Muscle-derived fibro-adipogenic progenitor cells for production of cultured bovine adipose tissue

Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat.


INTRODUCTION
Cultured meat (also known as 'cell-based' or 'cultivated' meat) is an emerging area of biotechnology that aims to address sustainability issues associated with traditional meat production, by leveraging the proliferation and differentiation capacity of stem cells to produce mature, edible tissues for human consumption in vitro [1][2][3] . Such technologies could have substantial environmental, sustainability and animal welfare benefits when compared to traditionally reared meat 1, [3][4][5] .
Traditional meat is composed primarily of skeletal muscle tissue, and prototype cultured meat products have thus focussed on the differentiation of stem cells (such as muscle satellite cells) to produce skeletal muscle 6 . Various animal-free scaffolding approaches have been developed to optimise the texture and protein expression of this cultured muscle 7,8 . However, inter-and intramuscular fat tissue (known as 'marbling') is also a key component of meat and contributes significantly to its flavour, texture and palatability [9][10][11] . Animal fat is characterised by a distinct lipid profile 12 and melting temperature, which is difficult to mimic using plant-based fats. Hence, methods for production of cultured fat are required, though key hurdles remain 13,14 . Current adipose tissue engineering protocols have so far proved unsuitable for production of cultured fat for several reasons, including limitations in adipogenic maturity 15,16 , the use of animal-based scaffolds 17,18 , and a lack of scalability 16,19 .
These FAPs play a poorly understood role in skeletal muscle biology, contributing to muscle regeneration under physiological conditions, but to fibrosis and fatty deposition during pathology 24,25,[31][32][33] . Nevertheless, FAPs offer several potential advantages when it comes to design of cultured meat bioprocesses, including ease of cell purification and accurate recapitulation of traditional intramuscular fat.
Here, we show that FAPs can be easily purified from bovine muscle samples, concurrently with SCs, using a simple FACS-based strategy. These cells can be efficiently expanded for many population doublings, and differentiated to produce cultured fat tissue which closely resembles traditional fat in terms of lipid profile and taste. FAPs therefore represent a viable strategy for the scalable production of cultured fat tissue.

RESULTS
FAPs can be purified from bovine muscle Whilst characterising satellite cells (SCs) from bovine muscle samples, we previously observed a substantial population of cells with a CD29+, CD31/45/56-immunophenotype 34 . In order to purify these cells, we employed a FACS strategy similar to that used for the isolation of SCs (Fig. 1a, b) 34 . Sorting this population yielded a culture of mononuclear cells which adhered readily to tissue culture plastic. These cells were larger and distinctly flatter than SCs in vitro (Fig. 1c), and based on previous reports we hypothesised that they might be fibro-adipogenic progenitors (FAPs) 24,25,27,29 . Using RT-qPCR, we found that the CD29+, CD31/ 45/56-population (which we subsequently refer to as FAPs) showed significantly increased expression of PDGFRA (a previously reported FAP marker 27,29 ), and significantly reduced expression of PAX7 35 and NCAM1 (CD56) 34 when compared to SCs (Fig. 1d). Both of these cell types were isolated with high yields, in the order of 10 5 cells per gram of skeletal muscle tissue.
FAPs are transcriptomically distinct from SCs Despite recent interest, FAPs remain poorly characterised from both a cellular and physiological perspective 36 . We thus used RNA sequencing to characterise broad transcriptomic differences between FAPs and SCs. Principal component analysis (PCA; based on the 500 most variably expressed genes between samples) demonstrated that samples clustered tightly by cell type (Fig. 2a). Differential expression analysis identified 3898 differentially expressed genes (27.8% of total identified genes; log2-FC > 1, FDR < 0.05) between SCs and FAPs, further demonstrating that these cell types are transcriptomically distinct (Fig. 2b). Previously reported SC markers, including PAX7 35 and NCAM1 34 , as well as other myogenic markers, were significantly downregulated in FAPs, as previously observed by RT-qPCR (Figs. 2c; 1d). In contrast, the adipogenic progenitor-related genes PPARG 37 and SCARA5 38 , as well as PDGFRA, were strongly upregulated in FAPs when compared to SCs (Fig. 2c).
Our transcriptomic comparison additionally identified several cell surface receptors differentially expressed between FAPs and SCs (Fig. 2b, c). We confirmed the selective expression of PDGFRα, c Brightfield microscopy images of sorted FAPs and SCs at subconfluence and confluence. Scale bars = 200 µm. d Expression of selected stem cell marker genes in FAPs and SCs after sorting, as measured by RT-qPCR. Data was normalised against expression in FAPs. All numerical data is shown as mean ± sd (n = 3); P-values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ITGA5, CD14, and CD61 at the protein level in sorted FAPs, as compared to SCs, by flow cytometric analysis (Fig. 2d). These data indicate that FAPs and SCs are indeed immunophenotypically distinct, potentially allowing further optimisation of respective purification strategies.

