A spontaneously immortalized muscle stem cell line (EfMS) from brown-marbled grouper for cell-cultured fish meat production

Primary cell culture and subculture

Healthy brown-marbled groupers (E. fuscoguttatus) with 3 ± 0.5 cm in length were purchased from a fish farm in Zhanjiang (Guangdong, China). The fish were pretreated in boiling-sterilized seawater containing 1000 IU mL⁻¹ penicillin and 1000 μg mL⁻¹ streptomycin for 24 h. The fish were then anesthetized by MS-222 (Sigma, USA) and disinfected by immersing in 75% ethanol for 2 to 3 min. The muscle tissues above the lateral line of the fish were aseptically dissected with a surgical scalpel and washed 3 times in D-hank’s solution (8 g L^-1 NaCl, 0.4 g L^-1 KCl, 0.06 g L^-1 KH₂PO₄, 0.08 g L^-1 Na₂HPO₄·12H₂O and 0.35 g L^-1 NaHCO₃, pH 7.0) supplemented with 10% penicillin-streptomycin mixture in a 50-mL centrifuge tube (Corning, USA) on ice. After that, the muscle tissues were transferred to a petri dish and cut into pieces with ophthalmic scissors, and then transferred to a 50-mL centrifuge tube again. Next, the tissue pieces were subjected to 2 mL L-15 medium supplemented with 2.5 mM CaCl₂, 1.25 mg mL^-1 dispase type II (Roche, Switzerland) and 5 mg mL^-1 collagenase D (Roche, Switzerland) and digested for 1.5 h at 28 °C on a water bath shaker at 170 rpm. The digested suspension was first filtered through a 70 μm filter membrane (BD-Pharmingen, USA) to remove large tissue blocks and then rinsed with 12 mL L-15 medium (Gibco, USA). After filtration, the filtrate was collected and centrifuged at 300 × g for 5 min. The supernatant was discarded and the cell precipitate was resuspended in 2 mL fish muscle stem cell medium (SCM-A) as listed in Table 1. The cells were counted using a hemocytometer, and a total of 1 × 10⁵ cells per dish were seeded into a 3.5-cm cell culture dish (Corning, Cat. 353001, USA). The cells were incubated at 28 °C in a biochemical incubator without CO₂. The medium was half changed every 48 h. The muscle stem cells were daily monitored and photographed under an inverted contrast phase microscope (Nikon, Japan).

Subsequently, the primary cell monolayer was sub-cultured using 0.0625% trypsin-EDTA solution and then a new marine fish muscle stem cell line of EfMS (E. fuscoguttatus muscle stem cell) was obtained. Cryopreservation of the cell line was conducted using a serum-free cryopreservation solution (Procell, China) at passages 5, 8, 10, 12, 15, and every 5 passages thereafter until passage 80. Two cryopreservation tubes (Coring, USA) were prepared during each cryopreservation: one for long-term storage and another thawed after one week to confirm viability. After passage 20, the concentration of trypsin-EDTA used for subculture was returned to 0.25%.

For the preparation of muscle extract, the adult brown-marbled grouper was anesthetized using MS-222. Once fully anesthetized, the fish was euthanized by a swift and forceful blow to the head using a hammer. Following euthanasia, the fish was descaled, and the skin was carefully removed. The dorsal muscle tissue was then dissected, ensuring that any bones, connective tissues, and skin remnants were removed. The extracted muscle tissues were rinsed with sterile PBS to remove blood and surface contaminants, then cut into small pieces for further processing. The muscle tissues of brown-marbled grouper were then homogenized in L-15 medium in a volume of 3 mL per g of tissue. After that, the homogenate was incubated in a 60 °C water bath for 1 h with inverted mixing every 10 min and then clarified by centrifugation at 8000  × g for 2 h at 4 °C. The supernatant was collected and adjusted to pH 7.0 with 0.1 g mL⁻¹ NaHCO₃, and sterilized by a 0.22 μm filter membrane (Sartorius, Germany). The protein content of the muscle extract was determined using the BCA Protein Assay Kit (Solarbio, China), with a concentration measured at 22.1 mg mL⁻¹.

