HUMAN PRACTICES
Engineering
Our BUCT-China team has always focused on the use of synthetic biology and tissue engineering techniques for the laboratory production of cell culture meat and provided the necessary technical support for its industrialization. So far, there is very little scientific research on commercial scaffolds that can be used for culturing muscle cells. So, the large-scale production of scaffolds suitable for cell culture meat production requires the identification of suitable biomaterials and further exploration and optimization of production methods.
Future production of cultured meat may involve the co-culture of multiple cell types to form structured tissues similar to real meat. This will require new scaffolds to support the differentiation of multiple cell types and allow for the spatial heterogeneity of the final product. These scaffolds will need to allow for precise fine-tuning of biomaterial properties such as stiffness and biochemical properties. However, due to diffusion limitations that limit media perfusion, these methods can only produce thin layers of tissue of approximately 100-200 μm. Therefore, overcoming the thickness limitation of cultured meat tissue remains a major challenge in this field.
In response to the above problems, our team innovatively used our production of fat-based materials to build scaffolds and borrowed the current emerging cell-microsphere co-culture system in the pharmaceutical industry for the production of artificial meat. This system improves the production process, and greatly reduces the technical operation difficulty of culturing cells into blocks, which saves a lot of human and material resources, and reduces the economic cost of artificial meat production. At the same time, it also provides technical support for industrial production as well as the industrialization of cultured meat.
Only a few commercially available materials exist in the market for stent materials with excellent performance, safety and non-toxicity, and high biocompatibility, so our project focuses primarily on the production of stent materials. We paid our attention to polyhydroxy fatty esters.
However, the biosynthesis of polyhydroxy fatty acid esters is an unnatural pathway, so we designed the following synthetic steps and expected to construct such a metabolic pathway in E. coli (Fig. 1), whose steps 1 and 2 are natural pathways that already exist in E. coli, and the hydroxylation reaction in step 3 has been proven feasible in a large amount of literature. So, the polymerization reaction of hydroxy fatty acids in this pathway is the key step, thus the study and optimization of steps 4 and 5 are the difficult points to achieve the construction of the whole metabolic pathway.
Fig. 1 Synthesis steps of PHFA
Based on last year's study, we initially constructed a CoA ligase/acyltransferase pathway with the exogenous addition of hydroxy fatty acids as substrate, but the degree of product polymerization and product yield was not high. Only small peaks were observed in the gas chromatogram (Fig.2), and almost no observable product appeared, indicating that this production method is not yet able to provide sufficient quantity and high-quality material for subsequent scaffold construction, so we must improve the original metabolic pathway in order to meet the requirements of the subsequent scaffold construction system.
Fig. 2 Gas chromatogram of last year's samples
Therefore, this year we focused our work more on the improvement of product yield and quality while validating our products with more characterization methods. We analyzed the constructed biological metabolic pathways and found that the factors affecting the products could be the following: 1) transmembrane transport of substrates; 2) activity of CoA ligase and acyltransferase; 3) metabolic viability of bacteria. So, we proposed some new designs for each of these issues and validated them.
Related studies have shown that the permeability of fatty acids through the cell membrane is low because the outer layer of the E. coli outer membrane, is composed mainly of lipopolysaccharides. And this may be the main barrier to the transport of hydrophobic compounds into E. coli, therefore the intracellular biotransformation of fatty acids may be slow due to supply limitations. And the expression level of the E. coli fatty acid transporter FadL has a more significant effect on its biotransformation. It is an essential carrier for the transport of exogenous fatty acids, and its presence facilitates the transport of fatty acids across the outer membrane (Fig. 3), especially those containing 10-18 C.
Fig. 3 Fatty acid transport mechanism
And the fadL expression level not only determines the long-chain fatty acids but also is one of the key factors in determining the whole cell biotransformation rate of hydroxy fatty acids. Also based on last year's study, we found that to increase the concentration of hydroxy fatty acids in cells, we need the assistance of FadL transporter protein, so this year, to achieve efficient hydroxy fatty acid transport, we still used overexpression of fadL to achieve it.
