Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation

Simple Summary Culturing of muscle precursor cells—satellite cells—is the basis for research on obtaining meat using in vitro techniques, which can become an alternative to traditional meat production. The available literature data do not give a clear answer as to the medium used at the proliferation stage of bovine satellite cells; therefore, there is a need to select the level of components of the growth medium. In our research, based on the fluorometric measurement of proliferating cell growth and qPCR analysis, we examined the effect of the three most common components: glucose, serum (bovine or horse), and bFGF—a mitogenic factor. The results show that the most critical component affecting the proliferation rate is the serum—the addition of bovine serum, followed by the addition of bFGF—at 10 ng/mL. In turn, the lower glucose content ensures the maintenance of cells at the early stage of myogenesis and, thus, the self-renewal of their population. In conclusion, a higher proliferation rate of bovine satellite cells is ensured under the conditions of bovine serum and a mitogenic factor at 10 ng/mL. Abstract It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells—being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor—bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.


Introduction
The world population is predicted to reach approximately 9.7 billion people in 2050 [1], so the demand for food-including meat-will continue to increase, contrary to the ability Table 1. Summary of compositions of media for BSCs cultivation.

Isolation of Muscle Satellite Cells
Satellite cells were isolated from musculus longissimus dorsi from female calf specimen as two separate isolations (described as R1 and R2). A sample of skeletal muscles was sourced from fresh slaughter waste and therefore required no ethical committee acceptance according to local law. The tissue was digested with collagenase II (2 mg (500 U)/mL; 2-3 mL/g tissue; Thermo Fisher Scientific, Waltham, MA, USA) in Phosphate Buffered Saline-PBS buffer pH 7.2 (Thermo Fisher Scientific) with 4% BSA (Thermo Fisher Scientific) for two hours at 37 • C. Then, cells were washed in PBS buffer and filtered through a 70 µm nylon strainer (Corning, New York, NY, USA). Cells were centrifuged for 10 min at 500× g, the supernatant was discarded, and cells were purified by pre-plating method, based on the rate of adhesion of different types of cells to the surface. Cells were seeded in Skeletal Muscle Cell Medium (Cell Applications, Inc., San Diego, CA, USA) with 1% of antibiotic Primocin (InVivoGen, San Diego, CA, USA) on non-coated culture dish. After 1 h, the medium containing non-adherent cells were collected, centrifuged, and the cells were plated in the same medium on a thin layer of 0,1% gelatin (Thermo Fisher Scientific) [23]. The procedure was repeated during the next cell split. Part of the cells was frozen in a medium DMEM (Thermo Fisher Scientific) with the addition of 20% fetal bovine serum (Thermo Fisher Scientific), 1% antibiotic Primocin (InVivogen), and 10% DMSO (Thermo Fisher Scientific).

Selection of the Components of the Tested Media
The experimental design was set to measure the synergistic, co-stimulatory effect of three medium components, which effect was evaluated on the proliferation rate of BSCs. Those stimulants were: A-Glucose content in the culture medium-DMEM medium with low glucose content (1 g/L) versus DMEM medium with high glucose content (4.5 g/L) (Thermo Fisher Scientific).
B-Type of serum-horse serum (hs) at a concentration of 20% versus fetal bovine serum (fbs) at a concentration of 20% (Thermo Fisher Scientific). It was hypothesized that fetal bovine serum would stimulate the proliferation of BSCs more efficiently [24,25].
C-Content of bFGF (Thermo Fisher Scientific) with known mitogenic effect at concentrations of 5 ng/mL and 10 ng/mL. Culture media was prepared fresh just before each medium change due to the instability of bFGF.
During the entire experiment, sera from one lot were used due to the possibility of differences in the concentration of individual components between batches.

