Another promising approach in cell agriculture involves self-assembling peptides. These peptides are studied as scaffolds for structural support and materials used in 3D bioprinting. The highly versatile nature of self-assembling peptides allows them to self-organize into complex three-dimensional structures, simulating the ECM. This property has enormous potential to create scaffolds that mimic the structure and function of animal tissues. Two mechanisms are known for the self-assembly process39. The first is the differential adhesion hypothesis, based on cell-cell bonding behavior and free energy minimizations, which drive self-assembly40. This interaction occurs because of the adhesion of proteins on the cell surface, creating a cell mass that moves together with a liquid, reducing its surface tension40. Cells with high surface tension tend to move to the center to improve intercellular adhesion, and similarly, in the self-assembly process, the non-adherent substrate feeds the cell population consistent with intercellular adhesion, leading to the minimization of free energy41. The second mechanism is based on the differential interfacial tension. The difference between this method and the previous one is the minimization of free energy, which is the cellular behavior, and not the substrate, with cell movement governed by forces created by the cell cytoskeleton in the cell membrane39. Cells with similar interfacial stresses will aggregate, while cells with different interfacial tension tend to remain separate42. An important advance in this area is the development of peptide coatings designed to control cell adhesion and detachment43. These coatings combine peptide sequences, such as arginyl-glycyl-aspartic acid (RGD), which promote cell adhesion with cleavage sites, allowing the controlled release of cells and, consequently, the continuous production of cells43. Innovative companies are leading this research and driving the use of these self-assembling peptides in practical applications. However, it is important to mention that the costs associated with manufacturing these peptides still pose a significant challenge for their large-scale adoption. Also, peptide scaffolds may not be suitable for long-term CM bioreactor culture, due to their generally poor mechanical properties. In this sense, optimizing current techniques and using recombinant organisms may be crucial to make these peptides viable in scaffolds for ECM44. Advancements in needle-free and multi-needle electrospinning technologies have demonstrated that the process can be scaled to industrial levels, resulting in a substantial increase in production rates45. Depending on the polymer and process parameters, industrial-scale electrospinning systems, such as needle-free systems, can attain production rates of up to 1 kg/h or more. Additionally, recent advancements in high-throughput electrospinning machines have made it feasible for commercial manufacturing to produce continuous fibers on a large scale. The potential for large-scale production of electrospun materials is underscored by these advancements, which bolster their viability for industrial applications.
Several techniques can be employed to achieve scaffolds, such as 3D bioprinting, electrospinning, microcarriers, and decellularization. Scaffold-free approaches are explained in this review as well. These techniques are presented in Fig. 3.
Crosslinking can be achieved through physical, chemical, or enzymatic means to form the scaffolds. Physical crosslinking arises from physical interactions, including ionic interactions, temperature-triggered mechanisms, and dehydrothermal crosslinking (DHT). Ionic interactions involve crosslinking agents forming ionic bridges with the polymer backbone. Temperature-triggered crosslinking relies on thermal behavior to form crosslinks, while DHT involves subjecting the polymer to high temperatures under vacuum to remove water and create crosslinks. Xiang et al., investigated physical crosslinking through steam sterilization or water annealing to create porous glutenin sponges and fibrous aligned scaffolds to support the proliferation and differentiation of C2C12 mouse skeletal myoblasts and bovine satellite cells (BSCs). These scaffolds obtained pore sizes ranging from 50 to 250 μm and showed good cell adhesion and proliferation without RGD motifs or the addition of extra ECM protein coatings. Also, the study demonstrated physical crosslinking methods based on hydrogen bonding, which resulted in structural stabilization due to the formation of β-sheet crystals. Physical crosslinking could improve food safety by avoiding additives compared to chemical crosslinking for structural stabilization.
