WO2023223083A1

WO2023223083A1 - Cultured meat production process, materials and equipment

Info

Publication number: WO2023223083A1
Application number: PCT/IB2022/054758
Authority: WO
Inventors: Anne-Sophie MERTGEN; Gricelda MENDIOLA-GARZA; Karen Ixchel BORRAYO-MONTANO
Original assignee: Micro Meat S De Rl De Cv
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-11-23

Abstract

A cultured meat production process comprising the steps of: a) mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) letting the cell-gel mixture drop into a cross-linking solution capable of cross-linking the cross-linkable gel material in the cell-gel mixture and of dissolving the second non-cross-linkable material; c) cross-linking the cell-gel mixture by keeping a suspension of cell-gel mixture droplets in the cross-linking solution until cross-linking is achieved thereby obtaining spherical cell-loaded edible scaffolds; d) removing the cross-linking solution with the non-cross-linkable gel material; e) growing the cells in the cell-loaded edible scaffolds in culture media; and f) removing the culture media to obtain tissue spheres, is provided. The invention further provides a cell-loaded edible scaffold and an equipment for enabling a cultured meat production process, as well as a muscular and/or adipose tissue particle and a meat product comprising said particles.

Description

CULTURED MEAT PRODUCTION PROCESS, MATERIALS AND EQUIPMENT

FIELD OF THE INVENTION

The invention pertains to the field of food production and tissue engineering and particularly refers to the production of meat by cell culture and the equipment required to implement the same.

BACKGROUND OF THE INVENTION

Meat culture has been developed over the years as an alternative to animal sacrifices by establishing processes that enable the growth of muscular tissue from stem cells that grow on structures or patterns so-called scaffolds that allow the adhesion of the differentiated cells and its growth to form tissue that is similar to the tissue of meat obtained from sacrificed animals, a process that corresponds to the field known as tissue engineering.

The current techniques of tissue engineering, however, have been developed mainly to enable tissue restoration for medical applications, the majority of which lead to a scaffold that has cells, embedded or adhered, capable of differentiating to the target tissue, such as bone, cartilage or organs such as kidney, liver, nerves, heart muscle or pancreas among others.

However, the nature of such techniques is not built for large scale production of tissues as needed by the meat industry, and therefore, the techniques are not suitable for the production of cultured meat for a variety of reasons, including edibility of the scaffolds or production rate and form of the scaffolds/tissue.

For instance, there has been described generally that the ability of the scaffolding material to receive cells within the material. That is the case of the scaffolds described in publication W02013/050921, where hollow microspheres are seeded with cells which are then used for detecting cancer by exposure to a test specimen. These microspheres cannot be applied to meat culture because they contain glass and are inedible.

Furthermore, in general, for the current techniques there is a need to have a process where the tissue is implanted into a person or an animal in order to continue the evolution of the cell growth in order to achieve the desired result, or in the case of the efforts to build full organs, the need to have very specific shapes and order for the growth of the cells in order to conform an organ-like structure.

For instance, a tissue that is often consumed as a kind of meat, liver tissue generation is of interest, such as the case of publication number JP2017085945. Although the document focuses in achieving liver function, it describes three-dimensional culturing of cells by inoculating a biodegradable scaffold material with a hepatocyte, and endothelial cell and a mesenchymal stem cell, and the culturing them therein under a simulated microgravity environment, which is also very complex to get for large scale production of meat. As observed, 3D scaffold printing is one of the most used techniques in tissue engineering, which includes typically the need to prepare an ink with proper materials through a mixing equipment and then extruding it onto a surface or a liquid to conform scaffolds or structures that will be used, once formed, to grow the cells of interest therein. Under these conditions cells are adhered to the outer surface of the scaffolds and even in porous materials they will not take advantage of the whole available holes or hollow spaces within the scaffold.

Accordingly, despite the large number of technologies developed in the field of tissue engineering and the advances in the techniques related thereto, technologies suitable for producing large volumes of tissue as necessary for the food industry are still insufficiently developed and scarce.

Amongst the few technologies that have been developed as an effort to increase the production volumes of meat cultures, it is found publication number W02021/250290 related to a method for the synthesis of a three dimensional porous, edible and sterilizable matrix for the production of cultured meat in large scale. The matrix described therein is made of biocompatible polymers with pores interconnected as a support material for the growth, proliferation and differentiation of adherent cells, which may be used to obtained tissue with nurturing content and/or cultivated meat. The biopolymer used therein as a scaffold may be sterilizable with steam and may be eaten by a person. Such scaffold is produced by depositing a drop of a polymer solution on a chilled surface, which will make it freeze into semi-spherical scaffold particles, which are then dried through lyophilization for use in a further process where the cells are put in contact with the so obtained scaffolds for obtaining the cultured meat. This process has several disadvantages for scaling up, including the use of lyophilization and the need to use equipment for cooling the bed where the biopolymer is dropped on, which makes it expensive, and it keeps the need to embed the scaffold with the cells for growth in a further processing step and limits the growth of the cells as these are typically adhered predominantly to the surface of the scaffold.

