Tissue Engineering Challenges for Cultivated Meat to Meet the Real Demand of a Global Market.

Cultivated meat (CM) technology has the potential to disrupt the food industry-indeed, it is already an inevitable reality. This new technology is an alternative to solve the environmental, health and ethical issues associated with the demand for meat products. The global market longs for biotechnological improvements for the CM production chain. CM, also known as cultured, cell-based, lab-grown, in vitro or clean meat, is obtained through cellular agriculture, which is based on applying tissue engineering principles. In practice, it is first necessary to choose the best cell source and type, and then to furnish the necessary nutrients, growth factors and signalling molecules via cultivation media. This procedure occurs in a controlled environment that provides the surfaces necessary for anchor-dependent cells and offers microcarriers and scaffolds that favour the three-dimensional (3D) organisation of multiple cell types. In this review, we discuss relevant information to CM production, including the cultivation process, cell sources, medium requirements, the main obstacles to CM production (consumer acceptance, scalability, safety and reproducibility), the technological aspects of 3D models (biomaterials, microcarriers and scaffolds) and assembly methods (cell layering, spinning and 3D bioprinting). We also provide an outlook on the global CM market. Our review brings a broad overview of the CM field, providing an update for everyone interested in the topic, which is especially important because CM is a multidisciplinary technology.

The interaction between aging and protein malnutrition modulates peritoneal macrophage function: An experimental study in male mice.

Malnutrition is considered one of the most common problems in the elderly population worldwide and can significantly interfere in health evolution in these individuals, predisposing them to increased infection susceptibility. The immune response triggered by infections comprises several mechanisms, and macrophages play important roles in this response. This study aimed to evaluate mechanisms related to macrophage function in a model of protein malnutrition in the elderly. Two age groups (young: 3-5 months and elderly: 18-19 months) male C57BL/6NTac mice were subjected to protein malnutrition with a low-protein diet (2 %). The nutritional status, hemogram and number of peritoneal cells were affected by both age and nutritional status. Additionally, the spreading capacity as well as the phagocytic and fungicidal activity of peritoneal macrophages were affected by the nutritional status and age of the animal. Interestingly, the percentages of F4/80+/CD11b+ and CD86+ cells were reduced mostly in elderly animals, while the TLR-4+ population was more affected by nutritional status than by age. The production of pro-inflammatory cytokines such as TNF-α, IL-1α, and IL-6 was also influenced by nutritional status and/or by age, and malnourished animals of advanced age produced higher amounts of the anti-inflammatory cytokine IL-10. Furthermore, the phosphorylation ratio of the transcription factor NFκB (pNFκB/NFκB) was directly affected by the nutritional status, independently of age. Thus, these results allow us to conclude that aging and protein malnutrition compromise macrophage function, likely affecting their immune function, and in aged protein-malnourished animals, this impairment tends to be more pronounced.

The influence of association between aging and reduced protein intake on some immunomodulatory aspects of bone marrow mesenchymal stem cells: an experimental study.

PURPOSE: Dietary protein deficiency is common in the elderly, compromising hematopoiesis and the immune response, and may cause a greater susceptibility to infections. Mesenchymal stem cells (MSCs) have immunomodulatory properties and are essential to hematopoiesis. Therefore, this study aimed to investigate, in an aging model subjected to malnutrition due a reduced protein intake, aspects related to the immunomodulatory capacity of MSCs. METHODS: Male C57BL/6 mice from young and elderly groups were fed with normoproteic or hypoproteic diets (12% and 2% of protein, respectively) and nutritional, biochemical and hematological parameters were evaluated. MSCs from bone marrow were isolated, characterized and their secretory parameters evaluated, along with gene expression. Additionally, the effects of aging and protein malnutrition on MSC immunomodulatory properties were assessed. RESULTS: Malnourished mice lost weight and demonstrated anemia, leukopenia, and bone marrow hypoplasia. MSCs from elderly animals from both groups showed reduced CD73 expression and higher senescence rate; also, the malnourished state affected CD73 expression in young animals. The production of IL-1ß and IL-6 by MSCs was affected by aging and malnutrition, but the IL-10 production not. Aging also increased the expression of NFκB, reducing the expression of STAT-3. However, MSCs from malnourished groups, regardless of age, showed decreased TGF-ß and PGE2 production. Evaluation of the immunomodulatory capacity of MSCs revealed that aging and malnutrition affected, mainly in lymphocytes, the production of IFN-γ and IL-10. CONCLUSION: Aging and reduced protein intake are factors that, alone or together, influence the immunomodulatory properties of MSCs and provide basic knowledge that can be further investigated to explore whether MSCs' therapeutic potential may be affected.