Chemical crosslinking typically involves covalent bond formation with polymer chains during scaffold synthesis. Chemical crosslinking creates more stable scaffolds than physical methods. Over time, several small crosslinking molecules, like genipin, dopamine, glutaraldehyde, and tannic acid, have become popular for scaffold synthesis. Although chemical crosslinking can achieve more stable scaffolds, they have some toxicity problems that should be considered. For example, glutaraldehyde (GTA) which is widely used in biological sample preparation, disinfection, and as a crosslinker for proteins and biomaterials, is associated with irritant and sensitizer, it can cause respiratory issues and skin irritation. Epoxy compounds (e.g., ethylene glycol diglycidyl ether), used to crosslink proteins, DNA, and other biomolecules may cause respiratory irritation, skin sensitization and carcinogenic effects. Diisocyanates (e.g., methylene diphenyl diisocyanate (MDI)), are used to produce polyurethanes and as crosslinkers in various industrial processes, causing occupational asthma, chronic lung disease, and skin irritation. Acrylamide and N,N'-methylenebisacrylamide (MBA), used often together to crosslink gels, are considered hazardous (neurotoxic) because they are linked to cancer. Another common example of chemical crosslinkers associated with toxicity is formaldehyde. While chemical crosslinkers are essential for creating stable scaffolds, many of them pose significant toxicity risks, affecting cell viability and human health. Alternatives, such as using less toxic crosslinkers or non-chemical methods (e.g., enzymatic crosslinking), are being explored to reduce these risks in applications requiring high biocompatibility. Conventional chemical crosslinking methods, such as photo-crosslinking and covalent crosslinking, lead to scaffolds with enhanced stiffness and rapid gelation times. To address the issue of cytotoxicity, enzyme crosslinkers have gained popularity, providing a potentially safer alternative and enabling a better microenvironment for artificial scaffold development. An example of chemical crosslinking involves using glutaraldehyde, to create glucuronoxylan-based quince seed hydrogels. The porosity of these hydrogels was measured before and after crosslinking. In the non-crosslinked scaffolds, the average pore size was about 99.85 μm with 22.52% porosity. After light crosslinking, the average pore size decreased to 76.59 μm with 18.36% porosity. The average pore size decreased to 56.04 μm for heavily crosslinked samples, with 13.58% porosity. This indicates that increased crosslinking creates a denser scaffold, reducing interconnected pore size and porosity. An interconnected and porous structure is a critical aspect of scaffold design, showing that the porous glucuronoxylan-based quince seed hydrogel has the potential for cellular agriculture applications.
First used in 1986 by Charles W. Hull, 3D bioprinting is a process that consists of creating a layer-by-layer model from a computer development. 3D bioprinting can be done using different methods: inkjet, laser-assisted bioprinting (LAB), laser-induced forward transfer (LIFT), EBB, and stereolithography (Fig. 3).
The different 3D bioprinting approaches require appropriate bioinks optimized to ensure the cellular fidelity of the printed scaffold while supporting cell viability. 3D bioprinting technology offers several advantages for food production, including the ability to customize food products' shape and composition. This technology also enables the fortification of foods, improving their nutritional profile to better meet specific dietary needs. Applications of 3D printing in food production include the creation of innovative shapes and complex geometries, such as structured cultured meat that resembles steaks. One of the current challenges with texture-modified foods is that the processes used to achieve a safe and desirable texture often compromise nutrient density and result in a lack of visual appeal, which can negatively impact appetite. In contrast, additively manufactured foods, due to the precision of the extrusion process, can achieve the desired texture, improve the nutritional profile, and offer a more visually appealing presentation. However, there is currently no research available on the nutritional quality, potential nutrient loss, or nutritional stability of these food products, particularly when production is scaled up. Bioinks used in food include biopolymers such as gelatin, agarose, cellulose, alginate, pectin, and plant proteins such as soy. These biopolymers have crosslinking mechanisms, allowing the formation of a stable hydrogel in the printed construction, while the bioink maintains its desired fluid properties. These polymers may also undergo thermal crosslinking, like in agarose or pH-based gelling, such as pectin. Cellulose can be cross-linked in different ways, such as UV radiation, enzymes, or calcium ionization. Another consideration that should be made in CM 3D bioprinting is the stability of the printed structure during further processing and the cooking processes. The versatility, precision, and reproducibility of 3D bioprinting show that it is a promising method for CM production (Table 1).
Although 3D printing has been demonstrated for various materials, the most relevant bioinks to CM are hydrogel-based. A hydrogel is a hydrophilic polymer matrix crosslinked by physical or chemical means and has a water retention capacity. Hydrogels are very important in cellular agriculture and must have indispensable requirements to be applied as scaffolds. For example, the polymer matrix must be cytocompatible and contain non-toxic biomaterials. Micronutrients and signaling molecules must also be able to reach cells throughout the tissue, and for this, the hydrogel diffusion kinetics must allow these molecules to penetrate the entire hydrogel thickness at the concentrations and rates required for support cells. Stiffness is an important factor for a hydrogel, as it can affect cell motility, proliferation, differentiation, and migration since cells must be able to reshape the hydrogel during tissue maturation. Finally, the degradation rate of the hydrogel should align with the cells' ability to remodel their microenvironment and deposit ECM components to compensate for scaffold loss. For example, the referenced study achieved a maximum ECM area of 61.08 mm² using a 75/25 PLGA/collagen scaffold seeded with C2 cells. Additionally, proteolytic sites should be incorporated into the hydrogel to facilitate cell adhesion and migration.
