Methods and use of serum-free medium for meat production

Abstract

In vitro method for producing meat by multiplying non-human fibroblasts, muscle cells, skeletal muscle stem cells and / or myosatellite cells by cultivation in serum-free medium, characterized in that the serum-free medium is free of blood serum and free of albumin, free of ROCK inhibitor, free of pro-inflammatory interleukins (IL), in particular free of IL-6, free of insulin-like growth factor (IGF) and free of vascular endothelial growth factor (VEGF), and the medium contains an inhibitor of the TGFβ signaling pathway which is an inhibitor of Smad2 / 3, and in that the medium contains TGFβ.

Description

[0001] The present invention relates to a method for cultivating animal cells and a medium for use in the method, wherein the cells are in particular induced muscle cells, skeletal muscle stem cells and / or myosatellite cells.

[0002] The method has the advantage of multiplying animal cells, e.g., mesenchymal cells from cattle, pigs, fish, poultry, molluscs, and mussels, in a serum-free medium, and using the cells thus produced as food, in particular as synthetically grown meat that can be further processed into food products. Accordingly, the invention relates to a method for producing artificial meat by serum-free in vitro cultivation of animal cells. Generally, animal cells are non-human cells. State of the art

[0003] WO 2019 / 140 151 A1 describes the production of beige adipocytes by contacting mesenchymal stem cells in medium with IL-4 and / or a TGF-β inhibitor, as well as a connection to adipogenic differentiation.

[0004] EP 4 023 669 A1 describes the cultivation of mesenchymal stem cells in serum-free medium in the presence of vitronectin and optionally a TGF-β inhibitor.

[0005] US patent 2020 0 172 861 A1 describes a medium containing, among other things, a TGF-β inhibitor, for the proliferation of epithelial stem cells in particular and for the production of organoids.

[0006] EP 3347450 B1 describes a serum-free medium containing human albumin for the cultivation of epithelial cells, with at least one ALKS inhibitor, which is A83-01, GW788388, RepSox or SB431542, an ALK4 inhibitor or ALK7 inhibitor.

[0007] WO 2021 / 158103 A1 describes the cultivation of bovine precursor cells in a medium that is serum-free and contains albumin and FGF and preferably IL-6. Object of the invention

[0008] The invention aims to provide a method by which animal cells, e.g. for use as food, can be cultivated, wherein the method can be carried out without animal blood serum, in particular without fetal serum. Description of the invention

[0009] The invention solves the problem with the features of the claims and in particular by a method for multiplying animal cells, especially animal mesenchymal cells, in a medium comprising as ingredients DMEM and / or Hams F12, L-Glutamine, at least one growth factor, preferably bFGF (basal fibroblast growth factor) and / or EGF (epidermal growth factor), an inhibitor of the TGFβ signaling pathway, which is an inhibitor of Smad2 / 3, additionally TGFβ, optional antibiotic contains and generally free of animal blood serum, or consists of the aforementioned ingredients.

[0010] Preferably, the method is an in vitro production process for meat by cultivating animal cells in a serum-free medium. The medium according to the invention is also free of animal components, e.g., free of blood serum and free of albumin. The growth factor, e.g., bFGF, EGF, TGFβ, is preferably produced by heterologous expression in microorganisms or plant cells.

[0011] The serum-free medium according to the invention contains or consists of Dulbecco's modified Eagle medium with the F12 additive, L-glutamine, at least one growth factor and an inhibitor of the TGFβ signaling pathway, and optionally an antibiotic, e.g., penicillin and / or streptomycin. The medium according to the invention is free of ROCK inhibitor, free of pro-inflammatory interleukins (IL), in particular free of IL-6, and optionally free of insulin-like growth factor (IGF) and free of vascular endothelial growth factor (VEGF).

[0012] Surprisingly, it has been shown that a concentration of TGFβ pathway inhibitor in a serum-free medium, especially DMEM with Hams F12 (DMEM-F12), which preferably contains only bFGF and / or EGF as additives and is, for example, free of insulin and interleukin, leads to a high proliferation of cells during cultivation, e.g., to a higher proliferation than in the otherwise identical medium with added fetal calf serum or human blood serum, but without TGFβ pathway inhibitor.

[0013] Preferably the serum-free medium contains DMEM and Hams F12, L-glutamine, bFGF and / or EGF, an inhibitor of Smad2 / 3, preferably LY2109761, in particular 1 to 100 ng / mL LY2109761, TGFβ, in particular 1 to 100 ng / mL, optionally an antibiotic, or consists thereof.