Prostaglandin F2α in vitro can affect basic inflammatory parameters of mesenchymal stem cells and slight modulating some of their immunomodulatory properties.

In the last decade, mesenchymal stem cells (MSCs) have been gaining attention due their ability to influence the function of other cells as well as modulate the inflammatory response. This occurs via their immunomodulatory functions,  acting through direct cell-cell interaction or by releasing a broad spectrum of bioactive factors such as cytokines and growth factors. In addition, prostaglandins are arachidonic acid metabolites that play a key role in the generation and modulation of the inflammatory response. Among the bioactive prostaglandins, PGF2α is able to stimulate cell proliferation as well as act to inhibit progenitor cell differentiation, but no information about this prostaglandin's action on the immunoregulatory function of MSCs has been reported. In this study we evaluate important aspects of the influence of PGF2α analog (17-phenyl-trinor PGF2α), which is a potent prostaglandin FP receptor agonist, on some mechanisms that control the main functions of MSCs. C3H10T1/2, a mesenchymal stem cell linage, was stimulated with PGF2α under inflammatory conditions trigged by LPS in order to investigate PGF2α inflammatory parameters as well as its ability to immunoregulate macrophages and lymphocytes. PGF2α has the ability to increase proliferation tax without altering the cell viability of LPS-stimulated MSCs, while also diminishing the phosphorylation of NFκB transcription factor leading to attenuation of IL-1ß and GM-CSF production. Additionally, MSC-s conditioned media from cells stimulated with PGF2α was able to increase the lymphocytes' IL-10 production. Overall, this study implied that PGF2α are able to modify some properties of MSCs.

Branched chain amino acids improve mesenchymal stem cell proliferation, reducing nuclear factor kappa B expression and modulating some inflammatory properties.

OBJECTIVES: The essential branched chain amino acids (BCAAs) valine, leucine, and isoleucine, are widely studied because of their effects on immunity and metabolism. Mesenchymal stem cells (MSCs) are a type of cell also studied due to their immunomodulatory properties. Since both BCAAs and MSCs have immunomodulatory capacity, the objective of this study was to evaluate the influence of BCAAs on some immunomodulatory aspects of MSCs. METHODS: MSCs were cultivated in BCAA-supplemented media to evaluate metabolic activity, including cell cycle, proliferative nuclear cell antigen, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, avian myelocytomatosis viral oncogene homolog, peroxisome proliferator activated receptor gamma, nuclear factor kappa B (NFкB), and signal transducers and activators of transcription 3 (STAT-3) expression. Additionally, some inflammatory mediators' synthesis, such as interleukin (IL) 1-beta, IL-10, granulocyte-macrophage colony-stimulating factor, transforming growth factor beta, nitric oxide, and prostaglandin E2, were also evaluated. RESULTS: Supplementation with BCAA led not only to increased MSC proliferation with more cells in the S, G2, and M cycle phases, but also to increased metabolic activity. BCAA supplementation also altered the immunomodulatory capacity of MSCs by decreasing the p-NFкB/NFкB and increasing the p-STAT-3/STAT-3 gene expression ratios, in addition to increasing synthesis of the antiinflammatory mediators transforming growth factor beta and prostaglandin E2. Finally, MSCs cultivated in BCAA-supplemented media was shown to decrease the IL-6 and tumor necrosis factor alpha production by macrophages. CONCLUSIONS: BCAA supplementation affected some immunoregulatory aspects of MSCs.

