Effect of whey protein hydrolysate isolation as a partial replacement of fetal bovine serum on proliferation and differentiation of Hanwoo primary muscle cells

TL;DR

This study shows that whey protein hydrolysate can replace fetal bovine serum in growing Hanwoo muscle cells, reducing costs and ethical concerns.

Contribution

The study introduces whey protein hydrolysate as a sustainable alternative to fetal bovine serum in muscle cell culture.

Findings

WPH at 0.9 mg/mL supported cell proliferation similar to 20% FBS.

WPH at 0.06 mg/mL enhanced differentiation with higher MyHC expression than FBS.

WPH offers a cost-effective and ethical replacement for FBS in cultured meat production.

Abstract

This study aimed to evaluate the potential of whey protein hydrolysate isolation (WPH) to partially replace fetal bovine serum (FBS) in Hanwoo primary muscle cell cultures, and to address issues related to cost, animal ethics, and undefined elements of FBS. WPH was prepared by enzymatic hydrolysis using bromelain. For proliferation, 10% FBS and 0.3–0.9 mg/mL WPH were used, and for differentiation, 1% FBS and 0.03–0.09 mg/mL WPH were applied. The results showed that cell proliferation increased with increasing WPH concentration, and the levels were similar to those of 20% FBS on days 4 and 5. Therefore, 0.9 mg/mL WPH was considered the most suitable concentration for proliferation. Differentiation initially increased and then decreased with increasing WPH concentration, but mRNA MyHC expression was higher than that of the 20% FBS group. Therefore, 0.06 mg/mL WPH was found to be the most suitable concentration for differentiation. These results suggest that WPH can effectively replace FBS to reduce the production costs of cultured meat and increase the value of by-product utilization, while providing a sustainable and ethical alternative source of nutrients.

Figures

Proliferative activity of Hanwoo satellite cells following 5 days of treatment with hydrolyzed whey protein (WPH). (A) Representative phase-contrast images of Hanwoo satellite cells cultured in the presence of hydrolyzed whey protein isolate (WPH) for 5 days. (B) MTS values of Hanwoo satellite cells cultured for 1, 2, 4, and 5 days with whey protein hydrolysate added. F0: Cells grown in medium containing 0% FBS, F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.3 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.6 mg/mL WPH, F10W9

Relative mRNA expression levels of (A) PAX7, (B), MYOD1 in Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 5 days. F0: Cells grown in medium containing 0% FBS, F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.3 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.6 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.9 mg/mL WPH. ^a–c^Means with different superscripts differ significantly (*p* < 0.05)

Relative mRNA expression levels of (A) *MYOG* and (B) *MyHC* in Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 4 days. F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.03 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.06 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.09 mg/mL WPH. ^a−c^ Means with different superscripts differ significantly (*p* < 0.05)

Full text

Introduction

In vitro cell culture is a technique applied across various fields, traditionally serving as a key tool in cytology and clinical experiments. In recently years, its use has expanded into agriculture, particularly livestock faming, where it plays an important role in reproductive science and meat science. Notably, cultured meat, produced by culturing muscle or fat cells in vitro, has emerged as a promising future food source (Kadim et al., 2015). To culture cells, synthetic media such as basal media, fetal bovine serum (FBS), and penicillin streptomycin antibiotics (PSA) are required. Among them, FBS is a substance that plays an important role in cell proliferation and differentiation. However, it not only accounts for 80% of the cost of synthetic media, but also continuously raises issues with animal ethics (Choudhury et al., 2020). Therefore, the need for a substance that can replace FBS is emerging.

As previously described, FBS contains nutrients such as hormones, growth factors, cytokines, and proteins essential for cell growth. Up to now, research on FBS substitutes aims to develop a mixed substance that can completely replace its main substances by replacing them one by one (Stout et al., 2023). To replace hormones, growth factors, and cytokines in FBS, the development of media with added substances such as FGF2, insulin, EGF, IGF, and TGF-β1 is mainly being conducted (Kolkmann et al., 2020; Lim et al., 2024). Other studies are being conducted using Chlorella vulgaris extract or microalgae such as cyanobacteria (Synechococcus sp. PCC 7002) as substitutes for nutrients such as proteins and lipids (Yamanaka et al., 2023; Haraguchi et al., 2023). Studies are actively being conducted by adding additives to cells and utilizing the protein synthesis mechanism.

