Production of Alternative Fat from Adipose-Derived Stem Cell from Bovine in 3D Culture

Abstract

Cultivated meat, developed through cell culture technology, is emerging as a promising solution that closely mimics both the flavor and nutrient profiles of conventional meat. One key component that contributes to the flavor of meat is fat content. In this study, bovine adipose-derived stem cells (bADSCs) were cultured for the production of alternative fat in vitro. The expression of mesenchymal stem cell (MSC) markers (CD29, CD73, and CD105) and colony forming efficiency were assessed to characterize bADSCs. bADSCs were differentiated into adipocytes to produce cultivated fat in 2D or 3D culture. The cultivated fat was analyzed by gas chromatography to verify the similarity of the fatty acids of animal-derived fat. Our results show that bADSCs have characteristics of MSC and could differentiate into adipocyte. The ratio of unsaturated fatty acids and saturated fatty acids in cultivated fat and adipose tissue was similar. Adipogenic differentiation of ADSCs using a textured vegetable protein (TVP) scaffold could form the lipid droplets in the TVP. This study demonstrated the establishment of a culture system for the fat production from bADSCs in vitro. The fat produced through bADSCs shows the potential to be used in the composition of hybrid-cultivated meat.

1. Introduction

Conventional meat production emits large amounts of greenhouse gases, which significantly contribute to global warming [1]. These emissions stem from activities such as feed production and manure management, collectively contributing to 14.5% of global greenhouse gas emissions [2]. Therefore, increasing meat production to meet rising demand is expected to further exacerbate environmental impact.

Presently, plant-based alternative meat is widely acknowledged and represents a popular choice among vegan alternatives [3,4]. Various types of plant-based meat continue to be produced, and textured vegetable protein (TVP) is mainly used as plant-based meat substitute [5,6,7,8]. However, plant-based meat has faced criticism for relying on potent food colorings, textural additives, causing deficiency in essential nutrients and elevated sodium content [9,10,11].

Cultivated meat, emerging as a sustainable future food source, is produced by growing animal cells in a controlled environment and is gaining attention as a sustainable and eco-friendly meat for the future [3,12]. Cell-based meat production is estimated to significantly reduce greenhouse gas emissions by decreasing land use, water consumption, and food-crop demand [13,14]. This mimics the natural in vivo of cells and resembles cell types arranged in structures as animal tissues, thereby replicating the nutritional/organoleptic profiles of conventional meat. However, less previous research is available in cultured meat on mimicking food-related characteristics, such as sensorial properties and nutritional value, compared to traditional meat studies.

Most research in the cultured meat field has concentrated on developing muscle tissue, the primary component of meat products. In contrast, few studies have addressed cultured fat tissue. Lipid composition of the fat tissue in the meat is critical in determining meat quality, affecting flavor, texture, and palatability [15,16]. A method development for generating cultured fat from animal cells is crucial for mimicking these important food-related attributes. Numerous researchers have sought to induce adipogenesis in animal-derived adult stem cells in vitro to obtain fat for cultured meat.

There are various types of adult stem cells in the body. Among them, mesenchymal stem cells (MSCs) have the capability to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes [17,18]. MSCs can be isolated from almost every tissue in the body, such as bone marrow, adipose tissue, trabecular bone, and umbilical cord blood [18,19,20]. The global application of MSCs for critical diseases demonstrates their safety and efficacy in the human body. However, challenges in stem cell harvest arise due to the limited quantity of tissues that can be collected. Thus, adipose tissue is emphasized due to its abundant availability and easy access. Adipose-derived stem cells (ADSCs) are MSC subsisted in adipose tissue. Large quantities of ADSCs can be easily harvested from abundant adipose tissue in the body, and these harvested ADSCs have potential to expand rapidly in vitro [21,22]. ADSCs have excellent capability to generate a multitude of growth factors and differentiate into adipocytes compared to MSCs from other tissues. These distinct characteristics of ADSCs led ADSCs to be the most promising stem cell population, and the utilization of ADSCs in multiple studies enhanced ADSCs value as a cell source for not only the pharmaceutical field but the cultivated meat industry. Recently, the possibility of using ADSCs for the food industry was suggested due to the gradual increase in ADSCs usage as a source for cultivated meat production.

The production of cultivated meat necessitates a significant quantity of cells, leading to elevated production costs [22,23,24]. In order to achieve a sufficient cellular product, suspension culture employing scaffolds is notable for its ability to support cell growth in a three-dimensional (3D) form [25,26,27]. Moreover, cultured meat should be produced in a 3D culture using supporting materials such as biomaterials, since tissues are in 3D structures composed of diverse cell types. Lately, scientists have unveiled methodologies for cultured meat production through diverse scaffold types, constructed by 3D printed bioink or textured soy proteins, to replicate the structural composition of meat.