All animal studies were performed in accordance with the Ethical Guidelines for the Use and Care of Laboratory Animals and were approved by the Animal Care and Use Committee of Ocean University of China. We have complied with all relevant ethical regulations for animal use. The fish used for cell isolation were one-month-old females, while those used for muscle extract preparation were six-month-old females.

DNA extraction and CO1 gene analysis

The species origin of EfMS cell line was determined by sequencing the mitochondrial CO1 (cytochrome oxidase subunit 1) gene. Total genomic DNA of the EfMS cells at passage 20 was extracted using a DNA extraction kit (Tiangen, China) and used as a template to amply the CO1 gene. The forward primer used was 5’-ATTGGCACCCTTTATCTTGTA-3’ and reverse primer was 5’-TGAGAGATAGCAGGGGGTTTTA-3’. The PCR amplification was carried out in a 25 μL volume containing 12.5 μL of 2×Taq Mix, 2.0 μL of template DNA (60 ng), 1.0 μL forward primer (10 μM) and 1.0 μL reverse primer (10 μM). The PCR reaction involved in an initial denaturation at 94 °C for 10 min, followed by 30 cycles of denaturation at 94 °C for 30 s, renaturation at 55 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 10 min. PCR products were analyzed by 1.0% agarose gel electrophoresis and then sequenced. The obtained sequences were aligned against known sequence of E. fuscoguttatus CO1 gene (GenBank No. NC_020046.1) deposited in the NCBI database.

Optimization of fish muscle stem cell medium (SCM)

To obtain an optimal SCM with a capability to support the active proliferation of EfMS cells along with undiminished stemness, this SCM was developed and optimized by successively analyzing the culture effects of varied concentrations of FBS (10% in SCM-E or 20% in SCM-A, B, C and D), grouper muscle extract (none in SCM-C, D and E or 10% in SCM-A and B) and growth factors (none in SCM-B, D and E or 20 ng mL⁻¹ bFGF and 20 ng mL⁻¹ EGF in SCM-A and C) on the EfMS cells. To do this, EfMS cell monolayers were prepared by seeding them into a 96-well culture plate (Corning, USA) at a density of 3 × 10³ cells/well. After incubation at 28 °C for 24 h, the old medium in each well was discarded and replaced with the tested 5 kinds of SCMs (A ~ E) as listed in Table 1, and continued to culture for another 4 days until confluency. After the addition of SCM, every 24 h, the cells in three wells were separately collected by trypsinization and counted with a hemocytometer and used to plot the growth curves for each tested SCM. The growth curve was plotted as EfMS cell number against culture time. The population doubling time (PDT) was calculated as follows: PDT = (ΔT×ln2) / [ln (Final cell number)–ln (Initial cell number)]

Screening of a substitute for fish muscle extract in SCM

The results previously obtained in this study have showed that the fish muscle extract supplemented in SCM played an important role in maintaining the stemness of EfMS cells. However, the use of muscle extract was labor- and time-consuming, with high-cost and undefined nutrient composition, thus unsuitable for the scale-up production of cell-cultured fish meat. To screen a substitute for fish muscle extract, we first analyzed the composition and content of the free amino acids in the muscle extract derived from the adult grouper. In brief, 2 mL of muscle extract was mixed with 2 mL of 5% sulfosalicylic acid solution and then centrifuged at 8000 g for 10 min at 4 °C. The supernatant was then filtered through a 0.22 μm water-based filter membrane into a vial. This sample was subsequently injected into a L-8900 amino acid analyzer (Hitachi, Japan) to determine the amino acid content. The analysis results revealed that the top two most abundant components in the muscle extract were taurine (554 μg mL^-1) and PEA (240 μg mL^-1) (Supplementary Fig. 2). Both of them are absent in L-15 medium.

Notably, taurine plays a significant role in skeletal muscle function, and PEA has been shown to modulate cellular autophagy, which can extend cell longevity^28,29. Based on this, as listed in Table 1, SCM-F and SCM-G were designed by replacing the muscle extract in SCM-A with taurine and PEA at their measured contents in the fish muscle extract, respectively. The culture effects of SCM-F and SCM-G were analyzed as described previously.