Fig. 4 plasmid profile with the fadL
Fig. 5 Comparison of product output
We designed the plasmid as above (Fig. 4) to overexpress the fadL gene in E. coli and verified by comparing the product yields obtained by overexpressing fadL with not overexpressing the fadL gene. And the results showed that the yield of polyhydroxy fatty acid lipids (PHFA) was increased nearly 1.7-fold in the engineered bacteria overexpressing fadL (Fig. 5). The results showed that the transporter protein expressed by fadL had a more significant effect on enhancing PHFA production.
In the unnatural synthetic PHFA pathway, we are trying to construct, the polymerization reaction of hydroxy fatty acids is a key step affecting the entire metabolic pathway (Fig. 6). So, the first important task is to investigate and improve this step.
Fig.6 Polymerization steps of hydroxy fatty acids
After reviewing the relevant literature, we found that WS2 has a broad substrate spectrum and different substrates have a low effect on the catalytic activity of the enzyme. Based on last year's study, we found that E. coli's own FadD enzyme has only very low activity. And the catalytic process of CoA ligase requires the participation of ATP and the hydroxylated lipoyl-CoA produced is in a high-energy state. So, we speculate that this step may be the key rate-limiting step in both reactions. Therefore, this year we analyzed and screened different sources of this enzyme to obtain a more active CoA ligase (Fig. 7).
Fig. 7 Comparison of CoA synthase activities from different sources
We finally selected ACOS5 from Arabidopsis thaliana, and we constructed it in a plasmid vector (Fig. 8) and expressed it in E. coli to verify its superior catalytic activity by measuring the product polymerization and product molecular weight.
Fig. 8 Plasmid profile containing fadD
Firstly, we explored the optimal induction conditions for WS2 and fadD genes, and we induced expression in E. coli (BL21) transfected with plasmids at 16°C and 30°C, respectively, and we also used different concentrations of IPTG for induction. The gel electrophoresis plots of the expressed proteins are shown in Fig. 9 and Fig. 10. The molecular weight of ACOS5 protein is about 63 kDa, and the molecular weight of WS2 protein is about 50 kDa.
Fig. 9 Protein gel electrophoresis under 30 ° C induction
The analysis revealed that the induction of different concentrations of IPTG had little effect on protein expression because the amount of protein expressed in the supernatant and precipitate was almost the same. In contrast, after induction at 30°C (Fig. 9), in comparison with the proteins expressed by the empty plasmid, we found that the number of inclusion bodies was significantly more in the precipitate than in the supernatant, indicating that induction at 30°C leads to the production of more inactive inclusion body proteins in bacteria, which in turn affects the efficiency of hydroxy fatty acid polymerization.
Fig. 10 Protein gel electrophoresis under 16 ° C induction
Because the IPTG induction concentration had little effect on protein expression, we searched for optimal conditions by varying only the induction temperature. We turned to 16°C for induced expression of chassis cells, and gel electrophoresis plots showed (Fig. 10) that compared with the proteins expressed by empty plasmid and those expressed at 30°C, there were significantly more ACOS5 protein and WS2 protein in the supernatant than in the precipitate, indicating that the production of inclusion bodies was significantly reduced and the number of active enzymes was higher under induction at 16°C. Thus, induction of expression at 16°C could enhance the polymerization efficiency of hydroxy fatty acids and improve the yield of PHFA.
Therefore, we selected 16°C to induce the expression of E. coli transferred with WS2 and ACOS5 genes, and after the bacteria were fully expressed, they were subjected to cell-breaking extraction to obtain the corresponding products for analysis and validation.
