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Efficient isolation of high-quality MSCs is essential for both basic research and for the development of therapies targeting muscle-related diseases and for advancing cultured meat production. Traditional isolation methods often use crude proteolytic enzymes, which can lead to contamination and cytotoxicity. This study aimed to improve the isolation of muscle stem cells by using recombinant proteolytic enzymes\u2014collagenase and thermolysin\u2014while comparing their performance to crude enzyme treatments. The isolation of MSCs from chicken muscle tissue was conducted using both crude pronase and recombinant enzymes. The effectiveness of each method was evaluated based on tissue dissociation efficiency and the quantity of MSCs isolated. The results showed that the combination of recombinant proteolytic enzymes significantly improved muscle tissue dissociation, resulting in a higher yield of MSCs compared to crude enzymes. Furthermore, the MSCs isolated using recombinant enzymes exhibited comparable or superior proliferation and differentiation characteristics, including myogenic gene and protein expression and creatine kinase activity. These findings suggest that recombinant proteolytic enzymes offer a safer and more efficient alternative to crude enzymes for isolating MSCs. This method has potential applications in muscle research and the production of cultured meat, improving both safety and efficiency in stem cell-based technologies and fostering advancements in these fields.<\/jats:p>","DOI":"10.1007\/s44463-025-00051-8","type":"journal-article","created":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T09:13:10Z","timestamp":1770801190000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Optimizing chicken muscle stem cell isolation using recombinant proteolytic enzymes for cultured meat production"],"prefix":"10.1007","volume":"46","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0375-8170","authenticated-orcid":false,"given":"Jeong Ho","family":"Lim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7489-2393","authenticated-orcid":false,"given":"Sibhghatulla","family":"Shaikh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0290-3161","authenticated-orcid":false,"given":"Woo-Jong","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2496-0463","authenticated-orcid":false,"given":"Eun Ju","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0884-5994","authenticated-orcid":false,"given":"Inho","family":"Choi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,2,11]]},"reference":[{"key":"51_CR1","doi-asserted-by":"publisher","DOI":"10.1186\/s41232-023-00308-z","volume":"43","author":"K Ahmad","year":"2023","unstructured":"Ahmad, K., Shaikh, S., Chun, H. 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