Whey-enhanced serum-free medium for bovine myoblast expansion

Abstract

Cell-based foods are anticipated to be a sustainable alternative to animal protein sources. The primary challenge is developing a food-grade serum-free medium (SFM) that is affordable, safe, acceptable to consumers, and available in large quantities. This study was conducted to develop such a medium using food ingredients dissolved in water that support the expansion of bovine myoblasts and maintain their differentiation capability. Through screening of various food ingredients, whey was identified as the most effective component. Whey improved myoblast proliferation in SFM to levels than those in a serum-containing medium. The differentiation capability of myoblasts cultured in whey-containing SFM was similar to that in serum-containing medium. Remarkably, whey could support myoblast proliferation even after heat treatment, allowing for the sterilization of whey-containing media by heat treatment, a common process in food processing. Hence, whey could be an ideal ingredient for affordable food-grade serum replacement in cell-based food production.

Introduction

Meat products are vital protein sources that contain more essential amino acids than plants or microorganisms. With the global population projected to reach 10 billion by 2050, meat demand is anticipated to surpass livestock production capacity (Alexandratos and Jelle, 2012). Although the livestock industry must increase productivity, environmental concerns such as land use, water use, and greenhouse gas emissions have been raised (Grossi et al., 2019). As these concerns grow, there is an increasing global demand for alternative proteins. Cell-based foods, prepared from animal cells cultured in vitro, are considered a promising future source of animal protein due to their potentially lower environmental impact than that of conventional meat (Kim et al., 2022). The mass production process of animal cells involves expanding cells in large-scale bioreactors, which has been developed in the pharmaceutical industry, but not the food industry (Garrison et al., 2022). Therefore, it is necessary to optimize the cell culture process for food production, especially for cost reduction and food safety.

In particular, the high cost of culture medium and ensuring its food safety are huge challenges (Ding et al., 2021). Myoblasts, the primary source of cells for cell-based food, differentiate into myotubes and lose proliferative activity under low nutritional conditions, making their proliferation highly dependent on fetal bovine serum (FBS).

FBS poses high cost, safety concerns, limited availability, animal welfare issues, and consumer acceptance issues (Lee et al., 2023). Even serum-free chemically defined media face challenges due to their high cost and the use of compounds with no history of safe use in foods, such as steroid hormones and small-molecule inhibitors (Parent et al., 2016; Kolkmann et al., 2022). Despite extensive research on the application of hydrolysates of foods and food processing by-products to serum replacement (Benjaminson et al., 2002; Andreassen et al., 2020; Flaibam et al., 2024), the complex pretreatment required for enzymatic hydrolysis and the risk of undesirable by-products in acid hydrolysis have limited the potential to scale-up production (Azmi et al., 2017). Therefore, it is necessary to develop more cost-effective and safer methods of reducing or replacing FBS.

Recently, rather than acid treatment or enzymatic hydrolysis, simply water-extracted food ingredients have demonstrated proliferative activity on bovine primary myoblasts (Park et al., 2023a). The interest lies in the simplicity of just dissolving them in water, which is a more promising method because of easily obtaining and ensuring food safety. To the best of our knowledge, there has been limited research on the efficacy of water extract for primary myoblasts under serum-free conditions.

Therefore, we conducted this study to develop a serum-free medium (SFM) supplemented with food ingredients dissolved in water that support the expansion of bovine myoblasts and maintain their differentiation capability. We present whey as an ideal food ingredient for proliferating bovine myoblasts. We anticipate that our findings would provide novel opportunities for the development of affordable and consumer-acceptable SFM.

Material and methods

Foods and chemical reagents used in media

The following commercially available food ingredients were tested in this study: soybean pulp (You-techs, Hokkaido, Japan), soybean powder (FUJI OIL, Osaka, Japan), wheat flour (Nisshin Flour, Tokyo, Japan), rice bran (Nichie, Nagoya, Japan), fish meal (NINBEN, Tokyo, Japan), fish gelatin (Nitta Gelatin, Osaka, Japan), egg white (Kewpie Corporation, Tokyo, Japan), whey (Kenkou-itiba, Nara, Japan), and pork gelatin (Morinaga, Tokyo, Japan).