The hydroxyhexadecanoic acid specimen, the cell-break extraction product, and the hydrolysis product after cell-break extraction were subjected to gas chromatography (Fig. 11), and the results showed that the monomer of our synthesized polymerization product was hydroxyhexadecanoic acid, thus proving that the product we obtained was a polymer made from hydroxyhexadecanoic acid.
Fig. 11 Gas Chromatogram of samples
(From top to bottom) The first line is the hydroxyhexadecanoic acid standard, which is the standard peak of the substrate and is used as a control; the second line is the product of cell-break extraction hydrolyzed with alkali and reduced with acid, then dissolved in trichloromethane for gas phase detection, which shows a clear peak of the substrate; the third line is the result of using the product of cell-break extraction directly without hydrolysis, and no clear peak of the substrate and product is detected. We analyzed that the reason for the above results may be that the unhydrolyzed product may not have a product peak due to the high degree of polymerization of the product and the maximum gasification temperature of the gas phase could not gasify the product. After hydrolysis, however, a clear product peak was obtained, confirming that our product was formed by the polymerization of added hydroxyhexadecanoic acid.
To determine the structure of the product as well as to detect the degree of polymerization of the product and the molecular weight size of the product, we used NMR (Fig. 12) and GPC (Fig. 13) assays to validate our product.
Fig. 12 NMR diagram of products
In the NMR spectra we observed hydrogen atom signals on carbon 20, 34, and 3 (15, 21, 33), proving that the product we obtained is compatible with the structure of the polymer we conceived and we were able to successfully produce the polymerized product of hydroxyhexadecanoic acid.
Fig. 13 GPC test results of products
We sent the obtained product for gel chromatography and the results showed that the number average molecular weight (Mn) was the highest at 3608, so it indicates that the system has more large molecules with higher polymerization degrees, and the molecular weight of our product was around 3600. And we can calculate that the polymerization degree of the polymer was around 13. This further shows that the efficiency and quality of the PHFA production system have been improved after the replacement of the FadD enzyme. We have successfully improved the polymerization pathway of hydroxy fatty acids. Meanwhile, the polydispersity coefficient of this gel chromatography was around 1.415 close to 1, which indicates that the substances were dispersed more uniformly during this assay and the results were more reliable.
Fig. 14 Alcohol precipitation diagram of products
Finally, we obtained the following PHFA products (in Fig. 14). We treated the fully expressed cells with cell disruption and extracted the corresponding products. The product morphology is shown in the above figure. The first sample (from left to right) is hydroxyhexadecanoic acid dissolved in ethanol; the second sample is the cell breakage extracted product dissolved in ethanol; the third and fourth samples are other impurities extracted from the cells dissolved in ethanol. From the comparison of the four samples, we can observe the product and determine that we obtained a significant yield of product.
However, our analysis revealed that the reason affecting the degree of product polymerization may also be the competition between CoA ligase and P450 enzymes, which compete for medium and long chain fatty acids (Fig. 15). And we speculate that the catalytic efficiency of CoA ligase may be much higher than that of p450 enzyme. So, when the two enzymes are present at the same time, it may make the fatty acid change to lipoyl coenzyme A first, and the hydroxylation enzyme P450 cannot recognize the substance and thus cannot achieve hydroxylation, which in turn leads to a significant decrease in the final PHFA yield.
Fig. 15 Possible Competitive Responses
Moreover, it has been shown in the literature that the use of static regulation strategies to regulate gene expression can limit the productivity, carbon yield, and product yield of cell factories, while artificial induction can accumulate excessive hydroxy fatty acids to a certain extent and have certain effects on cells. Therefore, we also wanted to further address the toxic effects of intermediates on cells as well as explore the effects of metabolic flow perturbations on the systematic synthesis of PHFA.
To sum up, to improve the first-generation system and enable our cell factory to produce the target compound PHFA with maximum yield, yield, and production capacity in the production process, we propose the idea of using the dynamic regulation system to regulate gene expression based on the above improvements.