As shown here, the edibility of the scaffolding material is one of the targets to achieve, and there are other scaffolds in the prior art that are biocompatible and might be edible, such as that described in publication CN106110402 where cells for human skin are constructed within a sodium alginate hydrogel scaffold and then grown in a layer structure as sheets for application to wounds.

From the bio-ink perspective, another applicable technique may be that described in publication CN101904775B, that describes a tissue-like manufacture die based on spheroid elementary unit and preparation process, which does not describe an application to meat culture but does refer to different applications as it uses a molding die for extrusion (die and press) in order to perform a forming operation based on an ink that comprises globules that are first formed and then grown to bigger cell clusters that will be in turn implanted. A bio-ink in this context is understood as a mixture of components that in addition to the scaffolding materials as described before, also includes a biological material before it is otherwise formed so that it is possible to provide a pattern for growth with the cells embedded therein.

Another approach to obtain hollow scaffolds and bio-inks has been also described in document published as W02021/062411. The methods described therein comprise providing a bio-ink composition and a fugitive ink composition followed by a chaotic printing process to generate a micro- structured precursor comprising a plurality of lamellar structures formed from the bio-ink composition in order to obtain fiber/filament shapes. The bio-ink composition is then cured to form a scaffold precursor and finally the fugitive ink is removed from the cured scaffold precursor, thereby forming a perfusable fiber-shaped scaffold. The difference of this procedure as compared to other procedures previously referred to, such as that of W02021/250290, is that the document describes the possibility of dispersing a population of cells in the bio-ink composition prior to the chaotic printing of the filament or fiber shape. However, the procedure described therein requires a careful control of the flow of the bio-ink and fugitive ink compositions through the process in order to achieve the laminar flow necessary to form the lamellar interfaces between the bio-ink and the fugitive ink, which in turn is achieved by co-extrusion of both inks under such controlled flow conditions. Such controlled conditions must remain throughout the filament/fiber formation process, as such fibers can be entangled as they become longer. Therefore, this technique is also very difficult to use in meat culture since the flow control required is a burden to obtain large volumes of the scaffold faster, even if the cells are diffused prior to the bioprinting process through extrusion. Furthermore, the same document describes a complex bioreactor structure for the scaffolds thereby obtained, which prevents the possibility of obtaining a high volume of meat as a result, as the lamellar materials thereby obtained require careful handling to keep its integrity after extrusion, and do not resist shear stresses for more efficient culture processes requiring mixing.

Another problem that prior art processes have not solved is the ability of cells to be fed from the culture media, which leads to a high rate of cell loss when scaffolds are thicker than 200 microns.

Given the current state of development of the technologies, cultured meat is still extremely expensive and viable only in small-scale processing, because the current technologies cannot be enabled in larger equipment to maximize the volume of tissue thereby obtained.

OBJECTIVES OF THE INVENTION

Considering the drawbacks and problems of the technologies related to tissue engineering that prevent them to be applied to meat culture in larger scales, it is an object of the present invention to provide a process for meat culture that is not dependent on a forming process such as die casting or extrusion.

It is another object of the present invention to provide a process for meat culture that is not dependent on a strict control of the flow rate of bio-inks or fugitive inks.

It is yet another object of the present invention to provide a process for meat culture that does not need forming a scaffold and cell embedding as sequential steps.

It an additional object of the present invention to provide a scaffold that allows cells to grow within the scaffold and not only on the surfaces that increase the shear stress resistance of the culture during cell growth.

It is a further object of the present invention to provide a scaffold that allows cell nutrients to penetrate within the scaffold to reduce cell death. It is an additional object of the present invention to provide equipment for enabling the process for meat culture of the present invention at large scale.

These and other objects are achieved through the cultured meat production process and equipment of the present invention.

BRIEF DESCRIPTION OF THE INVENTION

Under the principles of the present invention, it is provided a cultured meat production process comprising the steps of: a) mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) letting the cell-gel mixture drop into a cross-linking solution capable of cross-linking the cross-linkable gel material in the cell-gel mixture and of dissolving the second non-cross-linkable material; c) cross-linking the cell-gel mixture by keeping a suspension of cell-gel mixture droplets in the crosslinking solution until cross-linking is achieved thereby obtaining spherical cell-loaded edible scaffolds; d) removing the cross-linking solution with the non-cross-linkable gel material; e) growing the cells in the cell-loaded edible scaffolds in culture media; and f) removing the culture media to obtain tissue spheres.

The cell-loaded edible scaffolds obtained by the process of the invention thereby obtained have preferably a diameter bigger than 3 mm which allows cells to grow within the scaffold and not only on the surface, thereby maximizing nutrient availability and therefore cell growth.

