Serum-free cultured meat production by using Pichia pastoris-derived recombinant albumin

Summary

Cultured meat production involves the growth, differentiation, and food processing of muscle satellite cells. However, its heavy reliance on fetal bovine serum (FBS) significantly increases production costs, which has prompted interest in developing alternative serum substitutes. In this study, we aimed to enhance the economic viability and scalability of cultured meat production by cultivating and differentiating bovine muscle satellite cells (bMuSCs) in media supplemented with Pichia pastoris (P. pastoris)-derived bovine recombinant albumin (Br-A) or porcine recombinant albumin (Pr-A). As a result, P. pastoris-derived recombinant albumin (rAlbumin) effectively supported the proliferation of bMuSCs while maintaining their differentiation potential. In addition, the expression of the satellite cell marker Paired-box7 (Pax7) was upregulated. Moreover, P. pastoris-derived rAlbumin effectively induced myotube formation in the differentiation process. These results highlight the potential of P. pastoris-derived rAlbumin as a cost-effective and scalable alternative to serum components, providing a promising solution for serum-free media in the cultured meat industry.

Graphical abstract

Highlights

• •Bovine or Porcine recombinant albumin was synthesized from Pichia pastoris
• •Bovine recombinant albumin promoted bMuSC maintenance and proliferation
• •Bovine recombinant albumin promoted bMuSC differentiation efficiency into myotubes

Biotechnology; Food biotechnology

Introduction

Cultured meat is produced by cultivating animal cells in a controlled environment to create edible muscle tissue.¹ This approach has the potential to meet the growing demand for meat while addressing environmental and animal welfare concerns associated with traditional livestock farming.^2,3,4 The production of cultured meat is based on principles and techniques from cell biology and tissue engineering and consists of four main steps: cell acquisition, proliferation, differentiation, and food processing.¹ During the proliferation and differentiation phases, the cultured media must contain essential nutrients to support the growth of muscle satellite cells (MuSCs).⁵ Fetal bovine serum (FBS) is the most widely used serum supplement for MuSC culture and accounts for the largest proportion of the cost of cultured meat production.³ However, FBS is difficult to standardize, may contain undesirable components, and raises animal welfare concerns, thereby hindering the industrial scalability of cultured meat.⁶ Therefore, developing a serum-free medium for the mass production of cultured meat is imperative.

Recently, an inexpensive serum-free medium for human induced pluripotent stem cell culture was described.⁷ This medium contained a simple mixture of glucose, amino acids, vitamins, salts, fatty acids, minerals, and proteins found in animal tissues. Expanding upon these findings, Stout et al. demonstrated that the addition of human recombinant albumin (Hr-A) to this serum-free medium enables cell proliferation and maintenance of bMuSCs.⁴ However, although Hr-A is produced using recombinant technology, its application in food products may raise ethical concerns regarding the incorporation of human proteins. Therefore, this study aimed to evaluate the proliferation and differentiation efficiency of bMuSCs cultured with Pichia pastoris (P. pastoris)-derived bovine- (Br-A) or porcine- (Pr-A) recombinant albumin, in order to develop a more practical and safe alternative for cultured meat production.

Proteins produced by microorganisms have been widely used in scientific research, pharmaceuticals, food, and various industrial fields.^8,9 Yeast is a eukaryotic cell widely used for protein production due to its protein expression mechanisms being similar to those of mammalian cells.^10,11 However, yeast has a different biological system from humans; it is less likely to be contaminated with substances that are pathogenic to humans and is likely to be safe for human health.¹² In addition, yeast can be mass-cultured within a relatively short period, which can reduce the cost of albumin production and contribute to improving supply stability.¹³ Currently, yeast-based albumin production methods commonly utilize various host systems, including Saccharomyces cerevisiae (S. cerevisiae),^14,15,16 Hansenula polymorpha (H. polymorpha),^17,18 and P. pastoris.^{19,20,21,22,23,24} Among these systems, P. pastoris is particularly notable for its high yield production of recombinant proteins and its ability to perform post-translational modifications similar to those in higher eukaryotes, which are important for protein functionality.²⁵ Furthermore, its ability to utilize methanol as a carbon source provides distinct advantages in inducing protein expression.^26,27,28,29 Based on these characteristics, P. pastoris has emerged as a preferred host for recombinant protein production due to its high-density fermentation, secretion of properly folded proteins, and eukaryotic posttranslational modification capabilities. Recent advances in strain engineering and bioprocess optimization have further enhanced its utility as a cost-effective and scalable platform for industrial applications.^30,31,32,33

In this study, we aimed to establish the serum-free cultured meat production system using rAlbumin derived from P. pastoris and to explore its potential for supporting the culture of bMuSCs. The results revealed that P. pastoris-derived Br-A and Pr-A effectively supported the proliferation of bMuSCs while maintaining their myogenic potential. Additionally, long-term culture was also viable, preserving the expression of muscle satellite cell marker Pax7. Finally, successful myogenic differentiation was achieved even with a low rAlbumin concentration. The results of this study not only contribute to reducing the cost of cultured meat production but also provide new insights into establishing a microbial expression system for key media components required for the maintenance and differentiation of bMuSCs.

Results

Recombinant albumin synthesis by Pichia pastoris

In this study, codon-optimized recombinant bovine serum albumin (rBSA) and recombinant porcine serum albumin (rPSA) genes were cloned downstream of the AOX1 promoter in the secretory expression vector pPICZaA and expressed as described in the Section: STAR Methods (Figure 1A). Transformants of the P. pastoris strain with high expression levels were selected for upscaled protein expression. After fed-batch fermentation, culture supernatants were collected and used for protein purification. The maximal production of the rBSA and rPSA were 14.8 ± 0.7 mg/mL and 13.3 ± 0.5 mg/mL each as determined at 72 h methanol induction. The purified rBSA and rPSA were analyzed by SDS-PAGE with commercial BSA (Figure 1B). Each recombinant protein showed a molecular weight of almost 66 kDa (black arrow), similar to that of commercial BSA used as the positive control. The purified recombinant proteins were stored at −20°C for further analysis.

