METHOD FOR THE SYNTHESIS OF AN EDIBLE AND STERILIZABLE POROUS 3D SCAFFOLD USEFUL FOR CULTURED MEAT LARGE-SCALE PRODUCTION

Abstract

The present invention relates to a method for the large-scale synthesis of an edible and hot steam sterilizable macroporous three-dimensional (3D) scaffold which comprises biocompatible polymers with interconnected pores as a support material for adherent cell growth, proliferation and differentiation, which may be used to obtain tissue with nutritive content and/or cultured meat. These scaffolds are suitable for supporting cell tissue growth for biomedical or food applications.

Claims

1. A method for obtaining a sterilizable macroporous three-dimensional (3D) tissue engineering scaffold which comprising a network of at least an inter-crosslinked biocompatible polymer, wherein the method comprises the following steps: a) Preparing a dissolution of the at least a biocompatible polymer, b) Pouring out the solution of step a) into molds and freeze, preferably at a temperature lower than the freezing temperature of the solution; c) Lyophilizing the freeze-scaffold obtained in step b) d) Curing the lyophilized scaffold of step c), and e) Sterilization of the scaffold by hot steam.

2. The method according to 1 wherein, the biocompatible polymer is selected from natural, synthetic polymer, or any variant thereof.

3. The method according to claim 2 wherein, the natural polymer or any variant thereof is selected from the list consisting of: dextran, alginate, chitosan, starch, heparin, heparin sulfate, pullulan, cellulose, hemicellulose, glucomannan, agar, chondroitin sulfate, gelatin, chitin, polynucleotides, a polysaccharide, a glycosaminoglycan, natural polyesters, or any combinations thereof, preferably, the natural polymer or any variant thereof is selected from chitosan and/or alginate.

4. The method according to claim 2 wherein, the synthetic polymer or any variant thereof is selected from the list consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, bioesters, or any combinations thereof.

5. The method according to claim 1 wherein, the polymer has a molecular weight which ranges from 1000 Da to 5000000 Da, preferably from 10000 to 1000000 Da, more preferably from 100000 to 500000 Da.

6. The method according to claim 1 wherein, in step a) the polymer is dissolved in a solvent selected from the list consisting of: aqueous solutions, organic solvents, culture media, or any combinations thereof.

7. The method according to claim 1 wherein, the percentage by weight/volume of the polymer in the solution of step a) ranges from 0.5% to 16%, preferably, between 1% to 8 %, more preferably between 1.5% to 4%.

8. The method according to claim 1 wherein, the pH of step a) ranges from 1 to 14, preferably from 2 to 10; and the temperature of step a) ranges from 22° C. to 180° C., preferably from 30° C. to 70° C.

9. The method according to claim 1 wherein the scaffold of step b) is formed by molding the dissolution via extrusion directly or by molding into molds of different shapes and sizes.

10. The method according to claim 1 wherein, the freezing temperature of step b) ranges from -15° C. to -80° C.

11. The method according to claim 1 wherein, the lyophilization of step c) is performed for at least a period which ranges from 16 h to 96 h, preferably from 24 to 72 h, more preferably from 18 to 24, at a pressure which ranges from 1 to 2.6 x 10.sup.-4 mbar, preferably from 1 to 0.4 mbar, more preferably to 0.263 mbar, and at a temperature which ranges from 25° C. to -100° C., preferably from 20° C. to -50° C., and more preferably from -15° C. to -45° C.

12. The method according to claim 1 wherein, the curing process of step d) is a thermal curing process performed at a temperature which ranges from 30° C. to 180° C. for a period of time which ranges from 1 min to 48 h.

13. The method according to claim 1 wherein, the sterilization of step e) can be performed optionally by a physical or chemical sterilization, preferably a physical sterilization process.

14. The method according to claim 1 wherein, optionally further comprises in step a) and/or in an additional step between step c) and d), the addition of at least one additive, at least an adhesion molecule, at least a cross-linker agent and/or any combinations thereof.

15. The method according to claim 14 wherein, the additive is selected from the list consisting of: flavoring, a flavor enhancer, a colorant, a color enhancer, salts, acidity regulators, thickeners, emulsifiers, stabilizer, a nutritional enhancer, probiotics, prebiotics, saponins, antioxidants, essential fatty acids, minerals, and any combinations thereof.