Mammalian target of rapamycin (mTOR) is an intermediate of the key translational control pathway that regulates cell cycle, proliferation, and growth (Li & Sumpio, 2005). When insulin, IGF-1, and leucine are attached to mTOR, the mTOR pathway is activated and p70s6k and 4E-BP1 are phosphorylated, which can induce cell growth and proliferation (Melnik et al., 2012). Consequently, supplements containing high levels of insulin or leucine are important for activating mTOR (Rehman et al., 2023). Whey protein is known to be rich in leucine, and research is actively being conducted on the use of this protein to treat sarcopenia and maintain muscle mass (Cereda et al., 2022; Camajani et al., 2022). Moreover, whey protein is known to have various roles because it is rich in glycomacropeptides and other trace proteins (bovine serum albumin, folate-binding protein, proteoglycan 3, osteopontin) (Mehra et al., 2021). Therefore, we conducted this study to determine whether whey protein containing a large amount of leucine could reduce the required concentration of FBS in cell culture while maintaining proliferation and differentiation effects comparable to those of 20% FBS.

Materials and methods

Cattle

Top round muscle samples were harvested from carcasses of Hanwoo (Korean native cattle) steers aged 33 and 34 months, respectively, at the FarmStory Hannaeng Central Factory (Cheongwon-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea). Animals, identified under tracking number ‘002–1317-3849-6’, were castrated males. All animal handling procedures and experimental protocols were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University (Approval No.: CBNUA-2107-23-01).

Hanwoo satellite cell and Whey protein hydrolysate Preparation

Hanwoo satellite cells (H-SC) were used as isolated and purified cells in a previous study (Park et al., 2024). The carcass used was the rump of a 33 and 34 months old Hanwoo (Korean native cattle). It was stored in liquid nitrogen at −196 °C immediately after collection and used in the experiment. Hydrolyzed whey protein was prepared using whey protein isolate (WPI) produced by Hilmar Ingredients (Hilmar, CA, USA; protein 91.7%, lactose 0.5%, fat 1.4%). WPI was mixed with water at a ratio of 1:10 (w/v) and stirred until it was sufficiently mixed. The temperature of the solution was increased to a constant level of 55 °C and the pH was adjusted to 7.0 (using 1 M NaOH solution). Bromelain enzyme (enzyme/substrate ratio = 1:20 w/w) was then added to the stirred WPI for hydrolysis. After mixing constantly for 1 h, the mixture was heated at 90 °C for 15 min to inactivate the bromelain enzyme. The inactivated hydrolysate was centrifuged at 6,000 rpm for 30 min to collect the supernatant, which was then freeze-dried and used in this study.

Hanwoo satellite cell proliferation culture and myogenic differentiation

H-SC stored frozen was thawed in a 37 °C water bath immediately before use in the experiment. Growth medium (GM) used in this study was Ham’s F-10 Nutrient Mix containing 1% PSA (Penicillin-streptomycin-amphotericin). The following groups were used: a positive control group (F20) containing 20% ​​FBS, a negative control group (F0) without adding FBS, a treatment group containing 10% FBS and 0.3 mg/mL hydrolyzed whey protein isolation (WPH; F10W3), a treatment group containing 10% FBS and 0.6 mg/mL WPH (F10W6), and a treatment group containing 10% FBS and 0.9 mg/mL WPH (F10W9). To induce differentiation, cells were cultured in the same GM (containing 20% ​​FBS; commonly used) until 70% confluence. The GM was then replaced with a differentiation medium (DM), which consisted of Dulbecco’s modified Eagle’s medium (DMEM/High Glucose) and 1% PSA. The following groups were used: a control group (F20) containing 2% FBS, a treatment group containing 1% FBS and 0.03 mg/mL WPH (F10W3), a treatment group containing 1% FBS and 0.06 mg/mL WPH (F10W6), and a treatment group containing 1% FBS and 0.09 mg/mL WPH (F10W9). For cell culture, 1,800 and 3,000 cells were seeded into 96-well plates and T25 flasks, respectively. Cells were maintained at 37 °C with 5% CO_2_. For proliferation, cells were cultured for 5 days. For differentiation, cells were cultured for another 4 days.

Cell proliferation assay

Proliferation of cells was measured using a CellTiter 96^®^ AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Cells were cultured in 96 wells for 4 days. Cell viability was measured on days 1, 2, 4, and 5. The last medium of each well containing cultured cells was removed. After washing with PBS, 3-[(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2 H-tetrazolium (MTS)] was added into each well (MTS was mixed with culture medium at a ratio of 2:10). After reacting at 37 °C with 5% CO_2_ for 2 h, the absorbance was measured at 490 nm.