Cell Culture and Estimation of Proliferation Rate and Visualisation
Satellite cell lines R1 and R2 (both-3rd passage since isolation) were plated in the 96-well multiwell plate at a density of 3500 cells/wells in Skeletal Muscle Cell Medium (Cell Applications, Inc.) supplemented with 1% addition of antibiotic Primocin (InVivo-Gen) on a thin layer of 0.1% gelatin used as a coating (Thermo Fisher Scientific). Each cell line consisted of four replicates per experimental group. After 24 h, the number of cells in each well was estimated based on a fluorimetric method-CellTiter Blue Assay (Promega, Madison, WI, USA). The CellTiter solution was prepared according to the manufacturer's protocol, then was added to the plate with treated cells and incubated for one hour in 5% CO 2 in humidified air at 37 • C (previously length of the incubation period and the number of cells/wells were optimized). Fluorescence intensity was measured at 535 nm excitation and 595 nm emission wavelength using PlateReader AF2200 (Eppendorf, Hamburg, Germany). After the measurement, in each group tested media were added containing an appropriate amount of tested factors. The number of proliferating cells was estimated, based on CellTiter Blue assay, after three, five, seven, and ten days of incubation in the tested medium. The measurement results were obtained in the RFU unit-relative fluorescence unit. RFU values from each measurement were normalized based on day 0 RFU values.
Alongside to described, microscopic observation of satellite cells was carried out. Pictures of the representative area were visualized in Opta-tech microscope using OptaView-IS programme. Further post-processing consisted of contrast enhancement for every picture using ImageJ CLAHE algorithm (blocksize 79, hist bins 50, max slope 2.5) [26,27].

Statistical Analysis
The results were analyzed using Python [28] with NumPy [29], SciPy [30], Statsmodels [31], Pandas [32,33], and Matplotlib [34]. A 2 3 full factorial design was applied to evaluate the effects of three factors (Xi) on experimental results. The high and low levels defined for the 2 3 factorial designs are presented in Table 2. The variables X 1 , X 2, and X 3 represented the levels of glucose, serum, and bFGF, respectively. The analysis was performed separately for R1 and R2 cell lines and on the subsequent day of the experiment (3, 5, 7, or 10), so eight designs of experiments (DoE) were created. Additionally, the design is replicated fourth time. The experimental conditions obtained for each reaction set by the full factorial design are shown in Table 3. Every treatment factor in an experiment has two levels. The higher level (maximum) was designated as (+1) and the lower value (minimum) was designated as (−1).   The coded mathematical model may be given as: where Y is the proliferation rate measured as RFU value, X i are the coded variables (−1 or +1), and α i represents the fitted constants. Additionally, in this notation the α ij constant represented the estimation of the interaction effect between factor i and j for the response.
The p-values are used as a tool to check the significance of each coefficient, which can indicate the pattern of the interactions between the independent variables. If the p-value is below 0.05, then the model is significant at the 95% confidence interval. Additionally, the Akaike Information Criterion (AIC), Bayesian Information Criterion (BIC), coefficient of determination (R 2 ), and adjusted coefficient of determination (R 2 adj ) were calculated to show the goodness of fit of models.
Pareto's charts were used to evaluate the meaning and type of effects. Pareto analysis indicates the relative importance of each independent model's parameter and their interactions on the proliferation rate measured as RFU value. The values displayed in the horizontal columns are the absolute values of t-Student test values for each effect and the vertical line indicates the minimal significant effect magnitude for a 95% confidence level.
At the end of the analysis, the statistical validation of the model was performed using an analysis of variance ( Table 4). The main effect is the mean of all of the responses produced by changing the level of a factor, i.e., glucose, serum, or bFGF. If the p < 0.05, then the source is significant.

Analysis of the Expression of Genes of Myogenesis
For the analysis of myogenesis gene expression, the effect of each component was examined individually. Satellite cells (3rd passage) were plated on 35 mm culture dishes at a density of 70,000 cells/dish in Skeletal Muscle Cell Medium (Cell Applications, Inc.) supplemented with 1% of antibiotic Primocin (InVivoGen) on a thin layer of 0.1% gelatin (Thermo Fisher Scientific), six replicates per group, within the tested factor. After 24 h, the medium was replaced with the tested medium depending on the group studied. After three days, three replicates from each group were harvested, and RNA was isolated according to the manufacturer's protocol using PureLink RNA Mini Kit (Thermo Fisher Scientific). The medium was changed to a new one in the residual culture plates and after five days, RNA was isolated as described above. RNA concentration was determined using a NanoDrop2000C (Thermo Fisher Scientific) spectrophotometer, and the integrity of the isolated RNA was checked electrophoretically on a 1.5% agarose gel. Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Before starting the analysis, all tested solutions were equilibrated, regarding RNA concentrations in RNase-free water. The qPCR method was used to determine the expression of Pax7, Myf5, MyoD, Myf6, and MyoG genes. The RPL27 and OAZ1 genes were the endogenous control. The primer sequences were designed in Primer3Web (v. 4.1.0, https://primer3.ut.ee/, accessed on 30 November 2022) and produced by Genomed (Warsaw, Poland) ( Table 5). Gene expression analysis was performed using the HS-PCR Mix SYBR according to the manufacturer's protocol (A&ABiotechnology, Gdansk, Poland) and QuantStudio 7 Pro System (Thermo Scientific).