16. The method according to claim 14 wherein, the adhesion molecule are selected from the list consisting of: inmuglobulin-superfamiliy proteins, cadherins, selectins or integrins; fibronectins, poly-L-omithine, collagen, vitronectins, lectin, poly-ornithine, poly-L-lysine, poly-D-lysine, cyclic peptides, RGD-containing peptides, RGDS-containing peptides or any combinations thereof, preferably the adhesion molecule is poly-e-lysine.

17. The method according to claim 14 wherein, the cross-linker agent is selected from the list consisting of amine groups, hydroxyl groups, carbonyl groups, aldehyde groups, carboxylate group, carbonate group, carboxyl groups, carboxamide groups, imine groups, imide groups, thiol groups, inorganic ions and any combinations thereof.

18. The method according to claim 14 wherein further comprises washed the scaffold and optionally repeating the steps b) to c).

19. A sterilizable by hot steam and edible macroporous 3D tissue engineering scaffold which comprising a network of at least a biocompatible polymer obtained by the method according to claim 1.

20. Use in vitro of the scaffold according to claim 19 in the production of a tissue and/or a cultured meat.

21. Use according to claim 20 wherein the cultured meat comprises a plurality of adherent cells, preferably muscle cells and optionally, further comprises a plurality of satellite cells, stromal cells, fibroblasts cells, myoblast cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or any combinations thereof.

22. Use according to claim 21 wherein the cells belong to an animal source selected from the list consisting of: mammals preferably porcine, bovine, ovine, horse, dog, cat; avian; reptile; fish; amphibians; crustaceans, cephalopods or any combinations thereof.

23. Use in vitro of the scaffold according to claim 19 as carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] To complete the description and in order to provide for a better understanding of the invention, a set of drawings is provided. Said drawings form an integral part of the description and illustrate an embodiment of the invention, which should not be interpreted as restricting the scope of the invention, but just as an example of how the invention can be carried out. The drawings comprise the following figures:

[0100] FIG. 1. Cylindrical scaffolds of different heights and diameters formed by molding. Scaffolds a), b) and c) corresponding to Example 1, 2 and 3, respectively.

[0101] FIG. 2. Schematic description of extrusion into a frozen surface. Inlet shows the final pellets that can be produced by this method.

[0102] FIG. 3. Phase diagram of water indicating the triple point; wherein the lyophilization process will occur below of this triple point.

[0103] FIG. 4. Scanning electron microscopy (SEM) images of porous scaffolds of Example 1 (4a), Example 2 (4b) and Example 3 (4c) before cell seeding. Tissue proliferation over scaffolds of Example 1 (4d), Example 2 (4e) and Example 3 (4f).

[0104] FIG. 5. Mercury (Hg) porosimetry studies before (A) and after (B) sterilization process of the scaffold disclosed in Example 1. Data of total porosity and total surface area.

[0105] FIG. 6. Mercury (Hg) porosimetry studies before (A) and after (B) the sterilization process of the scaffold disclosed in Example 2. Data of total porosity and total surface area.

[0106] FIG. 7. Mercury (Hg) porosimetry studies before (A) and after (B) sterilization process of the scaffold disclosed in Example 3. Data of total porosity and total surface area.

[0107] FIG. 8. FTIR (Fourier Transform-Infrared Spectroscopy). spectra of samples before (top line) and after (bottom line) sterilization procedure corresponding to scaffolds from Example 1 (8A), 2 (8B) and 3 (8C), respectively.

[0108] FIG. 9. Figure shows glucose consumption (expressed al g/L) at different assay times (expressed as days) by the mammal cells seeded in the scaffold obtained in the Examples 1 to 3 of the invention (Ex 1, Ex 2 and Ex 3, in the legend) in comparison to the initial glucose (initial, in the legend) and the cell culture controls in the absence of the scaffolds of the invention (control, in the legend).

EXAMPLES

[0109] Following are examples of the invention by means of assays carried out by the inventors, which evidence the effectiveness of the product of the invention. The following examples serve to illustrate the invention and must not be considered to limit the scope thereof.

