SERUM-FREE MEDIUM FOR CULTURING A BOVINE PROGENITOR CELL

Abstract

A method for culturing a bovine progenitor cell, comprising the step of: culturing a bovine progenitor cell in a serum-free medium for culturing a bovine progenitor cell, wherein said serum-free medium comprises an albumin; and a fibroblast growth factor (FGF).

Claims

1. A method for culturing a bovine progenitor cell, comprising the step of: culturing a bovine progenitor cell in a serum-free medium for culturing a bovine progenitor cell, wherein said serum-free medium comprises an albumin; and a fibroblast growth factor (FGF).

2. The method according to claim 1, wherein said serum-free medium further comprises one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; and one or more cytokines and/or growth factors selected from the group consisting of a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin 6 (IL-6).

3. The method according to claim 2, wherein said serum-free medium comprises as said one or more cytokines and/or growth factors: an IL-6; an IL-6 and an IGF; an IL-6, an IGF and an HGF; an IL-6, an IGF, an HGF and a PDGF; an IL-6, an IGF, and a VEGF; an IL-6, an IGF and a PDGF; an IL-6, a PDGF and a VEGF; an IL-6, an IGF, a PDGF and a VEGF; or an IL-6, an IGF, an HGF, a PDGF and a VEGF.

4. The method according to claim 1, wherein said serum-free medium further comprises ascorbic acid or a derivative thereof; an insulin; a somatotropin; and a hydrocortisone; and a PDGF, a VEGF, an HGF, an IGF and an IL-6.

5. The method according to claim 1, wherein said serum-free medium further comprises a basal medium; a source of glucose; a source of glutamine; a source of fatty acids; a source of iron or an iron transporter; and/or sodium selenite.

6. The method according to claim 5, wherein the basal medium comprises (i) DMEM and/or Ham's F12 medium, preferably DMEM and Ham's F12 medium, for instance in a ratio of 1:10 to 10:1, more preferably a 1:1 ratio, respectively, (ii) RPMI medium and/or (iii) alpha-MEM medium.

7. The method according to claim 5, wherein said source of fatty acids comprises α-linolenic acid.

8. The method according to claim 1, wherein said serum-free medium further comprises a protein hydrolysate, preferably a soy protein hydrolysate.

9. The method according to claim 1, wherein said serum-free medium further comprises a biogenic amine, preferably an ethanolamine, putrescine, spermidine and/or spermine.

10. A method for culturing a bovine progenitor cell, comprising the step of: culturing a bovine progenitor cell in a serum-free medium for culturing a bovine progenitor cell, wherein said serum-free medium comprises an albumin; a fibroblast growth factor (FGF); one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; one or more cytokines and/or growth factors selected from the group consisting of a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin 6 (IL-6); a basal medium; a source of glucose; a source of glutamine; a source of fatty acids; a source of iron or an iron transporter; and sodium selenite; and optionally a protein hydrolysate, a biogenic amine and/or an attachment factor.

11. The method according to claim 1, wherein said albumin, said FGF, said one or more cytokines and/or growth factors, said one or more vitamins and/or hormones, said basal medium, said source of glucose, said source of glutamine, said source of fatty acids, said source of iron or said iron transporter, said sodium selenite, said protein hydrolysate, said biogenic amine and/or said attachment factor are present in an effective amount for proliferation of a bovine progenitor cell in culture.

12. The method according to claim 1, wherein said serum-free medium is a serum-free medium for proliferation of a bovine progenitor cell.

13. A composition comprising a serum-free medium as defined in claim 1 and a bovine progenitor cell.

14. A serum-free medium for culturing a bovine progenitor cell, comprising an albumin; and a fibroblast growth factor (FGF).

15. The serum-free medium according to claim 14, wherein said serum-free medium further comprises an IL-6.

16. The serum-free medium according to claim 14, wherein said serum-free medium comprises one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; and one or more cytokines and/or growth factors selected from the group consisting of a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin 6 (IL-6).

