The Effect of Long-Term Passage on Porcine SMCs' Function and the Improvement of TGF-ß1 on Porcine SMCs' Secretory Function in Late Passage.

Cultured meat is one of the meat substitutes produced through tissue engineering and other technologies. Large-scale cell culture is the key for cultured meat products to enter the market. Therefore, this study is aimed to explore the effect of long-term passage in vitro on smooth muscle cells (SMCs) and the effect of transforming growth factor-ß1 (TGF-ß1) on SMCs in the late passage. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage. Transcriptome results showed that the ECM pathway and aging-related signaling pathways were significantly up-regulated in the late passage period. TGF-ß1 did not promote SMCs of late passage proliferation at the proliferation stage but promoted the gene and protein expression of collagen as the main protein of the extracellular matrix proteins at the differentiation stage. In addition, proteomic analysis revealed that TGF-ß1 promoted the expression of cell adhesion molecules which activate the Hippo signaling pathway and the HIF-1 signaling pathway and further promoted the production of collagen-containing extracellular matrix proteins. This could provide ideas for large-scale production of cultured meat products using SMCs.

Identification of porcine adipose progenitor cells by fluorescence-activated cell sorting for the preparation of cultured fat by 3D bioprinting.

Cultured meat is an emerging technology that is friendly for the environment and animal welfare. As a novel food ingredient, cultured fat is essential for the flavor and nutrition of cultured meat. In this study, we purified adipose progenitor cell (APC) from freshly isolated porcine stromal vessel fraction (SVF) by fluorescence-activated cell sorting (FACS) and identified the transcriptome characteristics of APC by RNA sequencing (RNA-seq). The results showed that APC had characteristics of high-efficiency proliferation and adipogenic differentiation and was distinct from SVF cell in transcriptome profiles. Subsequently, APC was used to prepare cultured fat by 3D bioprinting and to evaluate the differences in fatty acid composition between cultured fat and porcine subcutaneous adipose tissue (pSAT). The results indicated that the fatty acid composition and content of cultured fat had a certain similarity with pSAT; specifically, the content of key monounsaturated fatty acid (MUFA) that create pork flavor in cultured fat, such as C18:1(n-12), C18:1(n-9) and C19:1(n-9)T, were close to that of pSAT. Therefore, this research indicated that APC is a promising candidate cell type for the production of cultured fat.

Quality evaluation of cultured meat with plant protein scaffold.

Cultured meat technology is a promising new technology to solve the negative problems brought by traditional animal husbandry. Cultured meat should be further developed to appear on consumers' tables as alternative protein product. Therefore, this study used food grade peanut wire-drawing protein as scaffold to culture smooth muscle cells (SMCs) in vitro to obtain cultured meat productions containing both animal protein and plant protein. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage, which means that the plasticity of cells decreased in the later stage of passage. SMCs can well adhere to the peanut wire-drawing protein scaffold and produce extracellular matrix protein and muscle protein, so as to form a cultured meat product with rich protein composition. This study provides a theoretical basis for the production of nutrient-rich cultured meat products.

Effects of selected flavonoids oncellproliferation and differentiation of porcine muscle stem cells for cultured meat production.

Stemness decline of muscle stem cells (MuSCs) is a significant problem in cultured meat processing. In the present study, three flavonoids (quercetin, icariin, and 3,2'-dihydroxyflavone) with multi concentrations were evaluated to promote the proliferation and differentiation of porcine muscle stem cells. In the proliferation phase, 3,2'-dihydroxyflavone (10 µM) significantly amplified the cells by 34% and up-regulated the expression of paired box transcription factor 7 (PAX7) by 60%, which was higher than quercetin (75 nM) and icariin (7.5 nM). In the differentiation phase, quercetin (50 nM) showed the best pro-differentiation effect and up-regulated the expression of myosin heavy chain (MYHC) by 4.73-fold compared with the control group. These results indicated that flavonoids had a significant impact on promoting the proliferation and differentiation of porcine MuSCs, and 3,2'-dihydroxyflavone (10 µM) for proliferation and quercetin (50 nM) for differentiation were the optimal combinations.

Production of cultured fat with peanut wire-drawing protein scaffold and quality evaluation based on texture and volatile compounds analysis.