Example 1. Synthesis of a Scaffold, by the Method of the Invention, Comprising Chitosan as Biocompatible Polymer

[0110] In a typical production method of the edible and sterilizable macroporous 3D scaffold that will be equally valid for other biocompatible polymers and mixtures thereof, commercial MEM Eagle (Minimum Essential Medium Eagle) w/Earle’s BSS w/Non-Essential aminoacids & L-glutamine, w/o Calcium in powder (ranging among 0-600 mg, to achieve 0-200% related to the chitosan weight, more preferably 75 mg, 10% related to the chitosan weight), was dissolved in acetic acid (0.1 M, 50 mL) and chitosan (750 mg, 1.5%) was slowly added while stirring. The mixture was stirred until homogeneous solutions were obtained (65° C., 2 hours) and the solution was poured into molds to give the desired shape and dimensions. All the samples were frozen at -20° C. for 24 hours and lyophilized at -40° C. during 72 hours, at a pressure of 0.263 mbar.

[0111] This is a method for manufacturing scaffolds with controlled shapes, which will be determined by the receptacle wherein the process is carried out.

[0112] The scaffolds were removed from the molds and put inside the oven at 135° C. for 1 hour without ventilation (the curing starts by pre-heating the oven at 45° C., as soon as the oven reaches 135° C. time starts; after 1 hour the scaffolds are left to reach room temperature inside the oven, it takes around 1h 30 minutes. At this stage, the scaffolds were washed with miliQ water (3x100 mL for 15 minutes), 0.1 M NaHCOs (3x100 mL for 15 minutes) and water (3x100 mL for 15 minutes).

[0113] The results show that the scaffold obtained by the method of the invention which comprises MEM powder (0%) loses its consistency when it is immersed in water. Therefore, it seems that the cross-linking temperature and time was not enough to obtain a scaffold with the desired properties regarding mechanical strength and appearance. However, the results show that the remaining scaffolds comprising different MEM concentrations (1%, 8%, 16%, 33%, 133%, 200% more preferably 8%) display good consistency and do not lose their shape. These data state that the components of the MEM are also acting as cross-linkers giving consistency to the otherwise breakable scaffolds.

[0114] Later, the scaffolds with an 8% MEM concentration were sterilized in an autoclave at 121° C., 1.05 bar, for 20 minutes and tested. The scaffolds are stable, and some degree of cross-linking has happened because the scaffolds do not swell in excess, this is the cross-linking reduces the swelling.

[0115] The macroporosity of these scaffolds obtained with a percentage by weight/volume of the chitosan polymer (1.5%), could be controlled and shows a high pore volume and total porosity of 90% giving rise to foam-like samples. The optimized scaffold exhibits a high volume and size of inter-connected macroporosity in the range 1-500 .Math.m as can be observed by Digital Imaging (FIG. 1a) and SEM (FIG. 4a).

[0116] In FIG. 5 it is show the Hg (mercury) intrusion porosity analysis of the scaffold obtained in the instant example. This technique is used to analyze the total connected porosity, volume of pores, pore size distribution, and surface area of solid and powder materials. The instrument, known as a porosimeter, employs a pressurized chamber to force mercury to intrude into the voids in a porous substrate. As pressure is applied, mercury fills the larger pores first. As pressure increases, the filling proceeds to smaller and smaller pores. Both the inter-particle pores (between the individual particles) and the intra-particle pores (within the particle itself) can be characterized using this technique. The volume of mercury intruded into the sample is monitored by a capacitance change in a metal clad capillary analytical cell called a penetrometer. The sample is held in a section of the penetrometer cell, which is available in a variety of volumes to accommodate powder or intact solid pieces. Sample size is limited to dimensions of approximately 2.5 cm long by 1.5 cm wide. This powerful technique was used to analyze the change on the porous nature of the scaffold before (FIG. 5A) and after (FIG. 5B) sterilizing the pieces. As can be seen in FIG. 5, results show a slight decrease of total pore volume and in the total surface area due to shrinking from the sterilization step. Still, the values after sterilization are more than highly suitable for the application showing the right pore volume range and high surface area available for cell colonization.