We were inspired by λ phage, a virus that infects Escherichia coli and when it infects the host E. coli, the cell will enter two different life cycles: the lysis cycle or the lysogenic cycle. This is due to the presence of the CI protein within the λ phage, whose early dynamic balance of separation or binding to the transcriptional promoter PR controls the lysogenic or lytic pathway of the phage (Fig. 16) (affinity of the CI protein for the manipulator: OR1>OR2>OR3), thus enabling the switch in the E. coli life cycle. The mechanism of interaction of this protein with the promoter is as follows (Fig. 17).
Fig. 16 Affinity of CI Protein
Fig. 17 Schematic diagram of CI protein
However, it has been found that the negative feedback regulation of CI is very little because the concentration of CI protein in the lysogenic state only reduces the activity of PRM by 5-20% from its maximum value. So, the negative auto-regulation of cI genes has almost no physiological role in the lysogenic state. This suggests that it is unrealistic to use the negative feedback regulation of CI proteins themselves to achieve our goal of controlling the order of appearance of different enzymes.
However, when OR2 is bound by a CI dimer and OR3 has no binders, transcription of CI is activated. At the same time the basal transcription rate after activation is about 10 times higher than the rate of inactivated transcription. This is because when the CI dimer is already bound to OR1, its affinity to bind to OR2 is enhanced, which is synergistic with adjacent site binding. Also, CI protein binding to the OR2 site acts as an anchor for RNA polymerase bound to the PRM promoter. Therefore, OR3 can be knocked out and this promoter can be modified to be activated by the CI protein, which can then be bound to other blocking systems to achieve the goal of regulating downstream gene transcription or not. Therefore, we used the modified promoter capable of being activated by CI proteins in combination with the Lac manipulator (Fig. 18) to achieve the goal of regulating dynamic gene expression (Fig. 19).
Fig. 18 Plasmid profile of dynamically regulated gene circuit
Fig. 19 Dynamic adjustment process
To improve the effectiveness of our validation system, we also found a tag that accelerates protein degradation to increase the match between the deterrent effect and the expression of the reporter protein. The LVA degradation tag we looked for, significantly increased the degradation rate of fluorescent proteins (Fig. 20)
Fig.20 Stability of Gfp variants in E. coli following a downshift
We transformed this designed plasmid into E. coli for expression and verified the dynamic regulation of the system by observing the fluorescence expression. Initially, we observed green fluorescence (Fig. 21), but no change in fluorescence occurred without the addition of the inducer IPTG, while red fluorescence appeared after the addition of the inducer IPTG (Fig. 21).
Fig. 21 Green fluorescence appears first, and red fluorescence appears after adding IPTG
It is possible that the rate of action of the protein of interest with the manipulator does not match the rate of degradation of the protein with the degradation tag; it is also possible that the transcription rate does not match the rate of repression due to the inefficient binding of RBS; it is also possible that the promoter is not strong enough or does not match the expression of other genes in the whole metabolic pathway, resulting in the inability to alternate the fluorescence. Therefore, the dynamic system we have constructed now is not stable enough, and we will further evaluate the match between the relevant proteins and the rate of manipulated action, the rate of degradation of the action protein, and the rate of degradation of the fluorescent protein based on the modeling data, and screen for a better promoter and RBS in the later stage.
The current study lacks commercial scaffolds that can be used for cultured meat production. Therefore, the establishment of suitable scaffolds for large-scale breeding meat production requires further exploration and optimization of methods and biomaterials, as well as new technologies to enhance the ideal characteristics of scaffolds (such as porosity and ligand availability). The future production of cultured meat may involve the co-culture of multiple cell types to form a structured tissue similar to real meat which requires new scaffolds to support the differentiation of multiple cell types and allows for the spatial heterogeneity of the final product. These scaffolds also should have the characteristics of precise fine-tuning of biomaterial properties, such as stiffness and biochemical properties. However, these methods can only generate about 100-200μm thin layers due to the limitation of diffusion of media infusion. Overcoming the thickness limitation of cultured meat tissue remains a major challenge in this field. The traditional method of seeding cells onto full-size scaffolds to form tissue structures cannot solve this problem.
