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We present a method of producing bulk cell-cultured fat tissue for food applications. Mass transport limitations (nutrients, oxygen, waste diffusion) of macroscale 3D tissue culture are circumvented by initially culturing murine or porcine adipocytes in 2D, after which bulk fat tissue is produced by mechanically harvesting and aggregating the lipid-filled adipocytes into 3D constructs using alginate or transglutaminase binders. The 3D fat tissues were visually similar to fat tissue harvested from animals, with matching textures based on uniaxial compression tests. The mechanical properties of cultured fat tissues were based on binder choice and concentration, and changes in the fatty acid compositions of cellular triacylglyceride and phospholipids were observed after lipid supplementation (soybean oil) during in vitro culture. This approach of aggregating individual adipocytes into a bulk 3D tissue provides a scalable and versatile strategy to produce cultured fat tissue for food-related applications, thereby addressing a key obstacle in cultivated meat production.

Cultured meat is an innovative meat-production technology that does not rely on animal husbandry. As a new food component, cultured fat is of great significance to cultured meat. In this study, we isolated adipose-derived stem cells (ADSCs) and identified the purity by immunofluorescence staining of ADSC-specific surface marker proteins CD44 and CD29 and showed that most of the cells were positive for CD29 and CD44. In addition, we detected the expression of FABP4 and Plin1 to confirm that ADSCs differentiated into mature adipocytes at 10 days post-induction. Subsequently, the culture conditions of ADSCs on microcarriers (MCs) were optimized and showed that cell density of living cells reached their highest after 5 days when continuously stirring at 50 rpm. Finally, the expression of FABP4 and PPARγ was detected to confirm the adipogenic differentiation ability of ADSCs on 2D and 3D culture systems and showed that ADSCs maintained their adipogenic differentiation ability after expansion on MCs. In conclusion, this research demonstrated that reliance on MCs to expand ADSCs was a promising approach for production of cultured fat. [ABSTRACT FROM AUTHOR]

Cultured fat is an important component of cell-cultured meat, which furnishes essential fatty acids and phospholipids, as well as being abundant in cholesterol. However, few studies have been on regulating the nutritional composition of cell-cultured meat, especially concerning the deposition of polyunsaturated fatty acids (PUFAs) and cholesterol. Here, we use sodium alginate-gelatin scaffolds to generate cultured fat, and find that phosphatidylcholine (PC) can promote the adipogenic differentiation of fibro-adipogenic progenitors (FAPs), accelerate lipid accumulation, and facilitate cultured fat production in the sodium alginate-gelatin 3D culture system. Interestingly, when PC is loaded into Euglena (Eg-PC) for co-culture, the triglycerides (TGs) content in fat is further increased. Multi-omics analysis reveals that PC treatment significantly alters the lipid composition of cultured fat produced by FAPs and increases the content of TGs, especially PUFAs (alpha-linolenic acid and linolenic acid). More importantly, PC treatment also reduces the total cholesterol content of cultured fat. This study provides innovative approaches for producing cultured fat with "high PUFAs and low cholesterol". [Display omitted] • Production of cultured fat using sodium alginate-gelatin composite hydrogel system. • PC promotes the adipogenic differentiation of fibro-adipogenic progenitors (FAPs). • Euglena-PC enhances the efficiency of PC in regulating FAPs differentiation. • PC contributes to cultured meat production with "high PUFAs and low cholesterol". • PC decreases cholesterol deposition by regulating HIF1A-Insig2-HMGCR pathway. [ABSTRACT FROM AUTHOR]