Hydrogels, despite their potential for applications in CM due to their ability to mimic the ECM and support cell growth, have some potential disadvantages. A major concern is in their mechanical properties; hydrogels typically lack the necessary strength and rigidity to fully replicate the texture and structural integrity of traditional meat, which can impact the whole consumer experience. Furthermore, the biocompatibility of certain hydrogels might provide challenges, as not all hydrogel materials are suitable for food-grade applications, necessitating careful selection and potential modification to ensure safety and regulatory compliance. Another drawback is the scalability and cost associated with the production of hydrogel. Many hydrogels utilized in research settings are expensive and challenging to produce at the necessary scale for commercial cultured meat production, which may hinder their feasibility in the cultured meat industry. Finally, some hydrogels may have limited capacity to support the intricate exchange of nutrients required for the growth of tissues on a large scale, which can hinder the development of thicker and more complex meat structures.
Recent studies have highlighted hydrogels used to create a 3D environment similar to that of the ECM, as a filler of 3D ECM within porous scaffolds as components of bioinks as thin membranes that can be micro-structured to produce cell alignment, or as source material to develop porous scaffolds. Guzelgulgen et al., used glucuronoxylan-based quince seed to fabricate a 3D hydrogel similar to the ECM. They created a porous and interconnected structure and tested cell culture and viability with NIH/3T3 cells. The ECM analysis occurred in inter/intracellular components for two months. Cell culture samples were evaluated via SEM and immunostaining methodologies; they observed that the spheroids were homogeneously scattered inside the quince seed hydrogel, where the average spheroid diameter was around 300 μm. Nuclear DAPI staining was done to investigate cellular units inside the spheroid structure. The results also confirm the homogeneous distribution of cells inside the spheroids, and ECM formation was confirmed via collagen secretion analysis. Therefore, these results prove that quince seed hydrogel is a novel scaffold material with suitable mechanical features, remarkable swelling capacity, and good biocompatibility. The use of 3D ECM as a filler is described by Chen et al.. The authors fabricated an AG/PAAM/chitosan/gelatin scaffold and tested the growth of MC3T3-E1 cells, testing the proliferation and differentiation. The filling of PAAM reduced the pores and thickened the pore walls of composite hydrogel scaffolds, which gave rise to the enhancement of stress support and stress transfer ability, thereby enhancing the mechanical properties of AG/PAAM/chitosan/gelatin composite hydrogel scaffolds. Park et al. used hydrogels as components of bioinks, creating a CBF with carrot tissues. The callus-based hydrogel showed a fully opened porous construction with macropores, essential for supplying oxygen and nutrients to the cells. Due to this porous structure, prolonged cell proliferation was observed during the overall incubation period, demonstrating that the cells could be successfully cultured in the hydrogel.
Synthetic hydrogels are commonly used for tissue engineering due to their inert biological properties, which prevent an immune response. On the other hand, composite hydrogels can better mimic the ECM and show improved properties compared to those composed of a single material. Hydrogels based on a blend consisting of AG/AGA/MC using EBB plotting of a basil cell-laden hydrogel were investigated for their potential use in food bioprinting applications. The blend was prepared and plotted with the Bioscaffolder 3.1 (GeSiM mbH, Radeberg, Germany). After that, an in vitro cell culture of basil (Ocimum basilicum L.var.purpurascens Benth. 'Cinnamon Basil') was used for plant cell bioprinting. They observed that the mixture of the basal cell agglomerates into the ALG/AGA/MC blend did not disturb the extrusion of homogeneous strands or the fabrication of stable scaffolds. During the EBB process, it was observed that the minimum inner needle size should be 610 μm, otherwise, smaller needle sizes blocked the process. Most of the cells survived the process of 3D plotting and cross-linking, and the size and shape of cell agglomerates were similar to those in suspension. The detection of living cells at later time points of cultivation until day 20 revealed that the cells could be cultivated within the plotted hydrogel matrix. Future studies can evaluate the same approach for cultivating animal cells. Figure 4 shows the plotted ALG/AGA/MC scaffolds after cross-linking.