Last year, our team used 3D printing technology to build cell growth scaffolds, although 3D biological printing can mix cells and scaffold biomaterials to layer them together forming meat tissue. But in fact, 3D biological printing for the development of cultured meat needs a simpler and lower cost technology than 3D printing in tissue engineering, because cultured meat does not require a complex vascular system like natural tissue. Meanwhile, this technology is not suitable for the scaling requirement, and cannot be used for mass industrial production. Future innovations in cell slice tissue engineering methods or designing small tissue-building units and assembling them into tissue structures may effectively address this challenge.
Given the above problems, we plan to use biodegradable polyester microspheres to construct scaffold materials providing a better cell culture environment for cell culture meat. Since microspheres have lower costs compared with 3D printing, and microspheres are commonly used in tissue engineering, the technology is more mature, and it is easier to expand the culture and industrial production.
2.1 Using the double lotion volatilization method to prepare Microspheres
We use the double lotion volatilization method to prepare microspheres (because the polyhydroxy fatty acid is amphiphilic). The parameters were successfully adjusted so that the medium and long-chain PHFA materials with strong hydrophobicity have appropriate physical and chemical properties. We adopt the method of "water in oil in water". The internal aqueous phase is ammonium bicarbonate, the oil phase is polyhydroxy fatty acid dissolved in the organic solvent, and the external aqueous phase is polyvinyl alcohol. First, the primary emulsion is obtained by mixing the oil phase with the internal water phase, and then the primary emulsion is added to the external water phase to mix evenly to obtain the secondary emulsion, which is stirred to form microspheres. Ammonium bicarbonate is decomposed into ammonia gas and carbon dioxide gas in the preparation process. The above two gases occupy part of the space of primary emulsion droplets, forming a porous structure.
According to the experimental scheme, PHFA microspheres with standard conditions of 6.25% W/V, 5wt% ammonium bicarbonate concentration, 0.25%PVA concentration, and 400rpm stirring speed [1].
Fig. 22: Example of microspheres.
Figure (a),(b) shows the microspheres in solution state with settling and suspension state,
Figure (c) shows the microspheres after lyophilization and vacuum-extraction of liquid nitrogen
Fig. 23: (a) and (b) show the microspheres under micropolariscope
2.2 Parameter regulation and surface modification of microspheres.
By adjusting the size, pore diameter, and particle size of microspheres under different parameters (changing the concentration of ammonium bicarbonate, PVA concentration, and stirring speed), we screened the most suitable microsphere scaffold system for cell attachment and growth. Moreover, the microsphere has a small diameter and a large specific surface area, which can effectively transport materials. The affinity to cells can be improved by surface modification. Adding collagen, RGD, and other substances allows for promoting the surface adhesion and structural stability of cell tissues, which is conducive to accelerating the cell growth rate. Then we can adjust the structural parameters and surface modification of the microsphere according to the cell growth. The surface of microspheres with - NH2 and - OH through the aminolysis of ethylenediamine solution. The aminated microspheres were further cross-linked with glutaraldehyde to make the surface of the microspheres contain bioactive collagen and RGD.
Through the experiment, we got the result below.
Speed adjustment results ×25, ×250, ×1000(a200rpm, b300rpm, c400rpm, d500rpm)
Fig. 24: (Figure a) shows the approximate particle size of ×25 magnification microspheres. (a1), (a2), (a3), and (a4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively. (Figure b) shows the particle size observation of a single microsphere at ×250 magnification. (b1), (b2), (b3), and (b4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively.
(Figure c) shows the pore distribution and pore size of ×1000 magnification microspheres. (c1), (c2), (c3), and (c4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively.