Figure 1.

Expression of recombinant bovine serum albumin (rBSA) and recombinant porcine serum albumin (rPSA) in Pichia pastoris

(A) Schematic diagram of rBSA and rPSA expression plasmids.

(B) SDS-PAGE analysis of purified rBSA and rPSA. M, protein molecular weight marker; lane 1, rBSA; lane 2, rPSA; lane 3, commercial BSA. The molecular weight of each band of the protein marker is labeled on the left of the panel.

Pichia pastoris-derived recombinant albumin supports short-term culture of bovine muscle stem cells

To assess the potential of P. pastoris-derived Br-A and Pr-A as alternatives to commercial Hr-A, a short-term culture analysis of bMuSCs was conducted. First, Hr-A, Br-A, and Pr-A were administered at concentrations of 800, 3,200, 6,400, and 11,200 μg/mL for 3 days. At all tested concentrations, Br-A and Pr-A maintained in vitro cell morphology similar to commercial Hr-A (Figures 2A and S1A). The cell viability assay results showed that Hr-A resulted in a higher viability than pure B8 at all tested concentrations. However, cell viability was found to decrease at concentrations of 11,200 μg/mL Br-A and 6,400 μg/mL and 11,200 μg/mL Pr-A. In contrast, cell viability was upregulated compared to the control at 800 μg/mL and 3,200 μg/mL Pr-A, as well as 800 μg/mL Br-A. The highest cell viability was observed at 3,200 μg/mL Br-A (Figures 2B and S1B). Therefore, 800 and 3,200 μg/mL were set as the optimal treatment concentrations.

Figure 2.

Short term culture of bovine muscle stem cells (bMuSCs) with recombinant albumin

(A) Bright field images of passage 3 bMuSCs cultured with 800 μg/mL and 3,200 μg/mL rAlbumin for 4 days. Scale bar: 200 μm.

(B) Cell viability of bMuSCs at short-term culture when cultured with 800 μg/mL and 3,200 μg/mL rAlbumin. The analysis was performed after treating with CCK-8 for 4 h. (n = 3). Different lowercase letters indicate significant differences (p < 0.05); the same lowercase letters indicate no significant difference (p > 0.05).

(C) Cell doubling of short term culture bMuSCs when cultured in different concentrations of recombinant albumin. (n = 3).

(D) Proportion of bMuSCs in the G0/G1, S, and G2/M phases by cell cycle analysis after 4 days of culture under the conditions as indicated. (n = 3).

(E) Representative images of MyHC immunofluorescence staining. Bovine muscle satellite cells were first expanded for 4 days under culture conditions of B8 and 800 μg/mL and 3,200 μg/mL of Hr-A, Br-A, and Pr-A and then transferred to 1% HS differentiation medium for 4 days to induce myotube formation. Red, MyHC; Blue, DAPI. Scale bar: 100 μm.

(F) qRT-PCR analysis for the expression of maintenance markers Pax7 and MyoD of bMuSC cultured with different concentrations of recombinant albumin. All numerical data are shown as mean ± SD (n = 3). Different lowercase letters indicate significant differences (p < 0.05); the same lowercase letters indicate no significant difference (p > 0.05).

Next, we investigated the effect of Br-A and Pa-A on bMuSC proliferation in short-term culture by measuring cell numbers daily from day 1 to day 4. In the control group cultured in pure B8 medium, cell numbers increased steadily from 4.6 × 10⁴ (day 1) to 20.3 × 10⁴ (day 4). The proliferation rates were slightly higher when treated with 800 and 3,200 μg/mL Br-A (20.8 × 10⁴ and 21.6 × 10⁴ on day 4, respectively), while Hr-A showed the strongest effect with 22.8 × 10⁴ and 25.2 × 10⁴ at 800 and 3,200 μg/mL, respectively. In contrast, Pr-A treated cells showed reduced proliferation throughout the culture period, reaching 14.8 × 10⁴ (800 μg/mL) and 18.5 × 10⁴ (3,200 μg/mL) on day 4. The complete time-course data for all conditions are presented in Figure 2C. Despite these differences, cell cycle analysis results showed no significant differences (Figure 2D).

To compare the maintenance of bMuSC stemness in short-term culture with Br-A and Pr-A supplemented media, the expression levels of Paired-box7 (Pax7) and myogenic differentiation 1 (MyoD1) were analyzed by qRT-PCR. The results indicated that Pax7 expression was upregulated in bMuSCs cultured in B8 medium supplemented with rAlbumin. The highest expression level of Pax7 was observed at 3,200 μg/mL Br-A. Noteworthily, the expression level of the MyoD1 gene was downregulated in all experimental groups, except those treated with Hr-A (Figure 2E).

In cultured meat production, MuSC cultivation is distinctly divided into proliferation and differentiation phases, each with clear objectives.¹ Therefore, differentiation experiments were performed to assess whether bMuSCs maintained their differentiation capacity after short-term culture in Br-A and Pr-A supplemented media. Immunofluorescence analysis showed that bMuSCs grown in Br-A and Pr-A-supplemented medium fused to form multinucleated myotubes and expressed MyHC,³⁴ indicating their myogenic differentiation ability (Figure 2F). Taken together, these results suggest that all three types of rAlbumin maintained the differentiation ability of bMuSCs. Moreover, P. pastoris-derived Br-A was more effective than commercial Hr-A in terms of cell viability, proliferation, and stem cell capacity maintenance. Although P. pastoris-derived Pr-A showed good cell survival and stem cell capacity maintenance, its proliferation was considerably lower than that of the Br-A.