FAPs can undergo adipogenic differentiation in vitro
To investigate the potential use of FAPs for cultured meat applications, we compared their potential for myogenic and adipogenic differentiation in vitro to that of SCs in 2D assays. After 96 h of serum starvation (traditionally used to induce myogenic differentiation in culture) 39,40 , SCs were observed to form large, desmin-positive myotubes, in contrast to FAPs (Fig. 3a). Myogenic fusion index (the percentage of nuclei present in multinucleated myotubes 41,42 ) was significantly reduced in FAPs when compared to SCs (2.4%, SD = 0.5%; 53.8%, SD = 2.3%; Fig. 3b). Expectedly, RT-qPCR analysis confirmed significantly greater upregulation of genes associated with myogenic differentiation in SCs when compared to FAPs (Fig. 3c). Conversely, after 14 days of treatment with an adipogenic differentiation medium (ADM, containing four adipogenic inducer molecules; Supplementary Table 1) 43 , FAPs showed significantly greater accumulation of lipid droplets (53%, SD = 1.1%, compared to 3.2%, SD = 0.7%) than SCs (Fig. 3a, d). genes, such as ADIPOQ (adiponectin), when compared to SCs (Fig. 3e), indicating that FAPs can differentiate into adipocytes, and suggesting that they warrant further investigation as a starting cell type for cultured fat.
FAPs can differentiate into mature adipocytes in 3-dimensional hydrogels Whilst FAPs were able to differentiate into adipocytes in a 2D system, large-scale production of cultured fat requires adipogenesis in the context of 3D tissue biomimetics. FAPs were thus encapsulated in alginate hydrogel and formed into microfibre constructs 15 . Culturing these fibres in ADM gave rise to highly differentiated adipocytes; after 28 days, most of the cells adopted the unilocular lipid droplet morphology characteristic of mature adipocytes (Fig. 4a), in comparison to 2D assays where cells exhibited an accumulation of multiple small fat droplets (Fig. 3a). Quantification of lipid content revealed significantly greater accumulation in ADM (Fig. 4b), although some FAPs All numerical data is shown as mean ± sd (n = 3); P-values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
differentiated spontaneously in the absence of inducers (control conditions; Fig. 4a). These differentiated microfibres stained strongly for two markers of mature adipocytes: perilipin-1 (PLIN1), at the surface of lipid droplets, and acetyl-CoA carboxylase (ACC), in the cytoplasm (Fig. 4c). Expression of fat-related genes was also highly upregulated upon induction of adipogenic differentiation (Fig. 4d), at significantly higher levels than observed after comparable time in 2D cultures (Fig. 3e). The capacity of lipid accumulation was largely conserved across FAPs derived from different donor animals, although there were significant differences between donors in some cases ( Supplementary Fig. 1). These results demonstrate that FAPs are amenable to differentiation in 3D culture, and are thus suitable for production of mature cultured fat tissue.
FAPs can be expanded effectively in long-term and upscaled cultures Alongside robust differentiation, efficient expansion of progenitor cells is a requisite for a cultured meat bioprocess. Assessing the long-term proliferative capacity in 2D culture vessels we found that FAPs were able to robustly proliferate for over 30 cumulative population doublings (PDs; Fig. 5a), showing little morphological change over this time ( Supplementary Fig. 2a). We also studied the effect of cell ageing on the potential of FAPs to differentiate in adipogenic microfibres. Adipogenesis dropped significantly between early, mid and late passage cells (Fig. 5b), as evidenced by an obvious visual reduction in the proportion of cells accumulating lipid droplets (Fig. 5c). Whilst substantial lipid accumulation was observable at 15 PDs, further work remains to understand and improve this loss of differentiation capacity. We next analysed the proliferative capacity of FAPs in conditions more suitable for upscaled cell culture. As a proof of concept, FAPs were expanded on Cytodex 1 microcarriers in 30 mL spinner flask cultures. Over a 110 h culture, microcarrier occupancy and confluency visibly increased, with cell density growing 26-fold (Fig. 5d, e). No lag phase was detectable, with an average doubling time of 22.6 h (SD = 2.0 h) during exponential growth (Fig. 5e). Spiking of waste metabolites demonstrated that FAPs were fairly tolerant of lactate and ammonium build-up (to levels of 20 and 3 mM, respectively; Supplementary Fig. 2b), well above the levels of lactate and ammonium we observed in spinner flask cultures (data not shown). Furthermore, after harvesting from microcarriers, FAPs treated with ADM were able to differentiate efficiently in adipogenic microfibres (Fig. 5f). Taken together, these results show that FAPs can be proliferated efficiently, for many PDs, in both planar and microcarrier-based systems suitable for upscaled cell culture.