Statistical Analysis of the Expression of Genes
Statistical analysis of the obtained results was carried out in the Statistica program (StatSoft, v.13). The normal distribution was tested with the Shapiro-Wilk test. The analyzes were based on the one-way analysis of variance (ANOVA) using the Bonferroni post hoc test. In the case of failure to comply with assumptions of Leavene's test, the Mann-Whitney U test was used.

Full Factorial Design of Tested Components
On the third day, in cell line R1 (Table 6 and Figure 1a), the concentration of bFGF in the medium (component C) showed the most robust effect strength of 6.92 × 10 3 value from among tested components. All the effects of the main tested, i.e., glucose (A), serum (B), and bFGF (C) components, were significant (p < 0.05). The interaction of the two A:B factors was significant (p < 0.05). The obtained results indicate that component C is a key factor in stimulating the proliferation of satellite cells and that its level is independent of the levels of other tested components on the third day. According to the data presented in Table 6 and the graphical Pareto chart shown in Figure 1a, the final first-order model for RFU in terms of coded parameters can be proposed. What is important, the nonsignificant effects were neglected to form the best-fitted regression equation, so the proposed model was: RFU value at 3rd day = 22,514.38 + 1642.69X 1 + 2438.38X 2 + 3458.25X 3 + 915.44X 1 X 2   The factors that occur with a p value less than 0.05 are considered to have a significant effect on the response significantly On days 5 and 10, the main effects of components B and C were significant (p < 0.05), an on day 7, all the tested components, i.e., glucose (A), serum (B), and bFGF (C) were signi icant (p < 0.05). On days 5, 7, and 10, the interaction of factors B:C was significant, and o day 7, the interaction of factors A:C was significant. On days five and seven, the interactio of all three factors A:B:C was significant. Thus, according to the data presented in Table  7-9 and in Figure 1b-  .375 values from among tested components, respectively. The type of serum was the most important component affecting the proliferation of BSCs. The factors that occur with a p-value less than 0.05 are considered to have a significant effect on the response significantly. On days 5 and 10, the main effects of components B and C were significant (p < 0.05), and on day 7, all the tested components, i.e., glucose (A), serum (B), and bFGF (C) were significant (p < 0.05). On days 5, 7, and 10, the interaction of factors B:C was significant, and on day 7, the interaction of factors A:C was significant. On days five and seven, the interaction of all three factors A:B:C was significant. Thus, according to the data presented in Tables 7-9 and in Figure 1b-  Four of the goodness of fit parameters, i.e., AIC, BIC, R 2 and R 2 adj were calculated. The model with the lowest AIC and BIC and with higher, closer to 1 R 2 and R 2 adj had better fit to data. In the case of R1 cell line, the obtained statistical parameters are presented in Tables 6-9 and show a better fit on the 3rd and 10th days.
On days 3, 5, 7, and 10, in cell line R2 (Tables 10-13 and Figure 2), the serum type (component B) showed the most robust effect strength of values of 1221.5, 4393.375, 6027.438, and 6903.375 among tested components, respectively. On days 3, 5, and 10, the main effects of components B and C were significant, and on day 7, the main effects of all components (i.e., A, B, C) were significant. On day three, the two interactions between factors A:B and A:C were significant, whereas on days 7 and 10, the interaction between factors A:C and B:C was significant. Moreover, on day seven, the interaction of the three factors A:B:C was also significant. The proposed equations of the final first-order models for RFU in term of coded parameters on each day were:    The goodness of fit parameters, i.e., AIC, BIC, R 2 , and R 2 adj of R2 cell line, are presented in Tables 10-13 and shows better fit on the 7th and 10th days.
In addition, for an evaluation of models and the relationship between factors A, B, C and response and cube plots, as shown in Figures 3 and 4, were generated. In cell line R1 (Figure 3), the highest RFU value was obtained for bovine serum and 10 ng/mL bFGF and on the third and seventh days also for high glucose content. On day three, higher RFU values were obtained primarily for the higher level of component C-10 ng/mL bFGF (p < 0.05). The level of component B-the type of medium, where bovine serum gave higher values (p < 0.05), and the level of component A-glucose level-where high glucose content resulted in higher values also had a significant effect (p < 0.05). On days 5, 7, and 10 higher RFU values were observed primarily in the group with a higher value of component B-bovine serum (p < 0.05). An important component was also bFGF (component C) where using a higher dose-10 ng/mL-resulted in higher RFU values (p < 0.05). On day seven, a significant effect was also shown by the glucose level in the medium-component A, where higher glucose content resulted in higher RFU values (p < 0.05). The goodness of fit parameters, i.e., AIC, BIC, R 2 , and R 2 adj of R2 cell line, are presented in Tables 10-13 and shows better fit on the 7th and 10th days.
In addition, for an evaluation of models and the relationship between factors A, B, C and response and cube plots, as shown in Figures 3 and 4, were generated. In cell line R1 (Figure 3), the highest RFU value was obtained for bovine serum and 10 ng/mL bFGF and on the third and seventh days also for high glucose content. On day three, higher RFU values were obtained primarily for the higher level of component C-10 ng/mL bFGF (p < 0.05). The level of component B-the type of medium, where bovine serum gave higher values (p < 0.05), and the level of component A-glucose level-where high glucose content resulted in higher values also had a significant effect (p < 0.05). On days 5, 7, and 10, higher RFU values were observed primarily in the group with a higher value of component B-bovine serum (p < 0.05). An important component was also bFGF (component C), where using a higher dose-10 ng/mL-resulted in higher RFU values (p < 0.05). On day seven, a significant effect was also shown by the glucose level in the medium-component A, where higher glucose content resulted in higher RFU values (p < 0.05).  In cell line R2 (Figure 4), the highest RFU value was obtained for bovine serum (com   In cell line R2 (Figure 4), the highest RFU value was obtained for bovine serum (com ponent B) and 10 ng/mL bFGF (component C), and on days 7 and 10 also for low gluco content (component A). Higher RFU values were obtained mainly for component B-b In cell line R2 (Figure 4), the highest RFU value was obtained for bovine serum (component B) and 10 ng/mL bFGF (component C), and on days 7 and 10 also for low glucose content (component A). Higher RFU values were obtained mainly for component B-bovine serum (p < 0.05), but the level of component C also had a significant effect-bFGF-at the level of 10 ng/mL (p < 0.05). On days 7 and 10, component A-glucose content also had a significant effect, where lower levels resulted in higher RFU values (p < 0.05).