Long-term growth of bovine muscle stem cells in Pichia pastoris-derived recombinant albumin

Subsequently, we investigated the effect of P. pastoris-derived Br-A and Pr-A on the long-term culture of bMuSCs for seven passages over 28 days. The morphology of passage 7 bMuSCs cultured in pure B8 and rAlbumin-supplemented medium is shown in Figure 3A. The cell cycle analysis results revealed that there were no significant differences between the experimental groups, consistent with the short-term culture results (Figure 3B). Cumulative population doubling level (CPDL) was analyzed over the cell culture period. Initially, the highest cumulative cell doubling was observed at 3,200 μg/mL Hr-A, but decreased after the six passages, and ultimately, 800 μg/mL Hr-A showed the highest efficiency, followed by 3,200 μg/mL and 800 μg/mL Br-A. Although Pr-A promoted cell proliferation, it did not enhance cell growth in long-term culture compared to pure B8 medium. After 28 days of culture, 800 μg/mL Hr-A continued to promote cell proliferation. In contrast, 800 and 3,200 μg/mL Pr-A did not promote additional proliferation after 20 days, while 3,200 μg/mL Hr-A and both concentrations of Br-A (800 and 3,200 μg/mL) did not support proliferation after 24 days of culture (Figure 3C). qRT-PCR analysis exhibited that Pax7 expression was highest at 800 μg/mL Hr-A, Br-A, and Pr-A. Pax7 expression levels at 3,200 μg/mL Hr-A and Br-A were similar to pure B8 medium, whereas 3,200 μg/mL Pr-A showed down-regulated Pax7 expression. MyoD1 expression was highest at 3,200 μg/mL of Hr-A, while 800 μg/mL of Hr-A and Br-A showed similar MyoD1 expression levels comparable to those of pure B8 medium. In contrast, both Pr-A at 800 and 3,200 μg/mL, and 3,200 μg/mL Br-A down-regulated MyoD1 expression (Figure 3D). Taken together, these results suggest that P. pastoris-derived Br-A improves the growth of bMuSCs while maintaining satellite cell identity at a level comparable to that of commercial Hr-A.

Figure 3.

Long term culture of bMuSCs with recombinant albumin

(A) Morphology of passage 7 bMuSCs cultured in different concentrations of recombinant albumin. Scale bar: 200 μm.

(B) Cell cycle of P7 bMuSCs cultured in different concentrations of recombinant albumin. (n = 3).

(C) Cell doubling of long term culture bMuSCs when cultured in different concentrations of recombinant albumin. (n = 3).

(D) qRT-PCR analysis of P7 bMuSCs for the expression of maintenance markers Pax7 and MyoD with different concentrations of recombinant albumin. All numerical data are shown as mean ± SD (n = 3). Different lowercase letters indicate significant differences (p < 0.05); the same lowercase letters indicate no significant difference (p > 0.05).

Low concentrations of P. pastoris-derived bovine recombinant albumin effectively promote the differentiation of bovine muscle stem cells as a substitute for fetal bovine serum.

Most in vitro cultured meat production methods rely on animal serum to stimulate myoblast division.^35,36,37,38 To explore a serum-free differentiation methods, we replaced the serum with P. pastoris-derived Br-A and Pr-A and determined the most effective concentration. As a control, serum-containing differentiation was performed using DMEM basal medium supplemented with 1% FBS. Serum-free differentiation media were prepared by supplementing DMEM basal medium with P. pastoris-derived Br-A or Pr-A at final concentrations equivalent to 110 μg/mL (0.5% FBS), 220 μg/mL (1% FBS), 660 μg/mL (3% FBS), and 1,100 μg/mL (5% FBS). After 3 days of differentiation in each medium, multinucleated myotube formation was observed under all culture conditions (Figure 4A), and MyHC expression was confirmed by immunocytochemistry (Figure 4B). qRT-PCR analysis revealed that bMuSCs cultured in BSC-GM did not express MyoG or MyHC. Regarding MyoG expression, all Br-A- or Pr-A-containing experimental groups except 1,100 μg/mL Pr-A showed higher expression levels than the 1% FBS condition. Specifically, 660 μg/mL Br-A showed a 3.4-fold higher expression, while 220 μg/mL Br-A and Pr-A exhibited a 2.6-fold increase in expression compared to 1% FBS. However, for MyHC expression, 220 μg/mL Br-A showed a 1.9-fold increase, 110 μg/mL Br-A resulted in a 1.6-fold increase. Additionally, 660 μg/mL Br-A and 220 μg/mL Pr-A showed a 1.3-fold increase in expression compared to 1% FBS. These results suggest that P. pastoris-derived Br-A or Pr-A have a more effective differentiation-inducing potential than FBS in bMuSC differentiation media, which offers the potential to reduce the cost of cultured meat production.

Figure 4.

Differentiation of bMuSCs in Pichia pastoris-derived Br-A- or Pr-A-supplemented serum-free differentiation media

(A) Bright field images of bMuSCs differentiated in P. pastoris-derived Br-A- or Pr-A-supplemented differentiation media. Scale bar: 200 μm.

(B) Representative images of MyHC immunofluorescent staining. Bovine muscle satellite cells were differentiated in P. pastoris-derived Br-A- or Pr-A-supplemented differentiation media. Red, MyHC; Blue, DAPI. Scale bar: 200 μm.

(C) qRT-PCR analysis for the expression of differentiation markers MyoG and MyHC differentiated with different concentrations of recombinant albumin. All numerical data are shown as mean ± SD (n = 3). Different lowercase letters indicate significant differences (p < 0.05); the same lowercase letters indicate no significant difference (p > 0.05).

