Heyman Emma, Devriendt Bert, De Vlieghere Elly, Goethals Klara, Van Poucke Mario, Peelman Luc, De Schauwer Catharina
Veterinary Stem Cell Research Unit, Ghent University, Merelbeke, Belgium.
Laboratory of Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
NPJ Sci Food. 2024 Oct 1;8(1):70. doi: 10.1038/s41538-024-00313-7.
Sustainable food provision for a continuously growing human population is one of the major challenges for the next decades. Cultured meat represents one of the alternatives which is currently extensively explored. Yet, the most appropriate cell type, capable of long-term proliferation and myogenic differentiation, remains to be identified. Bovine mesenchymal stromal cells (MSCs) are considered as a promising cell source. Within the context of cultured meat production, it is mandatory to maximize cell yield per tissue source. Although many enzymatic methods to isolate MSCs from adipose tissue (AT) have been described, cell yield has never been compared. In this study, we evaluate 32 isolation conditions including four enzyme mixtures (Collagenase type I, Collagenase type I + Trypsin, Liberase and Collagenase type IV) at varying concentrations and incubation times, regarding their efficiency to isolate MSCs from bovine subcutaneous AT. The highest cell yield in combination with a low population doubling time was obtained using Liberase at a concentration of 0.1% for 3 h. MSC identity of the cells was confirmed by tri-lineage differentiation potential and cell surface marker expression. Subsequently, isolated cells were myogenically differentiated using 5-aza-2'-deoxycytidine and galectin-1. mRNA levels of the myogenic regulatory factors (MRF) myogenic factor 5 (MYF5), myogenic differentiation 1 (MYOD1), MYF6, and myogenin (MYOG) were increased, while less paired box 3 (PAX3) mRNA expression was observed when compared to undifferentiated MSCs. The presence of desmin (DES), tropomyosin (TM), and myosin heavy chain (MyHC) in myogenically differentiated bovine AT-MSCs was confirmed using immunofluorescence stainings. When considering MSCs from bovine AT as potential cell source to produce cultured meat, it is recommended to use 0.1% Liberase for 3 h to ensure a high cell yield.
为持续增长的人口提供可持续的食物供应是未来几十年的主要挑战之一。培养肉是目前正在广泛探索的替代方案之一。然而，能够长期增殖和进行肌源性分化的最合适细胞类型仍有待确定。牛间充质基质细胞（MSCs）被认为是一种有前景的细胞来源。在培养肉生产的背景下，必须最大限度地提高每个组织来源的细胞产量。尽管已经描述了许多从脂肪组织（AT）中分离MSCs的酶法，但从未对细胞产量进行过比较。在本研究中，我们评估了32种分离条件，包括四种不同浓度和孵育时间的酶混合物（I型胶原酶、I型胶原酶+胰蛋白酶、 Liberase和IV型胶原酶），以研究它们从牛皮下脂肪组织中分离MSCs的效率。使用浓度为0.1%的Liberase孵育3小时可获得最高的细胞产量以及较短的群体倍增时间。通过三系分化潜能和细胞表面标志物表达确认了细胞的MSCs特性。随后，使用5-氮杂-2'-脱氧胞苷和半乳糖凝集素-1对分离的细胞进行肌源性分化。与未分化的MSCs相比，肌源性调节因子（MRF）肌源性因子5（MYF5）、肌源性分化1（MYOD1）、MYF6和肌细胞生成素（MYOG）的mRNA水平升高，而配对盒3（PAX3）mRNA表达降低。使用免疫荧光染色证实了肌源性分化的牛脂肪组织来源的MSCs中存在结蛋白（DES）、原肌球蛋白（TM）和肌球蛋白重链（MyHC）。当将牛脂肪组织来源的MSCs视为生产培养肉的潜在细胞来源时，建议使用0.1%的Liberase孵育3小时以确保高细胞产量。