EBB is the most used 3D printing method because it is a versatile, simple, and low-cost method in which the bioink is released by a computer-controlled robotic system, resulting in the precise and continuous deposition of cylindrical filaments. The limiting factor of this method is the slow printing time and the lower return of cell viability compared to the other methods, which are between 40% and 86%. EBB can be done with different techniques. The extruded gel often spreads after deposition, requiring an adhesion method to ensure the printed structure's stability. These adhesion methods can be photopolymerization or immersion of the printed material in a crosslinking agent solution. The EBB technique was used to obtain HAP porous scaffolds.
Although HAP microspheres possess great bioactivity, biocompatibility, biodegradability, absorbability, and compressive properties, the high brittleness and low toughness of pure porous HAP materials have limited their practical applications. Fortunately, combining HAP nanoparticles as fillers and ductile polymer as the matrix for preparing nanoparticle/polymer composite porous scaffolds provides a promising way to overcome the shortcomings of pure porous HAP materials. PCL is another polymer that has received much attention owing to its favorable biocompatibility, biodegradability, and processability.
In the 3D inkjet printing method, small drops of liquid ink are produced and deposited on the substrate. This method is considered versatile, affordable, accurate, and achieves good resolution, which returns cell viability between 70% and 96%. Still, there are limitations on its use, especially concerning the bioink used, which must have low viscosity and a crosslinking mechanism to stabilize the printed structure. Another limitation is the small nozzle, which leads to clogging and impairs cleaning, which makes production difficult and reduces efficiency in large-scale manufacturing. This method is based on printing multiple and not continuous drops, and this lack of mechanical integrity makes it not favorable to its use for printing. On the other hand, Chen et al. demonstrated that the production of scaffolds by PLGA created an environment that provides the appropriate conditions for the growth, differentiation, and survival of C2C12 myoblasts, simulating the complex structure of the ECM. The results showed that in 3D printed scaffolds, the survival rate was higher than in the control made in films and PLGA spheres. In addition, the scaffold with a 50 µm fibrillar gap was the most suitable because it demonstrated increased cell adhesion and proliferation compared to the others. Thus, it was observed that these scaffolds have a controlled and uniform architecture, proving that 3D inkjet printing is a suitable tool for manufacturing cell culture scaffolds with defined structures.
LAB is based on the deposition of liquid bioink on a metal-coated surface, followed by laser-induced cavitation on the tape, which forms micro bioink droplets. LAB preserves cell viability by more than 95% and is compatible with bioinks with a large viscosity range (1-300 mPa/s), and because their deposition is done without a nozzle, clogging is not a problem for this method. Using LAB combined with other methods is another possibility. Nawroth et al. applied ultraviolet laser-activated photosensitizer (UVL) to create hydrogel patterns. This technique returned a short manufacturing time and high standardization volume compared to conventional methods.
Another laser-assisted manufacturing technique is laser-induced forward transfer (LIFT) bioprinting. This method comprises an upper layer designed for energy absorption, a middle layer acting as the donor, and a lower layer consisting of the bioink. LIFT entails the vaporization of the donor layer upon exposure to a laser beam directed at predetermined points, inducing the generation of high-pressure bubbles at the interface. This pressure triggers the bioink transfer to the collection phase, culminating in a three-dimensional model creation. The advantages of using LIFT include the high rate of cell viability and utilization of highly viscous materials. One of the drawbacks is the laser cost and its difficulty in control, besides the metallic residue in the final product, which is a concern regarding the final product safety, restricting the use of LAB for the production and marketing of CM.
Stereolithography uses a matrix of digital micromirrors to adjust the intensity of the visible or UV light beam, curing photosensitive polymers layer by layer. This method is fast, inexpensive, and returns cell viability above 85%. One of the limitations of its use is that light-blocking agents are used for photoresist standardization and are not suitable for food applications because they are toxic and carcinogenic. On the other hand, callus-based food inks (CBF) were formulated for stereolithography. Ratios of CBF to AG were tested at 1:2, 1:1, and 2:1 (w/w). Shear-thinning behavior was observed across all scaffolds, indicating a decrease in viscosity as shear rates increased, a critical property for effective 3D printing. This shear-thinning characteristic allows CBF to flow through a fine nozzle at high speeds during the printing process. The 1:2 and 1:1 CBF formulations displayed fine resolution regarding layer width and pore diameter, resulting in well-defined printed structures with dimensions close to their intended targets. However, interlayer adhesion became apparent at higher cell concentrations, such as in the 2:1 CBF sample, possibly due to its lower alginate content and higher cell density. In cell-laden ink systems used for 3D printing, an increase in cell concentration typically results in more cells aggregating at the liquid-liquid interface, thus reducing the surface tension and total free energy. This reduced surface tension causes the CBF to flow quickly through the nozzle during the printing process and spread, leading to issues with interlayer adhesion. The curing test results proved this observation, showing that the 2:1 CBF sample, which had lower alginate content, did not bind sufficiently to Ca in the gelatin slurry, leading to inadequate gel strength. This finding suggests that cell concentration is crucial to improve printing accuracy and maintain structural integrity during incubation. Cell growth within the CBF lattice scaffold was assessed by culturing for 35 days. The printed lattice scaffold had a fully open porous structure with macropores, providing the necessary pathways for oxygen and nutrient delivery to the cells. This porous architecture enabled sustained cell proliferation throughout the incubation period, indicating that the cells could successfully grow in the CBF gel.