[Display omitted] • A combination of differentiation-inducing factors is obtained in order to produce high-quality cultured fat by single-factor experiment and orthogonal experiment. • Cultured fats with collagen as scaffolds are successfully prepared. • Cultured fats contain forty-eight fatty acids, mainly C16:0, C18:0, C20:0, C16:1, C18:1n-9, C18:1n-7, C20:4, C22:5n-3, and C22:6, and cultured fat and porcine subcutaneous fat are similar in fatty acid composition. • The ratio of ω-6 fatty acids content/ω-3 fatty acids content in cultured fat is 1.23:1, which is good and recommended for human health. Cultured fat is an important part of cultured meat, and the ability of adipose-derived mesenchymal stem cells (ADSCs) to differentiate into mature adipose tissue affects the quality of cultured fat. Thus, the primary aim of this study was to screen for combinations of differentiation-inducing factors (DIF) using single-factor experiment and orthogonal experimental design under two-dimensional culture conditions for ADSCs. The results showed that a combination of DIF consisting of 1 μmol/L dexamethasone, 0.1 mmol/L 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, 0.1 mmol/L indomethacin, and 2 μmol/L rosiglitazone was a good choice for the differentiation of ADSCs. An combination of DIF was applied to the preparation of cultured fat with collagen as scaffolds. Forty-eight fatty acids were detected in cultured fat by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Among them, the content of twenty-one fatty acids in cultured fat was significantly higher than that of conventional porcine subcutaneous adipose tissue (P < 0.05), and the content of 14 fatty acids was not significantly different (P > 0.05). The ratio of ω-6 polyunsaturated fatty acids content to ω-3 polyunsaturated fatty acids content was 1.23:1, which meant cultured fat was beneficial for human health. This study provides a method to improve the differentiation ability of ADSCs while also providing a reference for indicating the nutritional value of cultured fat. [ABSTRACT FROM AUTHOR]

Alternative technology for cultured meat production holds the potential to alleviate ethical, environmental, and public health concerns stemming from conventional husbandry. As a key food component, fat plays a crucial role in the sensory characteristics and nutritional value of meat products. However, current adipose tissue engineering protocols have so far proven unsuitable for edible cultured fat production. In this study, hybrid hydrogels based on Κ-Carrageenan (Car) and konjac glucomannan (KGM) were designed as a culture scaffold for adipocytes. The edible hydrogel with excellent biocompatibility, food safety, and low cost was demonstrated to support cell proliferation and allow the formation of tissue-like cell spheres. Furthermore, both porcine subcutaneous pre-adipocytes (PreAs) and fibro-adipogenic progenitors (FAPs) acquired mature adipocyte characteristics and higher lipid accumulation in Car-KGM (CK) hydrogel, suggesting its superior ability to support in vitro adipogenesis than conventional monolayer culture and alginate (Alg)-based 3D culture. Taken together, the excellent nature of CK hydrogel makes it a versatile platform for improving the molecular and cellular phenotypes of a variety of adipocyte models. The established spheroid model opens possibilities for broad applications in cultivated fat and represents a viable strategy for the manufacture of 'snowflake pork'. A schematic illustration showing the manufacturing process of cultured fat based on CK scaffolds. 2D culture: Methods for preparing immortalized cell lines involving steps of mechanical mincing, enzymatic digestion, and the transfection of SV40T virus. The proliferation and adipogenic differentiation ability was subsequently explored under 2D culture conditions. Preparation of CK mixed hydrogel: Car and KGM solutions were well-mixed after heating and cooling, followed by cross-linked with K+, to prepare mixed hydrogels with various mechanical properties. 3D culture: PreAs and FAPs suspended in medium are seeded in fabricated 3D constructs, respectively, and subsequently underwent a proliferation and differentiation protocol. Development of the cultured fat model based on CK scaffolds has provided important clues for functional studies on adipose tissue biology and the production of "snowflake pork". 2D, two-dimensional; 3D, three-dimensional; Car, Κ-carrageenan; CK, carrageenan-konjac glucomannan; FAPs, fibro-adipogenic progenitors; KGM, konjac glucomannan; PreAs, porcine subcutaneous pre-adipocytes. [Display omitted] • Edible κ-Carrageenan/konjac glucomannan (CK) hydrogel is developed and characterized. • The developed CK scaffold supports the proliferation and three-dimensional adipogenic differentiation of adipocytes. • CK scaffold exhibits better potential to support the formation of cultured fat compared with the 2% alginate. [ABSTRACT FROM AUTHOR]

Cultured meat is an innovative meat-production technology that does not rely on animal husbandry. As a new food component, cultured fat is of great significance to cultured meat. In this study, we isolated adipose-derived stem cells (ADSCs) and identified the purity by immunofluorescence staining of ADSC-specific surface marker proteins CD44 and CD29 and showed that most of the cells were positive for CD29 and CD44. In addition, we detected the expression of FABP4 and Plin1 to confirm that ADSCs differentiated into mature adipocytes at 10 days post-induction. Subsequently, the culture conditions of ADSCs on microcarriers (MCs) were optimized and showed that cell density of living cells reached their highest after 5 days when continuously stirring at 50 rpm. Finally, the expression of FABP4 and PPARγ was detected to confirm the adipogenic differentiation ability of ADSCs on 2D and 3D culture systems and showed that ADSCs maintained their adipogenic differentiation ability after expansion on MCs. In conclusion, this research demonstrated that reliance on MCs to expand ADSCs was a promising approach for production of cultured fat.