[0014] The inhibitor of the TGFβ signaling pathway can be an inhibitor of transforming growth factor beta (TGFβ), e.g., an anti-TGFβ antibody, a TGFβ ligand, or optionally an ALKS inhibitor, e.g., A83-01, GW788388, RepSox, or SB431542. Preferably, the inhibitor of the TGFβ signaling pathway is an inhibitor of Smad2 / 3. Particularly preferred is the inhibitor of the TGFβ signaling pathway LY2109761 (4-[5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-[2-(4-morpholinyl)ethoxy]quinoline, CAS number: 700874-71-1), e.g., at a concentration of 1 to 100 µM in the medium.

[0015] In one embodiment, iPSCs or muscle satellite cells are cultured in a medium according to the invention that does not contain TGFβ and are subsequently differentiated into muscle cells in a medium containing TGFβ. For this purpose, after cultivation to proliferate the cells in a medium that does not contain TGFβ, TGFβ can be added to trigger differentiation into muscle cells.

[0016] Preferably, cells produced using this method are separated from the culture medium after cultivation and further preferably washed to remove the medium, in particular the inhibitor of the TGFβ signaling pathway and growth factors, from the cells. The cells can be washed, for example, by suspending them in a washing solution, such as physiological saline solution, phosphate buffer, or water, optionally by incubation, followed by separation from the washing solution.

[0017] As an alternative to adding an inhibitor of the TGFβ signaling pathway, preferably a Smad2 / 3 inhibitor, the cultured cells exhibit genetic manipulation that inhibits or interrupts the TGFβ signaling pathway, e.g., inhibition of Smad2 / 3. The genetic manipulation can induce the expression of an siRNA that inhibits or knocks out the expression of the TGFβ receptor or that preferentially inhibits or knocks out the expression of Smad2 / 3.

[0018] In one embodiment, the medium contains TGFβ and the cells are genetically modified so that the expression of Smad2 / 3 is inhibited or switched off, e.g. by a nucleic acid construct from which an siRNA is expressed against the Smad2 / 3 encoding mRNA.

[0019] The invention will now be described in more detail using examples relating to the figures shown in - Fig. 1 the relative multiplication of animal cells cultivated according to the invention, - Fig. 2A, Fig. 2B, Fig. 2C the relative proliferation of animal cells cultured according to the invention with increasing concentrations of TGFβ and inhibitor, - Fig. 3. Show the senescence of animal cells cultivated according to the invention. Example 1: Multiplication of animal cells from muscle tissue

[0020] As a preferred example of animal cells, cells are obtained from muscle tissue and multiplied in serum-free medium. A sterilely extracted sample of muscle tissue from a biopsy or a slaughtered animal is mechanically and enzymatically comminuted and filtered through a 70 to 100 µm cell sieve to separate tissue debris from small cell clusters and individual cells. The suspended cell clusters and individual cells are cultured in the medium according to the invention under cell culture conditions (37°C, 5% CO2 atmosphere) with regular medium changes and / or addition of fresh medium, under static conditions, preferably with agitation of the medium, e.g., by a stirrer or by pumping the medium, and bubble-free aeration with oxygen, e.g., aeration with air or oxygen through a membrane.

[0021] As an alternative to suspended cell clusters and single cells, sterile pieces can be cut from muscle tissue, e.g., with an edge length of 1 to 2 mm, which are then cultured in the medium according to the invention. Muscle stem cells grow or migrate from these pieces, proliferate in the medium, and differentiate into muscle cells through TGFβ. The medium can initially be free of TGFβ to allow muscle stem cells to proliferate, which are then differentiated into muscle cells by the subsequent addition of TGFβ.