Electrospinning is a simple, inexpensive method already used in various industrial branches, such as the textile industry, nanotechnology, tissue engineering, and cellular agriculture. The products generated by electrospinning can be made on an industrial scale. However, although it is versatile and has the potential for large-scale production, its application in food systems has not yet been fully elucidated.
The electrospinning process uses electrostatic force to stretch droplets of a polymer in solution to their potential point, forming a structure called a Taylor cone. Upon reaching a critical value above the droplet surface stress, a polymer solution jet is released, diluting simultaneously as the solvent evaporates, forming submicrometric or nanometric solid fibers constantly deposited in a grounded collector. The filaments produced vary in size and microstructure to fit the desired application. This adjustment can be made depending on the polymer, chosen solvents, environmental factors (temperature, humidity), and process parameters.
Fibrous scaffolds are made through electrospinning, which can produce nanofibers with various useful properties for CM. Some of these properties are the ability to support cell adhesion, perform the diffusion of oxygen and nutrients, and produce aligned fibers that promote muscle fiber maturation. Polymeric materials for spinning techniques include PCL and cellulose acetate (CA), as shown in Table 1. Also, common material combinations that can improve scaffold properties, such as PVP + PGS and melanin+PHB, are also shown. Plant/fungi-based material by electrospinning, such as fungal mycelial mats with chitin-glucan polysaccharide cell walls and CA + SPH are promising.
Even though it is a promising technology aimed at large-scale production, producing scaffolds for CM using electrospinning has some challenges that need to be overcome. One of the main challenges is the need to use non-edible solvents, such as fluorine-alcohols, hexafluoro isopropanol and 2,2,2-trifluoroethanol, which denature proteins and provide elastic, viscous properties, allowing fiber formation during electrospinning. These solvents have a high evaporation rate and partially denature biopolymers made by electrospinning, breaking hydrophobic interactions, and hydrogen bonds. They are considered toxic and unsafe for food because they can leave residue even if they are quickly evaporated. As an alternative for the electrospinning of edible biopolymers, high ionic force aqueous solutions or benign solvents, such as ethanol, formic acid, or acetic acid can be used, which are classified as Class 3 solvents by the Food and Drug Administration (FDA). Also, they are less toxic, have lower risks to humans, and can be included in food in restricted quantities by good manufacturing practices.
Another solution is the addition of carrier polymers, which increase the spinning capacity of the electrospinning solution by improving its viscoelastic properties. They must be degradable in the human digestive system, and the concentration of the polymer and its degradation products should be atoxic, as determined by authorities such as the FDA. Poly (ethylene oxide) (PEO), PCL, and PLA are used as carrier polymers in the electrospinning of edible biomaterials. Among these polymers, only PEO is approved by the FDA as an indirect food additive. In addition, these synthetic polymers do not provide nutritional benefits or support cellular adhesion, which is essential for cellular scaffolds. Therefore, these polymers are undesirable for direct consumption, and alternatives should be investigated to develop CM scaffolds.
Ahn et al. used CA as a carrier polymer, which increases fibroblasts' proliferation, growth, migration, and infiltration. Using jet electrospinning, they built a plant-based scaffold made from hydrolyzed SPH and CA. The RJS system's polymer concentrations significantly influenced the spinnability and beading of CA and SPH nanofibers (w/v%). The SPH has bioactive peptides similar to the proteins that make up the ECM, which promote cell adhesion, proliferation, and migration to tissue regeneration, but SPH itself could not be spun into nanofibers because its molecular weight is too low. The short chains of SPH molecules cannot overlap and entangle, suggesting that SPH would require a co-spinning polymer with longer chains. Ten w/v% of CA was therefore selected as the carrier polymer for SPH. The developed continuous nanofibers had an intercalated structure that resembled the native ECM. The composite scaffold showed lower cytotoxicity when compared to nanofibers made of PCL or only CA.