This study aimed to generate fat, a key component of cultivated meat, by cultivating bADSCs and combining with TVP as a scaffold during differentiation period. To obtain bADSCs in vitro, stromal vascular fraction (SVF) was isolated from bovine subcutaneous fat tissue and subsequently cultured. Cellular characterization was performed using immunocytochemistry, Western blot, and flow cytometry. Adipogenic differentiation was carried out in both 2D and 3D culture systems. Subsequently, the cellular components of adipocytes were analyzed by gas chromatography to determine whether the ADSCs-derived adipocytes have a fatty acid composition similar to animal-derived fatty acids.

2. Materials and Methods

2.1. bADSC Preparation and Culture

Intermuscluar adipose tissues were immediately harvested from 29- to 33-month-old castration male or female Hanwoo cattles slaughtered at a local slaughterhouse (Kwell LPC, Hongcheon, Republic of Korea). Subsequently, the retrieved adipose tissues were used as donors of bADSCs. All experimental procedures were conducted to comply with the Animal Care and Use Guidelines of Kangwon National University and approved by the Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval no. KW-240809-5).

The excised adipose tissue was then immersed in a sterile solution of Dulbecco’s phosphate-buffered saline (DPBS, LB001-02, Welgene, Gyeongsan, Republic of Korea) supplemented with 1% Antibiotic-Antimycotic solution (A/A; Corning, 30-004-Cl, Corning, NY, USA). The adipose tissue was minced on a culture dish using sterile forceps and scissors, and subsequently incubated with 0.1% collagenase type 1 (Gibco, 17101015, Waltham, MA, USA) diluted in DPBS at 37 °C for 1 h. To inactivate collagenase activity, an equal volume of complete culture medium consisting of DMEM (Gibco, 11995073) supplemented with 1% A/A and 10% fetal bovine serum (FBS; Gibco, 16000044) was added. The sample was centrifuged at 350× g for 5 min, and the supernatant was discarded. The pellet was resuspended in DPBS and then resuspended cells were passed through a 100 μm mesh filter to ensure homogeneity and remove debris. After centrifugation, the supernatant was removed and the SVF pellet was resuspended in DMEM supplemented with 10% FBS and 1% A/A. SVF was cultured at 37 °C under 5% CO₂. After 24 h of incubation, the culture medium was changed to remove unattached cells. At approximately 90% confluence, the cells were harvested using 0.25% trypsin-EDTA (Gibco, 25200072) and then were subcultured at a density of 2 × 10³ cells/cm².

2.2. Immunofluorescence Staining

Cells were seeded in cover slip on 24-well well plate at 4 × 10⁵ cells/well and incubated overnight. Then cells were washed using DPBS and fixed with 4% formaldehyde solution (Sigma Aldrich, F8775, St. Louis, MO, USA) for 20 min. Non-specific binding was blocked with 20% normal goat serum in PBS for 1 h at RT. Afterwards, the cells were stained with anti-CD90 antibody (1:100; Proteintech, bs-0778R, Rosemont, IL, USA), anti-CD73 antibody (1:100; Proteintech, 12231-1-AP, Rosemont, IL, USA), and anti-CD105 antibody (1:100; Proteintech, 10862-1-AP, Rosemont, IL, USA) for 2 h at 37 °C, followed by fluorescence-conjugated anti-mouse secondary antibody (1:200; Invitrogen, A-11001, Waltham, MA, USA) or rabbit secondary antibody (1:200; Invitrogen, A-11012). Counterstaining was carried out with DAPI for 5 min at RT. Immunofluorescence images were captured using Carl Zeiss confocal microscope (Carl Zeiss, Oberkochen, Germany) and data were analyzed using the ZEN Microscopy software V2.0 (Carl Zeiss, Oberkochen, Germany).

2.3. Western Blot

The cell lysates were denatured and electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Thermo Fisher Scientific, 88518, Waltham, MA, USA). Blocking of the membrane was performed with 5% bovine serum albumin (Sigma-Aldrich, 126575, St. Louis, MO, USA) for 1 h. After blocking, the membranes were incubated with the anti-CD73 (1:200; Proteintech, 12231-1-AP, Rosemont, IL, USA), CD90 (1:200; Proteintech, bs-0778R, Rosemont, IL, USA), and CD105 (1:200; Proteintech, 10862-1-AP, Rosemont, IL, USA) antibodies, followed by anti-Ig G horseradish peroxidase-conjugated secondary antibody. The blots were visualized using chemiluminescence (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).

2.4. Flow Cytometry

In total, 1 × 10⁶ Cells were resuspended in FACS buffer (Invitrogen™, 00-42222-26) and stained with Alexa 647-conjugated anti-CD73 antibody (1:100; Bioss Inc., bs-4834R-A647, Woburn, MA, USA), Alexa 488-conjugated anti-CD90 antibody (1:20; Novus Biologicals NBP2-47755AF488, Centennial, CO, USA), anti-CD105 antibody (1:100; Proteintech, 10862-1-AP, Rosemont, IL, USA), and PE-conjugated anti-CD45 antibody (1:100; Invitrogen, MA1-81458) for 30 min at RT. Isotype control was stained with AF488-conjugated anti-mouse secondary antibody. Cells were then washed twice in the buffer. For cell acquisition, BD FACSymphony AI flowcytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used. Data analysis for these experiments was performed using FlowJo software (Version 11, Tree Star, Ashland, OR, USA).