[0022] Example 2: Increased proliferation of animal cells in the medium according to the invention. As a representative of animal cells, hCSC (human cardiac stem cells), or alternatively mCSC (mouse cardiac stem cells), were first pre-cultured in TC25 cell culture flasks (Sarstedt AG and Co., Nürmbrecht, Germany) and, after detachment with trypsin-EDTA (Sigma Aldrich, St. Louis, MO, USA), 1×10 3Cells per cavity were seeded into a 96-TC plate (Sarstedt AG and Co., Nümbrecht, Germany) coated with 0.1% gelatin type B (Sigma Aldrich, Taufkirchen, Germany) in hCSC medium consisting of DMEM-F12 (Life Technologies, Darmstadt, Germany), 10% fetal calf serum, L-glutamine (2 mmol / L), bFGF (5 ng / mL) (Miltenyi Biotech, Bergisch Gladbach, Germany), EGF (10 ng / mL) (Preprotech, Hamburg, Germany) and penicillin / streptomycin (10 mg / mL) (Sigma Aldrich, Taufkirchen, Germany). After 24 hours, the medium was replaced with a serum-free medium consisting of DMEM-F12 (Life Technologies, Darmstadt, Germany), L-Glutamine (2 mmol / L), bFGF (5 ng / mL) (Miltenyi Biotech, Bergisch Gladbach, Germany), EGF (10 ng / mL) (Preprotech, Hamburg, Germany) and penicillin / streptomycin (10 mg / mL) (Sigma Aldrich, Taufkirchen, Germany) and cultured for 72 hours.

[0023] In serum-free medium, the effect of TGFβ1 at increasing concentrations of 1 ng / ml, 5 ng / ml, and 10 ng / ml (Peprotech, Hamburg, Germany) and the effect of TGFβ in combination with 50 µM LY2109761 were investigated. After cultivation for 72 hours at 37 °C and hypoxic conditions with 5% CO2 and 5% O2 at 37 °C, 10 µL of Orangu Cell Counting Solution (Cell Guidance Systems, Cambridge, UK) was added to each cavity for 2 hours at 37 °C. Absorbance was measured at 450 nm, and cell count was calculated using a formula derived from a predefined calibration curve. For calibration, hCSCs from the same suspension were seeded into a TC-96 plate coated with 0.1% gelatin type B with 0, 250, 500, 750, 1000, 1500, 2000, 2500 and 3000 cells per cavity.After the cells had settled for 1.5 hours, 10 µL of Orangu Cell Counting Solution (Cell Guidance Systems, Cambridge, United Kingdom) was added for 2 hours and the absorption was measured at 450 nm.

[0024] The Fig. Figure 1 shows the cell proliferation of cultured cells relative to cell proliferation in serum-free medium without additives as a control, in serum-free medium with 50 µM LY2109761 (control + inhibitor), in serum-free medium with the addition of 10 vol% human blood serum (serum), and in serum-free medium with the addition of 10 vol% human blood serum and 50 µM LY2109761 (serum + inhibitor).

[0025] The results of Fig. Figure 1 shows that the addition of the Smad2 / 3 inhibitor LY2109761 to serum-free medium (control + inhibitor) alone leads to a three-fold increase in cell proliferation compared to serum-free medium (control), while the presence of blood serum (serum) results in a smaller increase in cell proliferation. This demonstrates that the inhibitor significantly increases cell proliferation when cultured in serum-free medium.

[0026] As a further comparison, even higher cell proliferation was observed in the serum-free medium with the addition of blood serum and the inhibitor (serum + inhibitor). The higher cell proliferation in the serum-containing medium with the inhibitor (serum + inhibitor) compared to the serum-containing medium (serum) demonstrates the surprising effect of the inhibitor in promoting cell proliferation during cultivation.

[0027] The Fig. Figure 2A shows cell proliferation in serum-free medium with the addition of 1 ng / ml TGFβ (TGFβ 1 ng / ml), in serum-free medium with the addition of 1 ng / ml TGFβ and 50 µM LY2109761 (TGFβ 1 ng / ml + inhibitor) in relation to cell proliferation in serum-free medium without addition (control). Fig. Figure 2B shows cell proliferation in serum-free medium with the addition of 5 ng / ml TGFβ (TGFβ 1 ng / ml), in serum-free medium with the addition of 5 ng / ml TGFβ and 50 µM LY2109761 (TGFβ 1 ng / ml + inhibitor) in relation to cell proliferation in serum-free medium without addition (control). Fig. 2C Cell proliferation in serum-free medium with the addition of 1 ng / ml TGFβ (TGFβ 1 ng / ml), in serum-free medium with the addition of 1 ng / ml TGFβ and 50 µM LY2109761 (TGFβ 1 ng / ml + inhibitor) in relation to cell proliferation in serum-free medium without addition (control).

[0028] The results of Fig. 2 show that in serum-free medium, the addition of the Smad2 / 3 inhibitor, even in combination with the addition of TGFβ, significantly increases cell proliferation, with the combination of the inhibitor with TGFβ increasing cell proliferation more than the addition of TGFβ alone.