Immunofluorescence staining

Cells that had completed proliferation and differentiation were fixed with 4% paraformaldehyde at 37 °C for 45 min, washed twice with PBS for 10 min each, and then used in this experiment. Fixed cells were permeabilized with 0.1% Triton-X (in PBS) for 20 min at room temperature, blocked with 2% bovine serum albumin (BSA) for 30 min, and washed with PBS. After permeabilization, cells were added to each well containing 2% BSA and primary antibodies (Pax7, PA5-68506, Invitrogen, Waltham, MA, USA; myosin, M4276, Sigma, St. Louis, MO, USA; diluted 1:100, respectively) and incubated at 4 °C overnight. To remove residual antibodies, cells were washed with 0.05% Tween-20 for 10 min and then reacted with secondary anti-rabbit IgG cross-adsorbed antibody (1:100, A21121, Invitrogen) at room temperature for 30 min. Afterwards, nuclei staining was performed using Hoechst 33,342 (1:2000; H3570, Invitrogen). After nuclear staining was complete, cells were washed with PBS to remove residual staining materials and images were taken. The number of nuclei inside and outside the root canal of the captured images was measured using ImageJ. Nuclei counts were calculated for proliferating cells. Fusion index and myotube area were calculated for ​​differentiated cells.

Quantitative real-time PCR (qRT-PCR)

Cells proliferated and differentiated in culture media added with WPH and 10% FBS were removed from the flask using Trizol reagent and used to extract mRNAs with a Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). Extracted mRNA was converted to cDNA according to the manufacturer’s instructions using a cDNA reverse transcription kit (ThermoFisher). PCR was performed using SYBR Green (ELPIS-BIOTECH, Daejeon, Korea). Primer sequences are listed in Table 1. PCR amplification used the following conditions: 50 ℃ for 2 min, 95 ℃ for 10 min, and 40 cycles of 95 ℃ for 15 s and 52–55 ℃ for 1 min. mRNA level was quantified using the 2^−ΔΔCT^ method.

Table 1. Sequences of primers used in RT-qPCROligo NameSequencePAX7_FAAT GCG AGG GTA TGT CCA AGPAX7_RATA CAG TGC CCG ATC TCG TCMYOD_FCTG GGG ACT TCA GCT GTT TCMYOD_RCCT GCC TGC CGT ATA AAC ATMYOG_FATG CCA GAC TAT CCC CTC CTMYOG_RTTC AGG GAG TGG ATT TGG AGMyHC2_FCTG GCT GGA GAA GAA CAA GGMyHC2_RCAC CGT CTG GAA AGA AGA GC

Statistical analysis

All data are expressed as mean and standard deviation. The experiment was repeated more than three times. Statistical analysis was performed using one-way ANOVA followed by Duncan’s multiple range test. The General Linear Model procedure of the SAS program (version 9.4, Cary, NC, USA) was used. Controls (FBS0, FBS20) and treatments (WPH3, 6, and 9) were compared. Treatment effects and comparisons were considered statistically significant at P < 0.05.

Results and discussion

Cell proliferation assay

Figure 1A is a picture showing images of treatment groups with whey protein added at various concentrations to culture medium containing 10% FBS, along with MTS results. Figure 1A shows cells from day 1, when cells begin to attach, to day 4 when cells begin to differentiate. Analysis results showed no difference between treatment groups on day 1. However, there was a clear difference between F0 and other treatment groups on day 4. Similar results were observed with naked eyes between F20 and treatment groups containing whey protein. It has been reported that whey’s antioxidant effect is higher when it is hydrolyzed, thereby enhancing antioxidant power within cells (Corrochano et al., 2018). Abadía-García et al. (Abadía-García et al., 2021) have reported that whey protein hydrolyzed with bromelain has high ACE inhibitory activity. Accordingly, it is believed that whey protein hydrolyzed with bromelain shows these results because its antioxidant capacity is increased in cells, which can relieve oxidative stress of cells and reduce damage to cells, including DNA damage and protein damage caused by reactive oxygen species.Fig. 1. Proliferative activity of Hanwoo satellite cells following 5 days of treatment with hydrolyzed whey protein (WPH). (A) Representative phase-contrast images of Hanwoo satellite cells cultured in the presence of hydrolyzed whey protein isolate (WPH) for 5 days. (B) MTS values of Hanwoo satellite cells cultured for 1, 2, 4, and 5 days with whey protein hydrolysate added. F0: Cells grown in medium containing 0% FBS, F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.3 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.6 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.9 mg/mL WPH. ^a−c^Means with different superscripts differ significantly (p < 0.05)