Bovine serum albumin, insulin–transferrin–selenium (ITS) supplement, and lipid mixture were obtained from Sigma-Aldrich (St Louis, MO, USA). Human recombinant fibroblast growth factor 2 (FGF-2), penicillin–streptomycin solution (PS), and penicillin–streptomycin–amphotericin B suspension (PSA) were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan).

Isolation of bovine myoblasts

Bovine myoblasts were isolated from fresh cheek muscle samples of 27-month-old first filial generation cattle (Holstein heifer and Japanese black cattle). The muscle samples were obtained from cheek beef, which was processed for human consumption according to the regulations of the Japanese Slaughterhouse Act at the slaughterhouse (Nippon Meat Packers, Inc., Aomori, Japan). Cells were isolated according to a previously described method (Miyake et al., 2012). Briefly, minced muscle tissue was digested in 0.2% collagenase II (Worthington, Lakewood, NJ, USA) and 76 U/mL dispase (GODO SHUSEI, Chiba, Japan) at 37 °C for 120 min. After digestion, the cell suspensions were incubated in uncoated tissue culture flasks (Corning, NY, USA) for 24 h at 37 °C with 5% CO₂. The plated suspensions (containing quiescent satellite cells) were collected and seeded onto collagen type I-coated flasks (AGC Techno Glass, Shizuoka, Japan). Quiescent bovine satellite cells were activated by culture medium containing 20% FBS (Biowest, Nuaillé, France), 5 ng/mL FGF-2, and PSA in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12; Thermo Fisher Scientific,Waltham, MA, USA). Myoblasts (active satellite cells) were cultured by changing the medium every 2 days until reaching ~ 70% confluency. Cells were rinsed with phosphate buffered saline (PBS) (Fuji Film Wako), harvested using TrypLE Express (Thermo), and cryopreserved in Cellbanker (ZENOAQ, Fukushima, Japan) at − 80 °C until use.

Culture of bovine myoblasts

Frozen cells were recovered in a 37 °C water bath; washed with DMEM/F12 containing 20% FBS, 5 ng/mL FGF-2, and 1% PS; and then seeded at 1.1 × 10⁴ cells/cm² on collagen type I-coated flasks. At 70% confluency, the myoblasts were collected and subcultured in test medium. The cell culture media listed in Table 1 was prepared by using sterile-grade products or solution sterilized by 0.2 μm filtration after dissociation. Sterile serum inactivated at 56 °C was used in all experiments. Therefore, additional sterilization was not conducted after media preparation. Commercial collagen I, fibronectin, and Matrigel-coated plates (Corning) were used for optimizing the extracellular matrix (ECM) coating of the culturing surface. For differentiation, cells were cultured to confluency, and the medium was changed to differentiation medium (DM) containing 2% horse serum (Thermo) and PS. All cell cultures were conducted at 37℃ under 5% CO₂. Cell counting was performed using a TC-2 automated cell counter (Bio-Rad Laboratories, California, USA).

Table 1.

Composition of the culture media used in the study

FBS-containing growth medium (GM)
DMEM/F12
FBS	10%
FGF-2	5 ng mL−1
PS	1%
Serum-free medium (SFM)
DMEM/F12
Insulin–transferrin–selenium (ITS)	1%
Bovine serum albumin	0.2%
Lipid mixture 1	0.1%
FGF-2	5 ng mL−1
PS	1%
Differentiation medium (DM)
DMEM/F12
Horse serum	2%
PS	1%

Preparation of culture media using food ingredients

For the evaluation of food ingredients, the test medium was prepared by adding the abovementioned food ingredients to SFM (Table 1). The prepared medium was vortexed for 5 min at room temperature before being filtering through a 0.45-μm filter. Except for whey, the powders did not fully dissolve in SFM and residues remained. These residues were subsequently removed via filtration. Unless otherwise stated, we used filtrate media that consisted of the water extract of the associated food ingredient.