Discussion

Cultured meat is a promising alternative to address global food demand and ethical concerns in animal agriculture. However, many challenges remain to be resolved, including high cost and ethical limitations of FBS, which is still widely used in MuSCs culture due to its rich composition of growth factors and nutrients.^{2,3,4,6,39,40} To overcome these limitations, active research has focused on developing serum-free media suitable for the proliferation and differentiation of MuSCs.^{4,41,42,43,44,45,46} In particular, developing a serum-free medium that is appropriate for the species (e.g., bovine, porcine, galline, and so forth) and cell types (e.g., muscle and fat) is essential for industrial applicability.

Albumin, a key serum protein, plays multiple roles in cell culture, including nutrient transport, growth factor stabilization, and antioxidant protection.^47,48 While Hr-A containing B8 media has been shown to support bMuSC proliferation and successfully reduce production costs up to 75% ($74–217 per liter) compared to FBS, depending on the mass order size,⁴ homologous (species-matched) albumins may yield more efficient and consistent outcomes in cultured meat production.⁴⁹ Br-A (bovine) and Pr-A (porcine), being derived from food-source species, may offer an advantage in regulatory approval and consumer perception over Hr-A.

Hr-A has been expressed using various hosts including bacteria,^50,51 yeast,⁵² and plants.^53,54 Despite intensive efforts to find better recombinant hosts for protein expression, bacteria and yeast are still the preferred hosts due to several reasons including well-developed genetic techniques, ease of manipulation, and cost-effectiveness. When plants were used as expression hosts, preliminary results showed that 8.0 ± 0.2 g of rHSA was produced from 1 kg of transgenic Bombyx mori cocoons⁵⁵ and 11.88 μg/mL of rHSA was produced in suspension cultures of transgenic tobacco,⁵³ but protein production took more than 10 days or required heating at 70 °C for 1 h during the purification step. In contrast, in the present study where serum albumin was expressed in P. pastoris, rBSA and rPSA were expressed at concentrations of 14.8 ± 0.7 mg/mL and 13.3 ± 0.5 mg/mL, respectively. In addition, it was confirmed that the α-signal peptides rBSA and rPSA were designed to be secreted into the medium, facilitating protein expression and purification, and exhibiting many advantages over existing expression systems. Furthermore, P. pastoris offers advantages for industrial scale protein production, cost-efficient expression, and food-grade safety.^{25,26,27,28,29,30,31,32,33}

Our results demonstrated that Br-A supported the short-term proliferation and myogenic differentiation under serum-free conditions. Br-A, in particular, promoted Pax7 expression, a key marker for stemness maintenance,^56,57 and exhibited robust induction of MyoG and MyHC, markers of early myoblast fusion and terminal muscle cell differentiation.^58,59 While Pr-A supported cell viability at lower concentrations, its overall performance was lower than Br-A, and cytotoxicity was observed at higher concentrations (>6 mg/mL). These effects may stem from post-translational modification issues,^{60,61,62,63,64} such as misfolding, or interspecies protein mismatch. Further biochemical characterization will be required to ensure the safety and consistency of P. pastoris-derived Pr-A in scalable applications. Although our study did not include a direct comparison with FBS, the results demonstrate that Br-A supports consistent proliferation and effective myogenic differentiation in serum-free conditions. These results align with previous findings using Hr-A,⁴ which showed reduced proliferation compared to FBS but preserved functional outcomes, such as myogenic differentiation.

In conclusion, this study demonstrates the potential of P. pastoris-derived recombinant albumins, particularly Br-A, as effective supplement for serum-free culture of bMuSCs. These findings contribute to the development of defined, scalable media for cultured meat production. Future research should explore large-scale validation, cost-performance optimization, and long-term culture strategies to enable practical industrial application.

Limitations of the study

Although MTT assays were used to assess cell viability, it should be noted that this assay reflects mitochondrial metabolic activity rather than actual cell number.^65,66 In this study, MTT and direct cell count data did not always correlate, suggesting that variations in metabolic activity rather than proliferation alone may explain the differences. This highlights the need for using multiple complementary assays to interpret cell growth under serum-free conditions.

Despite the promising findings, this study has several limitations. First, direct comparison with FBS and other commercial albumins is necessary to quantitatively evaluate substitution potential. Second, while Br-A supported proliferation over seven passages, the proliferation rate declined in later stages, which may reflect the inherent challenges in long-term maintenance of bMuSCs in vitro culture conditions.^67,68 Further studies should investigate long-term stability, genomic integrity, and differentiation capacity across extended passaging. In addition, the structural and functional stability of Br-A and Pr-A under stress conditions (e.g., freeze-thaw, heat exposure, and long-term storage) needs to be evaluated, as it remains an important consideration for industrial applications. Lastly, although P. pastoris enables scalable production, detailed cost analysis and purification strategy optimization will be necessary to ensure feasibility at an industrial scale.

Resource availability

Lead contact

• •Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Hyun Woo Choi (choihw@jbnu.ac.kr).

Materials availability

• •There are restrictions to the availability of recombinant bovine serum albumin (rBSA) and recombinant porcine serum albumin (rPSA) because of the lack of an external centralized repository for its distribution and our need to maintain the stock. We are glad to share rBSA and rPSA with reasonable compensation by requestor for its processing and shipping.
• •The rBSA and rPSA generated in this study will be made available on request, but we may require a payment or a completed materials transfer agreement if there is potential for commercial application.
• •All rBSA and rPSA generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

• •This article does not report original code.
• •Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the ‘High Value-added Food Technology Development Program’ and was funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322006-05-CG000).