Results of the MTS experiment, which can indicate the degree of cell proliferation, are shown in Fig. 1B. MTS is a colorimetric analysis test method that stains mitochondria in living cells and measures absorbance. It is known to be a simple and precise way to measure cell proliferation (Kamiloglu et al., 2020). As shown in Fig. 1A, there was no significant difference in cell proliferation between days 1 and 2. On day 4, F20 and treatment groups showed significantly higher values ​​than F0 (P < 0.05), whereas there was no significant difference between F20 and treatment groups (P > 0.05). Atherton et al. (Atherton et al., 2010) have reported that L-leucine (Leu) plays a role in cell promotion by increasing mTOR pathway activation. In particular, F20 and F10W9 showed similar values, suggesting that 0.9 mg of hydrolyzed whey protein could replace 10% FBS to have an effect similar to 20% FBS. On the 5th day, F20 and treatment groups showed significantly higher values ​​than F0 (P < 0.05), whereas F20 and F10W9 did not show a significant difference (P > 0.05). Muscle protein synthesis has been reported to be induced by protein sources such as whey, casein, and soybean (Tang et al., 2009). Moro et al. (Moro et al., 2019) have found that WPH intake can lead to transport of amino acids into muscles, which can activate mTORC1 signaling and increase the muscle protein synthesis rate. Therefore, WPH used in this study might have activated mTOR, leading to greater proliferation.

Proliferation ability of Hanwoo satellite cells when FBS is partially replaced with WPH

Results of analyzing effects of adding whey protein at various concentrations to replace 10% FBS on proliferation of Hanwoo satellite cells are shown in Fig. 2. Images of cells stained with PAX7 are shown in Fig. 2A. The number of nuclei measured based on images is shown in Fig. 2B. As shown in Fig. 2A, treatment groups showed significantly more cells than F0. More cells appeared when WPH level was increased. Although this increase seemed to be a small amount compared to F20, it was confirmed that F10W9 had proliferated significantly even when cells were viewed under a microscope. Paradkar et al. (Paradkar et al., 2019) have reported that FBS 10% and FBS 1% + bovine whey protein 9% show similar growth rates, similar to results of the present study. As shown in Fig. 2b, F20 also had a significantly higher value than other treatment groups. All treatment groups showed significantly higher values ​​than F0 (P < 0.05). One study has found that whey protein hydrolysate is rapidly absorbed and digested in the body to induce skeletal muscle protein anabolism, which might have affected cellular protein synthesis because whey protein hydrolysate contains a lot of branched amino acids (BCAA) (Morifuji et al., 2010). As a result, among treatment groups, F10W9, which contained the highest amount of hydrolyzed whey protein, showed a significantly higher value (P < 0.05), confirming that F10W9 showed the most effective proliferative capacity among treatment groups and displayed results comparable to higher FBS levels.Fig. 2(A) Representative immunostained images of Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 5 days, showing Pax7 expression, nuclear staining (DAPI), and merged channels. (B) Cell counts based on nuclear staining in Hanwoo satellite cells cultured with WPH for 5 days. F0: Cells grown in medium containing 0% FBS, F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.3 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.6 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.9 mg/mL WPH. ^a−d^Means with different superscripts differ significantly (p < 0.05)

PAX7 and MYOD1 mRNA expression levels are shown in Figs. 3A and 3B, respectively. PAX7 mRNA is known to be a transcription factor that plays a key role in the definition and maintenance of muscle satellite cells and skeletal muscle regeneration (Rahman et al., 2023). PAX7 mRNA expression level was higher than that in F0 but lower than that in F20. F10W6 and F10W9 did not show a significant difference in PAX7 mRNA expression level from F20 (P > 0.05). These results indicate that the addition of WPH at 0.06–0.09 mg/mL to 10% FBS achieved a comparable level of PAX7 expression to that of 20% FBS, suggesting that partial replacement of 10% FBS with WPH is feasible. Therefore, it is thought that hydrolyzed whey protein can affect the maintenance of muscle satellite cells and skeletal muscle formation. MYOD1 mRNA showed similar values ​​between F20 and F10W9. The remaining treatments showed significantly lower MYOD1 mRNA levels ​​(P < 0.05). MYOD1 mRNA is known to be a factor that regulates the proliferation of myoblasts and directed development of muscle cell lines (Zhou et al., 2022). As a result, it is expected that the expressed MYOD1 mRNA will help in the development of cells into muscles. The appropriate amount of hydrolyzed whey protein to replace 10% FBS is thought to be 0.9 mg/mL.Fig. 3. Relative mRNA expression levels of (A) PAX7, (B), MYOD1 in Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 5 days. F0: Cells grown in medium containing 0% FBS, F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.3 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.6 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.9 mg/mL WPH. ^a–c^Means with different superscripts differ significantly (p < 0.05)