Preparation of culture media for thermal stability assessment

To evaluate the effect of whey heat treatment on myoblast proliferation, we dissolved whey powder in water, heat-treated the resulting mixture, then prepared it as a culture medium. 1% (W/V) whey solution dissolved in water and FBS was then incubated in a boiling (~ 100 °C) water bath for 30 min. Samples were then centrifuged at 2500 × g for 10 min to remove visible protein aggregation that was generated upon boiling. This was removed to prevent it from interfering with cell proliferation. Subsequently, the solution was diluted 1:10 with SFM and DMEM/F12 containing FGF-2 to prepare whey-containing serum-free medium (W/SFM) and growth medium (GM), respectively.

5-Ethynyl-2-deoxyuridine (EdU) staining

EdU staining was conducted using the Click-iT EdU Imaging Kit (Thermo) according to the manufacturer’s instructions. After culturing myoblasts for 24 h in the test medium, EdU was added at a final concentration of 10 μM. The myoblasts were then cultured for an additional 3 h. Total cell nuclei were visualized by Hoechst 33,342 staining. The ratio of EdU⁺ cells was defined as the number of EdU⁺ nuclei divided by the total number of nuclei using the ImageJ software (National Institutes of Health, USA).

Immunostaining

Cells were washed once in PBS and fixed with 4% paraformaldehyde (Fuji Film Wako) for 60 min at room temperature, followed by permeabilization with 0.1% Triton X-100 (Bio-Rad) at room temperature for 10 min. The permeabilized cells were blocked with ImmunoBlock (KAC, Kyoto, Japan) for 30–60 min after washing twice in tris buffered saline (TBS). Fixed cells were incubated with primary antibodies diluted in blocking solution for 12–18 h at 4 °C and washed twice in tris buffered saline with 0.1% Tween 20 (TBS-T). Secondary antibodies and Hoechst (1:20,000; Life Technologies, California, USA) diluted in blocking solution were added, and the samples were incubated for 1 h at room temperature under a light shield, followed by washing two to three times in TBS-T. The primary and secondary antibodies used in this study were mouse anti-human MHC monoclonal antibody (1:400, #MAB4470, R&D Systems, Minneapolis, USA) and goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 488 conjugate (1:500, #A-11001, Invitrogen; Thermo), respectively. Fluorescent images were analyzed using an all-in-one fluorescence microscope (BZ-X800) with an image cytometer module (Keyence, Osaka, Japan). The fusion index was calculated by dividing the number of nuclei within MHC-positive myotubes containing two or more nuclei by the total number of nuclei in five randomly chosen fields, termed the fusion ratio.

Reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted using the TRIzol Reagent Kit (Thermo) and reverse-transcribed using the PrimeScript RT reagent kit (Takara-bio, Shiga, Japan). RT-qPCR was performed using SYBER Green Master Mix (Takara-bio) and the primer set described below using the CFX Real-time PCR System (Bio-Rad). The results of all experiments were analyzed using the ∆Ct method. RT-qPCR for myogenic gene expression was performed using the following primer pairs: myogenin: Fwd 5′-AGCCTCCAAATCCACTCCCTGAAA-3′ and Rev 5′-AGCCACTGGCATAGGAAGAGATGA-3′; desmin: Fwd 5′-GGAAGCCGAGGAATGGTACA-3′ and Rev 5′-TCGATCTCGCAGGTGTAGGA-3′; MHC: Fwd 5′-AGAGCAGCAAGTGGATGACCTTGA-3′ and Rev 5′-TGGACTCTTGGGCCAACTTGAGAT-3′; and GAPDH: Fwd 5′-TCCCAACGTGTCTGTTGTGGATCT-3′ and Rev 5′-TGTTGAAGTCGCAGGAGACAACCT-3′. All primers were synthesized at Eurofins Japan (Tokyo, Japan).

Statistical analysis

Data were expressed as mean ± standard error of mean of at least three independent experiments using a two-sided Student’s t-test. Differences were considered significant at p < 0.05.

