Novel Microcarriers for Scalable Clean Meat Production

2013年，第一个‘洁净肉’汉堡以25万美元的价格被生产、销售和在拍卖会上食用。生产汉堡的成本是惊人的，它只含有肌肉细胞，而且在制剂的某些部分使用了不可食用的材料。然而，这成为细胞农业领域的一块踏脚石，世界各地的研究人员和企业家开始试验新的细胞类型、生物材料、制造技术和生物工艺，以使清洁肉类原型更接近现实。尽管该领域取得了巨大进步，但清洁肉类行业仍处于初级阶段，仍需做大量工作才能使其成为负担得起和可持续的选择。持续存在的众多挑战之一是设计一种可扩展的过程，用于肉类中发现的细胞的扩增和分化。为此，必须考虑材料的成本、耐久性和机械性能，以及用于制造微载体的技术，以便它们能够承受搅拌槽生物反应器(STR)的条件，并促进细胞附着、生长和分化。通过将牛骨髓间充质干细胞(BMSC)分化为天然肉类中的组织(即肌肉、脂肪、结缔组织)，复制肉类的生物学特性也是很重要的。目的总体目标是设计和开发适合于可扩展和低成本的生物过程的可食用微载体，以生产肉末状产品，同时减少在细胞恢复阶段对有害的酶处理的需要。目的1)从BMSCs的黏附和扩增方面探索和寻找合适的可食用材料和制造技术；2)优化STR中微载体的操作条件，以支持细胞附着和维持细胞生长；以及3)评估新制造的微载体促进骨髓间充质干细胞向脂肪和肌肉细胞分化的潜力，这些细胞将构成肉末类产品的基础。微载体提供了细胞附着和生长的表面，因此在设计新型载体时需要考虑的重要因素是：足够的机械强度，使它们在STR中混合时不会破裂；生产成本低，便于放大；以及生物兼容材料，促进细胞附着和扩张。我们的目标是测试不同的技术来制造可食用的支架。更具体地说，我们将重点介绍静电纺丝和薄膜挤出的使用。一旦产生的微载体达到所需的标准，将首先在静态条件下测试它们维持脂肪来源的BMSC生长的能力，然后在旋转烧瓶中进行搅拌。我们将研究与生物反应器条件、微载体所承受的机械压力以及细胞的生长和存活有关的参数。在这项研究中，我们将集中于我们的可食用微载体支持BMSCs分化为肌肉和脂肪的能力。我们将在静态条件下进行小范围的工作，并进行诱导成肌和成脂分化的初步研究。我们的结果将为未来研究可食用微载体在搅拌釜式反应器中的性能和潜在的生物反应器放大奠定基础。成果在该项目结束时，我们将朝着创建上游细胞扩增和分化平台流程更近一步，该流程可以成为以培育的肉末为基础的产品的低成本商业生产的一部分。“

"In 2013, the first ever ''clean meat'' burger was produced, sold, and eaten at auction for $250,000. The cost of producing the burger was phenomenal, it only contained muscle cells, and inedible materials had been used in parts of the preparation. However, this became a steppingstone in the field of cellular agriculture and researchers and entrepreneurs across the world started to experiment with new cell types, biomaterials, manufacturing techniques, and bioprocesses to bring clean meat prototypes closer to reality. Despite huge advances in the field, the clean meat industry is still in its infancy, and a lot of work still needs to be done to make it an affordable and sustainable option.One of the many challenges that persists is in designing a scalable process for the expansion and differentiation of cells found in meat. To this end, it is imperative to consider the costs of materials, their durability and mechanical properties, as well as techniques used to manufacture the microcarriers so they can endure the stirred tank bioreactor's (STR) conditions and promote cell attachment, growth and differentiation. It is also important to replicate the biological features of meat too by differentiating bovine mesenchymal stem cells (bMSC) into tissues found in natural meat (ie muscle, fat, connective tissue).AimsThe overall aim is to design and develop edible microcarriers suitable for a scalable and cost-efficient bioprocess to produce minced meat-like products, while alleviating the need for detrimental enzymatic treatment at the cell recovery step.ObjectivesThis will be achieved by 1) exploring and identifying suitable edible materials and manufacturing techniques in terms of adherence and expansion of bMSCs; 2) optimising the operating conditions used in the STR for microcarriers to support cell attachment and sustain cell growth; and 3) evaluating the potential of the newly fabricated microcarriers to promote bMSC differentiation to fat and muscle cells that will form the basis of the minced meat like products. The microcarriers provide the surface on which the cells adhere and grow, and thus important factors to consider when designing new types are: adequate mechanical strength, so that they will not break up during mixing in the STR; low cost of production, to enable ease of scale up; and, biocompatible material, to promote cell attachment and expansion. We aim to test different techniques to fabricate the edible supports. More specifically, we will focus on the use of electrospinning and membrane extrusion. Once the microcarriers are generated to the required standards, they will be tested for their ability to sustain adipose-derived bMSC growth, first in static conditions and then under agitation in spinner flasks. We will investigate parameters in relation to the bioreactor conditions, the mechanical stresses endured by microcarriers, and the growth and viability of cells.In this study, we will concentrate on the ability of our edible microcarriers to support the differentiation of the bMSCs into muscle and fat. We will work at small scales under static conditions and carry out preliminary studies inducing myogenic and adipogenic differentiation. Our results will form the basis for future studies on the edible microcarrier performance in stirred tank reactors and potential bioreactor scale up. OutputAt the end of this project, we will be a step closer to creating an upstream cell expansion and differentiation platform process that can be part of the low-cost commercial manufacture of cultivated minced meat-based products. "