17. The serum-free medium according to claim 14, wherein said serum-free medium further comprises: a protein hydrolysate, preferably a soy protein hydrolysate; a basal medium comprising DMEM and Ham's F12 medium; a somatotropin; a hydrocortisone; spermine; and/or spermidine.

18. The method according to claim 1, wherein said bovine progenitor cell is a bovine muscle progenitor cell or a bovine fat (adipose tissue) progenitor cell.

19. A method of producing a cell culture-based meat product for human consumption comprising culturing a bovine muscle progenitor cell in the medium according to claim 14.

Description

FIGURE LEGENDS

[0133] FIG. 1.

[0134] The comparison of F10 and DMEM/F12 media used as a base in a serum-free medium of the invention. Bovine muscle progenitor cells were cultured in collagen-coated 96 wells plate in basal medium F10 or DMEM/F12 1:1 for 4 and 7 days (n=3). Cell numbers were measured with a proliferation assay MTS kit (Promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)).

[0135] FIG. 2.

[0136] The growth of bovine muscle progenitor cells when cultured in the serum-free medium for 4 days in different combinations of growth factors compared to the control serum based medium. The serum-free medium control contains DMEM/F12 1:1, supplemented with 1% penicillin/streptomycin/amphotericin (PSA), 2 mM L-alanyl-L-glutamine, 5 pg/ml bovine serum albumin, 75 ng/ml IL-6, and 5 μg/l FGF. The serum-containing medium control contains DMEM/F12 1:1 with 20% FBS.

[0137] The concentrations of the growth factors tested were: IGF1 at 100 μg/l, HGF at 5 μg/l, VEGF at 10 μg/l, PDGF-BB at 10 μg/l.

[0138] FIG. 3.

[0139] Long-term proliferation of bovine muscle progenitor cells in a serum-containing growth medium (Control GM) and a serum-free medium of the invention (SFM1). The tested serum-containing growth medium contains DMEM/F12+20% fetal bovine serum +5 ng/ml bFGF +1% Penicillin-Streptomycin-Amphotericin B) and the tested serum-free medium (SFM1) contains: albumin (5 mg/ml), somatotropin (2 ng/ml), L-Ascorbic acid 2-phosphate (50 μg/ml), hydrocortisone (36 ng/ml), a-linolenic acid (1 μg/ml), insulin (10 μg/ml), transferrin (5.5 μg/ml), sodium selenite (0.0067 μg/ml), ethanolamine (2 μg/ml), L-alanyl-L-glutamine or glutamine (2 mM), IL-6 (5 ng/ml), FGF2 also referred to as bFGF (10 ng/ml), IGF1 (100 ng/ml), VEGF (10 ng/ml), HGF (5 ng/ml), PDGF-BB (10 ng/ml) and DMEM/F12 basal medium.

[0140] The cells were cultured for several weeks in collagen coated tissue culture vessels and passed when 80%-90% confluent. At each passage, the cells were counted with a countess automated cell counter. Proliferation is shown in total population doublings (PDs).

[0141] FIG. 4.

[0142] The growth of bovine muscle progenitor cells in the serum-free medium (control)) with different concentrations of several components. The cells were cultured in a collagen-coated 96 wells plate for 4 days (n=4) with different concentrations of the following components: recombinant human albumin (2, 1, 0,5 or 0 mg/ml), fibronectin from bovine plasma (5, 2, 1 or 0 pg/ml), recombinant human IL-6 (25, 10, 5, 2 or 0 ng/ml), recombinant human insulin-like growth factor 1 (25, 10, 5, 2 or 0 ng/ml) and recombinant fibroblast growth factor 2 (5, 2, 1 or 0 ng/ml). Cells were counted by the high content analyser ImageXpress Pico Automated Cell Imaging System. It is shown that the absence of albumin and FGF provides for highly reduced growth of bovine muscle progenitor cells.

[0143] FIG. 5.