The equipment of the present invention consists of a modular production equipment comprising: a) a mixer of gel material capable of mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) an open container with stirring means for cross-linking solution and/or culture media with separation means to remove the cross-linking solution and/or the culture media; c) dropping means located above the open container capable of supplying drops of the cell-gel mixture. Finally, the equipment and process of the present invention allow obtaining edible spherical tissue clusters as culture meat product.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel and unique aspects of the invention shall be specified in the appended claims. However, some embodiments, features and advantages of the same shall be better understood in connection with the detailed description when read in relation to the appended figures, wherei Fig. 1 shows metabolic activity through 14 days of culture on three different scaffolds: filled fibers, hollow fibers and hollow spheres, which were embedded with C2C12 cells.

Fig. 2 shows metabolic activity through 14 days of culture on two different scaffolds: hollow fibers and hollow spheres, which were embedded with porcine satellite cells (pSC). Fig. 3 shows cell proliferation per area unit through 14 days of culture within three different scaffolds: filled fibers, hollow fibers and hollow spheres, which were embedded with C2C12 cells.

Fig. 4 shows cell proliferation per area unit through 14 days of culture within two different scaffolds: hollow fibers and hollow spheres, which were embedded with porcine satellite cells (pSC).

Fig. 5 shows cell doubling per area unit on three different scaffolds: filled fibers, hollow fibers and hollow spheres, which were embedded with C2C12 cells, wherein the cell doubling data was obtained through final comparison on cell numbers at the peak of the culture and initial cell numbers within the scaffolds.

Fig. 6 shows cell doubling per area unit on two different scaffolds: hollow fibers and hollow spheres, which were embedded with porcine satellite cells (pSC), wherein the cell doubling data was obtained through final comparison on cell numbers at the peak of the culture and initial cell numbers within the scaffolds.

DETAILED DESCRIPTION OF THE INVENTION

The cell-gel mixture is preferably of the type generally considered a bio-ink as it comprises a gel material and a live component. The cells loaded in the into de cross-linkable edible gel material are suitable for meat culture, preferably selected from myoblasts, myocytes, adipocytes, fibroblasts, satellite cells and any stem cells capable of differentiating into muscular or adipose tissue. In a preferred embodiment, the cells are selected from animals susceptible of human or animal consumption, preferably selected from mammals, birds, fish and shellfish. More preferably, the cells are capable of developing into animal muscular or adipose tissue selected from fish, crustacean, mouse, bovine, cattle, swine and poultry.

The cross-linkable edible gel material comprises an organic polymer able to provide firmness to the scaffold and anchorable sites for cell adhesion, more preferably a polysaccharide, protein, peptide or a combination thereof; a cross-linkable hydrogel, preferably a photo-cross-linkable hydrogel; a cell-proliferation stimulant; and, a buffer solution, preferably phosphate based.

More specifically, the organic polymers are selected from alginate, hyaluronic acid, agarose, gelatin, collagen or a combination thereof. The polymer preferably has such flow properties to allow its lamellar flow through the mixer. Moreover, in a preferred embodiment, the mixer comprises two inlets to properly mix components, and therefore the selected hydrogels preferably have similar viscosity to enable equal lamellar flow and proper internal microstructure formation. However, the polymer and hydrogel rheology values of the present invention shall not be limited but by the nature of the cells that will be grown therein, as long as the rheological properties are such that will allow mixing and dropping from the mixer.

The cross-linkable hydrogel is preferably selected from gelatin methacryloyl, collagen, fibrin, silk, hyaluronic acid, agarose, chitosan, cellulose, alginate, soy, extracellular matrix (ECM, such as but not limited to Matrigel ®), elastin, gelatin, polyethylene glycol (PEG), polyprolylene fumarate - polyethylene glycol (PPF-PEG), polyethylene oxide (PEO), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol diacrylate (PEGDA), and functionalized versions thereof, and it is further preferred to use gelatin methacryloyl.

Finally, regarding the hydrogel cross-linking agent, calcium ions can be used for polymers such as alginate, preferably in the form of a calcium chloride solution. When the hydrogel is photo-cross- linkable, such as gelatin metacryloyl, it is preferred using a photocrosslinker/initiator such as Irgacure®, or Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) together with UV light, more preferably LAP with UV light.

The buffer solution is selected from Dulbecco’s Phosphate Buffered Saline (DPBS), phosphate buffered saline (PBS), sodium phosphate, tris(hydroxymethyl)aminomethane buffer (Tris), Hank's balanced salt solution (HBSS), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffer, Sodium bicarbonate, Ethylenediaminetetraacetic acid (EDTA) buffer, and mineral buffers, and more preferably it is used Dulbecco's Phosphate Buffered Saline (DPBS).

The proportions of the components of the cross-linkable edible gel material are adjusted according to the quantity and type of cells to be seeded therein, and according to the nature of such components, but in a preferred embodiment, the organic polymer is added in 2 to 3% w/v, preferably 2% w/v; and the hydrogel is added in an amount of 0.1% to 3% w/v, preferably 3% w/v.