Exogenous glutamine impairs neutrophils migration into infections sites elicited by lipopolysaccharide by a multistep mechanism.

Glutamine (GLN) is the most abundant free amino acid in the body, and is considered as a conditionally essential amino acid under stress conditions, acting as an important modulator of the immune response. We here investigated the role of exogenous GLN treatment on leukocyte migration after the onset of endotoxemia and the intracellular mechanisms of GLN actions on neutrophils. Two in vivo models of endotoxemia caused by lipopolysaccharide of Escherichia coli (LPS) injection were carried out in male outbred Balb/C mice 2-3 months old, as follow: (1) LPS (50 µg/kg) was intravenously injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 2 h later to investigate the role of GLN on the acute lung inflammation; (2) LPS (1 mg/kg) was intraperitoneally injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 18 h later to measure the effects of GLN on local and later phases of inflammation in the peritoneum. Results showed that GLN administration reduced the number of neutrophils in the inflamed lungs, partially recovery of the reduced number of leukocytes in the blood; reduced adhesion molecules on lung endothelium and on circulating neutrophils. Moreover, GLN treatment diminished the number of neutrophils, levels of chemotactic cytokine CXCL2 in the inflamed peritoneum, and neutrophils collected from the peritoneum of GLN-treated mice presented lower levels of Rho, Rac, and JNK. Together, our data show novel mechanisms involved in the actions of GLN on neutrophils migration.

Glutamine metabolism and its effects on immune response: molecular mechanism and gene expression

This article aims to review glutamine metabolism and its effects on the immune response. Selected topics are addressed, particularly the effect of glutamine on cell survival and proliferation, as well as its importance in some biochemical pathways. The impact of glutamine on muscle, intestine, and liver metabolism are described, and a special section about glutamine regulation of the immune response is included. In this context, the modulation of glutamine on relevant signaling pathways as nuclear factor kappa B (NF-kB), mitogen-activated protein kinases (MAPKs), and heat shock protein and the influence of this amino acid on cell migration and adhesion molecules are highlighted. Some important immune response pathways modulated by glutamine were described as its action incritically ill patients. In summary, this review describes some important actions of glutamine, and a range of reactions and modulatory effects in different organs, which may inform new therapeutic strategies. However, further studies are necessary to provide information about glutamine use, especially about situations in which it can be better used as well as fine-tuning dose and administration.

Intravenous Glutamine Administration Modulates TNF-α/IL-10 Ratio and Attenuates NFkB Phosphorylation in a Protein Malnutrition Model.

Protein malnutrition (PM) is a major public health problem in developing countries, affecting the inflammatory response and increasing susceptibility to opportunistic infections. For this reason, an adequate nutritional intervention can improve the quality of life of patients. Glutamine (GLN) is a nonessential amino acid, but can be considered "conditionally essential" for macrophage function in stress situations, in which it plays a role in the improvement of the inflammatory response. Concerning this issue, in the current study, it was of interest to evaluate some biological aspects of peritoneal cells from a protein malnutrition (PM) mouse model challenged with lipopolysaccharide (LPS) and treated intravenously with GLN. Two-month-old male Balb/c mice were subjected to a low-protein diet (2 % protein) and stimulated intravenously with LPS 1 h prior to the injection of 0.75 mg/kg GLN. Malnourished animals showed a reduced number of total peritoneal cells. Malnourished animals stimulated with LPS or LPS plus GLN did not show differences in peritoneal cell counts; however, the control group showed increased cellularity after LPS stimulus, which was reversed after GLN injection. Further, in the animals from both groups stimulated with LPS, GLN decreased the circulating levels of TNF-α and the levels of TNF-α produced by peritoneal cells; additionally, GLN decreased the IL-10 circulating levels in the malnourished animals stimulated with LPS. In addition, peritoneal cells of the control and malnourished groups stimulated with LPS showed a negative modulation of the NFkB signaling pathway after GLN injection. In conclusion, this study shows that GLN has the capacity to reduce TNF-α synthesis as well as to act as a negative regulator of NFkB phosphorylation, leading to a positive outcome in the control of TNF-α production.