[0144] The effect of different concentrations of spermidine and a soy hydrolysate in the form of Soy Hydrolysate UF Solution on proliferation of bovine myosatellite cells. The cells were grown for 4 days (n=2) on collagen coated 96 wells plates with different concentrations of spermidine (0, 1, 2, 3, 4, 5 or 10 μg/ml) and different concentrations of soy hydrolysate (0, 0.05, 0.1, 0.15, 0.2 and 0.25% (w/v). Cells counted by the high content analyser ImageXpress Pico Automated Cell Imaging System. It is shown that spermidine and soy protein hydrolysate enhance the cell growth rate.

[0145] FIG. 6.

[0146] Short-term culture comparison of the growth of adipose-tissue progenitor cells, i.e. stromal vascular fraction cells (from Bos taurus), in a serum-containing medium or serum-free medium with or without polyamine supplementation. The cells were seeded in 48 wells plates in a serum containing medium (DMEM/F12+10% fetal bovine serum +2 ng/ml bFGF), serum-free medium (SFM1) or a serum-free medium (SFM1) supplemented with 1 ug/ml spermidine (SPMD) and 10 μg/ml spermine (SPMN). Following 6 days in culture, the cells were counted by the high content analyser ImageXpress Pico Automated Cell Imaging System.

[0147] FIG. 7.

[0148] Short-term culture of satellite cells and adipose-tissue progenitor cells, i.e., stromal vascular fraction cells (SVF), all from Bos taurus, in a serum-free medium of the invention wherein the basal medium type was varied. The cells were seeded in collagen-coated 96 wells plate in a serum-free medium of the invention in which different basal media were included (i.e. RPMI medium, alpha-MEM medium, F12 medium, and DMEM/F12 medium at 1:10, 1:1, and 10:1 ratios). Following 6 days in culture, the cells were counted by the high content analyser ImageXpress Pico Automated Cell Imaging System.

EXAMPLES

Example 1. Production of a Serum-Free Medium of the Invention

[0149] Materials and methods

[0150] Bovine muscle progenitor cells were isolated from a bovine muscle tissue (Bos taurus) and sorted based on their positive expression of CD29 as previously described (Ding et al., Sci. Rep., 17(8): 10808 (2018)).

[0151] Cells were cultured in collagen-coated 96 well plates in basal medium DMEM/F12 1:1, supplemented with 1% PSA, 2 mM L-alanyl-L-glutamine, 5 μg/ml bovine serum albumin and 5 μg/l FGF (recombinant human protein FGF basic (FGFb), 233-FB from R&D Systems) for 5 days (n=3). This medium constitutes the serum-free control as indicated in the figure legend of FIG. 2. For the serum-based control, the DMEM/F12 1:1 medium was supplemented with 20% FBS. For the tested conditions, the medium was supplemented with IGF1 (human recombinant IGF (291-G1 from R&D Systems)) at 100 μg/l, HGF (recombinant human HGF (294-HG from R&D Systems)) at 5 μg/l, VEGF (recombinant human VEGF (293-VE from R&D Systems)) at 10 μg/l and PDGF-BB (human recombinant PDGF (220-BB from R&D Systems)) at 10 μg/l, either alone or in combination. A two level, full factorial design of experiments (DoE) was performed to test the effect of different combinations of additional growth factors to the proliferation of bovine muscle progenitor cells. Cells were cultured at 37° C., 5% CO.sub.2 for 4 days. After 4 days they were fixed with paraformaldehyde 2% and stained with Hoechst staining. The number of cells per well was determined with the high content analyser ImageXpress Pico Automated Cell Imaging System. Statistical analysis was performed (JMP software) to investigate main effects and interaction effects between the growth factors tested (IGF1, HGF, VEGF and PDGF-BB).

[0152] Results

[0153] The relative (expressed as a percentage) growth of the cells as compared to the serum containing control is shown in FIG. 2. Statistical analysis of these results showed a significant positive effect of IGF1 and VEGF addition as well as a positive synergy of IGF1 with HGF. The serum free medium supplemented with IGF1 at 100 μg/l, HGF at 5 μg/l, VEGF at 10 μg/l, and PDGF-BB at 10 μg/l showed 1.5 fold increase in growth when compared to the serum contained control and a 4.7-fold increase of growth when compared to the serum-free based control (only containing 5 ng/mL FGF and 75 ng/ml IL-6 as growth factors).