Sensory and lipidomic analysis of cultured fat
In order for FAP-derived cultured fat to achieve consumer acceptance, it needs to mimic traditional animal fat from a sensory perspective 44 . Cultured fat tissue was produced from FAPs using the alginate microfibre method, on a gram scale, and compared to traditional subcutaneous beef fat (Fig. 6). Macroscopic observation showed clear similarities in appearance and structure between cultured and traditional fat, as compared to the empty alginate hydrogel, although the cultured fat had a more distinct yellow hue, potentially due to accumulation of carotenoid pigments (Fig. 6a). We then performed scanning electron microscopy (SEM) to compare the structures of these samples at the microscale. After 28 days of differentiation, cultured fat cells were considerably larger than at Day 0 (due to lipid accumulation), approaching the size of some cells observed in traditional fat (Fig. 6b). Interestingly, however, subcutaneous fat tissue displayed a branched network of extracellular matrix (ECM) attached to the cells that was not observed at comparable levels in cultured fat. Upon cooking (pan-frying without oil, for 3-5 min), both the traditional and cultured fat samples left oily residues on the pan, though cultured fat also released some water (presumably due to the alginate hydrogel). A panel of three volunteers assessed the sensory properties of the cultured fat tissue, including appearance, aroma, taste, and texture, reporting that the cooked cultured fat had a creamy consistency typical of animal fat, with a discernible 'beefy' flavour.
We next performed lipidomic analysis to obtain a quantifiable comparison of cultured fat with traditional bovine subcutaneous fat and muscle tissues. Expectedly, the total lipid content of cultured fat tissue increased significantly during adipogenic differentiation when normalised to protein content (Fig. 6c). During the course of adipogenic differentiation, the triglyceride composition of cultured fat became increasingly similar to that of subcutaneous fat ('Fat') and skeletal muscle ('Muscle'; Fig. 6d), likely reflecting the reported similarity in taste. Interestingly, after 28 days of differentiation, cultured fat contained lower levels of palmitic (C16:0) and linoleic acid (C18:2, an essential fatty acid), and higher levels of oleic acid (C18:1), compared with traditional fat and muscle, which has been reported to correlate positively with palatability (Fig. 6d) 45 . Indeed, the proportion of monounsaturated fatty acids (MUFAs; correlated with desirable flavour attributes 46 ) was increased in cultured fat, whilst saturated and polyunsaturated fatty acids were reduced (Fig. 6e). Acyl chain length within the triglyceride class was largely unchanged (Fig. 6f). These results suggest that FAP-derived cultured fat is able to closely mimic traditional beef fat in terms of lipid content and taste.