[Display omitted] • Residues of adipogenic differentiation cocktail in cultured fat were detected by high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) for evaluating the food safety of cultured fat. • The residues of dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone were not detected in the cultured fat on Day 10 of adipogenic differentiation. • Phosphate buffer solution (PBS) rinsing could reduce insulin content in cultured fat. Cultured fat is inducing adipose progenitor cells (APCs) to differentiate into mature adipocytes for consumption. The traditional adipogenic differentiation cocktail, including insulin, dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone, has potential food safety problems in cultured fat. Therefore, the detection of these residues is necessary to ensure food safety. In this research, a method of high performance liquid chromatography (HPLC) was established to quantitatively analyze the potential residual content of dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone in cultured fat and medium. Quantitative analysis showed that the content of four residues in cultured fat decreased to zero on Day 10. Subsequently, enzyme-linked immunosorbent assay (ELISA) was performed to detect the insulin content in the cultured fat and found that the insulin content in the cultured fat on Day 10 was 2.78 ± 0.21 μg/kg. After soaking with phosphate buffered saline (PBS), the insulin content decreased to 1.88 ± 0.54 μg/kg. In conclusion, this research provided an effective approach to clarify the content of potential residual components in cultured fat and it will provide reference for the safety of cultured fat in the future. [ABSTRACT FROM AUTHOR]

We present a method of producing bulk cell-cultured fat tissue for food applications. Mass transport limitations (nutrients, oxygen, waste diffusion) of macroscale 3D tissue culture are circumvented by initially culturing murine or porcine adipocytes in 2D, after which bulk fat tissue is produced by mechanically harvesting and aggregating the lipid-filled adipocytes into 3D constructs using alginate or transglutaminase binders. The 3D fat tissues were visually similar to fat tissue harvested from animals, with matching textures based on uniaxial compression tests. The mechanical properties of cultured fat tissues were based on binder choice and concentration, and changes in the fatty acid compositions of cellular triacylglyceride and phospholipids were observed after lipid supplementation (soybean oil) during in vitro culture. This approach of aggregating individual adipocytes into a bulk 3D tissue provides a scalable and versatile strategy to produce cultured fat tissue for food-related applications, thereby addressing a key obstacle in cultivated meat production., Competing Interests: JY, MS, NX, BB, HD, CL, SZ, MR, EL, KZ, GL, JP, DK No competing interests declared, (© 2023, Yuen Jr et al.)

Cultured meat is rapidly developing as an emerging meat production technology. Adipose tissue plays an essential role in the flavor of meat products. In this study, cultured fat was produced by cultured adipose-derived stem cells (ADSCs) based on collagen in vitro, with a 3D model. The research showed that ADSCs could attach to collagen hydrogels and differentiate into mature adipocytes. Texture analysis demonstrated that the springiness, cohesiveness, and resilience of cultured fat were consistent with porcine subcutaneous fat. Moreover, 28 volatile organic compounds (VOCs) were detected by headspace gas chromatography-ion mobility spectrometry. The relative contents of 17 VOCs in cultured fat were significantly higher than porcine subcutaneous fat and empty collagen hydrogels, and the relative contents of 5 VOCs in cultured fat were not significantly different from porcine subcutaneous fat. These findings assert the promising application of cultured fat in cultured meat production.

[Display omitted] • Porcine adipose progenitor cells (APCs) were identified by fluorescence-activated cell sorting (FACS). • RNA-seq demonstrated that ITGA5, CD44 and TNC may be possible marker genes of APC in pigs. • APC can be used as a candidate cell type for 3D bioprinting to prepare cultured fat. • The fatty acid composition of cultured fat had a certain similarity with porcine subcutaneous adipose tissue (pSAT). 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Basic fibroblast growth factor (bFGF) promotes the proliferation of porcine adipose-derived mesenchymal stem cell (ADSC) for large-scale cultivation. • Peanut wire-drawing protein (PWP) is a suitable three-dimensional cell scaffold to produce cultured fat. • The cell density, which is important for ADSC-derived cultured fat, was determined and showed that 1.4 × 105 cells/mm3 improved the adipogenic differentiation of ADSC. • Cultured fat showed decreased texture properties. • Cultured fat improved the volatile compounds of the original protein scaffolds. 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. [ABSTRACT FROM AUTHOR]

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field. [ABSTRACT FROM AUTHOR]