Results and discussion

Proliferation of bovine myoblasts in SFM

Bovine primary myoblasts were enzymatically isolated from cattle meat obtained from a commercial slaughterhouse. At passage 0, most isolated bovine cells (95%) were positive for Pax7, a muscle progenitor cell marker (data not shown), indicating low level of fibroblast contamination. Therefore, the cells were used as myoblasts in this study. In SFM, we included the minimum essential compounds required for the survival of animal cells. These compounds listed in Table 1 are known to exert effects in the absence of serum. For instance, ITS, lipids, and albumin provide critical support for mammalian cell metabolism, membrane integrity, and nutrient transport, respectively (Devireddy et al., 2019; Takii et al., 2022), and FGF-2 supports the survival and proliferation of myoblasts (Stout et al., 2023).

To examine the proliferation capability of SFM, cell numbers were counted every 24 h for 5 days after seeding. Myoblasts cultured in GM proliferated over time, whereas those cultured in SFM demonstrated minimal proliferation. Myoblast numbers in GM were significantly higher than those in SFM (Fig. 1a). We evaluated the proliferative abilities by measuring DNA synthesis via EdU incorporation (Fig. 1b). The proportion of EdU⁺ myoblasts in SFM (24.3% ± 5.3%) was significantly lower than that in GM (36.2% ± 5.0%) (p < 0.02) (Fig. 1c). These results indicate that SFM reduces myoblast proliferation and generates few progenies. Hence, it is considered necessary to add any growth-promoting components to restore cell proliferation in SFM. Therefore, we next attempted to identify food-grade growth-promoting components in SFM containing several compounds.

Fig. 1.

Proliferation of myoblasts in serum-free medium. (a) Growth curve of bovine myoblasts in GM and SFM from day 0 to day 5 of culture. *p < 0.05, ***p < 0.01 (Student’s t-test) at each time point, n = 3. (b) EdU staining of bovine myoblasts in GM and SFM at 24 h of culture. Scale bar, 100 μm. (c) Ratio of EdU⁺ cells. **p < 0.02 (Student’s t-test), n = 7. GM FBS-containing growth medium, SFM serum-free medium, EdU 5-ethynyl-2-deoxyuridine

Screening of food powders with proliferative activity

To identify food ingredients with cell proliferative activity in SFM, a screening was conducted using the food ingredients listed in Material and Methods section. We then selected nine commercially available dried food powders derived from plants, animals, and fish. Myoblasts were counted on day 4. Among the tested foods, only whey significantly promoted proliferation under serum-free conditions (p < 0.02) (Fig. 2a). The proliferation-promoting effect of whey was also confirmed in myoblasts derived from another individual animal (data not shown). Compared with egg white, whose water extract was found to promote the growth of bovine myoblasts (Park et al., 2023a), the proliferation effect of whey was significantly high (p < 0.05) (Fig. 2a). The whey used in this study was a dried product from liquid whey obtained from the general cheese manufacturing process, where raw milk is heat-pasteurized and then separated into solid curds and liquid whey using lactic acid bacteria and rennet. The whey was spray-dried without any additives to produce the dried product, suggesting that the whey itself, rather than any impurities, exhibits proliferative activity. Nevertheless, even in the presence of whey, the cell count was approximately 50% of that in GM (Fig. 2a), indicating the need for further improvements.

Fig. 2.

Screening of food ingredients supporting bovine myoblast proliferation. (a) Bovine myoblast counts at day 4 in SFM with various 0.1% food ingredients. SFM and GM serve as controls. *p < 0.05, **p < 0.02 (Student’s t-test). n = 4. (b) Bovine myoblast counts at day 3 with varying whey concentrations. **p < 0.02, ***p < 0.01 (Student’s t-test). n = 3. (c) Bovine myoblast counts at day 3 on collagen and fibronectin with 0.2% whey in SFM. GM serves as a control. ***p < 0.01, ns p ≥ 0.05 (Student’s t-test). n = 3. (d) Bovine myoblast proliferation in GM and 0.2% whey in SFM from day 0 to day 4. *p < 0.05, ***p < 0.01 (Student’s t-test at each time point); n = 4