To overcome the solvent problem, Narayanan et al. used β-mercaptoethanol (BME) and observed improvements in hemocompatibility and biocompatibility once BME conferred scaffolds made of fungus adhesion and proliferation of keratinocytes. BME is toxic in high concentrations. In cell culture, it is often used in very low concentrations (e.g., 0.1 mM or less) to minimize toxicity while still maintaining its reducing properties. Even at low concentrations, residual BME could pose health risks if it remains in the final product. Therefore, it would be advisable to remove or neutralize BME before the cells are harvested for meat production and implement careful control measures to ensure it does not remain in the final product. Further investigation must be done on the applicability of CM scaffolds. The authors demonstrated the manufacture of a cross-linked scaffold of chitin-glucan polysaccharides made of fungi using electrospinning. Fungi are a group of eukaryotes; they have cell walls composed of chitin, are highly branched from hyphae, and grow as rigid structures, very similar to micro and nanofibers made by electrospinning. Still, on the cell walls of filamentous fungi, they are constituted by several linear structures and branched polysaccharides, as well as proteins modified after translation and lipids. This mycelial organization in filaments offers mechanical resistance and promotes interactions with the host elements, justified by using these biomaterials to construct scaffolds.
For application in scaffolds, the nanofibrous pores mimic the morphology and structure of ECM tissues and have a large surface area, making them ideal for adhesion and proliferation. The orientation of the electrospun fibers can be adjusted to control the morphology of cells grown on the scaffold. For example, electrospun fibers can be aligned, which induces the alignment of seeded cells and promotes the stretching of muscle cells andyogenesis. Cell-loaded polymer solutions can be electrospun, and the micro-pattern of the electrospun filaments can guide cell growth, resulting in homogeneous cell distribution and greater accessibility of nutrients throughout the scaffold.
In addition, electrospun blankets may undergo post-processing modifications, such as chemical or physical crosslinking, to improve their mechanical properties. Some of the protein crosslinks usually used are considered toxic, including formaldehyde. Therefore, these crosslinkers should be avoided in food applications. Non-toxic crosslinkers, crosslinking enzymes, or physical crosslinking modes using pH or temperature can be used instead to obtain scaffolds of superior mechanical properties.
Microcarriers are made from the growth of adherent cells in small suspended particles. The microcarriers are mainly made of PE, crosslinked dextran, cellulose, gelatin, or polygalacturonic acid (PGA), coated with collagen or peptides containing adhesion or positive charges to promote cell adhesion. The diameter of a microcarrier is between 100 and 200 μm. Bodiou et al. describe existing microcarrier production technologies and how they can be adapted as CM scaffolds. Three possibilities for using microcarriers were raised. The first would be as a temporary carrier aimed at supporting cell proliferation and being removed before processing. Secondly, the temporary carrier is dissolved or degraded to release the cells. Finally, the microcarriers are an edible scaffold incorporated into the final product. Examples in Table 1 include the temporary carrier being dissolved or degraded approach and microcarriers as an edible scaffold incorporated into the final product. It is common to use microcarriers to scale cell proliferation in bioreactors, as they provide anchorage for suspended cells. For this reason, decellularized plant-based microcarriers can serve as a key factor in scale-grown and affordable meat production. Although microcarriers offer a relatively simple solution to expand mammalian cells on a large scale and require little space, they have limitations regarding cell dissociation and separation costs, the cost of the microcarriers themselves, the maximum cell densities that can be achieved, and potential impacts on the nutritional and sensorial properties of the final product.
One of the techniques that can be explored in microcarriers is microfluidic (Fig. 3). Microfluidic technology is dedicated to studying and manipulating all fluid volumes in miniature systems, using channels with dimensions between 10 and 10 l. These channels combine chemical compounds to synthesize and separate substances through a pumping technique. Unlike macro scales, where physical characteristics and mass transfer based on diffusion are linearly scalable, these properties cannot be extrapolated directly at the microscale. The main advantage of the microfluidic technique is obtaining a laminar flow, which is an impossible phenomenon to achieve in large-scale devices due to the predominance of viscous forces.