The loading of cells into the cross-linkable edible gel material is made by simply adding the material to the cells, preferably in darkness conditions in the embodiment where the material is set for photo-cross-linking. The count of cells to load is set according to the growth rate of the cells and on the application and type of cell used. Mostly, cells require proximity to other cells for successful communication in order to perform desired functions such as proliferation or differentiation. In a preferred embodiment, the minimum load of cells is O.lxlO⁶ cells/mL of cross-linkable edible gel material, and more preferably of at least 3xl0⁶ cells/mL of cross-linkable edible gel material. Upper limits for cell concentrations are defined by the minimum concentration of polymers in the solution to still be able to perform the cross-linking into a stable hydrogel.

As used here, the term edible refers to the fact that the material can be generally safely eaten by a healthy people or mammals, that is, without affecting their health condition. Regarding the second non-cross-linkable gel material, it comprises an innocuous water- soluble polymer enabling the formation of hollow structures within the scaffold for enhanced nutrient perfusion upon removal, preferably selected from cellulose derivatives. In a preferred embodiment the non-cross-linkable gel material is hydroxyethyl cellulose (HEC), or any other polymer that has similar rheological properties and does not cross-link with the mechanism used for crosslinking the stable hydrogel, such as Poloxamer 407.

As used here, the term innocuous refers to the fact that the material or traces thereof do not affect the health condition of healthy people or mammals if ingested.

The mixing step of the first cell-loaded cross-linkable edible gel material with the second non-cross-linkable gel material to obtain the cell-gel mixture is performed through any technique known in the art for bio-inks in bioprinting processes, for example those described by Ravanbakhsh, H., Karamzadeh, V., Bao, G., Mongeau, L., Juncker, D., Zhang, Y. S., Emerging Technologies in Multi-Material Bioprinting. Adv. Mater. 2021, 33, 2104730; or by Zhang, Y. S., Oklu, R., Dokmeci, M. R., & Khademhosseini, A. (2018). Three-Dimensional Bioprinting Strategies for Tissue Engineering. Cold Spring Harbor perspectives in medicine, 8(2), a025718, and more preferably is made through mixing equipment and mixture delivery fixtures allowing flow rates enough to achieve dropping of the mixed cell-gel. In a preferred embodiment of the invention, an extrusion equipment equipped with a head or dice for allowing droplets to fall is used.

In a further embodiment of the mixing step, a plurality of mixers and/or mixture delivery fixtures are configured in parallel so that a plurality of droplets with a minimum diameter of 1 mm, preferably of a diameter of 3 mm, is produced, in order to maximize the flow of cell-gel droplets thereby obtained for mass production.

The drops thereby obtained are let drop into a container with the solution for crosslinking, wherein the solution comprises an alkaline or alkaline earth metal cross-linker, such as calcium ions, preferably calcium chloride (CaCh) and water. At the same time, such solution dissolves the second non-cross-linkable material in the cell-gel droplets, in order obtain spherical cell-loaded edible scaffolds which have only the cross-linked material as a structure with the embedded cells therein. The cross- linking/dissolution process preferably lasts around 1 to 2 minutes, preferably 2 minutes.

The cross-linking solution is then removed from the container along with the non-cross- linkable gel material dissolved therein, while the cell-loaded edible scaffolds are transferred into a culture vessel or bioreactor for cell growth in a culture media.

Notably, well-known in the art bioreactors may be used under the principles of the present invention because the spherical cell-loaded edible scaffolds obtained by the process of the present invention do not require special configurations to keep fibers untangled or with a determined shape for growth, unlike the scaffolds obtained in the prior art.

Accordingly, the bioreactor where the cell culture is performed under the principles of the present invention are non-restricted. Active processes, such as those employed by standard stirred tank or rocker reactors, as well as passive, static culturing can be utilized. Reactors at a minimum must provide a sterile environment, temperature controlled between 32°C to 40°C, preferably 37°C, and exist at a minimum of approximately 1 atmosphere of pressure.

The cell growth step is preferably performed in a culture media suitable for the cells to be grown and is performed at a temperature and during a time according to the kinetics of the growth of such cells. Typically, the cell growth step will be performed at 37°C, for at least 48 hours, preferably under inert atmosphere. Preferred culture media is selected according to the conditions required for the growth of the cells in the cell-loaded edible scaffold to be processed and any suitable basal culture medium well known in the art can be used, but in a preferred embodiment, there is used Dulbecco's Modified Eagle's Medium - high glucose (DMEM-hg) supplemented with 10% Fetal Bovine Serum and 1% Anti-Anti (Penicillin 10,000 units/mL - Streptomycin 10,000 pg/mL- 25 pg/mL of amphotericin B). For induction of a myogenic program, natural origin compounds are preferred, more particularly curcumin, gingerol, dehydrocorydaline (from Corydalis tuber), tetrahydropalmatine (from Corydalis turtschaninovii), (-)- epicatechin gallate and (— )-epigallocatechin-3-gallate (from green tea). Furthermore, induction can begin by addition of surface receptor ligands, by using insulin+LPA+transferrin, by formulating differentiation medium fusing neurobasal + Leibovitz's - 15 medium (L15) + Insulin- like Growth Factor (IGF) + Epidermal Growth Factor (EGF), or by increasing the net, overall scaffold stiffness.

