Conditioned serum-free culture medium accomplishes adhesion and proliferation of bovine myogenic cells on uncoated dishes

Isolation of primary bovine myogenic cells

Primary bovine myogenic cells were isolated and cultured using previously reported methods by our group⁴⁵. Briefly, bovine myogenic cells were isolated from cheek skeletal muscle tissues of Japanese Black cattle provided by Tokyo Shibaura Organ (Japan). The meat surface was disinfected with 0.5% chlorhexidine gluconate (5% Hibitane, Sumitomo Dainippon Pharma, Japan) and cut with a scalpel into blocks of ~3 × 4 × 0.5 cm. The samples were disinfected with a povidone-iodine solution (Meiji Seika Pharma, Japan) and ethanol (Yoshida Pharmaceutical, Japan), followed by Hanks’ balanced salt solution (HBSS) (FUJIFILM Wako Pure Chemical, Japan). The samples were then minced into 2 mm pieces using a scalpel and scissors. The minced sample 2 g was placed in a 50 mL centrifuge tube, and 10 mL HBSS with 1 mg/mL pronase (pronase from Streptomyces griseus, Sigma-Aldrich, USA) was added to the tube. After the centrifuge tubes containing the samples were treated with the enzyme by shaking in a 37 °C thermostatic bath for 1 h, 10 mL HBSS with 10% FBS was added. Cells were dispersed in solution by pipetting 10–20 times with a 25 mL pipette and 10–20 times with a 10 mL pipette. The suspension sample was filtered using a 40 μm cell strainer and centrifuged at 1000 × g for 10 min. After removing the supernatant, precipitated pellet was dissolved in 10 mL Dulbecco’s Modified Eagle Medium (DMEM, FUJIFILM Wako Pure Chemical Corporation, Japan) + 10% FBS + 1% penicillin-streptomycin (PS, FUJIFILM Wako Pure Chemical Corporation, Japan) with 10 ng/mL basic fibroblast growth factor (bFGF, KAKEN PHARMACEUTICAL, Japan). To exclude non-myogenic cells that initially adhered, such as fibroblasts, the cell suspension was incubated in an uncoated culture dish at 37 °C for 1 h, and then the supernatant was collected. The collected myogenic cells were seeded at a dilution ratio of 1:5 on a culture dish coated with recombinant laminin 511 E8 fragment (Easy iMatrix-511, Nippi Inc., Japan). When the primary bovine myogenic cells became confluent on day 6 or 7 after seeding, the cells were collected and frozen. For freezing, rapid cryopreservation at –80 °C was performed using CELLBANKER 1 (ZENOGEN PHARMA CO., LTD, Japan), and the cells were stored in the vapor phase of a liquid nitrogen tank overnight. In this study, the bovine myogenic cells (P1), which were primary cultured primary bovine myogenic cells and then passaged once, were used as cell samples.

Fabrication of serum-free co-culture medium conditioned by HepG2 and NIH/3T3 cells

Figure 1 shows the fabrication of serum-free co-culture medium conditioned by the human hepatocellular carcinoma-derived cell line HepG2 and the mouse embryonic fibroblast cell line NIH/3T3. HepG2 and NIH/3T3 cells were suspended in the adhesion medium described below and seeded at a density of 2.0 × 10⁵ cells/cm² respectively (seed ratio to 1:1) in a ϕ 10 cm culture dish. The adhesion medium was prepared using DMEM + 10% FBS + 1% PS. The cells were incubated at 37 °C for 5 h, and then the adhesion medium was aspirated and discarded after initial cell adhesion was confirmed. The culture dishes were rinsed with 10 mL of D-PBS(-) (FUJIFILM Wako Pure Chemical Corporation, Japan) for three times.