Adjustment results of ammonium bicarbonate ×100, ×250, ×1000, sections (a5wt%, b6.25wt %, c8wt %, d10wt %)
Fig. 25: (Figure a) shows the approximate particle size of the ×100 magnification microspheres. Batches of (a1), (a2), (a3), and (a4) are 5wt%, 6.25wt%, 8wt%, and 10wt%, respectively.
(Figure b) shows the particle size observation of a single microsphere at ×250 magnification. Batches of microspheres (b1), (b2), (b3), and (b4) are 5wt%, 6.25wt%, 8wt%, and 10wt%,respectively.
(Figure c) shows the distribution of holes and pore size of ×1000 magnification microspheres. Batches of microspheres (c1), (c2), (c3), and (c4) are 5wt%, 6.25wt%, 8wt%, and 10wt%, respectively.
(Figure d) shows the observation of holes in the section, (d1), (d2) shows the distribution of holes and pore size in the section of microspheres at ×250 magnification, and (d3), (d4) shows the distribution of holes and pore size in the section of microspheres at ×1000 magnification.
Adjustment Results of PHFA concentration×25、×250、×1000(a6.25wt%、b5%wt、c3.13wt%、d1.56wt%)
Fig. 26: (Figure a) shows the approximate particle size of the microspheres at ×25 magnification. Batches of microspheres (a1), (a2), (a3), and (a4) with PHFA concentrations of 6.25wt%, 5wt%, 3.13wt% and 1.56wt%, respectively.
(Figure b) shows the particle size observation of a single microsphere at ×250 magnification. Batches of microspheres (b1), (b2), (b3), and (b4) with PHFA concentrations of 6.25wt%, 5wt%, 3.13wt% and 1.56wt%, respectively.
(Figure c) shows the pore distribution and pore size of ×1000 magnification microspheres. Batches of microspheres (c1), (c2), (c3), and (c4) with PHFA concentrations of 6.25wt%, 5wt%, 3.13wt% and 1.56wt%, respectively.
Finally, we selected the microspheres with a PHFA concentration of 6.25%, ammonium bicarbonate concentration of 10%, an average particle size of about 300μm, and average pore size of about 20μm at 400 rpm, which are the most favorable microspheres for cell attachment.
2.3 Linker (connecting cells to microspheres)
In order to achieve a better cell attachment rate, the linker should hold the following properties 1) firmly interact with cells and microspheres; 2) Independent and stable existence. From the article, we acknowledge that α5β1 integrins are stably expressed during muscle formation. In the process of mouse myogenesis, various integrins are expressed differently at separate periods and interact with different ECM components respectively, but a few types of integrins retain certain content in the whole process [18]. Although the precise spatiotemporal expression of integrins in chicken muscle formation still needs further study, based on the above examples, we selected α5β1[19] and α2β1[20], which have been verified to be expressed in chicken muscle formation. Apart from that, the selection of supplements for surface modification of microspheres, is shown in Table 1[21]. With considerations of the bio-affinity of materials and experimental feasibility, peptide GRGDSP is selected as our linker. It can form amide bonds with the -OH groups on the microsphere surface, meanwhile having the strongest binding force with α5β1 (see Table 2 for details). According to our experiment, it holds a better performance as a linker compared to collagen.
Fig. 27: Left: Schematic diagram of the interaction between RGD and integrin [21]; Right: Schematic Diagram of Collagen and Integrin [22]
Table 1: Different RGD derivative and Integrins α5β1 Effect [13]
Through the experiment, we got the result below.
According to the Fourier infrared spectroscopy, we compared the effect of collagen on physical and chemical methods founding that both methods were combined with collagen, yet we could tell that the chemical method was better via the analysis of key peaks.