Microscopic Observations of Proliferating BSCs
Microscopic observation showed that in the group treated with bovine serum and 10 ng/mL bFGF addition, cell differentiation was lower than in the group with horse serum and 5 ng/mL bFGF addition ( Figure 5). There formation of myotubes was observed already on the fifth day of the experiment. at the level of 10 ng/mL (p < 0.05). On days 7 and 10, component A-glucose content also had a significant effect, where lower levels resulted in higher RFU values (p < 0.05).

Microscopic Observations of Proliferating BSCs
Microscopic observation showed that in the group treated with bovine serum and 10 ng/mL bFGF addition, cell differentiation was lower than in the group with horse serum and 5 ng/mL bFGF addition ( Figure 5). There formation of myotubes was observed already on the fifth day of the experiment.

Gene Expression: Genes Involved in Myogenesis
For the groups differing in bFGF content, no significant differences were found in the relative expression of the examined genes on the third and fifth days ( Figure 6). For groups differing in glucose content, significantly higher levels of relative expression of Pax7 and Myf5 genes-genes expressed at the early stage of myogenesis-were found in the group supplemented with lower glucose content on the third day (p < 0.05). On the fifth day, no significant differences in the relative expression of the tested genes were observed between the study groups (Figure 7). For the groups differing in the type of serum, no significant differences in the relative expression of the examined genes were found on the third day. However, large standard deviations were observed in the group treated with horse serum. On the fifth day, a significantly higher relative level of Pax7 and Myf6 gene expression was observed in the bovine serum-treated group and a significantly higher relative expression level of Myf5 gene expression in the horse serum-treated group (p < 0.05) (Figure 8).