Author contributions

Conceptualization, Y.R.K., S.M.P., and H.W.C.; methodology, Y.R.K., and D.Y.H.; Investigation, Y.R.K., and D.Y.H.; writing—original draft, Y.R.K., and D.Y.H.; writing—review and editing, Y.R.K., S.M.P., and H.W.C.; funding acquisition, S.M.P., and H.W.C.; resources, Y.R.K., D.Y.H., J.H.H., G.R.N., J.H.P., and G.H.T; supervision, S.M.P., and H.W.C.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-MyHC antibody	DHSB	RRID: AB_2147781
Alexa Fluor 568-labeled anti-mouse IgG	Invitrogen	RRID: AB_2534072
Bacterial and virus strains
E. coli TOP10	Carlsbad	Cat#: C404003
P. pastoris X33	Carlsbad	Cat#: C18000
Biological samples
Korean native cattle muscle tissue-derived satellite cell	This study	N/A
Chemicals, peptides, and recombinant proteins
XbaI restriction enzyme	Promega	Cat#: R6185
XhoI restriction enzyme	Promega	Cat#: R6165
PmeI restriction enzyme	Promega	Cat#: R1852
T4 DNA ligase	Takara Bio	Cat#: 42011A
Luria-Bertani (LB) medium	BD Biosciences	Cat#: 244620
Yeast extract-peptone-dextrose (YPD) medium	BD Biosciences	Cat#: 242820
D-sorbitol	Daejung	Cat#: 7653-4400
Zeocin	Thermo Fisher	Cat#: R25001
Phosphate-buffered saline (PBS)	Biosesang	Cat#: RP2007-000-00
Phosphate-buffered saline (PBS)	Gibco	Cat#: 10010-072
Methanol	Samchun	Cat#: M1448
Ammonium hydroxide	Samchun	Cat#: A3780
Coomassie Blue R-250	BYLABS	Cat#: C0110R
Pre-stained protein ladder (10–180 kDa)	Proteintech	Cat#: 26617
Bovine Serum Albumin (BSA)	Sigma-Aldrich	Cat#: A8806
Antibiotic-antimycotic solution (A/A)	Gibco	Cat#: 15240-112
75% EtOH (1.00983.1011; Sigma-Aldrich)	Sigma-Aldrich	Cat#: 1.00983.1011
Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)	Gibco	Cat#: 11320-082
Penicillin-Streptomycin	Lonza	Cat#: 17-745E
Trypsin-EDTA (TE)	Gibco	Cat#: 25200-072
Collagenase Type II	Worthington	Cat#: CLS-2
Dispase II	Roche	Cat#: 4942078001
Fetal Bovine Serum (FBS)	Gibco	Cat#: 26140-079
Red Blood Cell (RBC) lysis buffer	Sigma-Aldrich	Cat#: R7757
Ham’s F-10	Gibco	Cat#: 15240-062
basic fibroblast growth factor (bFGF)	R&D Systems	Cat#: 223-FB-500/CF
Primocin	InvivoGen	Cat#: ant-pm-2
Gelatin	Sigma-Aldrich	Cat#: G1319
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat#: D2650
Dulbecco’s phosphate buffered saline (DPBS)	Gibco	Cat#: 14190-136
Paraformaldehyde	Biosesang	Cat#: PC-2031-050-00
BSA	Bovogen	Cat#: BSAS 0.1
RNase A	Sigma-Aldrich	Cat#: 70856
Propidium iodide (PI)	BioLegend	Cat#: 421301
EDTA	Invitrogen	Cat#: 15575020
HEPES (pH 7.0)	Sigma-Aldrich	Cat#: H6147
CaSO4 · 2H2O	Sigma-Aldrich	Cat#: C3771
MgSO4 · 7H2O	DAEJUNG	Cat#: 5513-4400
KOH	DAEJUNG	Cat#: 6584-4400
K2SO4	DAEJUNG	Cat#: P1193
H3PO4	DAEJUNG	Cat#: 6532-4100
Glycerol	DAEJUNG	Cat#: G0270
Casamino acid	Thermo Fisher	Cat#: 223050
CuSO4· 5H2O	DAEJUNG	Cat#: 2588-4405
KI	DAEJUNG	Cat#: 6599-4405
MnSO4 · H2O	Sigma-Aldrich	Cat#: M7634
Na2MoO4 · 2H2O	Sigma-Aldrich	Cat#: M1003
H3BO3	JUNSEI	Cat#: 69025-0301
ZnSO4 · 7H2O	Sigma-Aldrich	Cat#: Z0251
FeCl3 · 6H2O	Sigma-Aldrich	Cat#: 5021-4405
CoCl2 · 6H2O	DAEJUNG	Cat#: 2570-4405
Biotin	Sigma-Aldrich	Cat#: B4501
H2SO4	Sigma-Aldrich	Cat#: 258105
Critical commercial assays
PCR purification & Gel extraction Kit	Favorgen	Cat#: FAGCK 001-1
Plasmid Miniprep Kit	Favorgen	Cat#: FAPDE 100
Pierce™ BCA Protein Assay Kit	Thermo Fisher	Cat#: 23225
HisTrap FF resin	Cytiva	Cat#: 17531803
XK 16/20 chromatography column	Cytiva	Cat#: 28988937
Pierce™ BCA Protein Assay Kit	Thermo Fisher	Cat#: 23225
Cell Counting Kit-8 (CCK-8)	Dojindo	Cat#: CK04-11
AccuPrep® Universal RNA Extraction Kit	Bioneer	Cat#: K-3141
AccuPower® CycleScript RT Premix	Bioneer	Cat#: K-2047-B
SYBR Green Master Mix	Applied Biosystems	Cat#: A25918
Deposited data
Amino acid sequences of bovine serum albumin (BSA)	UniProt	Cat#: P02796
Amino acid sequences of porcine serum albumin (PSA)	UniProt	Cat#: P08835
Oligonucleotides
Presented in Table S3
Recombinant DNA
pPICZaA integrative expression vector	Carlsbad	V19520
Software and algorithms
SAS version 9.4	SAS Institute Inc
Other
0.2 cm electroporation cuvette	Bio-Rad	1652086
0.2 μm MCE membrane filter	SciLab	SL.MCE02047A
100 μm cell strainer	SPL	93100
40 μm cell strainer	SPL	93040