After the washing treatments, serum-free culture medium DMEM + 1% PS 13 mL was added, and the cells were incubated at 37 °C for 3 days. The duration for a general conditioning culture medium is commonly set to within 1 day^{46,<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 47" title="Jain, R. K., Vakil, D., Cunningham, C. & Sidhu, K. Human mesenchymal stem cells conditioned media promotes the wound healing process – An in vitro study. J. Stem Cell Ther. Transplant. 3, 028–030 (2019).” href=”#ref-CR47″ id=”ref-link-section-d394049975e1215″>47} or 3 days^48,49. However, the previous studies demonstrating that the co-culture of HepG2 and NIH/3T3 cells enhances the liver-associated function of HepG2 cells showed that albumin secretion levels continued to increase until day 11 of culture^17,18. Taking these findings into consideration, we hypothesized that maximizing the co-culture duration of HepG2 and NIH/3T3 cells for conditioning the medium could lead to an increase in liver-associated secretions, and thus, we set the conditioning duration to 3 days.

After incubation, the supernatant was collected in a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 min. The supernatant of the centrifuge tube was collected as a serum-free co-culture medium conditioned by HepG2 and NIH/3T3 cells. For comparison, a culture medium conditioned only by HepG2 or NIH/3T3 cells was also prepared. These media were prepared by collecting the supernatants according to the protocol described above from the culture dish in which HepG2 or NIH/3T3 cells were seeded at a density of 2.0 × 10⁵ cells/cm² in a ϕ 10 cm culture dish, respectively.

Evaluation of the cell adhesion and cell proliferation of bovine myogenic cells under the developed serum-free co-culture medium

Using the developed serum-free co-culture medium, bovine myogenic cells were seeded at a density of 2.0 × 10⁴ cells/cm² on uncoated culture dishes (Greiner Bio-One, CELLSTAR 12 well plate, Austria). The effects on cell adhesion and proliferation of the bovine myogenic cells were evaluated by measuring the cell density at 24 and 72 h of culture, respectively. For quantitative cell counting, the cell nuclei were fluorescently stained with Hoechst 33342 (Thermo Fisher Scientific, H3570, U.S.A.). At the end of culture, the cells were fixed with 4% paraformaldehyde (Muto Pure Chemicals, 33111, Japan) for 15 min and washed three times with PBS. After washing, Hoechst 33342 solution diluted 1000-fold in PBS was added into the samples and the cells were allowed to stand for 15 min. The cells were then washed three times with PBS. Stained cell nuclei were observed using a fluorescence microscope BZ-X810 (Keyence, Japan). The fluorescence microscope comprised of a CCD camera and a 4X objective lens, and fluorescence-stained images of the entire well were captured by tiling imaging.

For quantitative cell counting, the number of cell nuclei was quantitatively measured from fluorescence microscope images using image analysis. Image preprocessing for noise reduction was performed using median and Gaussian filters, and Hoechst-positive region was extracted by binarization using the Otsu algorithm. After the extraction, segmentation was performed using the watershed method, and then the number of cell nuclei was counted. These image analysis algorithms were applied to whole tiled images repeatedly and then calculating the average cell density. For the image analysis, custom codes implemented in Python (version 3.10.9) using NumPy (version 1.23.5), SciPy (version 1.9.3), and scikit-image (version 0.19.3) were used.

To assess the culture function of the developed serum-free co-culture medium in detail, serum-containing culture medium DMEM + 10% FBS + 1% PS was prepared as the control (+) group, serum-free culture medium DMEM + 1% PS as the control (-) group, and serum-free culture medium conditioned by only HepG2 or NIH/3T3 cells as the HepG2 group and NIH/3T3 group, respectively. The developed serum-free co-culture medium conditioned by HepG2 and NIH/3T3 cells was designated as the co-culture group.