Optimization of culture conditions using whey

To improve the proliferation-promoting effect of W/SFM, we tested the effects of the optimization of whey concentration and ECM coating. Cell proliferation was evaluated by counting cell numbers on day 3. As shown in Fig. 2b, myoblasts proliferated in a whey concentration-dependent manner, with 0.2%–0.5% whey being optimal. For ECM, we examined the effect of fibronectin and Matrigel on myoblasts, as they are known to improve the proliferation of muscle progenitor cells (Zhang et al., 2021). As depicted in Fig. 2c, myoblast proliferation significantly improved in W/SFM on fibronectin compared with that on collagen (p < 0.01). In contrast, we could not quantify myoblast numbers on Matrigel due to detachment difficulties with TrypLE Express. Microscopic observation suggested no significant difference compared with that on collagen (data not shown). The requirement of fibronectin in W/SFM is related to a previous report that demonstrated that fibronectin was essential for the adhesion and proliferation of fibroblasts in milk-containing serum-free culture media (Steimer et al., 1981).

On the basis of these results, we compared myoblasts cultured on fibronectin-coated dishes in W/SFM containing 0.2% whey with those cultured in GM for 4 days. The resulting growth curve revealed that myoblast proliferation in W/SFM improved significantly to > 80% of that in GM (Fig. 2d), indicating that myoblast proliferation in W/SFM is almost comparable to that in GM. Hence, in subsequent experiments, we used W/SFM containing 0.2% whey and fibronectin-coated dishes.

In a previous study, sufficient proliferation-promoting effect of milk-derived components was observed only in the presence of serum even in cell lines that were less serum-dependent than primary myoblasts (Belford et al., 1995). Nevertheless, the present study demonstrates a novel finding, i.e., serum-free proliferation of primary myoblasts was achieved using whey and only the components listed in Table 1. In addition, although proteins isolated from whey are known to promote cell proliferation and differentiation (Takada et al., 1996), achieving sufficient growth with crude whey that was simply dissolved without any special extraction operations is significant from the perspective of simplification of the manufacturing process.

Differentiative characteristics of myoblasts cultured in W/SFM

We next explored whether myoblasts that proliferated in W/SFM retained their myogenic differentiation capability. We evaluated differentiation using the morphology of cultured myoblasts, expression of differentiation markers, and differentiation into myotubes. As illustrated in Fig. 3a, bovine myoblasts were cultured in GM, W/SFM, and SFM for 4 days and then switched to DM to induce differentiation into myotubes. Morphology and differentiation were evaluated by optical microscopy. Representative brightfield images at 2 and 6 days after seeding are shown in Fig. 3b.

Fig. 3.

Differentiation of myoblasts in W/SFM, SFM, and GM. (a) Schematic of the experimental schedule. (b) Brightfield images of myoblasts in GM and SFM with 0.2% whey at days 2 and 6. Scale bar, 100 μm. (c) RT-qPCR of myogenic gene expression in myoblasts at day 2. Expression in GM-cultured cells was set to 1.0 as the control; other media were expressed as a ratio. Data are mean ± SD from three independent experiments, each in technical triplicate. *p < 0.05, **p < 0.02, ***p < 0.01 (Student’s t-test, n = 3). (d) RT-qPCR of desmin expression in myoblasts, which were cultured in SFM for 2 day followed by in each medium for 1 day. Expression in SFM-cultured cells at day 2 was set to 1.0 as the control; expressions at day 3 were expressed as a ratio. Data are mean ± SD from three biological triplicates, each in technical triplicate. *p < 0.05 (Student’s t-test, n = 3). (e) Fluorescent images of differentiated myoblasts at day 7. Green, MHC; blue, DAPI. Scale bar, 100 μm (f) Fusion index ratio at day 7. ns p ≥ 0.05 (Student’s t-test, n = 4)

