Investigating proliferation and differentiation capacities of Hanwoo steer myosatellite cells at different passages for developing cell-cultured meat

The aim of study was to investigate proliferation and differentiation capacities of Hanwoo myosatellite cells for the development of Hanwoo cell cultures. From P1 to P19, the number of live cells decreased and the cell size increased. It was confirmed that the PAX7 mRNA was higher in P3 than P6 and P9 (p < 0.05). The maximum differentiation score was measured from P1 to P12. The maximum differentiation score maintained high from P1 to P10. Immunostaining was performed for both P1 and P10 cells to investigate differentiation characteristics. And there were no significant differences in differentiation characteristics between P1 and P10 cells. MYOG mRNA was low, whereas C-FOS mRNA was high (p < 0.05) in the late passage. Myosin and Tom20 protein also showed low values in the late passage (p < 0.05). In conclusion, our results suggest that it is appropriate to use P1 to P10 for the production of cultured meat using Hanwoo muscle cells. If cell culture meat production is performed without differentiation, the passage range may increase further. These results provide basic essential data required for further development of Hanwoo cell cultures, which could provide a valuable source of protein for human populations in the future.


Introduction
Today's animal husbandry techniques must mass produce high-quality, inexpensive meat, milk, and eggs through eco-friendly, socially responsible, and economically sustainable production systems 1 .In short, the livestock industry contributes strongly to solving global food security problems, as well as provides economic value and improves national well-being through increased food security and nutrition 2,3 .The world population was 6.9 billion in 2010, 7.7 billion in 2020, and is expected to be 9.1 billion in 2050 3 .Globally, average personal income and population growth are increasing the meat consumption per capita 4 .Even if meat consumption declines in developed countries, global meat consumption will likely continue to rise as developing countries such as China, India, and Russia increase their meat consumption with rising personal incomes 5,6 .As the world population grows, meat production increases to meet the growing demand for meat.The problem here is the steep increase in livestock production.
Animal agriculture contributes about 14.5% of global greenhouse emissions and occupies about 77% of the earth's habitable land to raise and fed livestock while only providing about 17% of global calory supply 7 .Another concern is that climate change issue will affect the sustainability of the livestock industry 8,9 .It will likely include decreased pasture productivity, increased instances of livestock parasites and diseases, and increased competition for land and water 10 .There are many countries vulnerable to future animal protein shortages due to climate change and populations growth 11 .To solve some of the environmental issues that come with livestock production, the livestock industry needs to make meat production more environmentally friendly.Additionally, the livestock industry needs to reduce the water and grain required to produce animal protein 5 .In light of these issues, research into more e cient protein production methods is ongoing and aims to comply with environmental and animal welfare issues while increasing food security 12 .Recently, the term 'cell agriculture' has been proposed for the eld of work that focuses on growing cells to be used as food, cosmetics, or ingredients 13 .Cultured meat technology is the in vitro production of cells from certain stem cells to produce proteins and structures similar to animalbased meat 14,15 .If there are appropriate strategies for realizing cultured meat products, they can be a solution to the problems of industrial livestock farming 16,17 .Consumers who want to consume animal protein and are responsible for the state of livestock production are also interested in cultured meat as a sustainable alternative [18][19][20] .In order for cultured meat to be accepted as a meat substitute and industrialized, technical challenges including regulation, cost, mimic and e ciency must be met 20,21 .One major technical challenge is to produce a product as similar to currently consumed meats as possible.This is done by creating complex structures containing organized myo bers, various fats and bones in cultured meat products 16 .The basic research and developments should be established before the eld can make a headway in providing consumers with new cultured meat products 20,21 .It is also important to maintain the ability to proliferate and differentiate cells used in cultured meat production.Hanwoo (Korean cattle) as an indigenous beef cattle are known to have an inferior ability to produce meat because of a low milk producing capacity and slow growth rate, while having a relatively favorable meat quality 22 .Hanwoo is the most popular beef consumed in Korea.Due to the genetic ability of Hanwoo, its deposition of fat is larger than that of European breeds.It is characterized by excellent eating quality and unique scent 23 .Therefore, the purpose of this study was to contribute to the development of cultured meat technology.There are few studies on cultured meat using Hanwoo satellite cells.In this experiment, the proliferation and differentiation of Hanwoo satellite cells was observed through subculture.And the experimental results are expected to become standard data for the production of cultured meat using Hanwoo muscle satellite cells.A range that can be used for the production of cultured meat of the estimated Hanwoo muscle cells was proposed.

Materials and Methods
All animal studies were approved and performed within the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University, Republic of Korea.