Cultured meat is an emergent technology that cultivates cells in three-dimensional scaffolds to generate tissue for consumption. Fat makes an important contribution to the flavor and texture of traditional meat, but there are few reports on cultured fat. Here, we demonstrated the construction of cultured fat by inoculating porcine adipose-derived mesenchymal stem cell (ADSC) on peanut wire-drawing protein (PWP) scaffolds. First, we demonstrated that basic fibroblast growth factor (bFGF) promoted cell proliferation and maintained adipogenic differentiation ability. Then, we generated cultured fat and found that cultured fat decreased the texture of PWP scaffolds. Moreover, 43 volatile compounds were detected by headspace gas chromatography-ion mobility spectrometry (GC-IMS), of which 17 volatile compounds showed no significant differences between cultured fat and porcine subcutaneous adipose tissue (pSAT), which indicated that cultured fat and pSAT had certain similarities. Collectively, this research has great promise for improving the quality of cultured meat.

Production of cultured meat by culturing porcine smooth muscle cells in vitro with food grade peanut wire-drawing protein scaffold.

Cultivating meat is a promising solution to the negative problems brought by traditional animal husbandry. To make cultured meat have the sensory and nutritional characteristics of conventional meat as much as possible, many studies have been conducted on various cell types and scaffold characteristics. Therefore, this study aims to produce a low-cost cultured meat with a quality closer to that of conventional meat. Tissue generation requires three-dimensional (3D) scaffolds to support cells and simulate extracellular matrix (ECM). Here, we used peanut wire-drawing protein (a biomaterial based on edible porous protein) as a new culture meat scaffold to culture cells. The scaffold can support cell attachment and proliferation to create 3D engineered porcine muscle tissue. The differentiation of smooth muscle cells (SMCs) was induced by a low serum medium to produce more extracellular matrix proteins. After differentiation, it was found that peanut wire-drawing protein scaffolds could be used for porcine smooth muscle cell adhesion and growth. The ECM protein and muscle protein produced by SMCs can endow cultured meat with better quality. This technology provides an innovative pathway for the industrialized production of cultured meat.

Evaluation of the effect of smooth muscle cells on the quality of cultured meat in a model for cultured meat.

While the research on improving the meat quality of cultured meat is in full swing, few studies have focused on the effect of smooth muscle cells (SMCs) on the meat quality of cultured meat. Therefore, this study aimed at building a cultured meat model containing smooth muscle cells, and further evaluating the effect of smooth muscle cells on the quality of cultured meat, so as to reveal the contribution of smooth muscle cells in the production of cultured meat. In this study, we isolated high purity of smooth muscle cells from vascular tissues. The addition of basic fibroblast growth factor (bFGF) to the medium significantly increased the growth rate of smooth muscle cells and the expression of extracellular matrix related genes, especially collagen and elastin. Smooth muscle cells were seeded in a collagen gel to construct a culture meat model. It was found that the pressure loss of the model meat significantly decreased from 98.5 % in control group to 54 % with the extension of culture time for 9 days, while the total collagen content of model meat increased significantly (P < 0.05). In addition, the hydrogel tissue with smooth muscle cells compacted more dramatically and were more tightly, accompanied by significantly increased hardness, springiness and chewiness compared to the control one (P < 0.05). These results indicate that smooth muscle cells can secrete extracellular matrix proteins such as collagen, which can significantly enhance the texture of cultured meat models prepared by hydrogel.

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In the winter-wheat and summer-maize multiple cropping system in lime concretion black soil of Huanghuaihai Plain, the effects of three tilling methods (conventional tillage, rotary tillage, subsoiling tillage) in wheat season coupling with three nitrogen treatments (120 kg·hm-2, 225 kg·hm-2, 330 kg·hm-2) before maize sowing on the activities of microorganisms and enzymes re-lated nitrogen transformation, and inorganic nitrogen content in the rhizosphere soil during the main growth stages of maize, as well as the yield were investigated. The results showed that the rotary tillage had the highest ammonification intensity, and the more nitrogen was put in, the higher were the activities of microorganisms and enzymes related to soil nitrogen transformation. The activities of nitrification, denitrification and urease of subsoiling tillage was significantly higher than those of conventional and rotary tillage. Furthermore, in subsoiling tillage treatment, increasing nitrogen fertili-zer could promote soil nitrogen transformation while excessive nitrogen input inhibited soil nitrogen transformation, though the latter had higher yield and soil inorganic nitrogen content. The treatment of subsoiling tillage coupling with 225 kg·hm-2 nitrogen, was best for soil nitrogen transformation while the treatment of subsoiling tillage coupling with 330 kg·hm-2 nitrogen, had the highest corn yield.