Finally, the culture media is separated in order to obtain meat-like tissue spheres.

According to the described process, in the step of growing the cells in the cell-loaded edible scaffolds in culture media, at least 100,000 cells per scaffold outer surface area unit are obtained. Furthermore, a minimum cell doubling of 2.5 per scaffold outer surface area unit is obtained.The cell- loaded edible scaffolds obtained by the process of the invention thereby obtained have preferably a diameter bigger than 1 mm, preferably 3 mm, which allows cells to grow within the scaffold and not only on the surface, thereby maximizing nutrient availability and therefore cell growth.

Another aspect of the invention comprises a cell-loaded edible scaffold having a substantially spherical shape of a diameter bigger than 1 mm, preferably 3mm, and comprising a cell-gel mixture made of a first cell-loaded cross-linkable edible gel material and a second non-cross-linkable gel material wherein the cell-loaded scaffold is capable of dissolving into a cross-linking solution capable of cross-linking the cross-linkable gel material in the cell-gel mixture and of dissolving the second non-cross- linkable material.

The equipment of the present invention consists of a modular production equipment comprising: a) a mixer of gel material capable of mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) an open container for cross-linking solution and/or culture media with separation means to remove the cross-linking solution and/or the culture media; c) dropping means located above the open container capable of supplying drops of the cell-gel mixture.

As mentioned before, the mixer may be of the type generally known in the art of bioprinting, but must comprise mixture delivery fixtures allowing low flow rates enough to achieve dropping of the mixed cell-gel. In a preferred embodiment of the invention, the mixer comprises an extrusion equipment further equipped with a head or dice for allowing droplets to fall.

The mixer may be selected from any commercially available capable of handling the first cell-loaded cross-linkable edible gel material and the second non-cross-linkable gel material according to their rheological properties, including but not limited to motionless static mixers.

In a further embodiment, a plurality of mixers and/or mixture delivery fixtures are configured in parallel so that a plurality of droplets from 1 mm of diameter, preferably 3 mm, is produced in order to maximize the flow of cell-gel droplets thereby obtained for mass production in a single open container.

The separation means of the open container are selected between sieves and valves configured to enable the cell-gel or the meat-like tissue spheres to be kept into the container while the solution or the cell culture is removed.The equipment and process of the present invention allow obtaining edible substantially spherical tissue clusters as cultured meat product. Unlike the prior art products obtained based on structured fiber-like scaffolds which may include tissue on the surface, the spherical tissue clusters of the present invention comprise the remaining edible cross-linked gel material as described and a meat tissue formed from the cultured cells, in both the surface and the inner body of the spheres.

More specifically, the present disclosure also refers to a muscular and/or adipose tissue particle comprising a cross-linked edible gel material that is substantially spherical, with a diameter bigger than 1 mm, and at least 100,000 cells per cross-linked edible gel material outer surface area unit.

In addition, a meat product made of such muscular and/or adipose tissue particles is also described.

Some specific embodiments that show how the invention can be practiced under the principles described herein and some advantages thereof over the prior art are described in the following examples, which must not be considered as the only possible embodiments of the invention, but only as exemplary of determined embodiments of the invention.

EXAMPLES

Example 1 - Cross-linkable edible gel materials

Example 1A. Cell-loaded cross-linkable edible gel material

Low viscosity alginate (LVA) was dissolved in Dulbecco's phosphate-buffered saline (DPBS) in a proportion of 2% w/v to which gelatin methacryloyl in 3% w/v and 0.3% w/v of LAP were also added. The mixture was heated to 70°C for 15 minutes and then sterilized through filtration with syringe filters and then tempered to 37°C for addition of 3xl0⁶ cells/mL of either immortalized mouse myoblast cell line ATCC Number CRL-1772 or a primary culture of porcine muscle satellite cells which are multipotent stem cells, thereby obtaining the cell-loaded cross-linkable edible material.

Example IB. Cross-linkable edible gel material (non cell-loaded) Low viscosity alginate (LVA) was dissolved in Dulbecco's phosphate-buffered saline (DPBS) in a proportion of 2% w/v to which gelatin methacryloyl in 3% w/v and 0.3% w/v of LAP were also added. The mixture was heated to 70°C for 15 minutes and then sterilized through filtration with syringe filters and then tempered to 37°C, thereby obtaining a cross-linkable edible material without cells loaded as used in the prior art.