Chromosomal analysis

EfMS cells at passage 30 were used for chromosome analysis. In brief, the EfMS cells were seeded into 25-cm² culture flasks and incubated at 28 °C for 24 h, and then the old medium was replaced with fresh medium containing 10 μg mL^-1 colchicine (Solarbio, China). After 2 h of incubation, the cells were collected by trypsinization and centrifugation at 70 × g for 3 min, and then exposed to 5 mL 0.075 M KCL solution for 30 min. After hypotonic treatment, the tubes were centrifuged again at 70 × g for 3 min and the cell pellet was subsequently fixed in freshly prepared, ice-cold methanol–acetic acid (3:1) for 15 min. The cell pellet was then resuspended in the fixative and spread onto a glass slide. After air-drying the slides, they were stained with a 5% Giemsa staining solution (Sigma, USA) at pH 6.8 for 15–20 min. Once stained, the slides were rinsed, air-dried, and the chromosome numbers of 100 metaphase-stage cells were counted under a microscope (Leica, Germany).

Semi-quantitative RT-PCR

Using semi-quantitative RT-PCR technology, the transcriptional expression levels of the muscle stemness marker gene (Pax7) in the EfMS cells cultured in various growth media during the process of passages 5 to 50 were monitored, the differential expressions of 8 kinds of oncogenesis-related genes (TP53, TP53I3, TP53RK, MYC, PTEN, EGFR, TERT and DKC1) in the EfMS cells between passages 20 and 80 were compared (Supplementary Fig. 3), and also, 3 kinds of myogenic differentiation-related genes (MHC, Myogenin and ACTB) (Fig. 3), 4 kinds of adipogenic differentiation-related genes (PPARγ, C/EBPα, LPL and Leptin) and 2 kinds of preadipocyte marker genes (CD73 and CD105) (Fig. 6) in the EfMS cells before and after they were induced to myogenic differentiation or adipogenic trans-differentiation were analyzed, respectively. The total RNAs of the tested EfMS cells were extracted using a total RNA extraction kit (Tiangen, China) according to the manufacturer’s instruction. Then the total RNAs (1 μg) were reversely transcribed into cDNAs using ReverAidTM First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA) and used as PCR templates. All the tested genes and their gene-specific primers were listed in Table 2. All the target gene fragments were amplified using the same reaction condition as described previously for the CO1 gene analysis. Semi-quantitative RT-PCR products were analyzed by 1.5% agarose gel electrophoresis, and gray scale analysis was performed by Image J software to quantitatively detect the differential gene expression.

Furthermore, we compared the transcriptional expression levels of TERT and DKC1 in 11 kinds of tissues (skin, gallbladder, fat tissues, kidney, spleen, heart, gills, intestines, muscle, fin, and liver) and EfMS cells at passage 20 using the same method described above (Supplementary Fig. 4).

Immunofluorescence staining

The expression of Pax7 and MyoD proteins in the EfMS cells were analyzed by immunofluorescence staining. In brief, the tested EfMS cells were fixed in immunostaining fixative (Beyotime, China) for 10 min and then permeabilized in 0.2% Triton X-100 (Sigma-Aldrich, USA) for 10 min. Next, the cells were blocked in blocking solution (Beyotime, China) for 1 h at room temperature. After that, the EfMS cells were incubated with the mixed primary antibodies of anti-Pax7 antibody (1:10, DSHB, clonePAX7-s, USA) and anti-MyoD antibody (1:100, Santa Cruz, sc-377460, USA) overnight at 4 °C. After triple washing in PBS (8.0 g L⁻¹ NaCl, 0.2 g L⁻¹ KCl, 1.44 g L⁻¹ Na₂HPO₄⋅12H₂O and 0.24 g L⁻¹ KH₂PO₄, pH 7.0), the corresponding two secondary antibodies were added and incubated for another 1 h at room temperature. Both the two secondary antibodies were purchased from Thermo Fisher Scientific with Alexa Fluor 488 goat anti-mouse IgG1 (1:500) for Pax7 and Alexa Fluor 555 goat anti-mouse IgG2b (1:500) for MyoD. Finally, the cell nuclei were counterstained with DAPI (1:1000 in water, Sigma, USA). Images were taken with an inverted fluorescence microscope (Nikon, Japan).