Experimental model and subject details

Cell, vector, host strains, and reagents for producing P. pastoris-derived recombinant albumin

E. coli TOP10 (C404003), P. pastoris X33 (wild-type, C18000), and P. pastoris integrative expression vector (pPICZaA, V19520) were purchased from Invitrogen (Carlsbad, CA, USA). All media used for the growth of E. coli and P. pastoris were prepared according to the manufacturer’s protocols (Invitrogen). The restriction enzymes, XbaI (R6185), XhoI (R6165) and PmeI (R1852) were purchased from Promega (Madison, WI, USA). The T4 DNA ligase (42011A) was purchased from Takara Bio (Ōtsu, Shiga, Japan). The polymerase chain reaction (PCR) purification & gel extraction (FAGCK 001-1), and miniprep kits (FAPDE 100) for plasmid extraction were obtained from Favorgen (Pingtung, Taiwan). The primers were synthesized by GenoTech (Daejeon, South Korea). Luria-Bertani (LB) medium (244620) for E. coli strains culture and Yeast extract-peptone-dextrose (YPD) medium (242820) for P. pastoris strains culture were purchased from BD Biosciences (San Jose, CA, USA). D-sorbitol (7653-4400) for P. pastoris transformation was purchased from Daejung (Siheung, South Korea).

Animal care

This study follows the recommendations of the ARRIVE guidelines. All animal procedures were approved by the Animal Ethics Committee of Jeonbuk National University (JBNU; NON2023-137), Republic of Korea. All experiments were performed in accordance with the ethical guidelines and regulations of the Jeonbuk National University.

Isolation and culture of bovine muscle stem cells

Bovine muscle samples were anonymized and untraceable to specific animals or farms, and no identificable information was provided, Primary bMuSCs were isolated as previously described.^35,36 Briefly, bMuSCs were isolated from Korean native cattle and transported to the cell culture laboratory on ice at 4 °C. Muscle tissues were washed twice using PBS (10010-072; Gibco, Carlsbad, CA, USA) containing 10% antibiotic-antimycotic solution (A/A, 15240-112; Gibco) after a 75% EtOH (1.00983.1011; Sigma-Aldrich, St. Louis, MO, USA) wash. The tissues were minced into a paste and digested with digestion medium containing Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (11320-082; Gibco), 10% penicillin-streptomycin (17-745E; Lonza, Basel, Switzerland), 0.25% trypsin-EDTA (TE, 25200-072; Gibco), collagenase 2 (CLS-2; Worthington, Lakewood, NJ, USA), and DispaseII (4942078001; Roche, Basel, Switzerland) for 30 min at 37 °C. The mixture was then vortexed for 5 min. The cells were neutralized using DMEM/F12 supplemented with 15% FBS (26140-079; Gibco) and 1% A/A. The mixture was centrifuged at 640 rpm for 3 min at 4 °C. The supernatant containing mononuclear cells was collected. The muscle fragment was triturated with neutralized media and centrifuged at 640 rpm for 3 min at 4 °C. The supernatant was collected and added to the former supernatant. Mononuclear cells were centrifuged at 2,260 rpm for 5 min and washed twice with PBS. Next, cells were filled with neutralized media and filtered through a 100 μm strainer (93100; SPL, Pocheon, South Korea) followed by straining through a 40 μm cell strainer (93040; SPL). The pellet was then reconstituted with Red Blood Cell lysis buffer (R7757; Sigma-Aldrich) to lyse red blood cells and neutralized using PBS containing 10% penicillin-streptomycin. Cells were then centrifuged again at 1,850 rpm for 5 min at 4 °C. Cells were then reconstituted in bovine satellite cell growth medium (BSC-GM) containing Ham’s F-10 (15240-062; Gibco), 20% FBS, 1% A/A, 5 ng/ml basic fibroblast growth factor (bFGF, 223-FB-500/CF; R&D System, Minneapolis, MN, USA), and primocin (ant-pm-2; InvivoGen, San Diego, CA, USA). Primocin was administered only for the first 7 d. The bMuSCs were separated using a preplating method. Cells were seeded in a 100 mm dish coated with 0.1% gelatin (G1319; Sigma-Aldrich) and incubated at 37 °C in a 5% CO₂ incubator for 1 h. After 1 h, the medium (suspension cell) was collected and transferred to a new 100 mm dish coated with 0.1% gelatin. All experiments were conducted using passage 3 bMuSCs. The bMuSCs were seeded in a 100 mm dish coated with 0.1% gelatin and incubated at 37 °C with 5% CO₂ incubator until a density of 70–80%. Subsequent passaging was carried out using 0.25% TE, followed by seeding the cells at a density of 8,150 cells/cm². For regular cell maintenance, cells were frozen in FBS containing 10% dimethyl sulfoxide (DMOS, D2650; Sigma-Aldrich) and preserved in liquid nitrogen. All experiments were conducted using bovine satellite cells obtained from a single animal, and cells were derived from the same isolation batch to ensure consistency.

Method details

Synthesis of bovine serum albumin (BSA) and porcine serum albumin (PSA) genes and construction of the expression vector

Amino acid sequences for BSA (P02796) and PSA (P08835) were obtained from UniProt (https://www.uniprot.org/). To improve PSA expression in P. pastoris, codon deviations of P. pastoris genes were optimized using the NCBI-related database at http://www.kazusa.or.jp/codon. The optimized codon sequences of BSA and PSA for P. pastoris were synthesized by Bioneer (Daejeon, South Korea), and to facilitate the upcoming purification of the recombinant BSA and PSA, a 6X His tag- encoding sequence was in-frame fused to the N-terminal end of the BSA and PSA coding sequences.