Example 2. Non-cross-linkable gel material

Hydroxyethyl cellulose (HEC) was prepared in DPBS at 0.8% w/v by dissolving powdered HEC under stirring and heating conditions at 70°C until a homogeneous solution was obtained. The mixture remains heated in a water bath at 70°C for lh for sterilization purposes and then tempered to 37°C for mixing with the cell-loaded cross-linkable edible material of Example 1.

Example 3. Scaffolding and cell growth

Based on the materials obtained in Examples 1 and 2 there were designed different experiments in order to compare the performance of the process of the present invention with the techniques of the prior art.

For all the examples a commercially available miniaturized mixer of 5.1-7.7cm length with four Kenic's elements was used to mix the cross-linkable edible gel material of Example 1 (A or B) with a second scaffolding material, including the material of Example 2 in some cases, and further obtaining either fibers or droplets to be cross-linked in a solution of 2% w/v CaCI2 in DPBS.

For the materials to be shaped as fibers, 5 mL of the mixed materials were extruded inside the cross-linking CaCh solution at a rate of 1.5 mL/min to avoid tangling or misshaping of the obtained fibers. The fibers thereby obtained were then taken out from the solution, and carefully organized and placed on petri dishes to be subject to UV light for 30 seconds for cross-linking and then placed in ultra-low adherence plates (Costar® 24 well plates Cat. Id 3473) for cell culture.

In turn, the materials shaped as droplets were processed similarly, but instead of extruding them directly into the cross-linking CaCh solution, they were simply let drop from above the solution container at a rate of 1.5 mL/min. The spheres obtained from the droplets were recovered and cross-linked at the same conditions than fibers, except that no measures were taken to handle or order the spheres.

Additionally, experiments regarding the spheres on a constant agitation environment (Spinner Flask) were made for both cell types: immortalized mouse myoblast C2C12 and primary cell culture of porcine Satellite Cells (pSC). Agitation conditions for the scaffolds were set on 50rpm throughout the 14 days of culture.

The obtained fibers or spheres were then incubated in Dulbecco's Modified Eagle's Medium - high glucose (DMEM-hg) supplemented with 10% Fetal Bovine Serum and 1% Anti-Anti (Penicillin 10,000 units/mL - Streptomycin 10,000 pg/mL- 25 pg/mL of amphotericin B) at 37°C under an atmosphere of 5% of carbon dioxide. The culture was performed for 14 days and cell viability was assessed with Presto Blue Assay in days 4, 7, 10, and 14. Cell count (CC) and doublings (CD) were assessed with CyQUANT assay on the same days under the following formula:

CD = 3.33

The different experiments and variables are summarized in Table 1 below.

Table 1. Example 3 scaffolding and cell growth experiments

For all the examples, metabolic activity was measured through PrestoBlue® cell viability kit on day 1, 4, 7, 10 and 14, setting day 1 as reference point of comparison. The results for Examples 3A to 3D are shown in Fig. 1, and the results for Examples 3E and 3F are shown in Fig. 2.

As it can be observed in Fig. 1, on day 10, cells growing on hollow scaffolds (3B, 3C and 3D) had a better metabolic activity than those growing on the filled scaffold (3A). From the cells growing on hollow scaffolds, those that were embedded in sphere scaffolds (3C and 3D) had a higher activity and, surprisingly, the cells corresponding to Example 3D showed the highest metabolic activity, despite of being under agitation conditions at 50 rpm, which was expected to jeopardize such activity.

On the other hand, according to Figure 2, for pSC, it was observed a higher metabolic activity for the cells within the hollow spheres, in comparison with the cells within the hollow fiber, on day 10 of culture thus confirming that with different cells the spheres of the present invention have a better performance than fibers.

In addition, cell proliferation within the scaffolds was measured by using CyQUANT® cell proliferation kit as explained before. The area of scaffolds was measured according to the following: a) for fibers, the area was calculated with the formula to calculate the total surface area of the cylinder: 2nrh-;-2nr², and considering that the diameter of each sample is 0.15 cm and the height is 2 cm; and b) for spheres, the surface area was calculated with the formula: 4nr², and considering a diameter of 0.4 cm. From this, it was found that fibers surface area measures 0.98 cm² while spheres surface area is 0.5 cm². Thus, the cell number per area unit (cm²) was calculated for each example. The results for Examples 3A to 3D are shown in Fig. 3, and the results for Examples 3E and 3F are shown in Fig. 4.

Said figures demonstrate growth or cell proliferation on two different cell types respectively: immortalized mouse myoblast C2C12 (Fig. 3) and primary culture of porcine Satellite cells (pSC) (Fig. 4). In this sense, both figures show cell growth through a 14-day incubation period, and for both cell types, best cell growth can be observed on hollow spheres scaffolds, under static conditions. For C2C12 cells, growth reached its peak on day 10 of culture when the scaffold was a sphere, and on day 14 when the scaffold was a fiber, while for pSC, the cell growth was reached on day 14 for both fibers and spheres. It is important to notice the difference of cell number within day 1 which varies according to the cell type and scaffold. In this sense, C2C12 being an immortalized cell line grows faster than primary cell culture of pSC. Additionally, the area of the fibers being analyzed is 2 to 3.5 times greater than spheres.