Fig. 28: In the infrared spectrogram, the amide bond characteristic peaks at 1592cm-1 and 3000cm-1 of the main peaks in the figure can be seen that The physical and chemical collagens were combined successfully after adding EDA; The results of other peak analyses showed that the effect of the chemical method was better than that of physical method
Our cell-related experiments mainly focus on the widely used cell growth、proliferation、differentiation by using cck8、 fluorescent staining、RT-qPCR. Therefore getting or finding a series of data or literature supports (chemical and biological similarly material): PHFA has properties of certain cell affinity、non-toxic、promoting the proliferation and differentiation of cells, which is a good biological material for cell culture in vitro. Later, we will continue to explore parameter optimization and product molding.
3.1 Selection with cell culture
In order to optimize the experimental techniques and improve the efficiency, we chose the C1C12 cell line as our first cell model. After the technique is mature, the muscle cells from the muscle of the chicken are used. For cell culture, DMEM (1% double antibody, 10%BMS) is used as the culture media, the cells were resuscitated and subcultured for one generation before inoculation on microspheres. The medium was replaced each 48h.
3.2 Cell seeding
We will first utilize the static culture method to demonstrate the feasibility of the scheme and optimize the experimental design based on the following. After the technology is further developed, the suspension culture method is used for quantitative production exploration.
Through the stationary culture method, we observed good growth of cells on the surface of the microsphere on the first day (see Figure 2), which preliminarily demonstrated that the cells of the material were non-toxic and had an affinity of cell.
Fig. 29: Microsphere-cells (inverted microscope)
3.3 Observation of cell culture to microspheres growth:
Fluorescence staining: After the sample is inoculated and cultured for a certain time (such as 24h, 48h, etc.), the cells need to be stained before the observation of cell morphology.
Using the live-dead cell staining method, we observed the cells growing on the surface of the microsphere under a confocal microscope, and the cell growth condition was good, which further intuitively demonstrated that PHFA has good cell affinity and can be used as a new biomaterial for cell culture in vitro.
Fig. 30: Cell adhesion growth map on microsphere surface (confocal microscopy)
Left: the scale bar is 200μm; Right: the scale bar is 100μm
3.4 Cytotoxicity test & cell growth test - cck8
cck8 was measured on the first, third, and fifth days after the cells were inoculated to the microsphere which was compared with the control group, and the growth rate of cells in the experimental group was calculated. Based on the experimental results, we obtained the relevant data on the third day when cells were cultured on PHFA microspheres and compared them with PLGA, which demonstrates the non-toxicity of PHFA materials.
Fig. 31: Cytotoxicity experimental data of the PHFA microspheres on the third day: -J is a microsphere modified with collagen
Fig. 32: CCK8 and microsphere-cell co-incubation
3.5 Cell differentiation assay: qPCR
After the cells covered 80% of the surface area, the differentiation medium was replaced, and the total RNA was extracted by the Trizol method on days 1, 3, or 7, respectively. After qualified detection, the RNA was reverse-transcribed into cDNA by a two-step method and then RT-qPCR was performed. The genes to be tested were MyoD, MyoG, and MRF.
3.6 Cross-linking: final product molding (using glutamine transaminase)
Since myosin and other proteins are expressed on the surface of myoblasts, glutamine aminotransferase was selected to cross-link the cell-microsphere complexes together.
Last year, our team used 3D printing technology to build cell growth scaffolds, although 3D biological printing can mix cells and scaffold biomaterials to layer them together forming meat tissue. But in fact, 3D biological printing for the development of cultured meat needs a simpler and lower cost technology than 3D printing in tissue engineering, because cultured meat does not require a complex vascular system like natural tissue. Meanwhile, this technology is not suitable for the scaling requirement, and cannot be used for mass industrial production. Compared to 3D printing, microsphere holds a lower cost. Apart from that, since microspheres are commonly used in tissue engineering, the related technology is more mature. What's more, culture meat using a microsphere scaffold has advantages for large-scale production, which is important for industrialization.