At day 2, myotubes began forming in SFM but were scarcely observed in GM and W/SFM. We next performed RT-qPCR for desmin, myogenin, and MHC as early, intermediate, and terminal differentiation markers, respectively, at day 2, which revealed higher mRNA expression of these genes in SFM-cultured cells than in GM-cultured cells (Fig. 3c). After adding whey to SFM, we detected reduced mRNA expression of these genes, indicating that whey inhibits the initiation of myogenic differentiation and maintains proliferative capability in SFM culture. To determine whether whey inhibits the progression of myogenic differentiation, SFM-cultured cells were further cultured for 1 day in SFM, W/SFM, and GM. The effect was assessed by RT-qPCR using desmin as a marker of mid-to-late myogenic differentiation. Desmin expression in myoblasts cultured in SFM was markedly increased on 1 day, whereas that of myoblasts cultured in W/SFM was significantly lower (p < 0.05) (Fig. 3d), indicating that whey suppresses the progression of the myogenic differentiation. As these findings confirm the suppression of both initiation and progression of myogenic differentiation, they suggest that whey effectively inhibits unintended differentiation during proliferative culture conditions.

At day 4, myoblasts were induced to differentiate into myotubes by switching to DM. By day 6, W/SFM-cultured myoblasts formed myotubes similarly to GM-cultured myoblasts, whereas SFM-cultured myoblasts remained almost unchanged (Fig. 3b). Immunostaining of MHC at day 7 (Fig. 3e) revealed that the ratio of MHC⁺ myotubes in W/SFM was almost equal to that in GM (Fig. 3f) (p > 0.05), indicating that whey exerts no adverse effect on terminal differentiation. As primary myoblasts are known to be more sensitive to culture conditions than myogenic cell lines (e.g. C2C12 and L6) (Lubna et al., 2017) and to begin differentiating upon the removal of serum, maintaining and controlling the myogenic capability of primary myoblasts in serum-free growth media is an important step forward.

Long-term culture in whey-supplemented SFM

To confirm the suitability of the medium for robust expansion, we examined the proliferative performance and differentiation capability of myoblasts with repeated passaging. Myoblasts at passage 3 (P3) were cultured in W/SFM and GM. Cells were seeded in fibronectin-coated plates and passaged every 3–4 days to maintain < 80% confluence for six passages. Cell numbers at P3, P5, P7, and P9 were counted for comparing proliferation in each medium. Proliferation in both media showed no significant differences from P3 to P9 (p > 0.05), whereas proliferation decreased at higher passages (Fig. 4a). Then, myoblasts at P5, P7, and P9 were induced to differentiate in DM at > 80% confluence and immunostained for MHC. The formation of myotubes was compared qualitatively based on microscopic observation due to insufficient cell numbers at higher passages. The formation of MHC⁺ myotubes appeared similar in each medium at each passage, with the differentiation capability significantly decreased after P7 (Fig. 4b). These results indicate that W/SFM maintains the proliferation and myogenicity of myoblasts over long-term culture, enabling expansion for at least six passages over 15 days under serum-free conditions.

Fig. 4.

Long-term culture comparison in GM and W/SFM (a) Myoblast proliferation rate at P3, P5, P7, and P9, with cells successively passage from P3 to P9. The initial number of seeded myoblasts, 1 × 10⁴ cells, was set to 1.0 at each passage. ns p ≥ 0.05 (Student’s t-test, n = 3). (b) Fluorescent images of differentiated myoblasts at P5, P7, and P9. Green, MHC; blue, DAPI. Scale bar, 100 μm

The loss of the differentiation capability of cells when they are still proliferative is consistent with a previous study that examined muscle aging in vitro (Park et al., 2023b); hence, it is considered a fundamental property of primary myoblasts. Because longer maintenance of myoblast differentiation capabilities is crucial for the efficient ex vivo production of muscle structures, it is essential to explore methods for suppressing the aging of primary myoblasts. As the P16^INK4a/Rb/E2F signaling pathway is known to drive the aging of muscle satellite cells (Sousa-Victor et al., 2014), there exists a need for an approach to modify this signaling pathway.