Primary Hanwoo muscle satellite cell isolation and uorescence-activated cell sorting (FACS)
The top round muscle tissues of a 33-month-old and 34-month-old Hanwoo (Korean native cattle) steer carcass were collected from FarmStory Hannaeng Central Factory in Cheongwon-gu, Cheongju-si, Chungcheongbuk-do.After storage in an ice box and transport to the lab, the muscle tissues were collected and dissociated with a collagenase mix (Worthington, Cat # LS004176).The connective tissue and muscle satellite cells were then separated for 5 min at 800 g using a centrifuge.The obtained Hanwoo muscle satellite cells were then nally stored in freezing medium to be stored in liquid nitrogen until the experiment (Cell culture freezing medium, Gibco, Cat # 12648010).These cells were considered passage 0.

Satellite cell proliferation culture and myogenic differentiation
The asks were coated with 0.05% bovine collagen type (sigma) for proliferation culture and coated with matrigel (Corning) for differentiation culture.After thawing the Hanwoo muscle satellite cells, they were cultured on collagen coated asks in GM and cultured on Matrigel coated asks in differentiation medium (DM, DMEM-Dulbecco's Modi ed Eagle Medium (Gibco, Cat # 11995065), 2% FBS, 1% PSA).The cells were stained with trypan blue for measurement via automated cell counter (Countess, Invitrogen).
Hanwoo satellite cells were then seeded 1800 live cells / cm 2 on asks and these were placed into an incubator at 37 ℃, 5% CO 2 .These cells were set passage 1.After 24 hours, the passage 1 cells were set to proliferation day 1 and incubated at 37 ℃, 5% CO 2 until 6th day.

Subculturing of Hanwoo muscle cells
On 6th day of passage 1 Hanwoo cells, the growth medium was removed from ask and the ask was washed with PBS before being treated with trypsin-EDTA to detach the cells and make a cell suspension.The cell suspension was then neutralized with TNS (Trypsin Neutralizer solution, 2% FBS in PBS), centrifuged at 350 g for 5 min, and supernatant was removed.After staining the cells with trypan blue, the number of living cells was counted using an automated cell counter (Countess, Invitrogen).Cells were then reattached by seeding 1800 live cells / cm 2 into newly prepared coated asks and the cells in those asks were considered to be in their second passage (passage 2).This process proceeded to passage 19.For the differentiation phases, the growth medium was replaced with the differentiation medium on day 6.After 24 hours, those cells were marked as differentiation day 1 and they were allowed to incubate for a full 6 days at 37 ℃, 5% CO 2 .

Differentiation image analysis from bright eld microscopy
For each passage, bright eld images were captured every 24 h to create for 6 days differentiation measurements.Images were scored by six independent observers based on a scoring system ranging from 0 (no differentiation) to 6 (high proportion of myotubes).Representative bright eld microscopy images became the criterion for the maximum differentiation score 25 .

Images acquisition and analysis
The proliferation and differentiation of Hanwoo muscle cells in all passages were observed with the EVOS M5000 imaging system (EVOS M5000, Invitrogen, Cat # AMF5000) (Figure 1, 2, 4).Four images of stained p1 and p10 cells were randomly acquired (Figure 5) and the average width of myotubes (myotube width ( )), the number of myotubes, myotube area ratio on the image (myotube area (%)), the number of nuclei, nucleus area ratio on the image (nucleus area (%)), and the fusion index (%) were measured by Image J (NIH, Bethesda, MD, United States) 26 .Myotubes were classi ed as elongated structures containing three or more nuclei within a single membrane structure.The number of times the width of myotubes is measured is the number of myotubes.The width of myotubes were measured both at the beginning of relatively thick myotubes, as well as at the beginning of the relatively thin myotubes that split from the thick myotubes on the image.The fusion index was calculated as the number of nuclei incorporated into myotubes expressed as a percentage of the total number of nuclei in the image frame 27,28 .

Real-time reverse transcription (RT)-quantitative PCR
The cultured cells were collected, and total RNA was extracted using the Total RNA Extraction Kit (iNtRON Biotechnology, Cat # 17221, Korea) according to the manufacturer's instructions.cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Cat # 4368814).Real-time quantitative PCR was performed using Fast qPCR 2x SYBR Green Master Mix (Cat # EBT-1821).
Ampli cation was conducted as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec, 52-55 °C for 1 min.The target genes were PAX7, MYOD, C-FOS, MYOG, and GAPDH 29 .The primers used to amplify each gene are shown in Table 1.The mRNA quantities were analyzed using the 2−ΔΔCT method 30