Due to their suitable physicochemical characteristics, monocrystalline silicon and borosilicate glass are commonly used to build microfluidic platforms. In addition, polymers have been widely used in manufacturing these devices, with polydimethylsiloxane (PDMS) being one of the most favorites. The PDMS can be easily shaped into channels with high accuracy in terms of micrometer size, transparency to light, and low water permeability. However, an important disadvantage of PDMS is its lack of resistance to organic solvents, such as amines, strong acids, and hydrocarbons, which led to the development of solvent-resistant microfluidic reactors.
To achieve better adhesion of the cells, the microcarrier should have a porous surface. Porous scaffolds have a sponge-like structure (Fig. 3), with a pore size in the micrometer range. This structure provides the mechanical stability necessary for cultured cells to form tissues. These scaffolds resemble the structure, mechanical properties, and composition of the connective tissue of the perimysium, considering that the scaffold would remain a component of mature tissue. Commonly used porous scaffold manufacturing techniques such as particle leaching, melt molding, freeze-drying, and gas foaming, usually use synthetic polymers, which must be replaced by edible structure for CM application. Pore size, porosity, and scaffold material are key factors affecting tissue development and cell survival. While pore size is important for cell culture, the integration of larger pores suitable for medium perfusion should also be considered for pseudo vascularization to enable the efficient transport of nutrients and oxygen in thicker scaffolds for CM.
Kankala et al. were motivated by the lack of research on the applicability of porous microcarriers, and, as a result, they manufactured microspheres with highly open pores using a microfluidic technique. These microspheres were designed to house skeletal structures in myoblast proliferation and were subsequently evaluated for their viability in cell delivery. The biocompatible microspheres produced had particle sizes between 280 and 370 µm and pores with dimensions between 10 and 80 µm. This structure provides a favorable microenvironment, allowing cells to be closely arranged in elongated forms with the deposited ECM, facilitating adhesion, proliferation, and increased myogenic differentiation of cells. Using PLGA to manufacture porous microspheres allowed a minimally invasive cell delivery system creation. The study demonstrated a high cell adhesion rate, continuous proliferation, and increased myogenic differentiation of C2C12 when organized in fibrous layers in porous microspheres. Additionally, the porous microspheres presented an established ECM and exhibited a strong potential for myoblast differentiation, which facilitated the growth of these skeletal muscle cells concomitantly with vascularization.
Lyophilization is another technique that can be used in microcarriers. It consists of a drying process in which a solvent, usually water, is removed from a product by sublimation. This process has already been used to manufacture porous scaffolds for cellular agriculture. The lyophilization process can be divided into three stages: solidification, primary, and secondary. In the first phase, solidification, the solution begins to be cooled to a temperature below its eutectic point, which is the point at which the entire sample is frozen. Subsequently, the vacuum is applied in the second phase to reduce the pressure and facilitate the sublimation process. The process transitions from solid to steam, beginning in the first phase. During the first drying, unbound water is removed from the material, leaving only a porous structure. In the third phase, the secondary drying, the sample is heated, facilitating the unbound water desorption. Still, in the freeze-drying stages, it is known that the primary drying is the slowest one. If the established time is inadequate in the primary drying, the removed solvent will be insufficient, and what remains in the sample will be heated during secondary drying, spoiling the sample. Temperature is also a determining factor in primary drying. Both temperature and time influence the crystal size formed, affecting the pore structure. The primary drying phase presents additional opportunities to control the physical properties of the scaffold by monitoring the interactions between the sample and the bound water. Previous studies have shown that manipulating these interactions, changing drying rates, and time considerably affect scaffold stiffness and secondary structure formation.
Although drying techniques like freeze-drying and spray-drying are well-established and optimized in pharmaceutical applications, their application in scaffold manufacturing is still emerging. Unique challenges, including variations in material composition, porosity, and mechanical stability, complicate the direct adaptation of these methods, highlighting the need for further research in this area. In their study, Abbott et al. demonstrated the influence of time and temperature on primary lyophilization drying to produce porous scaffolds. Four different solutions were tested, varying the concentration of water/volume of solution in 3%, 6%, 9%, and 12%, using three distinct protocols: long hold, slow ramp, and standard. The long hold and slow ramp protocols resulted in scaffolds from all concentrations, while the standard did not work well for 9% and 12% concentrations. In order to investigate the use of different scaffolds, a live cell of the HepaRG line was grown on scaffolds of all concentrations made by the Long Hold protocol. Initially, the scaffolds of each concentration showed variations in lipid accumulation, cell growth, and metabolic activity, but these differences were no longer observed after the 28th day of culture. It was possible to conclude that by modifying the parameters of the primary drying and the concentration of the solutions, it is possible to obtain lyophilized scaffolds with suitable properties for cellular agriculture.