Evaluation of proliferation-promoting activity of heat-treated whey

In general, whey is processed under unsterile conditions; however, sterilization is essential to use it as a component of cell culture medium. To investigate the applicability of heat sterilization for W/SFM, we evaluated the effect of heat-treated whey on cell proliferation. Accordingly, heat treatment of whey solution and FBS was performed by boiling. Heat-treated FBS showed marked turbidity due to protein aggregation, whereas whey exhibited less cloudiness. In addition, protein aggregation was more prominent in FBS, including the presence of large visible aggregates. After centrifugation at 2500 g to remove large aggregates that may interfere with cell proliferation, W/SFM and GM were prepared by adding boiled whey solution and serum to SFM and DMEM/F12 supplemented with FGF-2, respectively. Whey solution and FBS without heat treatment were used as controls. Cell numbers were counted 3 days after seeding. Heat-treated serum significantly decreased the proliferation of cells in GM (p < 0.02) (Fig. 5a). Conversely, there was no significant change in cell proliferation in W/SFM (Fig. 5b) (p > 0.05). These findings indicate that whey can support cell proliferation after boiling. Therefore, whey-containing medium can be sterilized by heat, which represents a simpler large-scale method than UV or filter sterilization (Walker, 2014). As whey can be easily extracted and withstand heat sterilization, this is a significant step forward that could enable the production of cell-based foods in food-grade operations. To the best of our knowledge, this is the first study to demonstrate that the proliferation-promoting component of whey is heat-resistant. However, further research is needed to identify this component and the underlying mechanisms. Previous studies have shown that whey proteins (WP) support muscle cell proliferation via PI3K/Akt and MAPK/ERK signaling, along with anti-apoptotic and antioxidant effects (Li et al., 2024; Sundaram et al., 2025). Given these findings, it is plausible that certain functional domains within WP remain active even after thermal denaturation. As there are many previous studied on the production of antioxidant peptides from WP (Saubenova et al., 2024; Knowles et al., 2019), whey-derived antioxidant peptides may be a promising key component.

Fig. 5.

Effect of heat treatment. (a) Relative myoblast number in unheated and heated GM at day 3. The medium was prepared by adding heated and unheated FBS to DMEM/F12 supplemented with FGF-2. The mean value of unheated GM is set to 1.0. ***p < 0.01 (Student’s t-test, n = 3). (b) Relative myoblast number in unheated and heated W/SFM at day 3. The medium was prepared by adding heated and unheated whey solutions to SFM. The mean value of unheated W/SFM is set to 1.0. ns p ≥ 0.05 (Student’s t-test); n = 3

We developed an SFM supplemented with food ingredients that support the expansion of bovine myoblasts and also maintain their differentiation capability. We found that whey was the most potent in promoting cell proliferation among the tested food ingredients (Fig. 2a). In W/SFM, bovine myoblasts proliferated by > 80% of that in GM (Fig. 2d). Furthermore, when differentiation was induced, the myoblasts cultured in W/SFM formed comparable myotubes to those cultured in GM (Fig. 3e, f). Even in long-term culture, the proliferation and differentiation capabilities between GM and W/SFM were not significantly different (Fig. 4). We also found that the proliferation-promoting activity of whey was heat-resistant (Fig. 5b), which can significantly benefit in being sterilized easily in food factory operations.

Therefore, we conclude that whey improves the proliferation of myoblasts in SFM and also maintains their myogenicity. Considering that whey, a by-product of cheese-making, is commonly consumed as a protein-rich food, easy to obtain, and rich in food consumption experience, our findings might significantly contribute to the production of cell-based foods in a more efficient and consumer-acceptable manner.

Future studies should aim at reducing the number of compounds used in SFM, listed in Table 1, to achieve a medium composition consisting of only food components. Combining other food ingredients with W/SFM could also be beneficial. In addition, the applicability to suspension and scale-up culture requires further investigation, as only adherent culture was conducted in this study.

Author contributions

Makoto Segawa conceptualized, conducted experiments, performed data analysis, designed figures and wrote main manuscript text. Eri Kitagawa assisted primary cell isolations. Yasutaka Nishiyama and Takanori Hasegawa contributed to conceptualize and scrutinize this manuscript. All authors approved the final version for submission.

Data availability

The data that support the findings of this study are available from the corresponding author, Takanori Hasegawa, upon reasonable request.

Declarations

Competing interests

This study was conducted with support from a research fund of the Research & Development Center, NH Foods Ltd. All authors are employees of NH Foods Ltd. The authors have no conflicts to declare.