Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jul;40(4):659-667.
doi: 10.5851/kosfa.2020.e39. Epub 2020 Jul 1.

Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro

Kwang-Hwan Choi  1 , Ji Won Yoon  1 , Minsu Kim  1 , Jinsol Jeong  1 , Minkyung Ryu  1 , Sungkwon Park  2 , Cheorun Jo  1   3 , Chang-Kyu Lee  1   3
Affiliations

Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro

Kwang-Hwan Choi et al. Food Sci Anim Resour. 2020 Jul.

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 (Landrace×Yorkshire×Duroc, LYD) and cultured in minimum essential medium-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, 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: EGF; dexamethasone; muscle stem cells; p38 inhibitor; pig.

PubMed Disclaimer

Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Fig. 1.
Fig. 1.. Pig muscle stem cells cultured in 10% fetal bovine serum (FBS)-containing minimum essential medium (MEM).
Muscle stem cells isolated from the biceps femoris muscle of 3-d-old LYD pigs were cultured with 10% FBS-supplemented MEM. (A) The morphological changes of the pig muscle stem cells during the in vitro long-term culture. (B) The expression pattern of myogenic marker genes during the in vitro long-term culture as measured using qPCR. LYD, Landrace×Yorkshire×Duroc. Scale bar=400 μm.
Fig. 2.
Fig. 2.. Optimizing culture conditions for supporting the stemness of pig muscle stem cells in vitro.
To find suitable in vitro culture conditions for pig muscle stem cells, various culture components were tested. (A) Comparative analysis of the proliferation rate of pig muscle stem cells cultured with 10% FBS-supplemented MEM and SkGM-2. (B) The effect of basal media on pig muscle stem cells during the in vitro long-term culture. (C) The effect of basal media on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. (D) The effect of SB203580 on pig muscle stem cells during the in vitro long-term culture. (E) The effect of SB203580 on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. * p<0.05, ** p<0.01, *** p<0.001. The significance of differences was determined between the control and treated groups. FBS, fetal bovine serum; MEM, minimum essential medium; P, passage. Scale bar=400 μm.
Fig. 3.
Fig. 3.. The myogenic potential of pig muscle stem cells cultured in SkGM-2-supplemented SB203580.
The myogenic ability of pig muscle stem cells cultured in SkGM-2-supplemented SB203580 was examined and defined using immunostaining of myosin heavy chain (MHC). Red and green fluorescence represent nuclei and MHC, respectively. Scale bar=400 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bentzinger CF, Wang YX, Von Maltzahn J, Soleimani VD, Yin H, Rudnicki MA. Fibronectin regulates wnt7a signaling and satellite cell expansion. Cell Stem Cell. 2013;12:75–87. doi: 10.1016/j.stem.2012.09.015. - DOI - PMC - PubMed
    1. Bernet JD, Doles JD, Hall JK, Kelly Tanaka K, Carter TA, Olwin BB. p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice. Nat Med. 2014;20:265–271. doi: 10.1038/nm.3465. - DOI - PMC - PubMed
    1. Campion DR, Richardson RL, Reagan JO, Kraeling RR. Changes in the satellite cell population during postnatal growth of pig skeletal muscle. J Anim Sci. 1981;52:1014–1018. doi: 10.2527/jas1981.5251014x. - DOI - PubMed
    1. Cao Y, Kumar RM, Penn BH, Berkes CA, Kooperberg C, Boyer LA, Young RA, Tapscott SJ. Global and gene-specific analyses show distinct roles for myod and myog at a common set of promoters. Embo J. 2006;25:502–511. doi: 10.1038/sj.emboj.7600958. - DOI - PMC - PubMed
    1. Chakravarthy MV, Davis BS, Booth FW. IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle. J Appl Physiol. 2000;89:1365–1379. doi: 10.1152/jappl.2000.89.4.1365. - DOI - PubMed