Gene Expression: Genes Involved in Myogenesis
For the groups differing in bFGF content, no significant differences were found in the relative expression of the examined genes on the third and fifth days ( Figure 6). For groups differing in glucose content, significantly higher levels of relative expression of Pax7 and Myf5 genes-genes expressed at the early stage of myogenesis-were found in the group supplemented with lower glucose content on the third day (p < 0.05). On the fifth day, no significant differences in the relative expression of the tested genes were observed between the study groups ( Figure 7). For the groups differing in the type of serum, no significant differences in the relative expression of the examined genes were found on the third day. However, large standard deviations were observed in the group treated with horse serum. On the fifth day, a significantly higher relative level of Pax7 and Myf6 gene expression was observed in the bovine serum-treated group and a significantly higher relative expression level of Myf5 gene expression in the horse serum-treated group (p < 0.05) (Figure 8).

Discussion
Recreating the process of myogenesis in vitro requires the provision of relevant factors supporting the proliferation of satellite cells, which are contained in the culture medium. Our research aimed to develop the composition of the culture medium that would most efficiently support the proliferation of BSCs. The experimental design was set to measure the synergistic, co-stimulatory effect of three medium components: glucose content, type of serum (bovine or horse), and mitogenic factor-bFGF-content. Each observation (except observation for 3rd day, cell line R1) showed that the type of serum had the most robust effect on the proliferation rate of BSCs amongst tested components. The main effect of the tested component was statistically significant (p < 0.05), and in addition, in the majority of the observations, the interaction between component B (serum type) and component C (bFGF) was statistically significant (p < 0.05). In the present study, bovine serum was proven to stimulate the proliferation of BSCs to a greater extent than horse serum. Our observations are consistent with the study by Woods et al. [35], which showed a higher proliferation rate of BSCs in a medium supplemented with bovine serum than horse serum 24 h after cell seeding. Research by Franke et al. [25] focusing on comparing the effect of bovine and horse serum on the proliferation and differentiation of equine bronchial fibroblasts showed that bovine serum more efficiently supported the proliferation process of fibroblasts by maintaining normal cell morphology and increasing the number of passages. Similar observations were made in the case of equine mesenchymal stromal cells, where cells cultured in bovine serum proliferated faster and secreted much more, e.g., bFGF and interleukins (IL-4, IL-5, IL-17) [24]. Gene expression analysis did not provide conclusive information on which of the sera maintained the expression profile characteristic of the early stage of myogenesis. A statistically higher expression level of Pax7 and Myf6 genes was observed in the group treated with bovine serum (p < 0.05). Pax7 and Myf6 genes expression occur at different stages of myogenesis, in the early and late stages, respectively. Quiescent satellite cells express Pax7, which maintains cells in an undifferentiated state and regulates genes involved in the initiation of proliferation and inhibiting differentiation [8,36]. The expression of Myf6 gene-supervises the inhibition of premature differentiation and is maintained at a state level [37,38]. However, it is worth

