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

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.

Purification of Pig Muscle Stem Cells Using Magnetic-Activated Cell Sorting (MACS) Based on the Expression of Cluster of Differentiation 29 (CD29)

“…Previously, we optimized the in vitro culture conditions for maintaining the stemness of pig muscle stem cells ( Choi et al, 2020 ). Unfortunately, approximately 30% of the nonmyogenic cells in the total cultured cells was observed without the purification process in our system, as determined by immunostaining for CD56 and CD29 ( Fig.…”

“…The cryopreserved muscle stem cells were thawed and cultured on Matrigel-coated dishes (Matrix TM ; SPL Life Science, Pocheon, Korea) in basic GM consisting of the Skeletal Muscle Cell Growth Medium-2 BulletKit TM (SkGM-2, Lonza, Basel, Switzerland) supplemented with 20 μM SB203580 (Cayman Chemical, Ann Arbor, MI, USA) as described previously ( Choi et al, 2020 ). The pig muscle stem cells were subcultured every 3 d. When the cells reached approximately 90% confluency, the cultured cells were dissociated using TrypLE™ Express (Gibco).…”

“…For producing cultured meat, it is crucial for muscle stem cells to be efficiently isolated and stably maintained in vitro at a large scale. In a previous study, we optimized the in vitro culture conditions to maintain the stemness of pig muscle stem cells for an expanded period ( Choi et al., 2020 ). For the purification of pig muscle stem cells, the density gradient centrifugation and preplating techniques have been widely used in pig studies.…”

Status of meat alternatives and their potential role in the future meat market — A review

“…Therefore, to launch cultured meat in the market, its production cost should be lowered, and quality characteristics should be improved. The optimization process should be preceded by the whole process of cultured meat [66]. Once cultured meat is produced with a similar quality to that of traditional meat, it may play an important role in increasing meat supplies because it will be the only actual meat that has animal protein [67,68].…”

Technical requirements for cultured meat production: a review

“…Food engineering has unlocked many such solutions till date, an alternative to animal meat being one of them. With the advent of breakthrough milestones in stem cell technology (Figure 2), it is now possible to direct stem cells towards highly differentiated cells, for instance, satellite cells (SCs) can be directed towards skeletal muscle cells in many animals [23,24]. A single cell can be used to produce massive quantities of skeletal muscle cells by way of stem cell technology which is better known as in vitro meat (IVM), in vitro meat agriculture (IMA), or cultured meat; this innovation is a promising alternative to livestock.…”