Optimization of culture conditions for maintaining pig muscle stem cells in vitro

Kwang-Hwan Choi¹^,^†, Ji Won Yoon¹^,^†, Minsu Kim¹, Jinsol Jeong¹, Minkyung Ryu¹, Sungkwon Park², Cheorun Jo¹^,³^,^*, Chang-Kyu Lee¹^,³^,^*

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¹Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

²Department of Food Science and Biotechnology, Sejong University, Seoul 05006, Korea.

³Institute of Green Bio Science and Technology, Seoul National University, Pyeong Chang, Kangwon-do 25354, Korea.

^*Corresponding Author: Cheorun Jo, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea. Phone: +82-2-880-4804. E-mail: cheorun@snu.ac.kr.

^*Corresponding Author: Chang-Kyu Lee, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea. Phone: +82-2-880-4805. E-mail: leeck@snu.ac.kr.

† These authors contributed equally to this work.

© Copyright 2020 Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Apr 29, 2020 ; Revised: May 18, 2020 ; Accepted: May 19, 2020

Published Online: May 21, 2020

Abstract

Muscle stem cells isolated from domestic animals, including cows and pigs, were recently spotlighted as candidates for the production of alternative protein resources, so-called cultured meat or lab-grown meat. In the present study, we aimed to optimize the in vitro culture conditions for the long-term expansion of pig muscle stem cells via the screening of various signaling molecules. Pig muscle stem cells were collected from the biceps femoris muscles of 3-d-old crossbred pigs (LYD; Landrace × Yorkshire × Duroc) and cultured in minimum essential medium (MEM)-based growth media. However, the pig muscle stem cells gradually lost their proliferation ability and featured morphologies during the long-term culture over two weeks. To find suitable in vitro culture conditions for an extended period, skeletal muscle growth medium-2 (SkGM-2), including epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580), was used to support the stemness of the pig muscle stem cells. Interestingly, pig muscle stem cells were stably maintained in a long-term culture without loss of the expression of myogenic marker genes as determined by PCR analysis. Immunostaining analysis showed that the stem cells were capable of myogenic differentiation after multiple passaging. Therefore, we found that basal culture conditions containing EGF, dexamethasone, and a p38 inhibitor were suitable for maintaining pig muscle stem cells during expanded culture in vitro. This culture method may be applied for the production of cultured meat and further basic research on muscle development in the pig.

Keywords: pig; muscle stem cells; EGF; dexamethasone; p38 inhibitor