To evaluate the effect of the fabrication method of the serum-free co-culture medium on the cell adhesion and proliferation of bovine myogenic cells, the co-culture medium prepared by the contact co-culturing described above was used as a control group, and a non-contacted co-culture group using a Cell Culture Insert was established (Fig. 1c). HepG2 cells were seeded on the bottom of the insert and NIH/3T3 cells were seeded on the bottom of the well plates and prepared as the non-contacted coculture (H-N) group. NIH/3T3 cells were seeded on the bottom of the insert and HepG2 cells were seeded on the bottom of the well plates and prepared as the non-contacted coculture (N-H) group. In the non-contacted coculture groups, the ratio of the number of cells in the culture medium was adjusted to that in the control group. In addition, we also fabricated the samples that were prepared by mixing the HepG2 supernatant and NIH/3T3 supernatant as mix group, and by twofold diluting of the co-culture medium with DMEM + 1% PS as diluted co-culture group.

To control for technical variations, technical triplicates were performed using three wells for each culture condition, and the average cell density was calculated as the representative value for each culture condition. To control for biological variation, biological triplicates with three different culture schedules were performed.

Evaluation of cell function by immunofluorescence and fluorescence staining

To evaluate the function of the bovine myogenic cells in a serum-free co-culture medium, protein expression at 24 and 72 h of culture was evaluated by immunofluorescence and fluorescence staining. To provide sufficient cells to constitute cultured meat, it is necessary to achieve expanded culture by cell proliferation while maintaining an undifferentiated state. Ideally, the adhesive and proliferative functions of cells in the developed serum-free co-culture medium should be comparable to those in serum-containing culture media and the undifferentiated state should be maintained. In the present study, we evaluated cell adhesion, proliferation, and myogenic differentiation of the bovine myogenic cells. To assess the cell adhesion function, CD29 and F-actin, which constitute the focal adhesion and cytoskeleton respectively, were immuno-fluorescently and fluorescently labeled^50,51. To assess cell proliferation function, Ki67, a prominent proliferation marker that is expressed in the cell cycles other than G0 phase, was immuno-fluorescently labeled^21,52. To assess myogenic differentiation in the myotube formation of bovine myogenic cells, Desmin, an intermediate filament of the cytoskeleton and a muscle-specific marker, was immuno-fluorescently labeled in accordance with previous reports^45,53,54,55. In all stains, the cell nuclei were fluorescently labeled with Hoechst 33342.

For fixation and permeabilization, all samples were fixed with 4% paraformaldehyde (Muto Pure Chemicals, 33111, Japan) for 15 min, washed three times with PBS, left to stand in PBS containing 0.5% Triton X-100 surfactant for 15 min, and then washed three times with PBS. To block the nonspecific binding of antibodies, 2% BSA solution, which was prepared using BSA powder (Sigma-Aldrich, A7906-50G, U.S.A) and PBS, were added to all samples, allowed to stand for 30 min, and then washed three times with PBS.

For the immunostaining of CD29, a mouse host CD29 monoclonal antibody solution (BioLagend, 303002, U.S.A.) diluted 100-fold with 0.1% BSA solution was prepared as the primary antibody solution. The primary antibody solutions were added, and then allowed to overnight (4 °C) on a shaker at 60 rpm. After washing three times with PBS, the CD29 immunolabeled samples were treated with Alexa Fluor 568 Goat anti-mouse IgG (H + L) antibody solution (Thermo Fisher Scientific, A11031, Polyclonal, U.S.A.) diluted 200-fold with 0.1% BSA solution, and then allowed to stand on a shaker at 60 rpm for 1 h (room temperature, shielded from light). After washing three times with PBS, the samples were treated with Phalloidin-iFluor 488 Reagent (Abcam, ab 176753, U.K.) diluted 1000-fold in 1% BSA solution was added for fluorescence staining of F-actin, and then allowed to stand on a shaker at 60 rpm for 90 min (room temperature, shielded from light). After washing three times with PBS, samples were treated with Hoechst 33342 solution (Thermo Fisher Scientific, H3570, U.S.A.) diluted 1000-fold in PBS for 5 min, and then washed three times with PBS.