Transformation of P.pastoris and screening of transformants with high expression capacity

P. pastoris was transformed by electroporation, as described in the Pichia manual (Invitrogen), with some modifications. PmeI-linearized pPICZA/BSA (5 μg) and pPICZA/PSA (10 μL) were mixed with 80 μL of competent P. pastoris cells, respectively. The cell mixture was then transferred to an ice-cold 0.2 cm electroporation cuvette (1652086; Bio-Rad, Hercules, CA, USA) and kept on ice for 5 min. The cell mixture was then pulsed at 2 kV, 25 μF, and 200 Ω for approximately 5 msec. Following electroporation, 1 mL pre-chilled sorbitol (1 M) was immediately added to the cuvette and incubated for 1 h at 30 °C without shaking. Finally, 200 μL aliquots were spread on separate YPD plates containing 100 mg/mL Zeocin (R25001; Thermo Fisher, Waltham, MA, USA) and 1 M sorbitol. Plates were incubated for 72 h at 30 °C until colonies formed. Recombinant-positive P. pastoris transformants were screened using PCR of colonies.

Fermentation of recombinant transformants using two different feeding strategies

Seed culture was performed in two steps. A single colony from the YPD agar plate was inoculated into 50 mL YPD media in 250 mL baffled flasks and grown overnight at 30 °C and 250 rpm until approximately an OD_600nm of 6–8. Next, 5 mL of these cultures was inoculated into 200 mL YPD media in a 1 L baffled flask until an OD_600nm of 6–8. The cultures were separated by centrifuging, and only the cells were resuspended in 10 mL phosphate-buffered saline (PBS, RP2007-000-00; Biosesang, Seongnam, South Korea).

Fermentation of recombinant transformants was performed using a 5 L bioreactor (Centrion, Daejeon, South Korea). A 2 L initial batch phase medium was prepared and cultivated until appearance of a dissolved oxygen (DO) spike.⁶⁹ The composition of batch phase medium are listed in Tables S1 and S2.

The glycerol fed-batch phase was then initiated with glycerol feeding with 50% (v/v) glycerol solution containing 50 mL trace element solution per liter. After the glycerol fed-batch phase, starvation was performed for 1 h and the methanol induction phase was initiated by methanol (M1448; Samchun, Pyeongtaek, South Korea) feeding with a 50 mL trace element solution per liter. The methanol induction phase lasted for approximately 72 h, with a total of 1 L of methanol used. The temperature was maintained 29 °C during fermentation. During fermentation, the pH was adjusted to 6.0 using ammonium hydroxide (A3780; Samchun). Agitation was set to 400 rpm, and air was mixed with oxygen and supplied at 2.0 SLPM. The cell density was measured using a UV-vis spectrophotometer (Klab, Daejeon, South Korea). Samples (1 mL each) were collected on Days 0, 1, 2, and 3 of the methanol induction phase. Supernatants were stored at –20 °C and were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The BCA assay was used to quantify the produced proteins in the supernatants using a Pierce™ BCA Protein Assay Kit (23225; Thermo Fisher). All experiments were performed in triplicate, and the results were shown as the mean ± SD from the independent experiments.

Purification of recombinant BSA (rBSA) and recombinant PSA (rPSA)

The protein purification was performed with AKTA purifier system (Cytiva, Marlborough, MA, USA) Affinity chromatography was performed using a column packed with 30 mL of HisTrap FF resin (17531803; Cytiva) on an XK 16/20 chromatography column (28988937; Cytiva). Before purification, the column was equilibrated with start buffer (0.5 M sodium chloride, 20 mM sodium phosphate, pH 7.4). Subsequently, the culture supernatants were filtered using a 0.2 μm MCE membrane filter (SL.MCE02047A; SciLab, Daejeon, South Korea) and loaded onto the column. The column was then washed using start buffer, and proteins within the chromatography medium were eluted with elution buffer (0.5 M sodium chloride, 20 mM sodium phosphate, and 500 mM imidazole, pH 7.4). The flow rate was maintained at 5 mL/min in all steps, and the protein was monitored by measuring the UV absorbance at 280 nm and collected by observing the protein absorption peak. The pooled elution fractions containing rBSA and rPSA from the affinity chromatography were analyzed by SDS–PAGE. The BCA assay was used to quantify the proportion of purified proteins among the eluates using a Pierce™ BCA Protein Assay Kit (23225; Thermo Fisher). Finally, the purified protein was dialyzed in PBS solution and stored at -20 °C for further analysis.

SDS-PAGE

Protein samples were analyzed by SDS–PAGE, according to the method of Laemmli with some modifications.⁷⁰ The culture supernatants were harvested and analyzed with SDS-PAGE. The SDS–PAGE analysis was performed using a 10% gel under reducing conditions and then visualized using Coomassie Blue R-250 (BYLABS, Hanam, South Korea) staining. Samples were mixed with 5X loading buffer and boiled 95-100 °C for 10min before electrophoresis. A pre-stained protein ladder 10–180 kDa (Proteintech, Rosemont, IL, USA) was used for estimating the molecular weight. The rBSA and rPSA were analyzed by SDS-PAGE with commercial BSA (A8806; Sigma-Aldrich) after purification.