Results
Timing to detach muscle cells for subculture Figure 1 shows the degree of con uence at day 5 and day 6 as a percentage of total coverage on passage 19.Anchorage-dependent cell lines growing in monolayers need to be subculture at regular intervals to maintain exponential growth.When anchorage-dependent cells are approximately 70 % to 90 % con uent, they are ready to be subculture.Cell lines that grow attached to the surface should be subculture regularly before the con uent period (the period when growth is stagnant) to ensure viability and genetic stability 31 .In this experiment, all passages were 80 % con uent on day 5 and 90 % on the day 6, and it was considered appropriate to subculture between day 5 and 6 (Fig. 1).In order to obtain a su cient number of cells, subcultures were performed only once the con uence reached 90 % 32 .

Maintaining the proliferation capacity of Hanwoo myosatellite cells
Hanwoo myosatellite cells were cultured for a total of 6 days between each passage (Fig. 2E).On day 1, it was con rmed that anchorage dependent Hanwoo myosatellite cells did attach to the ask.On day 6 of culture, Hanwoo myosatellite cells were con uent up to 80-90 % for all passages with which the experiment was conducted.In this experiment, the same subculture method was performed for all passages in order to maintain constant proliferation of Hanwoo myosatellite cells.Image data were gathered for all passages to determine the overall trend.Figure 2A, 2B, 2C, and 2D are representative image data.It shows the proliferation trend of P2, P5, and P10 among subcultures from P2 to P19.Among passages surveyed, the highest number of cells was found in P3 (Fig. 2F).The trend line of live cells tended to decrease gradually from the early passage to the late passage (until P19).It was con rmed that the proliferation potential decreased as the subculture progressed.In contrast, the cell size increased (Fig. 2F).Decreased proliferation capacity and increased cell size might have been related to replication aging 33 . [

Fig 2 site]
The proliferation potential was con rmed through biomarkers related to the proliferation of early (P3), middle (P6), and late passage (P9).It was con rmed that the PAX7 mRNA level was higher in P3 than in P6 and P9 (Fig. 3A) (p < 0.05).It suggests that the proliferation potential was con rmed through biomarkers related to proliferation of early, middle, and late passages.PAX7 mRNA levels decreased signi cantly at late passage compared to those at early passage (Fig. 3A), suggesting that quiescent satellite cells were signi cantly reduced during the subculturing process.The difference in the number of stationary satellite cells between early passage (P3) and late passages (P6 and P9) was expected to be large.MYOD mRNA levels remained unchanged from P3 to P9 (Fig. 3B).From early passage to passage 9, differentiation potential was expected.The increase in C-FOS mRNA from passage 3 to passage 9 appeared to be related to the maintenance of live cells until late passage (Fig. 3C).Downregulation of C-FOS mRNA is essential for terminal differentiation and myotube formation 34,35 .Downregulation of proliferation-promoting transcription factors such as c-fos is a prerequisite for initiation of myocyte differentiation.The fact that C-FOS mRNA was not reduced in the late passage might affect the differentiation potential.