For Ki67 immunostaining, a mouse host Ki67 monoclonal antibody (Exbio Praha, 11-155-C100, Czech Republic) diluted 500-fold with 0.1% BSA was used as the primary antibody solution. The primary antibody solutions were added, and then allowed to overnight (4 °C) on a shaker at 60 rpm. After washing three times with PBS, the Ki67 immunolabeled samples were treated with Alexa Fluor 488 Goat anti-mouse IgG (H + L) antibody solution (Thermo Fisher Scientific, A11029, Polyclonal, U.S.A.) diluted 200-fold with 0.1% BSA solution, and then allowed to stand on a shaker at 60 rpm for 1 h (room temperature, shielded from light). After washing three times with PBS, the samples were treated with the Hoechst 33342 solution diluted 1000-fold in PBS for 15 min and then washed three times with PBS.

For Desmin immunostaining, a mouse host Desmin monoclonal antibody (Thermo Fisher Scientific, MA5- 13259, U.S.A.) diluted 200-fold with 0.1% BSA was prepared as the primary antibody solution. After washing three times with PBS, the Desmin immunolabeled samples were treated with the Alexa Fluor 488 Goat anti-mouse IgG (H + L) antibody solution diluted 200-fold with 0.1% BSA solution and then allowed to stand on a shaker at 60 rpm for 1 h (room temperature, shielded from light). After washing three times with PBS, the samples were treated with the Hoechst 33342 solution diluted 1000-fold in PBS for 15 min and then washed three times with PBS.

To visualize F-actin and CD29, fluorescence microscopy images of F-actin and CD29 were captured using an ECLIPSE Ti2 fluorescence microscope (Nikon, Japan) equipped with a CMOS camera and a 20x objective lens. To visualize Ki67 and Desmin, fluorescence microscopy images of Ki67 and Desmin were captured using a BZ-X810 fluorescence microscope (Keyence, Japan) equipped with a CCD camera and a 10x objective lens. To suppress the variation in evaluation indices due to bias in the imaging position, fluorescence microscopy images were acquired at random positions 10 times (F-actin and CD29) or 40 times (Ki67 and Desmin). To suppress the measurement bias, the exposure time, analog gain, and other measurement conditions were consistently unified for all imaging conditions.

Quantitative image analysis of the fluorescence microscopy images was performed to quantitatively evaluate cell function. For the image analysis, custom codes implemented in Python (version 3.10.9) using NumPy (version 1.23.5), SciPy (version 1.9.3), and scikit-image (version 0.19.3) were used.

To quantify the cell adhesion function, the CD29-positive area ratio of the entire microscope image was evaluated. To quantify the CD29-positive area ratio, image preprocessing for noise reduction was performed using median and Gaussian filters. Background subtraction was performed using the Rolling-ball algorithm to improve the signal-to-noise ratio of the image. Subsequently, the CD29-positive regions were extracted by binarization using the Otsu algorithm. After extraction, segmentation processing was performed using the watershed method. To eliminate detection errors caused by image noise, only regions with an area of CD29-positive regions between 10² and 5.0 × 10⁵ pixels were extracted. These exclusion criteria were determined by qualitative evaluation of the analysis results. Finally, the CD29-positive area ratio was calculated by dividing the summed areas of CD29-positive regions by the entire image area.

To quantify cell proliferation function, the Ki67-positive area ratio of the entire microscope image was evaluated. To quantify the Ki67-positive area ratio, image preprocessing for noise reduction was performed using median and Gaussian filters, and Ki67-positive regions were extracted by binarization using the Yen algorithm. After extraction, segmentation processing was performed using the watershed method. To eliminate detection errors caused by image noise, only the regions with an area of Ki67-positive regions between 10¹ and 10² pixels were extracted. These exclusion criteria were determined by qualitative evaluation of the analysis results. Finally, the Ki67-positive area ratio was calculated by dividing the summed areas of Ki67-positive regions by the entire image area.