Likewise, for all the examples, cell doubling within the scaffolds was measured through a comparison on cell numbers at the peak of the culture and initial cell numbers within the scaffolds. The results of cell doubling per area unit (cm²) obtained for scaffolds loaded with C2C12 cells are shown in Fig. 5 and the results obtained for pSC are shown in Fig. 6.

According to these figures, best doubling outcomes can be observed on hollow spheres scaffolds, under static conditions, in both cell types being studied.

As for cell viability, hollow spheres scaffolds were embedded with C2C12 at a density of 3 million cells per mL of cell ink. Scaffolds were later incubated in a Spinner Flask at 50 rpm for two weeks. At day 14 samples were stained with fluorescent markers for cell viability (Live-Dead) and fixed for immunofluorescence staining of Actin -DAPI. 2.5X scale bar: 500 pm, 10X scale bar: 100 pm. In order to prove that the scaffolds of the instant invention reduce cell death through cell nutrients penetration, samples corresponding to day 14 of hollow spheres scaffolds embedded with C2C12 cells at a density of 3 million cells per mL of cell ink and incubated on Spinner Flask at 50 rpm for two weeks, were stained with fluorescent markers for cell viability (Live-Dead) and fixed for immunofluorescence staining of Actin- DAPI, observing high cell viability on hollow spheres scaffolds even after being subjected to 50 rpm throughout the whole two weeks of incubation (data not shown). Moreover, C2C12 differentiation was observed by cells arrangement through their Actin filaments stained by red dye whereas nuclei are seen by blue DAPI staining (not shown in figures).

Example 4. Process-ability of spheres and fibers. For the materials of examples 3B and 3C, different processing flows were compared for fibers and spheres at different rates. In addition, Live-Dead staining as explained above was made instantly after extrusion to verify cell viability post-extrusion (day 0). The results were as follows:

Table 2. Effect of flow rates

S=successful; F=fragile post-extrusion with low survival time; N=not processable; H=Processable but heterogeneous sizes

These results demonstrate the absence of strict flow rate parameters through the extrusion process in the present invention as opposed to the strict flow control and handling required for fibers. Table 2 provides a detailed description of hollow scaffold achievement where fibers extruded at 2 mL/min or higher are extremely fragile and thus do not survive longer incubation periods. In contrast, spheres do maintain their shape and viability through normal incubation periods and viability through fluorescence microscopy can be seen easily at day 7.

According to what has been described, it shall be observed that the process and equipment of the present invention has been designed to overcome the scalability problems of the prior art by providing a process that can be implemented in large scale in high volume tanks without the need of flow control or scaffold special handling conditions, and it shall be evident to anyone skilled in the art that the embodiments described and illustrated in the examples are only illustrative but do not limit the applicability of the teachings of the description, as there are possible numerous changes in its details within the scope of this description, such as the cell, scaffolding, solution and culture materials.

Therefore, the present invention should not be considered restricted except as required by the prior art or the scope of the appended claims.

Claims

1. A cultured meat production process comprising the steps of: a) mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) letting the cell-gel mixture drop into a cross-linking solution capable of cross-linking the cross-linkable gel material in the cell-gel mixture and of dissolving the second non-cross-linkable material; c) cross-linking the cell-gel mixture by keeping a suspension of cell-gel mixture droplets in the crosslinking solution until cross-linking is achieved thereby obtaining spherical cell-loaded edible scaffolds; d) removing the cross-linking solution with the non-cross-linkable gel material; e) growing the cells in the cell-loaded edible scaffolds in culture media; and f) removing the culture media to obtain tissue spheres.

2. The process according to claim 1, wherein the cells loaded into de cross-linkable edible gel material are selected from myoblasts, myocytes, adipocytes, fibroblasts, satellite cells and any stem cells capable of differentiating into muscular or adipose tissue.

3. The process according to claim 2, wherein the cells are selected from animals susceptible of human or animal consumption, preferably selected from mammals, birds, fish and shellfish.

4. The process according to claim 3, wherein the cells are capable of developing into animal muscular or adipose tissue selected from fish, crustacean, mouse, bovine, cattle, swine and poultry.

5. The process according to claim 1, wherein the cross-linkable edible gel material comprises: 2 to 3% w/v of an organic polymer able to provide firmness to the scaffold and anchorable sites for cell adhesion; 0.1% to 3% w/v of a cross-linkable hydrogel; and, a buffer solution.

6. The process according to claim 5, wherein the organic polymer is a polysaccharide, protein, peptide or a combination thereof.

7. The process according to claim 6, wherein the organic polymer is selected from alginate, hyaluronic acid, agarose, gelatin, collagen or a combination thereof.