Enrione et al. also used the lyophilization technique to produce porous scaffolds. Four polymeric solutions were created: all containing salmon gelatin and sodium alginate, two with agar, two with agarose, and one in each of these two groups with glycerol. The concentrations of each component were not varied. These scaffolds were tested for cell line cultivation of myoblasts C2C12. The most promising scaffold contained salmon gelatin, sodium alginate, agarose, and glycerol. The pore size obtained for this scaffold was around 200 µm in diameter, the biocompatibility and adhesion of myoblast cells were around 40%, and it took around 24 h to double the growth rate. The biodegradation profile of scaffolds was lower than 25% after 4 weeks; they also had adequate myogenic response, high cell proliferation and viability, and adequate cell distribution.
Decellularized structures derived from plants or fungi can also produce scaffolds. It can provide natural 3D structures, which facilitate the transport of oxygen and nutrients essential for cell growth. Using these natural structures as scaffolds can reduce the complexity of the manufacturing process and increase the efficiency of CM production. The process of tissue decellularization can be carried out by physical, chemical, and enzymatic methods. The most commonly used physical methods are faster freezing or freezing-thawing. In freezing, intracellular ice crystals are formed in tissues, disrupting cell membranes and triggering cell lysis. In freezing-thawing, there must be precise control over temperature because it affects the size of the ice crystals formed, a factor that can degrade the ECM.
The lack of native cell adhesion molecules (fibronectin, integrins, and collagen, which are natural in animal tissues and crucial for cell attachment and signaling in animal cells) in plant-derived scaffolds and the biochemical incompatibility with animal cells may hinder cell attachment and growth. The limitations of those scaffolds include their mechanical stiffness and the absence of essential cell adhesion sites, which makes it difficult for animal cells to recognize and adhere to plant-based surfaces. Therefore, surface modification is necessary because plant-based scaffolds do not inherently possess the biochemical and physical properties required to support the attachment and growth of animal cells. By modifying the surface, it is possible to create a more favorable environment for cell adhesion, thereby improving the functionality of plant scaffolds in CM applications. Thus, to overcome these limitations, it is possible, for instance, to introduce biochemical cues and topographical features (micro- and nano-patterns), in order to improve cell attachment, proliferation, and differentiation, besides functionality on scaffolds.
Chemical decellularization typically employs detergents, acid/alkaline solutions, and chelating agents. In a study on chemical decellularization, researchers explored the use of decellularized apples coated with alginate/gelatin as a bioscaffold for CM production. They created two types of 3D scaffolds, uncoated (A) and coated (CA), varying pore size distributions ranging from 100 to 250 μm. The decellularization process involved treating thinly sliced apples with SDS, washing them, and then placing them in a beaker with SDS, maintaining the solution at 25 °C with 150 rpm agitation for five days. After decellularization, the scaffolds were crosslinked with a gelatin and alginate polymer blend and then freeze-dried. This crosslinked polymer coating increases the surface area for cellular metabolic activity and can contribute to the scaffold's meaty texture. Satellite muscle cells were seeded onto scaffolds to test cell support, and coculture of NIH/3T3 cells and muscle satellite cells was established to assess cell growth. Co-culturing these two cell types was successful on both scaffolds, but it was more pronounced on the CA scaffolds, likely due to the polymer coatings enhancing cell adhesion (Fig. 5). The coculture remained viable for seven days, indicating that both muscle cells and NIH/3T3 fibroblast cells could sustain growth in this medium.
Enzymatic decellularization offers some advantages, such as reducing cellular residues, but removing enzymes after the completion of the process is difficult, imposing limitations on the utility of enzymatic treatments. Enzymes used for decellularization include trypsin and pepsin. In a recent investigation by Thyden et al. rapid decellularization of broccoli was achieved, demonstrating the capacity of decellularized broccoli to support the adhesion and viability of BSCs within a dynamic reactor environment. Furthermore, decellularized broccoli exhibits physical and nutritional attributes that can offer advantages in both the production and consumption of CM.
Studies have investigated the use of spinach for scaffolds based on its wide availability, dense vascularization, and wide petiole (stem that connects the leaf to the stem). Another example of a vegetable that has been explored is apple. The authors demonstrated that decellularized apples can support the adhesion and survival of C2C12 myoblasts during a two-week culture period and promote binding and proliferation of induced pluripotent stem cells (iPSCs) differentiation in bone tissue. These characteristics make plants an attractive and sustainable alternative for the manufacture of scaffolds.