To quantify the differentiation function of myogenic cells, the Desmin-positive area ratio of the entire microscope image was evaluated. To quantify the Desmin-positive area ratio, image preprocessing for noise reduction was performed using median and Gaussian filters, and the Desmin-positive regions were extracted by binarization using the Yen algorithm. After the extraction, segmentation processing was performed using the watershed method. To eliminate detection errors caused by image noise, only regions with Desmin-positive regions between 10² and 10³ pixels were extracted. These exclusion criteria were determined by qualitative evaluation of the analysis results. Finally, the Desmin-positive area ratio was calculated by dividing the summed areas of the Desmin-positive regions by the entire image area.

These image analyses were repeated for all randomly sampled images, and the median value of the quantitative index was calculated to obtain a representative value for each biological sample. The experimental conditions were the same as those used to evaluate the performance of serum-free co-culture supernatants, and biological triplicates were performed to control for biological variations.

Characterization of the contact co-culture methods

To understand the mechanism of the stable culture effects similar to those of the serum-containing culture medium, we evaluated the characteristics of the contact co-culture method for generating a serum-free co-culture medium conditioned by HepG2 and NIH/3T3 cells. Specifically, we evaluated the morphological characteristics of the serum-free co-culture system and the culturing stability of the serum-free co-culture medium.

To evaluate morphological characteristics, immunofluorescent staining for albumin and E-cadherin was performed. After collecting supernatants from the co-culture system, all samples were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, left to stand in PBS added with 0.5% Triton X-100 surfactant for 15 min and then washed three times with PBS. After the washing, the samples were left to stand in 2% BSA solution for 30 min and then washed three times with PBS. For the immunostaining of albumin, rabbit host Albumin monoclonal antibody (Abcam, ab207327, U.K.) diluted 500-fold in 0.1% BSA was used as the primary antibody solution. The primary antibody solutions were added, and then allowed to overnight (4 °C) on a shaker at 60 rpm. After washing three times with PBS, the albumin immunolabeled samples were treated with Alexa Fluor 488 Goat anti-rabbit IgG (H + L) antibody solution (Thermo Fisher Scientific, A11034, Polyclonal, U.S.A.) diluted 200-fold with 0.1% BSA solution, and then allowed to stand on a shaker at 60 rpm for 1 h (room temperature, shielded from light). After washing three times with PBS, the samples were treated with the Hoechst 33342 solution diluted 1000-fold in PBS for 15 min, and then washed three times with PBS. For the immunostaining of E-cadherin, a mouse host E-cadherin monoclonal antibody (Abcam, ab231303, U.K.) diluted 1000-fold in 0.1% BSA was used as the primary antibody solution. The primary antibody solutions were added, and then allowed to overnight (4 °C) on a shaker at 60 rpm. After washing three times with PBS, E-cadherin-immunolabeled samples were treated with Alexa Fluor 488 Goat anti-mouse IgG (H + L) antibody solution (Thermo Fisher Scientific, A11029, Polyclonal, U.S.A.), diluted 200-fold with 0.1% BSA solution, and then allowed to stand on a shaker at 60 rpm for 1 h (room temperature, shielded from light). After washing three times with PBS, the samples were treated with the Hoechst 33342 solution diluted 1000-fold in PBS for 15 min, and then washed three times with PBS.

To evaluate the culturing stability of the serum-free co-culture medium, a passage culturing test to verify whether bovine myogenic cells could be cultured passingly was performed. In the passaging test bovine myogenic cells were seeded at a density of 2.0 × 10⁴ cells/cm² on a uncoated ϕ 3,5 cm culture dishes with each culture medium. After 2 days of culture at 37 °C, the culture medium was removed, and the sample was washed with D-PBS(-) 2 mL. After the washing, samples were incubated with 0.25 w/v% trypsin-1mmol/L EDTA-4Na solution 500 μL (FUJIFILM Wako Pure Chemical Corporation, 209-16941, Japan) for 3 min at 37 °C, and then samples were tapped to detach cells from the culture dish. The cell suspension was collected and mixed with DMEM + 10% FBS + 1% PS 500 μL. The mixed cell suspension was collected in a 1.5 mL microtube and centrifuged at 6200 × rpm for 5 min. After discarding the supernatant from the tube, the precipitated cells were suspended in D-PBS(-) 1 mL. After suspension, the number of cells were counted using a hemocytometer. To passage, the cell suspension was centrifuged again at 6200 rpm for 5 min, the supernatant was removed, and the precipitated cells were resuspended in each culture medium, and reseeded in a new culture dish. This process was repeated twice, with the samples being cultured at 37 °C for 2 days and passaged.