Measurement the differentiation capacity of Hanwoo myosatellite cells
Major components of Matrigel, an ECM used for the differentiation of various cells, were found to be laminin and enactin, which share relatively few common peptides with collagen 36 .It is known that the growth capacity of cells in matrigel is higher than that in collagen and that α7β1 binding sites in Matrigel are related to differentiation 37 .Unlike collagen coating in a proliferative culture, this experiment used Matrigel as an ECM for observing the differentiation of Hanwoo myosatellite cells.The presence of myosin is often considered evidence that muscle cells have differentiated.This can be con rmed through immuno uorescence analysis 38 .Previous studies have demonstrated that serum can promote the growth of muscle stem cells, but inhibit muscle differentiation at high concentrations 39 .In this experiment, FBS was mixed with the differentiation medium at a smaller ratio than the growth medium in order to prevent inhibition of muscle cell differentiation Differentiation phases were monitored for 6 days (Fig. 4E).On differentiation day 1, su cient cells proliferated, and differentiation started.It was con rmed that most myotubes were formed between 2 and 4 days of differentiation.Our data clearly showed that differentiated myotubes detached on differentiation day 5 -day 6 (Fig. 4A, 4B, 4C, 4D).Hanwoo satellite cells were subculture from passage 1 to passage 12 in order to observe the differentiation level by passage.The formation of myotube decreased signi cantly after passage 12. Thus, differentiation culture was not performed for subsequent passages (Fig. 4E, 4F).Image data were acquired during each passage to better determine the overall trend.Figure 5 shows images of myosin and nucleus uorescence staining of Hanwoo skeletal muscle cells.To characterize morphological changes, myotubes are often quanti ed via immuno uorescence-staining of myotube cytoskeletal markers (e.g., desmin or myosin heavy chain) along with DAPI 40,41 .The diameter, surface area, and nuclear fusion index (de ned as the number of nuclei incorporated into myotubes, expressed as a proportion of the total visible nuclei in each eld of view) of myotubes could be measured manually using a public-domain software such as ImageJ 42 .Capacities for differentiation of cells at passages 1 and 10 are shown in Figure 5. Table 2 shows results of comparing differentiation characteristics of cells at passages 1 and 10.There were no signi cant differences in myotube width, myotube area, number of nuclei, or nucleus area between passages.The fact that there were no signi cant differences in Hanwoo muscle differentiation phenotype characteristics between passages 1 and 10 suggested that the differentiation capacity was not signi cantly lost based on the expression of myotubes throughout passages from 1 to 10.   4-p1(a,b,c,d) or p10(a,b,c,d).All images were measured by trained analysts using ImageJ.There were no signi cant differences between the groups in these data.
Fig. 6A, 6B shows MYOG and C-FOS mRNA levels in Hanwoo muscle cells differentiated for 4 days at passages 3, 6, 9, and 12.At passages 3, 6, and 9, MYOG mRNA level was signi cantly higher than that at passage 12 (Fig. 6A).The amount of C-FOS mRNA, a transcription factor for promoting proliferation, was higher at passages 6, 9, and 12 than that at passage 3 (Fig. 6B).Fig. 6C, 6D, and 6E shows protein expression levels and band of Myosin and Tom20 in Hanwoo muscle cells differentiated for 4 days at passages 3, 6, 9, and 12. Myosin protein expression level decreased signi cantly as passages increased (Fig. 6D).Tom20 protein level was the highest in passage 3.There was no difference of Tom20 protein levels in passages 6, 9 and 12 (Fig. 6E).The number of mitochondria related to skeletal muscle protein and energy metabolism decreased as late passage progressed.

[Fig 6 site] Discussion
Hanwoo cattle has maintained its character as a purebred from the end of 1900 to the present 22 .Hanwoo cattle has undergone long-term arti cial selective breeding as part of a national breeding program aimed at improving their meat qualities and characteristics.Genetic improvements of Hanwoo cattle has been carried out using traditional selection methods based on phenotypic information such as the Korean proven bull number (KPN).Although the nature of genes that in uence economically important traits is not generally known, arti cial selective breeding in this way has been successful.Hanwoo cattle has less diversity and relatively xed genetic variation than other breeds of cattle [43][44][45] .Since Hanwoo breeds have relatively less genetic diversity than other small species, the characteristics of cells isolated from Hanwoo muscle tissue will also be relatively small.
Most of cells used in the production of cultured meat are adult stem cells and primary cells isolated from livestock.The proliferation potential and differentiation potential are important requirements for producing cultured meat because of nite cell replication.This senescence is mediated by the shortening of chromosomal telomeres that occurs along with cell division 46 .In addition, it has been reported that muscle stem cells cultured in vitro can gradually lose stemness due to a lack of signaling molecules 47 .
Subculturing is a good method to identify the potential of cell proliferation and differentiation by causing continuous cell replication.In general, proliferation and myotube formation decrease as the passage number increases 47 .This might be related to replicative senescence cells 33 .In addition to previous studies, by measuring the number and size of live cells at each passage in this experiment, it can be inferred that cell senescence progressed in the later passages through a decrease in proliferation capacity and an increase in cell size.The decrease in PAX7 mRNA level after passage 3 indicated a decrease in quiescent satellite cells, which meant that the growth potential of Hanwoo muscle cells had decreased.At passages 3, 6, and 9, there is no signi cant difference in the amount of MYOD mRNA level known to affect differentiation.Thus, the reduction in differentiation capacity could not be explained by difference in MYOD mRNA level.Myotubes are multinucleate cell produced by the differentiation of myoblasts into myocytes and the fusion of several clusters.Myotubes have rows of centrally located nuclei and peripheral masses of forming contractile myo laments and cytoskeleton that soon become oriented into sarcomeres and myo brils with restoration of cross-striations in the immature myo bers 48 .Hanwoo muscle cells maintained myotube phenotype and characteristics of early passage until passage 10.
There was no signi cant difference in myotube morphology or fusion index between passage 1 and passage 10.The maximum differentiation score remained high until passage 10.These indicate that the maximum differentiation score can be used to determine the range of available Hanwoo muscle cells for e cient cultured meat production.In addition, the rapid differentiation capacity and myotube formation were steadily maintained in the late passage, and then there was a rapid decrease at a speci c point.In order to differentiate muscle cells, proliferation inhibition must be performed as a prerequisite.In addition, c-fos, a transcription factor known to promote proliferation, must be down-regulated to induce differentiation of myocytes.The C-FOS mRNA level was the lowest at passage 3, indicating that differentiation and myotube formation in Hanwoo cells were higher at passage 3 than those at later passage.Myogenin is known to affect myotube formation by inhibiting myoblast proliferation 35 .It was con rmed that MYOG mRNA decreased in the late passage.Myosin protein values of Fig. 5 and Fig. 6D were slightly different in both experiments.In the phenotype analysis of protein through staining, it was determined that myosin maintained skeletal formation up to p10, but it does not appear to be measured the amount of protein.Phenotype analysis by protein staining and protein amount measurement seem to have different meanings.Judging by the results, phenotype analysis by protein staining con rms the skeleton muscle structure and formation characteristics, and protein amount analysis by western blotting measures the density of myosin protein.Therefore, it is believed that there was a difference in the tendency of the two experiments.The mitochondria density in myotube, which is muscle contraction, is higher than myoblasts 49,50 .Therefore, the amount of Tom20 protein, which is mitochondrial protein, became an indicator of relative myotube and differentiation.The differentiation capacity of Hanwoo muscle cells was con rmed through Myosin and Tom20 protein expression.Decreases of myotube cytoskeletal and mitochondrial protein levels of Hanwoo cells in the late passage meant decreased differentiation capacity.It is judged that muscle cell transcriptional regulatory factor of proliferation and differentiation comprehensively decreased differentiation capacity at a speci c point.Due to the low genetic diversity of Hanwoo, the common tendency of Hanwoo muscle cells may be that their proliferation and differentiation capacity rapidly decreases at certain points.If these points are con rmed through more advanced studies, there is a possibility that the culture period of Hanwoo muscle cells for the production of cultured meat may be easily de ned.