Example 2. Culturing a Muscle Progenitor Cell in a Medium of the Invention

[0154] Materials and Methods

[0155] Bovine muscle progenitor cells were isolated from a bovine muscle tissue (Bos taurus) and sorted based on their positive expression of CD29 as previously described (Ding et al., Sci. Rep., 17(8): 10808 (2018)).

[0156] Muscle progenitor cells were seeded at a density of 3000-5000 cells/cm.sup.2 in the serum-free medium of invention (i.e. SFM1 medium) or in the serum containing medium (DMEM/F12 supplemented with 20% fetal bovine serum, 5 μg/ml bFGF and 2 mM L-alanyl-L-glutamine) in an appropriate collagen-coated cell culture vessel. The cells were passaged upon reaching 90% confluency. Briefly, the cells were rinsed once with phosphate buffer saline (PBS, 20012027 from ThermoFischer Scientific) followed by the addition of trypsin (25200072 from ThermoFischer Scientific). Once the cells were detached, trypsin was neutralised by the addition of trypsin inhibitor from Glycine max (T6522 from Sigma Aldrich), the cells collected into PBS and centrifuged at 350 g. The supernatant was aspirated and the cell pellet resuspended as needed. The cells were maintained in a 95% air/5% CO.sub.2 humidified atmosphere at 37° C.

[0157] Results

[0158] Muscle progenitor cells cultured in the serum-containing medium had an initial higher rate of growth that decreased with time, whereas the serum-free grown cells had a lower growth rate that remained relatively constant (more stable). With time, the total growth of cells in the two media was comparable. See FIG. 3.

Example 3. Culturing Satellite Cells and Adipose-Tissue Progenitor Cells in a Serum-Free Medium of the Invention with Different Basal Media

[0159] Materials and Methods

[0160] Satellite cells and adipose-tissue progenitor cells, e.g. stromal vascular fraction (SVF) cells, all from Bos taurus, were seeded in collagen-coated 96 wells plate and cultured for six days in a 95% air/5% CO.sub.2 humidified atmosphere at 37° C. in a serum-free medium of the invention composed of albumin (5 mg/ml), somatotropin (2 ng/ml), L-Ascorbic acid 2-phosphate (50 μg/ml), hydrocortisone (36 ng/ml), a-linolenic acid (1 μg/ml), insulin (10 μg/ml), transferrin (5.5 μg/ml), sodium selenite (0.0067 μg/ml), ethanolamine (2 μg/ml), L-alanyl-L-glutamine or glutamine (2 mM), IL-6 (5 ng/ml), FGF2 also referred to as bFGF (10 ng/ml), IGF1 (100 ng/ml), VEGF (10 ng/ml), HGF (5 ng/ml), PDGF-BB (10 ng/ml), with a different basal medium (i.e. RPMI medium (11875093, Thermo Fischer Scientific), alpha-MEM medium (12561056, Thermo Fischer Scientific), F12 medium (11765054, Thermo Fischer Scientific), DMEM/F12 1:1 medium, DMEM/F12 10:1 medium and DMEM/F12 1:10 medium) all made using F12 above and DMEM (A1443001, Thermo Fischer Scientific). Following 6 days in culture, the cells were counted by the high content analyser ImageXpress Pico Automated Cell Imaging System. The relative growth rate was calculated by dividing the number of cells in a certain medium by the number of days and normalizing to the control (growth in DMEM:F12).

[0161] Results It was observed that advantageous growth (proliferation) rates of satellite cells and adipose-tissue progenitor cells were achieved with a serum-free medium of the invention comprising a spectrum of different basal media, and that growth (proliferation) rates of such cells do not depend on a specific basal medium (FIG. 7).