Differentiation ability of Hanwoo satellite cells with FBS partially replaced by WPH

Figure 4 is a drawing analyzing the effect of adding whey protein at various concentrations to replace 10% FBS on differentiation of Hanwoo satellite cells. Differentiation analysis was performed excluding F0, where differentiation did not occur. Immunofluorescence analysis results confirmed that F20 was similar to F10W3 and F10W6, whereas F10W9 exhibited lower differentiation ability compared with the other groups. According to Bar-Peled and Sabatini (Bar-Peled & Sabatini, 2014), leucine plays an important role in promoting protein synthesis in myocytes by activating mTOR1. This research result supports results of the present study. Figures 4B and 4C show myotube area and fusion index based on images, respectively. Myotube formation showed a significantly higher value in F10W6 than in F20. However, it decreased sharply in F10W9 (P < 0.05). Fusion index also showed the highest value in F10W6, whereas F20 showed no significant difference (P > 0.05). One study has found that β-hydroxy-β-methylbutyrate (HMB), a leucine metabolite, can promote muscle protein synthesis and muscle hypertrophies (Jackman et al., 2017). Therefore, it could be judged that differentiation was well achieved due to the presence of leucine metabolite in hydrolyzed whey protein. On the other hand, a lower result in F10W9 was thought to be because protein metabolism control mechanism was disrupted by excessive nutrient content (Goldberg, 2003). According to Johnson et al. (Johnson et al., 2013), nutrients abundant in the medium can activate mTOR and promote cell growth and proliferation, which can inhibit differentiation.Fig. 4(A) Representative immunostained images of Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 4 days, showing myosin expression, nuclear staining (DAPI), and merged channels. (B) and (C) represent myotube area and fusion index, respectively, as measured using ImageJ software. F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.03 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.06 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.09 mg/mL WPH. ^a−b^ Means with different superscripts differ significantly (p < 0.05)

Levels of MYOG and MyHC mRNAs as genetic traits related to differentiation are shown in Fig. 5A and 5B, respectively. MYOG mRNA did not show a difference between control and treatment groups. However, it showed a high value in the treatment group. Muscle synthesis is known to depend mainly on cellular pathways that regulate protein turnover (Figueiredo, 2019). Sahin et al. (Sahin et al., 2024) have reported that whey protein can activate mTOR to improve muscle protein synthesis. This is similar to results of the present study. On the other hand, HyHC mRNA showed a significantly higher value in F10W6 than in other treatment groups. F20 showed significantly lower HyHC mRNA level (P < 0.05). F10W9 showed a lower result, similar to fluorescent staining results. This might be because high concentrations of leucine could lead to overexpression of mTOR, which can inhibit Akt/FoxO, consequently leading to lower differentiation (Saxton & Sabatini, 2017).Fig. 5. Relative mRNA expression levels of (A) MYOG and (B) MyHC in Hanwoo satellite cells cultured with hydrolyzed whey protein isolate (WPH) for 4 days. F20: Cells grown in medium containing 20% FBS, F10W3: Cells grown in medium containing 1% FBS and 0.03 mg/mL WPH, F10W6: Cells grown in medium containing 1% FBS and 0.06 mg/mL WPH, F10W9: Cells grown in medium containing 1% FBS and 0.09 mg/mL WPH. ^a−c^ Means with different superscripts differ significantly (p < 0.05)

Conclusions

This study was conducted to determine whether WPH could replace FBS to produce cultured meat. In this study, WPH was added to the culture medium to partially substitute for 10% FBS, aiming to achieve a comparable effect on cell proliferation and differentiation. Results confirmed that using 0.9 mg/mL WPH and 0.06 mg/mL WPH resulted in a high proliferation rate and myotube area, respectively. Based on the present finding, the use of WPH allowed comparable cell proliferation and differentiation even when the FBS concentration was reduced from 20% to 10%. This suggests that WPH supplementation could be a practical approach to reduce the reliance on FBS by at least half. Reducing 10% FBS cannot only decrease cost, but also use natural products for cell culture. It is expected to have a positive impact on consumers in the future. In addition, the use of whey protein, a by-product, can contribute to the creation of high-value-added products with WPH. Further studies are warranted to investigate whether the FBS concentration can be further decreased to 1% or even eliminated.

IACUC

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