DISCUSSION
Cultured meat must accurately mimic both the skeletal muscle and intramuscular fat elements of traditional meat 44 . However, cultured fat has thus far been an often-neglected component in this field 6,23 . In this study, we aimed to address this issue by exploiting the advantageous properties of bovine muscle-derived FAPs, which have not previously received significant attention, for the production of edible cultured fat tissue.
Alongside mature multinuclear muscle fibres, skeletal muscle contains a large number of different cell types 47,48 . Whilst purifying satellite cells by FACS 34 , we had previously observed a large population of CD29+, CD31/45/56-cells. Sorting this population yielded a culture of mononuclear cells possessing distinct morphology from SCs (Fig. 1). Hypothesising that these cells might be FAPs, we confirmed their expression of PDGFRα (CD140a; a tyrosine kinase receptor previously reported to label FAPs in human 26,29 , mouse 24,25 and bovine 27,28 ), as well as other factors previously implicated in adipogenesis, including PPARɣ 37 and SCARA5 38 (Fig. 2). These FAPs showed lipid droplet accumulation, as well as strong induction of adipocyte marker genes, upon treatment with a differentiation medium containing a cocktail of adipogenic inducers (Fig. 3). Since FAPs represent the primary source of intramuscular fat deposits in vivo 49,50 , we postulated that cultured fat derived from FAPs might mimic traditional fat tissue more closely than that produced from other cell types, such as subcutaneous fat-derived MSCs. It will be interesting to compare lipid accumulation rates for these bovine cell types, as has been performed for humans 29 . In addition, FAPs are easy to acquire concomitantly with SCs from muscle samples, supporting the development of a commercially viable bioprocess for cultured meat, and are widely conserved across agriculturally relevant species 51 . Our characterisation of FAP cell surface immunophenotypes will help to further optimise sorting strategies for these cell types; for example by combining positive selection for PDGFRα or ITGA5 with known SC markers such as CD56 (Fig. 2) 52 .
Previous studies aiming to produce cultured fat have relied on animal-derived hydrogels or scaffolds 17,18 . Here, we employed alginate-based hydrogels to produce edible cell-laden microfibres 15 , which supported the 3D culture and adipogenesis of FAPs (Fig. 4). The biocompatibility, low cost, and ease of gelation of alginate enable economic scalability 53 , and fibres could feasibly be fabricated as meter-scale constructs 17,54 , cultured in bioreactors to permit efficient nutrient exchange. This hydrogel supported the robust differentiation of FAPs over a four week culture, as evidenced by the presence of unilocular lipid droplets characteristic of white adipocytes ( Fig. 3; Fig. 4) 55 . Nevertheless, further understanding and optimisation of cell-biomaterial, cellcell, and cell-medium interactions will be required to maximise the adipogenic capabilities of FAPs, and to identify a fully-food compatible adipogenic differentiation medium. The tissue origin of FAPs also offers intriguing possibilities for the co-culture of multiple muscle-derived cell types. FAPs reportedly help to promote myofibre repair after muscle injury through the release of paracrine factors, such as IGF-1 and IL-6, that stimulate satellite cell differentiation 25,56 . FAPs also have the capacity to enter a fibroblastic lineage, and the ability to manipulate the differentiation of these cells could permit the production of native extracellular matrix (which we did not observe at considerable levels in this study) to construct more complex tissue mimetics 25,32 . FAPs, like SCs, are a population of adult stem cells, and as such would be expected to have limited proliferative capacity. However, observed proliferation rates were promising, with FAPs demonstrating robust doubling times even after 20 population doublings (Fig. 5). This may reflect an intrinsic property of the cells, given the propensity for intramuscular fat to increase during aging 9,10 , and is highly advantageous from the perspective of a cultured meat bioprocess 57 . FAPs were also able to proliferate rapidly in microcarrier-based spinner flask cultures at rates similar to those previously observed in 2D culture 58 , whilst retaining their capacity for adipogenic differentiation. Further studies will be required to maximise obtained cell densities, eliminate FBS from proliferation medium formulations, and to fully assess the effects of prolonged proliferative phases in upscaled culture vessels 2 , particularly given the drop in differentiation capacity we observed during long term passaging (Fig. 5). It will be interesting to discern the pathways involved in this loss of stemness, perhaps using transcriptomic approaches. While such studies should allow the useful cellular lifespan to be extended, allowing production on a kilogram scale from single tissue biopsies, frequent biopsying will nonetheless be required, and careful selection of stem cell donors necessary to reduce donor-to-donor variability (which was observed to some extent with respect to differentiation in this study; Supplementary Fig. 1). The biology of adipogenesis changes during aging 59 , whilst differences in fatty acid composition have also been observed between muscles 12,60 , offering intriguing possibilities for donor optimisation.
FAP-derived cultured fat tissue appeared visually similar to traditional animal fat on both a macroscopic and microscopic level, albeit with some colour difference. Lipidomic analysis of mature cultured fat revealed that the triglyceride composition (the most prevalent lipid class, known to be crucial for flavour 45,46 ) was similar to that of traditional animal fat tissue (Fig. 6), likely reflecting the close similarity in taste reported in our volunteer testing (although further blind tastings are certainly also required). The majority of the most prevalent fatty acid species have been correlated with palatability of meat, although it is unclear to what extent fatty acid composition per se, compared to the levels of intramuscular fat, contribute to this 60 . Comprehensive understanding of the adipogenic biology of FAPs may in future allow engineering of cultured fat products with specifically altered fatty acid compositions; for example to favour polyunsaturated fatty acids, which have been associated with reduced risk of cardiovascular disease 61 , or to increase levels of essential fatty acids (such as linoleic acid).
Collectively, this work demonstrates that FAPs are a candidate cell type for the production of cultured fat tissue as a novel food ingredient, offering potentially significant advantages with respect to ease of acquisition, mimicry to traditional fat, and co-culture with satellite cells to produce complex tissues. Key challenges remain, including the maintenance of differentiation potential at higher PDs, and the transition to protocols that are fully compatible with mass-market food production. Nevertheless, this represents an important step towards making cultured meat a reality.