[0117] In order to prove that non-chemical modifications have occur during hot steam sterilization procedure that could compromise scaffolds suitability for cell culture, scaffolds before and after sterilization have been analyzed by FTIR (Fourier Transform-Infrared Spectroscopy). FTIR is an analytical technique used to identify organic (and in some cases inorganic) materials. This technique measures the absorption of infrared radiation by the sample material versus wavelength. The infrared absorption bands identify molecular components and structures. As it can be seen in FIG. 8A there are no changes on the band spectrum shape and position before and after the sterilization process proving the suitability of the chemical identity of the scaffolds.

Example 2. Synthesis of a Scaffold, by the Method of the Invention, Comprising Alginate as Biocompatible Polymer and Coated With Poly-ε-Lysine.

[0118] Alginic acid sodium salt (Sigma- Aldrich) at a concentration of 2.0 g was slowly added under vigorous stirring to water (200 mL). The mixture was stirred at 50° C. for 3 hours until a homogeneous and transparent and clear solution was obtained. The solution was left to reach room temperature.

[0119] Later, the solution was poured into molds to give the desired shape and dimensions. All the samples were frozen at -20° C. for 24 hours and lyophilized at -40° C. for 72 hours at a pressure of 0.263 mbar.

[0120] The lyophilized scaffolds obtained were removed from the molds and immersed in a 2% aqueous solution of calcium chloride (4 g, 200 mL) (any other aqueous solution can be used, such as, calcium sulfate, calcium carbonate, calcium gluconate, calcium citrate) in miliQ water (200 mL) for 15 minutes to provoke the crosslinking of the polymer. In this solution, the alginate scaffolds gelled immediately and washed extensively with miliQ water to remove the unbound calcium ions. The pellets were then immersed in a 0.2% poly-epsilon-lysine solution (1.0 g in 500 mL) for 16 hours, and then washed extensively with miliQ water. The scaffolds were frozen again at -20° C. for 24 hours and lyophilized at -40° C. for 72 hours and a pressure of 0.263 mbar.

[0121] The lyophilized scaffolds were later heated in an oven at 115° C. for 1 hour without ventilation (the curing starts by pre-heating the oven at 45° C., as soon as the oven reaches 115° C. time starts; after 1 hour the scaffolds are left to reach room temperature inside the oven, it takes around 1h 30 minutes), and later, stored until used.

[0122] Later, the scaffolds were sterilized in an autoclave at 121° C. for 20 minutes at 1.05 bar.

[0123] The optimized scaffold exhibits a high volume and size of inter-connected macroporosity in the range 1-500 .Math.m as can be observed by Digital Imaging (FIG. 1b) and SEM (FIG. 4b).

[0124] As shown in FIG. 6, the Hg (mercury) intrusion porosity analysis, before (FIG. 6A) and after (FIG. 6B) sterilizing the pieces, shows a slight decrease of total pore volume and in the total surface area. Still, the values after sterilization are highly suitable for the application showing the right pore volume range and high surface area available for cell colonization.

[0125] As it can be seen in FIG. 8B there are no changes on the band spectrum shape and position before and after the sterilization process proving the suitability of the chemical identity of the scaffolds.

Example 3. Synthesis of a Scaffold, by the Method of the Invention, Comprising Chitosan as Biocompatible Polymer and Coated With Poly-ε-Lysine

[0126] Food grade chitosan (9.0 g) was mixed with acetic acid (0.1 M, 600 mL) under gentle stirring at 40° C. until chitosan was dissolved. Poly-ε-L-lysine (360 mg powder, M.sub.w:1000-300000) was portion wise added to the previous solution under vigorous stirring and the mixture was warmed to 65° C. for 2 hours and 30 minutes to obtain a homogeneous and clear solution.

[0127] The previous solution was left to reach room temperature and it was poured into the molds to give the scaffolds the desired shape and size (FIG. 1c). The solution inside the molds was frozen at -20° C. for 24 hours and lyophilized at -40° C. for 72 hours and a pressure of 0.263 mbar.