Fig. 33:3D printing to microsphere culture
Based on the experience of our first-generation culture meat, we explored the application of glucose-synthesized PHFA as 3D-printing biocompatible scaffold raw materials, and preliminarily achieved culture meat based on 3D printing, which laid a feasible foundation for further optimization and improvement of our cultured meat project. The scaffold system for cultured meat is often high-cost, meanwhile having difficulties in recovery. Based on this, in this year's project, our team used engineered bacteria with the self-directed dynamic regulation system to synthesize PHFA with superior performance. Then, the synthetic PHFA was used to develop a porous microsphere scaffold that holds a large specific surface area, and a series of cell-microsphere culturing experiments were carried out to explore large-scale production of culture meat.
Compared with cell aggregates methods, the advantages of the cell-microsphere complex are that it has a higher surface-volume ratio, the pH can be easily adjusted, the concentration of gases and metabolites in the media can be controlled, and it has great potential for large-scale operations. Apart from that, the surface of the microsphere can be modified by adding supplements such as collagen or RGD, creating an ECM-like microenvironment, which can improve the cell affinity and benefit its growth. In addition, the porous surface and cytotoxic supplements strengthen cell adhesion, improving its tolerance to shear stress. From the above, we can see that the microspheres stimulate cell adhesion in vivo in a variety of ways.
Considering large-scale production, cell aggregates methods have little control over the size of an aggregate, and the oversized aggregates cause a gradient in the concentration of nutrients and O2 inside, which caused a necrotic core. Apart from that, compared with culture using fixed bed reactors, microspheres are easier to control and monitor, with less need for manpower and less capital investment for equipment.
In addition, the characteristics of the microsphere method allow wider choices of reactors, it can be operated in stirred tanks, fluidized beds, filled beds, and gas-filled reactors. What's more, the cells can migrate to the new microspheres added into the system during operation, the so-called "sphere to sphere transfer". (Some near-fused MSs(10-25%) are transferred to loaded fresh MSs, reducing MSs fusion).
In the preparation stage of the microsphere scaffold: we have mastered the mature technology for preparing the microsphere scaffold and found the most suitable microsphere state for cell growth through the adjustment of rotation speed, PHFA concentration, and ammonium bicarbonate concentration, developing porous microspheres system with a large specific surface area. In addition, the microspheres have a small diameter, large specific surface area, and unique porous microstructure, aiming to optimize the transport of gas and nutrients. New microspheres can also be added to the culture medium for cell transfer between microspheres in order to achieve large-scale production. Moreover, we modified the surface by adding collagen, and the coating of collagen on the surface greatly enhanced the cell culture.
In the cell culture stage: Firstly, we verified the non-toxicity of the material. We measured the cell proliferation on the surface of the microsphere by the CCK-8 method, to judge the activity of the microsphere material by whether there are viable cells and the number of viable cells. We successfully observed that the cell proliferation on the surface of the microspheres was well, which provided a strong demonstration for the edible of the subsequently cultured meat scaffolds from a certain level. In the future, we hope that the microsphere-cell system could have a richer taste and a more meat-like texture, and the quality of taste depends on the differentiation ability of the cells, thus we observed and recorded the cell system continuously. Then we found that myogenic cells differentiated significantly in the laboratory culture stage. It indicates that the microsphere-cell system has a nice differentiation capacity and has the potential to reach the differentiation goal of the finished cultured meat eventually. We constructed and cultured the entire microsphere-cell system using various innovative approaches such as GRGDSP-modified microspheres. In order to test the effectiveness of our innovative culture, we use cytological experimental indicators such as live-dead cell staining for evaluation. The evaluation process also argued the excellent nature of our constructed cell-microsphere system and the rationality of the culture method from a certain perspective. Such advantages enable us to achieve better cell adhesion, growth, and value addition on the microsphere surface. Eventually, our team laid the foundation for further industrialization by cultured meat crude products.
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