[0029] To determine senescence, 1 × 10 5hCSCs were seeded into TC100 trays coated with 0.1% gelatin type B. After 24 hours, the medium was replaced with serum-free medium consisting of DMEM-F12, L-glutamine (2 mmol / L), bFGF (5 ng / mL), EGF (10 ng / mL), and penicillin / streptomycin (10 mg / mL), and cultured for a further 72 hours. Subsequently, the serum-free medium was replaced with serum-free medium containing either 10% human blood serum, 10% blood serum in combination with 50 µM LY2109761, or 10 ng / mL TGFβ1, and incubated for 72 hours at 37 °C under hypoxic conditions with 5% CO2 and 5% O2. After six washes with PBS, the cells were transferred to 300 µL of 1x lysis buffer containing 5 mM CHAPS, 40 mM citric acid, and 40 mM sodium phosphate. 0.5 mM benzamidine and 0.25 mM PMSF were lysed. The cells were separated by scraping, transferred to a 1.5 mL reaction tube, and frozen at -80 °C.After thawing, the samples were centrifuged at 12,000 x g for 5 min, and 100 µL of the lysate was mixed with 100 µL of 2x reaction buffer containing 40 mM citric acid, 40 mM sodium phosphate, 300 mM NaCl, 300 mM β-mercaptoethanol, 4 mM MgCl₂, and 1.7 mM MUG ((4-methylumbelliferyl-B-D-galactoside)). After incubation for one hour at 37°C, 50 µL of the reaction mixture was added to 150 µL of 400 mM sodium carbonate stop solution. 150 µL of this mixture was then transferred to a black 96-well plate, and quantification was performed using a TECAN reader with an excitation frequency of 360 nm, an emission frequency of 465 nm, and an integration frequency of The assay was performed for 40 µs. The senescence-associated β-galactosidase activity was normalized to the amount of total isolated protein.

[0030] The Fig.Figure 3 shows the suppression of senescence by adding the exemplary inhibitor of the TGFβ signaling pathway to the serum-free medium, based on the measured values. Compared to senescence when cultured in serum-free medium (control), the addition of 10 vol% human blood serum (serum) reduces senescence. Adding 10% blood serum in combination with 50 µM LY2109761 (serum + inhibitor) further drastically reduces senescence. The addition of 10 ng / ml TGFβ1 reduced senescence, but to a lesser extent than serum or serum in combination with the inhibitor.

Claims

[1] In vitro methods for producing meat by multiplying non-human fibroblasts, muscle cells, skeletal muscle stem cells and / or myosatellite cells by cultivation in serum-free medium, characterized by , that the serum-free medium is free of blood serum and free of albumin, free of ROCK inhibitor, free of pro-inflammatory interleukins (IL), in particular free of IL-6, free of insulin-like growth factor (IGF) and free of vascular endothelial growth factor (VEGF), and that the medium contains an inhibitor of the TGFβ signaling pathway, which is an inhibitor of Smad2 / 3, and by the fact that the medium contains TGFβ. [2] Method according to claim 1, characterized by , that iPSCs or myosatellite cells are cultured in the medium that does not contain TGFβ, and subsequently differentiated into muscle cells in medium that does contain TGFβ. [3] Method according to any of the preceding claims, characterized bythat the medium contains bFGF and / or EGF. [4] Method according to any of the preceding claims, characterized by , that the inhibitor is an anti-TGFβ antibody, a TGFβ ligand, an ALKS inhibitor, LY2109761 or an siRNA expressed in the animal cells during propagation that is specific for TGFβ, the TGFβ ligand, ALK5 or Smad2 / 3. [5] Method according to any of the preceding claims, characterized by that the medium consists of DMEM with Hams F12 (DMEM-F12) and only bFGF and / or EGF as an additive. [6] Use of serum-free medium in a method according to any of the preceding claims, characterized by that the medium is free of blood serum and free of albumin, free of ROCK inhibitor, free of pro-inflammatory interleukins (IL), especially free of IL-6, free of insulin-like growth factor (IGF) and free of vascular endothelial growth factor (VEGF) and characterized bya level of inhibitor of the TGFβ signaling pathway, which is an inhibitor of Smad2 / 3, and that the medium contains TGFβ. [7] Use of serum-free medium according to claim 6, characterized by , that the inhibitor is an anti-TGFβ antibody, a TGFβ ligand, an ALKS inhibitor, LY2109761 or a nucleic acid construct for the expression of siRNA in animal cells, wherein the siRNA is specific for TGFβ, the TGFβ ligand, ALK5 or Smad2 / 3.

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