Cell isolation
Fresh bovine skeletal muscle and subcutaneous fat samples were obtained from a registered abattoir according to national guidelines on animal tissue handling. Ethical approval was not required for acquisition of muscle and fat samples from commercially slaughtered cattle. Samples were acquired and transported in accordance with Dutch national guidelines on handling of animal material. Mosa Meat B.V. has been granted license to handle Category 3 animal material.
Muscle-derived cells were isolated from the semitendinosus muscle of commercially slaughtered Belgian Blue cattle (both male and female, aged from 1 to 7 years) as previously described 34 . Briefly, muscle was minced and dissociated with collagenase (CLSAFA, Worthington; 1 h, 37°C). Cell slurries were filtered through a 100 μm cell strainer and incubated in ammonium-chloride-potassium (ACK) erythrocyte lysis buffer (1 min, room temperature (RT)). Cells were resuspended in growth medium (GM; Supplementary Table 1), and filtered through a 40 μm strainer prior to culture.

Cell culture
Cells were cultured on collagen-coated flasks in GM at a seeding density of 2 × 10 3 cells/cm 2 . For myogenic differentiation, cells were plated at 5 × 10 4 cells/cm 2 on 0.5% Matrigel in GM for 24 h, and differentiation initiated by switching to myogenic differentiation medium (Supplementary Table 1). For adipogenic differentiation, cells were seeded at 4 × 10 4 cells/cm 2 in GM and after 24 h switched to adipogenic differentiation medium (ADM) containing four inducer molecules. After 3 days, medium was exchanged for ADM containing only rosiglitazone and insulin (ADM; Supplementary Table 1). Medium exchanges were performed every three days. Cells treated without inducers served as a control for adipogenic differentiation. Brightfield microscopy images were captured with an EVOS M5000 microscope (Thermo Fisher).

RT-qPCR
RNA was isolated using the E.Z.N.A MicroElute Total RNA Kit (proliferative and myogenic samples) or E.Z.N.A Total RNA kit II (adipogenic samples; Omega Bio-tek, R6831, or R6934, respectively). Fig. 6 Lipidomic analysis of FAP-derived cultured fat. a Macroscopic photographs of empty alginate hydrogel, cultured fat (after 28 days of differentiation), and bovine subcutaneous fat. Scale bar = 5 mm. b SEM images of cultured fat after 0 and 28 days of differentiation, and bovine subcutaneous fat. Scale bar = 10 μm. c Absolute quantification of total lipid content (normalised to mass of protein) of cultured fat after 0, 7, and 28 days of differentiation. d Breakdown of relative percentages of fatty acid species within the triglyceride lipid class in cultured fat samples after 0, 7, and 28 days of differentiation, and bovine subcutaneous fat ('Fat') and skeletal muscle ('Muscle') control samples. Legend denotes C:D numerical annotation (where C indicates number of carbon atoms, and D number of double bonds, in the acyl chain). e Proportion of triglyceride species with acyl chains containing 0 to 6 unsaturations, for three of the samples shown in d. Statistically significant differences to both fat and muscle are indicated. f Proportion of triglyceride species with acyl chains containing 16, 18, 20, or 22 carbon atoms for the same samples shown in e. All numerical data are shown as mean ± sd (n = 3). P-values: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, 1708891) according to the manufacturer's instructions. RT-qPCR was performed using iQ SYBR Green Supermix (Bio-Rad, 1708880) with primer pairs detailed in Supplementary Table 2. 2 ΔCt values for genes of interest were normalised to the average of three housekeeping genes (B2M, RPL19, and RPLP0 for myogenic samples; RPL19, RPLP0, and UXT for adipogenic samples).