Optimization of myogenic differentiation medium

Commonly used myogenic differentiation medium for mammalian muscle stem cells are not suitable for fish muscle stem cells. Thus, a new myogenic differentiation medium specific for EfMS cells was developed in this study by optimizing the basic medium of L-15 or MEM supplemented with varied concentrations of vitamin C (Solarbio, China), vitamin D (Solarbio, China), insulin (Sigma-Aldrich, USA), horse serum (Absin, China) or FBS (special for mesenchymal stem cells, BD-Pharmingen, USA). A total of 8 kinds of different myogenic differentiation media, designated as DM-A ~ DM-H as listed in Table 3, had been prepared and compared in their myogenic differentiation efficiencies. To do this, the EfMS cells were seeded into 6-well cell culture plates (Corning, USA) at a density of 1.5 × 10⁵ cells/well in 2 mL medium of SCM-A and cultured until the cell confluency reached 90–95%. Then, the old medium was discarded and the cell monolayer was washed once with PBS. Next, the medium in each well was replaced with 2 mL differentiation media tested to induce the myogenic differentiation of EfMS cells. The differentiation status of the cells was morphologically recorded daily and analyzed by Phalloidin staining (F-actin in green).

Phalloidin staining

Phalloidin staining was used to detect the F-actin formation in the EfMS cells after induced by different myogenic differentiation media. After 3 days of differentiation induction, the old medium was discarded and the cell monolayers were washed with PBS. Then, the cells were fixed with 4.0% paraformaldehyde for 10–30 min at room temperature, followed by another wash with PBS. Next, the cells were permeabilized by 0.1% Triton X-100 for 3–5 min at room temperature, and then washed with PBS. After that, phalloidin staining solution (Absin, China) was added into the wells and the cells were stained for 20 to 90 min, and then washed once with PBS. Then, the nuclei of EfMS cells were counterstained with DAPI, and then the cell monolayers were washed once again with PBS. Images were captured under an inverted fluorescence microscope (Nikon, Japan).

Induction of adipogenic trans-differentiation of EfMS cells

To trans-differentiate the EfMS cells into adipocytes, an adipogenic trans-differentiation medium was designed based on the mammalian adipogenic differentiation medium³¹. This medium consisted of 1 μM dexamethasone, 0.45 mM 3-isobutyl-1-methylxanthine (IBMX), 10 μg mL⁻¹ insulin, 500 μM palmitic acid, 500 μM oleic acid, and 20% FBS (BI, Israel, Cat. 04-400). To induce the adipogenic trans-differentiation, the EfMS cells were plated into 6-well culture plates at a density of 1 × 10⁵ cells per well and cultured in the adipogenic trans-differentiation medium for 3 days. The medium in each well was replaced every 2 days. Finally, the adipogenic differentiation effects of the induced EfMS cells were analyzed by Oil Red O staining.

Oil Red O staining

The Oil red O working solution was prepared by diluting the stock solution (Solarbio, China) with ddH₂O at a ratio of 3: 2 and then filtered with 0.45 μm membrane. The EfMS cells were firstly washed three times with PBS and then fixed with 4% paraformaldehyde at 28 °C for 20 min. Then the cells were washed with PBS again and rinsed with 60% isopropanol for 1 min. Next, the cells were covered with the filtered Oil Red O working solution in dark at 28 °C for 30 min. After that, the Oil Red O was removed and the cells were washed with PBS and then treated with hematoxylin (Beyotime, China) in dark at 28 °C for 5 min. At last, the cells were washed with PBS and photographed under an inverted phase contrast microscope.