8. The process according to claim 5, wherein the cross-linkable hydrogel is a photo-cross- linkable hydrogel.

9. The process according to claim 5, wherein the cross-linkable hydrogel is selected from gelatin methacryloyl, collagen, fibrin, silk, hyaluronic acid, agarose, chitosan, cellulose, alginate, soy, extracellular matrix (ECM), elastin, gelatin, polyethylene glycol (PEG), polyprolylene fumarate - polyethylene glycol (PPF-PEG), polyethylene oxide (PEO), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol diacrylate (PEGDA), and functionalized versions thereof.

10. The process according to claim 9, wherein the cross-linkable hydrogel is gelatin methacryloyl.

11. The process according to claim 5, wherein the buffer is phosphate based.

12. The process according to claim 5, wherein the buffer is selected from Dulbecco’s Phosphate Buffered Saline (DPBS), phosphate buffered saline (PBS), sodium phosphate, tris(hydroxymethyl)aminomethane buffer (Tris), Hank's balanced salt solution (HBSS), 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffer, Sodium bicarbonate, Ethylenediaminetetraacetic acid (EDTA) buffer, and mineral buffers.

13. The process according to claim 5, wherein the polymer and the hydrogel have similar viscosity to enable equal lamellar flow and proper internal microstructure formation.

14. The process according to claim 1, wherein the minimum load of cells is O.lxlO⁶ cells/mL of cross-linkable edible gel material.

15. The process according to claim 1, wherein the non-cross-linkable gel material comprises an innocuous water-soluble polymer.

16. The process according to claim 15, wherein the non-cross-linkable gel material is selected from cellulose derivatives.

17. The process according to claim 1, wherein the non-cross-linkable gel material is hydroxyethyl cellulose (HEC) or any other polymer with similar rheological properties and which does not cross-link with the mechanism used for crosslinking the stable hydrogel.

18. The process according to claim 1, wherein step a) is made through mixing equipment and mixture delivery fixtures allowing flow rates enough to achieve dropping of the mixed cell-gel.

19. The process according to claim 18, wherein step a) is made through an extrusion equipment equipped with a head or dice for allowing droplets to fall.

20. The process according to claim 18, wherein a plurality of mixers and/or mixture delivery fixtures are configured in parallel.

21. The process according to claim 1, wherein the droplets have a minimum diameter of 1 mm.

22. The process according to claim 1, wherein the cross-linking solution comprises an alkaline or alkaline earth metal cross-linker.

23. The process according to claim 22, wherein the cross-linking solution comprises calcium ions, preferably calcium chloride (CaCh) and water.

24. The process according to claim 1, wherein the cross-linking/dissolution lasts from 1 to 2 minutes.

25. The process according to claim 1, wherein step e) comprises transferring the cell- loaded edible scaffolds into a culture vessel or bioreactor for cell growth in a culture media.

26. The process according to claim 1, wherein in step e), at least 100,000 cells per scaffold outer surface area unit are obtained.

27. The process according to claim 1, wherein in step e), a minimum cell doubling of 2.5 per scaffold outer surface area unit is obtained.

28. A cell-loaded edible scaffold having a substantially spherical shape of a diameter bigger than 1 mm and comprising a cell-gel mixture made of a first cell-loaded cross-linkable edible gel material and a second non-cross-linkable gel material wherein the cell-loaded scaffold is capable of dissolving into a cross-linking solution capable of cross-linking the cross-linkable gel material in the cellgel mixture and of dissolving the second non-cross-linkable material.

29. A muscular and/or adipose tissue particle comprising a cross-linked edible gel material that is substantially spherical, with a diameter bigger than 1 mm, and at least 100,000 cells per crosslinked edible gel material outer surface area unit.

30. A meat product comprising muscular and/or adipose tissue particles of claim 29.

31. An equipment for enabling the cultured meat production process of claim 1, comprising: a) a mixer of gel material capable of mixing a first cell-loaded cross-linkable edible gel material with a second non-cross-linkable gel material to obtain a cell-gel mixture; b) an open container for cross-linking solution and/or culture media with separation means to remove the cross-linking solution and/or the culture media; c) dropping means located above the open container capable of supplying drops of the cell-gel mixture.

32. The equipment according to claim 31, wherein the mixer comprises mixture delivery fixtures allowing low flow rates enough to achieve dropping of the mixed cell-gel.

33. The equipment according to claim 32, wherein the mixer comprises an extrusion equipment further equipped with a head or dice for allowing droplets to fall.

34. The equipment according to claim 32, wherein the mixer is a motionless static mixer.

35. The equipment according to claim 32, wherein a plurality of mixers and/or mixture delivery fixtures are configured in parallel.

36. The equipment according to claim 31, wherein the separation means of the open container are selected between sieves and valves configured to enable the cell-gel or the meat-like tissue spheres to be kept into the container while the solution or the cell culture is removed.