Repeated and long-term cryopreservation of primary bovine myogenic cells to maintain quality in biomimetic cultured meat
Repeated and long-term cryopreservation of primary bovine myogenic cells to maintain quality in biomimetic cultured meat
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DOI：
10.3389/fsufs.2023.1023057
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发表时间：
2023-02
期刊：
影响因子：
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通讯作者：
Roka Kakehi;Azumi Yoshida;Hironobu Takahashi;Tatsuya Shimizu
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作者：
Roka Kakehi;Azumi Yoshida;Hironobu Takahashi;Tatsuya Shimizu
使用细胞培养技术生产的培养肉可以潜在地减轻与传统牲畜肉生产相关的许多伦理、环境和公共卫生问题。培养肉的工业化广泛采用需要新的方法来有效地收集高质量的细胞并保持其细胞质量。冷冻保存是一种广泛使用的技术，能够长期储存细胞而不会造成严重损害。在本研究中，我们专注于冷冻保存的可行性，以保持细胞质量的牛肌原性细胞从牛肉收获的基础上，我们独特的原代培养方法。将原代牛细胞在培养皿中孵育，然后在−80°C下冷冻保存1周或1年。解冻后，将细胞进一步培养几代以评价细胞增殖或分化成肌管的能力。此外，将细胞反复冷冻保存1周，每次以研究反复冻融的影响。因此，在-80 °C下长期（1年内）或重复（最多3次，每次1周）冷冻保存不会导致细胞增殖或分化的能力下降，这对培养肉生产很重要。我们还证实，冷冻保存不需要任何独特的细胞冷冻培养基。此外，基于我们的组织工程技术，我们冷冻保存的牛肌原细胞即使在冷冻1年后也具有形成肌节结构和产生肌肉收缩的能力。虽然这里描述的牛肌肉组织需要更成熟的结构和功能，以密切模仿天然肌肉组织，我们认为，肌原细胞的功能成熟是必不可少的，以产生一个“组织工程肉”，将有天然的营养，质地和味道，消费者将在未来的期望。这些结果揭示了冷冻保存质量控制的牛肌源性细胞的潜力，有助于培养肉生产的高质量细胞的稳定供应。
Cultured meat produced using cell culture technology can potentially alleviate many of the ethical, environmental, and public health concerns associated with conventional livestock meat production. The industrialization of cultured meat for wide-spread adoption requires new methods to efficiently collect high-quality cells and to preserve their cell quality. Cryopreservation is a widely used technique to enable the long-term storage of cells without causing severe damage. In this study, we focused on the feasibility of cryopreservation to maintain cell quality for storage of bovine myogenic cells harvested from bovine meat based on our unique primary culture method. Primary bovine cells were incubated in a culture dish and then cryopreserved at −80°C for 1 week or 1 year. After thawing, the cells were further cultured for several passages to evaluate the abilities of the cells to proliferate or differentiate into myotubes. Furthermore, the cells were repeatedly cryopreserved for 1 week each time to investigate the impact of the repeated freezing and thawing. Consequently, long-term (within 1 year) or repeated (up to 3 times for 1 week each) cryopreservation at −80°C caused no degradation in the abilities of the cells to proliferate or differentiate, which is important for cultured meat production. We also confirmed that the cryopreservation did not require any unique cell freezing media. Moreover, based on our tissue engineering technique, our cryopreserved bovine myogenic cells had the ability to form sarcomere structures and produce muscle contractions even after they were frozen for 1 year. Although the bovine muscle tissues described here require more mature structures and functions in order to closely mimic native muscle tissue, we believe that the functional maturation of myogenic cells is essential to produce a “tissue-engineered meat” that will have native-like nutrients, texture, and taste that consumers will expect in the future. These results reveal the potential of cryopreserving quality-controlled bovine myogenic cells to contribute to a stable supply of high-quality cells for cultured meat production.