Calcein-AM and propidium iodide (PI) staining

Calcein acetoxymethyl ester (Calcein-AM) is a non-fluorescent compound that can penetrate live cells due to its lipophilicity. Inside live cells, Calcein-AM can be converted by esterase into fluorescent Calcein, which is retained within the cells due to its non-lipophilic nature, thereby marking them as viable with a green fluorescence. Propidium iodide (PI) is a red-fluorescent dye that cannot penetrate live cells because of its impermeability, but can enter dead or dying cells with damaged membranes, and bind to nucleic acids, and emit red fluorescence, marking these cells as non-viable. To monitor the viability of the EfMS cells during 3D culture, the old medium in the culture plate was removed and the Calcein-AM and PI (Beyotime, China) working solution (1 μM) diluted in L-15 medium was added into each well and incubated at 28 °C in the dark for 45 min. Next, the cells were washed once with PBS and fresh medium was added, and observed under an inverted fluorescence microscope.

3D cell culture on microcarriers and production of fat-free and fat-containing cell-cultured fish meats

Edible 3D microcarriers fabricated from squid gelatin were kindly provided by Qingdao Institute of Marine Bioresources for Nutrition & Health Innovation (China). For 3D culture, the microcarriers in powder form were first added into a 48-well ultralow-binding cell culture plate (Corning, USA) at an amount of 20 mg/well. Next, the EfMS cells were harvested by trypsinization and centrifugation, and then suspended in the taurine-containing growth medium of SCM-G (Table 1) to a concentration of 1 × 10⁷ cells mL⁻¹, and seeded into the plates preloaded with microcarriers at a volume of 500 μL per well and incubated at 28 °C for the production of fat-free or fat-containing cell-cultured fish meat. The medium in each well was changed daily.

To assess the proliferation of EfMS cells, the 3D microcarriers with attached cells were first harvested at days 1, 3, and 5 by centrifugation, and then washed with 2 mL of PBS. After that, 1 mL of 0.25% trypsin-EDTA was added, and the mixture was incubated at 28 °C for 1 min to detach the cells from the microcarriers. The detached cells (with a diameter of 10–20 μm) were efficiently separated from the microcarriers (with a size of 150–300 μm in diameter) by filtering the cell-microcarrier suspension through a 70 μm filter membrane. Following filtration, the cells were counted using a hemocytometer. The relative proliferation rate was calculated as the ratio of the number of cells counted to the number of cells on day 1.

To produce the fat-free cell-cultured fish meat, the EfMS cells were allowed to proliferate on microcarriers for 5 days, then the SCM-G medium was replaced with the myogenic differentiation medium of DM-H (Table 3) and the myogenic differentiation of the EfMS cells on microcarriers was induced. On the third day of differentiation induction, after the removal of medium, a total of 10 wells of microcarriers carrying EfMS myofibers were harvested and used to produce the centimeter-scale cell-cultured fish meat (Fig. 4).

To produce the fat-containing cell-cultured fish meat, the EfMS cells were first allowed to proliferate on microcarriers for 5 days, and then, the SCM-G media in 12 wells were replaced with the myogenic differentiation medium of DM-H (Table 3), respectively, whereas the SCM-G media in another 8 wells were replaced with the adipogenic trans-differentiation medium, and both myogenic and adipogenic differentiation of the EfMS cells were separately induced as described previously. On the third day of differentiation induction, all the 12 wells of microcarriers carrying EfMS myofibers and all the 8 wells of microcarriers carrying EfMS cell-derived adipocytes were harvested, respectively, and used to produce the fat-containing cell-cultured fish meat. In detail, the 12 wells of microcarriers carrying EfMS-derived myofibers were evenly divided into 3 portions, and the 8 wells of microcarriers carrying EfMS-derived adipocytes were divided into 2 portions. Next, these 5 portions were then arranged alternately in a muscle-fat-muscle pattern to produce the fat-containing cell-cultured fish meat (Fig. 6).

