝胶胶囊培养复杂的牛组织，在生物反应器中生长，用于培养肉的规模化生产。等（2018）食品科学与技术趋势2 Hanga MP等（2020）生物技术生物工程; 117（10）：3029-3039。

The way we feed ourselves is becoming a growing problem, growing population and income growth in many developing countries is fueling an increase in global meat demand. Animal agriculture has been responsible for around 14.5% of total man-made greenhouse gas emissions, takes up 70% of arable land and 27% of fresh water usage for maintaining livestock and feed production1. Traditional meat production and its role in perpetuating the climate crisis is a clear problem which is currently affecting the climate and is set to get worse. Cultured meat represents a solution to this problem, by producing laboratory grown animal tissue from a small cell sample using laboratory techniques rather than livestock rearing and slaughtering. This technology has many benefits over livestock farming with drastic reductions in land and fresh water requirements as well as greenhouse gas emissions. Further, being manufactured under controlled conditions, the meat can be produced free from bacteria and viruses which could infect consumers. This is achieved without the excessive antibiotic usage that occurs in livestock farming which perpetuates the rise of antibiotic resistant diseases. Controlled manufacturing conditions also support customisable nutrient profiles which can be enriched in desired nutrients to eliminate deficiencies and have reduced levels of certain compounds such as cholesterol which are potentially harmful.Cultured meat has come a long way since the first 250,000 Euro burger in 2013 with a recent Good Food Institute report depicting a growing industry with 366 million dollars of investment in 2020 alone. With the company Good Meat selling cultured chicken in a Singapore restaurant the race to bring commercially viable large scale cultured meat to consumers is under way. Despite the GFI reports encouraging statement that there are no more fundamental technological breakthroughs required for commercially viable large scale cultured meat, there is still much research needed to tackle the biochemical and engineering challenges of increasing affordability, scaling up production and mimicking the texture and nutritional profile of slaughtered meat.This project aims to produce cultured meat tissues by engineering hydrogel capsules that will act as a 3D scaffold on which cells can grow into a 3D network. These encapsulated cells can be cultured within a bioreactor and the co-culture of different cell types can help replicate the complexity of meat tissue. In order to imitate livestock meat, cultivated meat will need a complex structure consisting of muscle, fat and connective cells which together provide the taste and nutritional value of meat. Bovine mesenchymal stem cells (bMSCs) will therefore be used in this project as not only are they easy and cheap to grow and easy to isolate but they also have the ability to differentiate into both muscle and fat cells, both of which are necessary for the co-culture of a complex meat-mimicking tissue2. The hydrogel capsules on which these bMSCs will be grown will need to fit a range of parameters. They will need to be edible and provide a 3D structure suitable to allow adhered cell growth, these characteristics will be defined by the hydrogels biochemical make up and to thoroughly investigate ideal growth conditions a range of hydrogels and capsule sizes will be explored. Capsule size can be altered through fine tuning of production parameters, such as hydrogel injection rate and shearing force, and specific size ranges can be selected using membrane extrusion techniques.This project aims to define an optimized process by which complex bovine tissue can be cultured using 3D hydrogel capsules, grown within a bioreactor for the scalable production of cultured meat.1Stephens, N. et al. (2018) Trends in Food Science & Tech2Hanga MP et al (2020) Biotech Bioeng; 117(10):3029-3039.