Conclusion
Using cells for cultured meat production is free from genetic recombination and ethical issues as food.For the production and commercialization of cultured meat, research on the utilization of livestock primary cells is essential.In this experiment, Hanwoo myosatellite cells showed a trend of decreasing cell counts at late passage.It is judged that muscle cell transcriptional regulatory factor of proliferation and differentiation comprehensively decreased differentiation capacity at a speci c point.Myotube phenotype of Hanwoo cells had no difference between passage 1 and passage 10.The maximum differentiation score remained high levels from passage 1 to passage Relative proliferation mRNA levels of Hanwoo muscle cells.Relative PAX7 and MYOD mRNA levels of Hanwoo myosatellite cells cultured for 6 days were compared among passages 3, 6, and 9. PAX7(A), MYOD (B) and C-FOS (C) mRNA levels were analyzed.GAPDH was used as an internal control for RT-PCR.Experiments were performed repeated three times (n=3).Mean ± standard deviation with differing letters differs signi cantly (p < 0.05).

Figure 4
is a representative image showing differentiation trend of passages 2, 5, 10, and 12 among subcultures from passage 1 to passage 12. Figure4Fshows results of the maximum differentiation score.Differentiation and myotube formation scores of Hanwoo myosatellite cells dropped signi cantly at passages 11 and 12 compared to passage 10.[Fig 4 site]

Figures Figure 1
Figures

Figure 2 Images
Figure 2

Table 1 .
. Sequences of the primers used in the RT-qPCR study Statistical analysis was conducted with analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the SAS program (Statistical Analysis System 2002, Cary, NC, USA), and the signi cance of the comparisons between the means of the treatment groups was veri ed (p < 0.05) using Duncan's multiple range test.

Table 2 .
Hanwoo cell differentiation characteristics 10.The amount of myotube formation and differentiation capacity decreased at later passage.Based on the results, Hanwoo muscle cells measured in this experiment are expected to signi cantly decrease the productivity of cultured meat after P10.It is judged that it is appropriate to use cells up to passage 10 when differentiated cells are used to produce cultured meat of Hanwoo muscle satellite cells used in this experiment.The results of this experiment are limited to two Hanwoo muscle cells.If Hanwoo muscle cells of another individual are used in experiments, the differentiation capacity may not be maintained up to P10.However, since the genetic diversity of Hanwoo is relatively low compared to other cattle breeds, similar trends can be inferred in other Hanwoo muscle cells.It is believed that the tendency of proliferation and differentiation capacity to gradually decrease is similar.If the differentiation process of Hanwoo myosatellite cells is omitted to produce cultured meat, the range of passages available will be higher.