Cell proliferation analysis

Cell proliferation analysis was conducted for short- (4 d) and long-term (28 d) growth. The Beefy-8 (B8) medium with various rAlbumin concentrations was prepared using a previously described formulation and preparation method (Table S4).⁷ Passage 3 bMuSCs were plated with BSC-GM in 12-well plates at a density of 8,150 cells/cm² with 0.1% gelatin coating. The seeding process was repeated thrice consecutively for each medium type (n = 3). After 24 h, BSC-GM was removed, cells were washed once with Dulbecco′s phosphate buffered saline (DPBS, 14190-136; Gibco) and new medium (e.g., B8 with rAlbumin) was added. The medium was changed on Day 3, and after 96 h of culture, cells were detached using 0.25% TE. The cell counts were determined using an inverted microscope equipped with a hemocytometer. The cells were then pelleted at 1,100 rpm, resuspended in B8 medium, re-counted, and seeded onto new 12-well plates at a density of 8,150 cells/cm². This process was repeated over seven passages. Throughout the culture period, cells were fed every 2 d. When seeding cells for passage 5, additional 6-well plates were used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.

MTT assay

The bMuSCs were seeded in 96-well plates at a density of 2,000 cells/cm². Cells were cultured in BSC-GM for 3 d as described above. Cell viability was detected using the Cell Counting Kit-8 (CCK-8, CK04-11; Dojindo, Kumamoto, Japan). Cells were treated with CCK-8 solution following the manufacturer’s instructions and incubated at 37 °C for 4 h. Cell viability was measured using a microplate reader (Multiscan Sky, Thermo Fisher) at a wavelength of 450 nm.⁷¹ The differentiation of bMuSCs occurs via two pathways: serum-containing (FBS) and serum-free (albumin).

Cell differentiation

For the differentiation experiments, P3 bMuSCs were seeded in four wells at a density of 8,750 cells/cm². The medium was changed every 2 d, and cells were cultured in BSC-GM until 80–90% confluence. The serum-containing differentiation medium consisted of DMEM supplemented with 1% FBS, and 1% A/A,⁷² while the serum-free differentiation medium included DMEM, rAlbumin at various concentrations, and 1% A/A (Table S5). Each type of differentiation medium was applied for 3 days.

Reverse transcription polymerase chain reaction (qPCR)

To assess relative gene expression level between bMuSCs cultured in various media, RNA was extracted from cells using an AccuPrep® Universal RNA Extraction Kit (K-3141; Bioneer) following the manufacturer’s protocol. Total RNA (1 μg) was reverse transcribed with an Accupower® CycleScript RT Premix (K-2047-B; Bioneer) according to the manufacturer’s instructions. Relative gene expression was measured in triplicate using Powerup SYBR Green Master Mix (A25918; Applied Biosystems, Foster City, CA, USA). The primer sequences are listed in Table S3. For the examination of expression level, the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an internal control. Reactions were run on the LightCycler® 96 system, and results were analyzed as 2^−ΔCt normalized to gene expression.⁶⁸

Immunofluorescence staining

Immunocytochemistry was performed to assess the phenotype of the differentiated cells in each medium. Cells were fixed with 4% paraformaldehyde (PC-2031-050-00; Biosesang) for 30 min at RT and washed three times in PBS. After fixation, cells were permeabilized in 0.3% Triton X-100 with 3% BSA (BSAS 0.1; Bovogen, Melbourne, Australia) with DPBS for 2 h. Cells were stained overnight with a primary antibody against myosin heavy chain (MyHC) (1:20, MF20-s; DHSB, Iowa, IA, USA) at 4 °C. Cells were washed three times in 0.3% Triton X with PBS and incubated with the Alexa 568 labeled anti-mouse (A11004; Invitrogen) secondary antibody at RT for 2 h. The cells were then stained with 4′6-diamidino-2-phenylindole (DAPI, 1 μg) for 10 min. After staining with DAPI, the cells were washed thrice with PBS.³⁶ They were then observed under a Leica DMi8 microscope and confocal laser-scanning microscope (LSM 880 with Airyscan; Carl Zeiss, Oberkochen, Germany) installed at the Center for University-wide Research Facilities (CURF) at Jeonbuk National University.

Cell cycle analysis

Bovine muscle satellite cells were collected at passages 3 and 7. The cells were seeded in 35 mm dishes coated with 0.1% gelatin at a density of 8,750 cells/cm². Cells were detached using 0.25% TE and neutralized with neutral media. Cells were then washed with cold PBS containing 1% BSA and fixed with 70% EtOH for 5 min at 4 °C. Cells were centrifuged at 2,080 rpm at 4 °C for 5 min. EtOH was removed, and cells were washed twice with PBS and 1% BSA. After washing, 100 μg/mL of RNase A (70856; Sigma-Aldrich) was added, and 25 μg/mL of propidium iodide (PI, 421301; Bio Legend, San Diego, CA, USA) was added with PBS.⁶⁸ Cells were analyzed using a FACSCalibur (Becton, Dickinson, Franklin, NJ, USA) with a blue laser (excitation 488 nm) installed at the Center for University Research Facility (CURF) at Jeonbuk National University.

FACS analysis and sorting

Samples were dissociated into single cells by treating with 0.25% TE. Cell pellets were then resuspended in PBS supplemented with 1% FBS, 1 mM EDTA (15575020; Invitrogen), and 25 mM HEPES (pH 7.0, H6147; Sigma-Aldrich). The samples were analyzed and sorted using a FACS Aria III (Becton, Franklin Lakes, NJ, USA) installed at the Center for University Research Facility (CURF) at Jeonbuk National University.

Quantification and statistical analysis

All experiments were performed in triplicate, and the data of all repetitions of each experiment were collated and expressed as means ± standard error (SE) of the mean. Statistical tests were conducted using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and statistical differences were analyzed using the Student’s t-test or analysis of variance (ANOVA), followed by Duncan’s Multiple Range Test for post hoc comparisons. A p-value < 0.05 was regarded as significant.

Published: July 29, 2025