[0128] The scaffolds were removed from the molds and they were jellified with ethanol absolute (2 litres) for 4 hours. The scaffolds were washed with high purity water and the scaffolds were immersed in a NaOH solution (1%) for 3 hours. The scaffolds were extensively washed with high purity water until the lixiviate reaches a neutral pH (6.5-7.5). The so formed scaffolds are frozen at -20° C. for 24 hours and lyophilized (72 hours, -40° C. and P = 0.263 mbar). The scaffolds were directly used without any further treatment or alternatively, it can be cured. Cured scaffolds were subjected to a heat treatment by introducing them inside an oven at 110° C. for 1 hour without ventilation (the curing starts by pre-heating the oven at 45° C., as soon as the oven reaches 110° C. time starts; after 1 hour the scaffolds are left to reach room temperature inside the oven, it takes around 1h 30 minutes). Later, the scaffolds were sterilized in an autoclave at 121° C. for 20 minutes at 1.05 bar. The optimized scaffold exhibits a high volume and size of inter-connected macroporosity in the range 1-500 .Math.m as can be observed by Digital Imaging (FIG. 1c) and SEM (FIG. 4c).

[0129] As shown in FIG. 7, the Hg (mercury) intrusion porosity analysis, before (FIG. 7A) and after (FIG. 7B) sterilizing the pieces, shows a slight decrease of total pore volume and in the total surface area. Still, the values after sterilization are highly suitable for the application showing the right pore volume range and high surface area available for cell colonization.

[0130] As it can be seen in FIG. 8C there are no changes on the band spectrum shape and position before and after the sterilization process proving the suitability of the chemical identity of the scaffolds.

Example 4. Culture the Scaffolds of Examples 1 to 3 Obtained by the Method Of the Invention With Non-Human Cells in a Bioreactor

[0131] To verify that the chitosan based-scaffolds (Examples 1 and 3) and alginate based-scaffolds (Example 2) obtained by the method of the invention are useful for obtaining cultured meat, the inventors cultured living cells along the mentioned scaffolds of the invention in a bioreactor.

[0132] To this end, the scaffolds of the Examples 1, 2 and 3 were sterilized in an autoclave at 121° C. for 20 minutes at 1.05 bar and sterilely immersed in a final volume of culture medium of 2 liters (Gibco™ OptiPRO™). Next, a known cell density (13.000 cells/cm.sup.2) was inoculated in the mentioned scaffolds and kept under culture for long periods of time (10 days or more) in a 48-hour perfusion bioreactor. The inoculated cells were primary porcine-myoblast cells, of proprietary extraction obtained from muscle biopsies and extracted following the protocol described in JM Spinazzola et al., Bio Protoc. 2017 November 5; 7(21). The culture conditions for adequate cell proliferation require maintaining a temperature of 37° C. and a pH value of 7.1-7.4. Additionally, a continuous supply of gases is maintained, such that the concentration of dissolved oxygen ranges between 20% - 30%. After a period of 10 days, the scaffolds were removed from the bioreactor and dehydrated with ethanol in order to perform SEM (Scanning Electron Microscopy) visualization. SEM images (FIGS. 4d, e and f ), show the full tissue proliferation over the scaffold of Example 1, Example 2 and Example 3, respectively.

[0133] During the incubation period of 10 days, glucose consumption was measured with a Bioprofile Analyzer with a Glucose biosensor. Glucose biosensors are amperometric electrodes that have immobilized enzymes in their membranes. In the presence of oxygen and the substrate being measured, these enzyme membranes produce hydrogen peroxide (H.sub.2O.sub.2), which is then oxidized at a platinum anode held at constant potential. The resulting flow of electrons and current change is proportional to the sample glucose concentration. Measurements were carried out at different assay times as an indirect measurement of the proliferation and formation of tissue over the scaffold of the present invention. The data obtained are shown in FIG. 9. The results show the existence of glucose consumption by the primary porcine-myoblast cells sown in the matrix of the invention over time (1-10 assay days), since a decrease in glucose concentration values is observed compared to the initial glucose levels of the culture medium (approximately 4 g/L). Positive controls were performed on each assay day.

[0134] Thus, the results show that the method of the present invention is useful to obtain a good scaffold for cell culture and for obtaining in vitro cultured meat.