Production of scaffold-free cell-cultured fish meat and its amino acid content analysis

For the production of scaffold-free cell-cultured fish meat, the EfMS cells in SCM-G medium (Table 1) were seeded into a 12-well blunt-bottomed ultralow-binding culture plate at a density of 2 × 10⁸ cells/well (Corning, USA). After 24 h of culture, the cells in each well spontaneously formed a cell ball. Next, the SCM-G medium in each well was replaced with myogenic differentiation medium of DM-H (Table 3) to induce the myogenic differentiation and myofiber formation within the EfMS cell ball. On the third day of differentiation induction, the differentiated cell balls were collected to produce the scaffold-free cell-cultured fish meat (Fig. 5).

To evaluate the nutritional value of cell-cultured fish meat, both the gross and free amino acid contents of the scaffold-free cell-cultured fish meats were analyzed and compared with those of natural fish meat, respectively (Fig. 5). For the gross amino acid content analysis, 100 mg of the scaffold-free cell-cultured fish meats or natural fish meat (i.e., grouper muscle tissue), were collected or dissected and then transferred to a 25-mL ampoule. After 10 mL of 6 M HCl (containing 5‰ mercaptoethanol) was added, the ampoule was subjected to hydrolysis at 110 °C for 22 h and then sealed under blowing nitrogen gas. The hydrolysate was then diluted in 0.02 M HCl to a final volume of 50 mL and then 1 mL of the hydrolysate was taken out and dried by blowing nitrogen gas. Next, 3–5 drops of ultrapure water were added and dried again to remove residual acid. Repeated this process three times, and then evaporated the solution to dryness. Finally, reconstituted the dried residue with 2 mL of 0.02 M HCl. Following reconstitution, filtered the solution through a 0.22 μm water-based filter membrane into a vial and injected it into a L-8900 amino acid analyzer (Hitachi, Tokyo, Japan) for the gross amino acid content analysis.

For the free amino acid content analysis, 100 mg of the scaffold-free cell-cultured fish meats or natural fish meat (i.e., grouper muscle tissue), were collected or dissected, respectively, and then transferred to a 2-mL Eppendorf tube. After adding two small steel balls and 1000 μL extraction solution (acetonitrile: methanol: water = 2: 2: 1, precooled at −40 °C and contained isotopically-labeled internal standard mixture), the sample was vortexed for 30 s, homogenized at 40 Hz for 4 min, and sonicated for 5 min in ice-water bath. The homogenate and sonicate circle were repeated for 3 times, followed by incubation at −40 °C for 1 h and centrifugation at 13,800 g for 15 min at 4 °C. A 100 μL aliquot of the clear supernatant was transferred to an auto-sampler vial for UHPLC-MS/MS analysis, performed by Shanghai Biotree Biotech co., LTD.

Flavor measurement

Flavor analysis of cell-cultured fish meat and natural fish meat were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) (Thermo Fisher Scientific, USA). Volatile compounds from the fish samples were extracted using chromatography-grade acetone, followed by qualitative and quantitative analysis based on the ion fragmentation spectra obtained. For sample preparation, 2 g of fish meat sample was placed in a 25 ml colorimetric tube, and 10 ml of acetone was added. The mixture was vortexed for 5 min and centrifuged at 6000 rpm min⁻¹ for 10 min. The supernatant was filtered and subjected to GC-MS analysis. The GC-MS system was equipped with an Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm) and an ISQ™ 7000 single quadrupole mass spectrometer. The injection port temperature was set at 220 °C, and 1 μL of sample was injected in a splitless mode. The column temperature program was as follows: initial temperature at 50 °C (held for 5 min); increased to 100 °C at 3 °C min^-1 (held for 2 min ); increased to 140 °C at 4 °C min^-1 (held for 1 min); and then increased to 250 °C at 5 °C min^-1 (held for 5 min). The SCAN mode was used for scanning within a range of 40–400 m/z. The ion source temperature was set to 240 °C, and the transfer line temperature was 220 °C (Supplementary Table. 1).

Statistics and reproducibility

Data were presented as mean ± SD (n = 3). Student’s t-test was used to determine the significance of the differences between two groups. Fluorescence intensity and gray scale were analyzed using Image J software. p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s42003-024-07400-1