Component analysis and metabolome analysis for identifying characteristic nutrients

To identify the characteristic nutrients in the developed serum-free co-culture medium, the nutritional composition analysis of the culture medium was performed. Our preliminary studies revealed that inorganic salts, amino acids and vitamins may contribute more to the cell proliferation of bovine myogenic cells than proteins such insulin recombinant full chain and AlbuMAX™ II in the absence of albumin (Supplementary Fig. 7). In addition, it was showed that the osmotic pressure was similar under all experimental conditions, and the total protein content of the HepG2, NIH/3T3 and co-culture groups was lower than that in the control(+) group (Supplementary Fig. 8). Based on these results, we studied the metabolites in the culture medium.

Analyses of the nutrient content and osmolality of the extracts were outsourced to the clinical laboratory company SRL Inc., in Japan. A list of the test contents is provided in Supplementary Table 1.

For the clustering analysis, we built a custom script using SciPy (version 1.9.3) and performed hierarchical clustering using the complete linkage method based on cosine similarity. For principal component analysis, we built a custom script using scikit-learn (version 1.2.0) and performed the analysis. In these analyses, the z-normalized values for each test component concentration were used. To identify the characteristic nutrients in each culture medium, multiple comparison tests were performed using the Tucky–Kramer method in R (version 4.0). P-value less than 0.01 was considered significant in the analysis. For the quantitative enrichment set analysis, MetaboAnalyst 5.0⁵⁶, a web-based metabolome analysis tool, and the KEGG database was used. In the analysis, all missing values were replaced with zeros.

Evaluation of the culture function of each characteristic nutrient

The culture functions of the identified characteristic nutrients (alanine, asparagine, asparatic acid, glutamic acid, ornithine, pyridoxal, and pyridoxamine) were evaluated using additive culture experiments.

The concentration of each component was set at five levels (Supplementary Table 2). These concentrations were determined to include the concentration in the developed co-culture medium and DMEM + 10% FBS + 1% PS. In this experiment, DMEM + 1% PS was used as the basal culture medium. Each nutrient was supplemented by filter sterilization using a sterile filter (Millex®-GV 0.22 μm PVDF, Merck & Co. Inc., U.S.A). The experimental conditions using bovine myogenic cells were the same as those used for the performance evaluation of the serum-free co-culture supernatant. Cell adhesion and proliferation were evaluated by capturing phase-contrast microscopy images at 24 and 72 h of culture. To control for biological variations, biological triplicates with three different culture schedules were performed.

Statistical analysis

In all experiments, at least three biological replicates were performed by repeating independent biological experiments conducted on different experimental dates. For each independent biological experiment, three samples from different test wells were prepared under the same conditions to obtain technical replicates. The average of the experimental values obtained in the technical replicates was used as the representative value for each biological sample in the independent biological experiments.

To visualize the experimental data, bar graphs representing the mean values were used. All sample points are plotted on the bar graphs, and the standard deviations of the samples are shown in the error bars.

Welch’s t-test was performed to test for the differences in means between the two groups. One-way ANOVA was performed for the analysis of variance. For multiple comparisons, Dunnett’s test was used to test the difference in the means of each sample group relative to the control group. The significance level was set at p < 0.05. Statistical analyses were performed using SciPy (version 1.11.4).

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• Source: https://www.nature.com/articles/s41538-024-00355-x