RNA sequencing
RNA was harvested from FAP and SC samples using the E.Z.N.A MicroElute Total RNA Kit. Sequencing libraries were prepared using the TruSeq stranded mRNA kit (Illumina), and sequenced on a high-output 75 bp NextSeq 500 (Illumina). STAR aligner 2.7 62 was used to align single-end reads to the reference genome bosTau9 (ARS UCD1.2.98). Subsequent analysis was performed in R (version 4.1.0). Gene counts based on the aligned reads were quantified using the FeatureCounts function of the Rsubread package 63 .
Principal component analysis was performed using the 500 most variable genes (based on variance of RPKMs across all samples). The limma package was used for differential expression analysis by following the authors workflow, with genes considered significantly differentially expressed above a log2-fold change cutoff of 1, and a false-discovery rate (FDR) below 0.05 64,65 .
Immunofluorescence images were captured, and adipogenic and myogenic differentiation quantified, using an ImageXPress Pico High Content Analyser (Molecular Devices, LLC). Nuclei counts were determined by quantifying Hoechst staining, and BODIPY immunoreactivity was assigned to the nearest nuclei using MetaXPress software. BODIPY positivity was then determined by quantifying BODIPY positive nuclei as a proportion of total nuclei. Fusion index was calculated as the percentage of nuclei located within desmin-stained myotubes 41,42 . For metabolite spiking experiments, cells were fixed, stained with Hoechst, and quantified using MetaXPress.

Immunofluorescence for adipogenic microfibres
Adipogenic microfibres were fixed in fixation buffer (4% formaldehyde, 66  Images were captured on a confocal microscope (TCS SP8, Leica Microsystems) using a 25×/1.00 objective lens and 5 µm Z-steps. Nuclei counts and lipid volumes were quantified using a custom python script (contact authors for details) for three separate images per sample, and the total quantified lipid volume divided by the number of nuclei to calculate lipid volume per cell.
For cell harvesting, microcarriers were washed with PBS and incubated in 2X TrypLE (Gibco, A12177; 10 min, 37°C, 200 rpm). Cells were separated from microcarriers using a vacuum driven 100 µm Steriflip filter unit (Merck Millipore, C3238) and several washing cycles. Cells were harvested by centrifugation (350 × g, 5 min), resuspended in GM and counted prior to subsequent handling or analysis.

Lipidomic analysis
10-30 mg samples were collected for lipidomic analysis. Lipids were extracted using a modified Bligh-Dyer protocol, and analysed by hydrophilic interaction liquid chromatography mass spectrometry (HILIC LC-MS/MS) 66 . Lipid quantities were normalised to the amount of protein present within the respective sample.
For data analysis, peak integration was performed with MultiQuantTM (version 3.0.3). Lipid species signals were corrected for isotopic contributions (calculated with Python Molmass 2019.1.1) and quantified based on internal standard signals as per the Lipidomics Standards Initiative (LSI).

Sensory analysis
Sensory analysis was performed by three consenting, unblinded volunteers, who each regularly consume beef. Cultured fat was frozen and stored (−20°C) prior to analysis. Frozen bovine subcutaneous fat and empty alginate hydrogel were provided as control samples. Samples were pan-fried (without oil) for 3-5 min, and samples scored numerically on a range of visual, olfactory and gustatory characteristics. All analyses were performed in accordance with EC Guidance on Ethics in Food Research 67 , and in compliance with Mosa Meat B.V. standard operating procedures. Ethical approval was waived under Dutch MREC/CCMO regulation.

Statistical analysis
Statistical analysis was performed using Prism 9.1.0 (GraphPad). Analysis between two groups was performed using a Student's t-test. Analysis of three or more groups was performed using one-way ANOVA with Bonferroni's multiple comparisons test against indicated control(s) (Fig. 4d; Supplementary Figs. 1b; 5b; Supplementary Figs. 2b; 6c-f). Analysis of three or more groups with two independent variables was performed using a two-way ANOVA with Bonferroni's multiple comparison test against indicated control(s) (Fig. 4b).