Discussion
Recreating the process of myogenesis in vitro requires the provision of relevant factors supporting the proliferation of satellite cells, which are contained in the culture medium. Our research aimed to develop the composition of the culture medium that would most efficiently support the proliferation of BSCs. The experimental design was set to measure the synergistic, co-stimulatory effect of three medium components: glucose content, type of serum (bovine or horse), and mitogenic factor-bFGF-content. Each observation (except observation for 3rd day, cell line R1) showed that the type of serum had the most robust effect on the proliferation rate of BSCs amongst tested components. The main effect of the tested component was statistically significant (p < 0.05), and in addition, in the majority of the observations, the interaction between component B (serum type) and component C (bFGF) was statistically significant (p < 0.05). In the present study, bovine serum was proven to stimulate the proliferation of BSCs to a greater extent than horse serum. Our observations are consistent with the study by Woods et al. [35], which showed a higher proliferation rate of BSCs in a medium supplemented with bovine serum than horse serum 24 h after cell seeding. Research by Franke et al. [25] focusing on comparing the effect of bovine and horse serum on the proliferation and differentiation of equine bronchial fibroblasts showed that bovine serum more efficiently supported the proliferation process of fibroblasts by maintaining normal cell morphology and increasing the number of passages. Similar observations were made in the case of equine mesenchymal stromal cells, where cells cultured in bovine serum proliferated faster and secreted much more, e.g., bFGF and interleukins (IL-4, IL-5, IL-17) [24]. Gene expression analysis did not provide conclusive information on which of the sera maintained the expression profile characteristic of the early stage of myogenesis. A statistically higher expression level of Pax7 and Myf6 genes was observed in the group treated with bovine serum (p < 0.05). Pax7 and Myf6 genes expression occur at different stages of myogenesis, in the early and late stages, respectively. Quiescent satellite cells express Pax7, which maintains cells in an undifferentiated state and regulates genes involved in the initiation of proliferation and inhibiting differentiation [8,36]. The expression of Myf6 gene-supervises the inhibition of premature differentiation and is maintained at a state level [37,38]. However, it is worth noting that the standard deviations within the group treated with horse serum were high, which may suggest that bovine serum provides more stable conditions for cell proliferation.
Basic fibroblast growth factor (bFGF) can regulate proliferation and myogenesis by stimulating or inhibiting muscle metabolic pathways [39]. Studies conducted on Jeju Black pig muscle cells showed that the presence of bFGF affected the rate of proliferation and, together with EGF, also the level of expression of the MyoD gene [40]. The present study confirmed the pro-mitogenic effect of bFGF on BSCs and after the serum type showed the most robust effect on the proliferation rate. Each observation showed that the factor's main influence was statistically significant (p < 0.05). Interactions between component C (bFGF) and component B (serum type) in most observations were statistically significant. In the R2 cell line, a statistically significant interaction between component A (glucose) and component C (bFGF) content was observed on the 3rd, 7th and 10th days (p < 0.05). In greater observations, the addition of 10 ng/mL bFGF resulted in a significantly higher rate of proliferation than the addition of 5 ng/mL. Higher concentrations of bFGF were not tested due to the need to select the two most commonly used levels by reason of the mathematical model used. Moreover, microscopic observations showed that in the group treated with bovine serum and 10 ng/mL bFGF differentiation was lower than in the group with horse serum and 5 ng/mL bFGF, which is consistent with the rest of the observations. This confirms the fact that using bovine serum together with bFGF at the level of 10 ng/mL more effectively supports the proliferation process.
Recent research shows that muscle cells' proliferative and self-renewal capacities are greater in a medium with a lower glucose content [41]. Moreover, high glucose concentrations may induce adipogenic differentiation in satellite cells [42]. This examination could be successfully used in a selection of the composition of the medium for co-cultures of satellite cells with adipocytes in seeking to obtain cultured meat. In this study, generally, we observed no significant effect associated with glucose content on the proliferation rate in BSCc compared to the other components. Notably, the expression of Pax7 and Myf5 genes-genes expressed at the early stages of myogenesis-was significantly higher in the group supplemented with lower glucose content on the third day. The observation is consistent with the research of Furuichi et al. [41] and proves the ability of satellite cells to self-renew in conditions of lower glucose content. These results would suggest using a medium with low glucose content, which provides higher expression levels of genes involved in the self-renewal of satellite cell populations.
It is worth noting that cell cultivation based on serum of animal origin is contrary to the ideology of cultured meat, raises many ethical controversies, and requires high financial investments. Research is still undertaken to find optimal substitutes for animal serum because serum-free cultures are still inefficient [43]. In 2022, Stout et al. [44] proposed the Beefy-9-B8 medium supplemented with recombinant albumin-which allowed the cultivation of BSCs for seven passages. Independently, in 2022, Kolkmann et al. [19] developed a serum-free medium (SFM) composition supporting proliferation almost as efficiently as the medium supplemented with animal serum. However, both solutions still require a lot of studies, although they optimistically approach us closer to serum-free cell cultivation, which is free of suffering for animals.

Conclusions
The component which most significantly affects the level of BSCs proliferation is the type of serum. Using bovine serum provides a higher rate of proliferation and based on qPCR analysis, it would seem that there are more stable growth conditions for cells than horse serum. The second significant component is bFGF, where adding 10 ng/mL significantly increased the proliferation rate. In most observations, glucose does not significantly affect the rate of proliferation. However, the low glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population, which is consistent with previous studies. The highest rate of proliferation measured as RFU value was observed, supposing the addition of bovine serum and bFGF at the concentration of 10 ng/mL.