Quantifying Biomass and Visualizing Cell Coverage on Fibrous Scaffolds for Cultivated Meat Production
Xinxin Li, Khin Thu Lwin, Hirunika U. Kumarasinghe, Lilianne Iglesias-Ledon, Eesha Bethi, Yushu Wang, Colin Fennelly, Ryan Sylvia, Sonja Hatz, Timothy Olsen, Thomas Herget, Ying Chen, David Kaplan
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引用次数: 0
Abstract
Cultivated meat represents a transformative solution to environmental and ethical concerns of traditional meat industries, replicating livestock meat's texture and sensory attributes in vitro with a focus on cost, safety, and nutritional quality. Central to this process are biomaterial scaffolds that support tissue development from isolated animal cells grown in or on these matrices. Understanding scaffold interactions with cells, including scaffold degradation and biomass production, is crucial for process design and for scaling-up goals. In this article, we outline comprehensive methods to quantify scaffold-cell interactions for such scenarios, focusing on biomaterial scaffold degradation and changes in cell biomass [measured by cell weight, extracellular matrix (ECM) deposition, and cell coverage] during cell culture. We introduce two methodologies for assessing cell coverage: fixation and staining for detailed imaging analysis, and non-invasive, real-time evaluation across scaffolds. Here we focus on fiber-based scaffolds, while the assessments can be extrapolated to 2-dimensional (2D; films), and in part to 3-dimensional (3D; sponge) systems. Utilizing the C2C12 mouse myoblast cell line as a gold standard, the protocols deliver precise, step-by-step instructions for preparing fiber scaffolds (using silk proteins here), seeding cells, and monitoring key parameters for cultivated meat production, providing a framework for advancing cellular agriculture techniques. © 2024 Wiley Periodicals LLC.
Basic Protocol 1: Fabrication and preparation of silk fiber scaffolds for cell seeding
Support Protocol 1: Cultivation of C2C12 cells and seeding onto fibrous scaffolds
Basic Protocol 2: Quantification of decellularized yarn scaffold degradation during cell culture
Basic Protocol 3: Quantification of biomass variation and ECM deposition on yarn scaffolds during C2C12 cell culture
Basic Protocol 4: Visualization of cell-laden yarn scaffolds and determination of cell coverage ratio using confocal microscopy
Support Protocol 2: Real-time imaging of cell-laden yarn scaffolds using Celigo system
Support Protocol 3: Applying green CellTracker fluorescent probes to C2C12 cells seeded on yarn scaffolds
栽培肉是解决传统肉类行业环境和道德问题的变革性方案,可在体外复制牲畜肉类的质地和感官特性,同时注重成本、安全性和营养质量。这一过程的核心是生物材料支架,它支持在这些基质中或基质上生长的分离动物细胞的组织发育。了解支架与细胞的相互作用,包括支架降解和生物质生产,对于工艺设计和扩大规模目标至关重要。在本文中,我们概述了在这种情况下量化支架-细胞相互作用的综合方法,重点是生物材料支架降解和细胞培养过程中细胞生物量的变化(以细胞重量、细胞外基质(ECM)沉积和细胞覆盖率衡量)。我们介绍了两种评估细胞覆盖率的方法:用于详细成像分析的固定和染色法,以及跨支架的非侵入式实时评估法。在此,我们将重点放在以纤维为基础的支架上,而这些评估可推广到二维(2D;薄膜)以及部分三维(3D;海绵)系统。利用 C2C12 小鼠成肌细胞系作为黄金标准,这些方案提供了精确的分步指导,用于制备纤维支架(此处使用丝蛋白)、播种细胞和监测培养肉生产的关键参数,为推进细胞农业技术提供了一个框架。© 2024 Wiley Periodicals LLC.基本方案 1:制造和制备用于细胞播种的丝纤维支架 支持方案 1:培养 C2C12 细胞并将其播种到纤维支架上 基本方案 2:细胞培养期间脱细胞纱线支架降解的量化 基本方案 3:C2C12 细胞培养期间纱线支架上生物量变化和 ECM 沉积的量化 基本方案 4:使用共聚焦显微镜观察细胞负载的纱线支架并确定细胞覆盖率 支持方案 2:使用 Celigo 系统对细胞负载的纱线支架进行实时成像 支持方案 3:对播种在纱线支架上的 C2C12 细胞使用绿色 CellTracker 荧光探针。
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