Bodiou Vincent, Kumar Anitha Ajith, Massarelli Edoardo, van Haaften Tessa, Post Mark J, Moutsatsou Panagiota
Mosa Meat BV, Maastricht, Netherlands.
CARIM (The Cardiovascular Research Institute Maastricht), Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands.
Front Bioeng Biotechnol. 2024 Nov 1;12:1443914. doi: 10.3389/fbioe.2024.1443914. eCollection 2024.
To bring cultivated beef to the market, a scalable system that can support growth of bovine satellite cells (bSCs) in a serum-free and preferably also animal-free medium is of utmost importance. The use of microcarriers (MCs) is, at the moment, one of the most promising technologies for scaling up. MCs offer a large surface to volume ratio, they can be used in scalable stirred tank bioreactors, where the culture conditions can be tightly controlled to meet the cells' requirements (temperature, pH, dissolved oxygen). The inherent capacity of the cells to migrate from one MC to another, also known as bead-to-bead transfer, facilitates a scale-up strategy involving MCs. Previous studies have shown growth of bSCs on three commercially available MCs in serum containing media. Unfortunately there is currently no information available regarding their growth on MCs in serum-free conditions.
In this study, we aimed to find suitable serum-free media, MCs and attachment promoting compounds (APCs) supporting the growth of bSCs. Initially, six commercial MCs and three serum-free media were evaluated. The effects of three APCs were compared (vitronectin, laminin and fibronectin). Subsequently, the effects of different concentrations and modes of addition of the best performing APC were investigated.
Our results showed that Cytodex 1, Synthemax II and CellBIND supported bSCs' growth in all serum-free media. Overall, better growth was observed with Cytodex 1 in serum-free proliferation media. We showed that the use of laminin or vitronectin with Cytodex 1 can significantly improve cell growth and purity. Laminin also allowed attachment and growth of bSCs on Plastic MCs which had been previously unsuccessful without APCs. Finally, we optimized the use of vitronectin from a sustainability and process perspective, and showed that it can be used solely as a coating for Cytodex 1 (16-100 ng/cm) MCs, instead of as a medium supplement, enhancing cell attachment and proliferation.
为了将培养牛肉推向市场，一个可扩展的系统至关重要，该系统能够在无血清且最好也是无动物成分的培养基中支持牛卫星细胞（bSCs）的生长。目前，使用微载体（MCs）是扩大规模最有前景的技术之一。微载体具有较大的表面积与体积比，可用于可扩展的搅拌罐生物反应器中，在其中培养条件能够得到严格控制以满足细胞需求（温度、pH值、溶解氧）。细胞从一个微载体迁移到另一个微载体的固有能力，即所谓的珠对珠转移，有利于涉及微载体的扩大规模策略。先前的研究表明bSCs在含血清培养基中的三种市售微载体上生长。不幸的是，目前尚无关于它们在无血清条件下在微载体上生长的信息。
在本研究中，我们旨在找到适合支持bSCs生长的无血清培养基、微载体和附着促进化合物（APCs）。最初，评估了六种市售微载体和三种无血清培养基。比较了三种附着促进化合物（玻连蛋白、层粘连蛋白和纤连蛋白）的效果。随后，研究了最佳性能附着促进化合物的不同浓度和添加方式的影响。
我们的结果表明，Cytodex 1、Synthemax II和CellBIND在所有无血清培养基中都支持bSCs的生长。总体而言，在无血清增殖培养基中使用Cytodex 1观察到更好的生长。我们表明，将层粘连蛋白或玻连蛋白与Cytodex 1一起使用可显著改善细胞生长和纯度。层粘连蛋白还能使bSCs在塑料微载体上附着和生长，而此前在没有附着促进化合物的情况下这是不成功的。最后，我们从可持续性和工艺角度优化了玻连蛋白的使用，并表明它可以仅用作Cytodex 1（16 - 100 ng/cm²）微载体的包被，而不是作为培养基补充剂，从而增强细胞附着和增殖。