2.5. Adipogenic Induction

For 2D culture, bADSCs were seeded at a density of 3 × 10⁵ cells per well in 6-well plates. Once the cells reached approximately 80% confluency, the medium was replaced with adipogenic induction medium (AIM), consisting of DMEM supplemented with 1% Antibiotic-Antimycotic (A/A), 5% fetal bovine serum (FBS), and the following adipogenic factors: 10 μM human insulin (Sigma-Aldrich, I2643, St. Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich, D4902, St. Louis, MO, USA), 5 μM troglitazone (Sigma-Aldrich, R2408, St. Louis, MO, USA), and 500 μM isobutyl methylxanthine (IBMX; Sigma-Aldrich, I7018, St. Louis, MO, USA). After 3 days of induction with AIM, the medium was replaced with adipogenic differentiation medium (ADM), composed of DMEM supplemented with 1% A/A, 2% FBS, 10 μM human insulin, and 5 μM troglitazone. Differentiation was continued for 18 days in ADM. The control group consisted of cultured cells with complete culture medium for 21 days. All mediums used during differentiation were changed every 3 days.

For 3D culture, autoclaved TVPs (3 cm²) were first immersed in a 0.2% gelatin solution (Sigma-Aldrich, G1890, St. Louis, MO, USA) and incubated overnight at 4 °C for coating. The coated TVPs were then washed twice with 5 mL of DPBS and placed in a 12-well plate. Subsequently, 50 μL of DMEM containing 6 × 10⁶ bADSCs was seeded onto the gelatin-coated TVPs (Hokyung-Tech, Anseong, Gyeonggi-do, Republic of Korea), and the cells were allowed to attach for 30 min in a 37 °C incubator. After cell attachment, 3 mL of complete culture medium was added to each well containing the bADSC-seeded TVPs. The constructs were cultured for 2 days to allow cell proliferation. After 2 days, the bADSC-seeded TVPs were transferred to a 100 mL sterile glass bottle containing 30 mL of AIM for adipogenic induction. The TVPs floated in the medium but remained fully submerged to prevent drying. After 3 days of induction, the medium was replaced with ADM and maintained for an additional 18 days, with medium changes every 3 days. Adipogenic differentiation was assessed over 21 days following the initiation of suspension culture.

2.6. Oil Red O Staining

Cells were rinsed twice with DPBS and fixed with 4% (w/v) paraformaldehyde for 20 min. The Oil Red O working solution was prepared by diluting the 0.5% (w/v) Oil Red O stock solution (in isopropanol; Sigma-Aldrich, O1391, St. Louis, MO, USA) with distilled water at a ratio of 3:2. The cells were then incubated with 60% isopropanol for 5 min and subsequently treated with the Oil Red O working solution for 15 min. Cells were washed with distilled water. To stain adipocytes in TVP, cells were fixed in 5% (w/v) paraformaldehyde for 30 min, embedded in optimal cutting temperature compound (OCT compound, Sakura Finetek USA, Inc., Torrance, CA, USA), and then were frozen for cryosection (5 µm of thickness). The sectioned sample was stained with Oil Red O solution and then mounted. Finally, the The samples were imaged using a inverted research microscope Eclipse TS100 (Nikon, Tokyo, Japan) and processed using the IMT iSolution Lite software ver 10.0 (IMT i-Solution Inc., Vancouver, BC, Canada). To quantify stained area, Oil Red O in lipid droplet was extracted by 2-propanol and then the optical density (O.D) was measured.

2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

To examine the concentration of bovine adiponectin, supernatant was collected and centrifuged to remove the debris. Adiponectin was quantified by bovine adiponectin ELISA kit (AssayGenie, BOEBO276, Dublin, Ireland), according to the manufacturer’s instruction. Optical density was determined using a microplate reader (Molecular device, SpectraMax iD3, San Jose, CA, USA) set to 450 nm.

2.8. Fatty Acid Analysis

The fatty acid composition of the cultivated fat and adipose tissue was compared. The fatty acids were measured by a two-dimensional gas chromatography–flame ionization detector (GCxGC/FID). The standard solution of fatty acids was a mixture of 37 fatty acid methyl esters (FAME) in hexane. The samples were methylated with 14% BF₃-Methanol after being extracted with methyl tert-butyl ether utilizing the Folch method. The columns used were Rxi-5MS (30 m × 0.25 mm × 0.25 μm, Restek, Lisses, France) and Rxi-17sil MS (2.0 m × 0.25 mm × 0.25 μm, Restek, Lisses, France). The carrier gas was Helium at 1.0 mL/min. 37 FAME were separated using temperature programming. The injector and detector were analyzed at 250 °C and 300 °C, respectively.

2.9. Statistical Analysis

Data are presented as three independent experiments’ mean standard deviation (SD). Probability values of less than 0.05 were considered statistically significant. An unpaired, two-tailed Student t-test was carried out for the statistical analysis of all data. * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results

3.1. Charaterization of Bovine ADSC

After isolating SVF from the initial 2 g of fat tissue, 1 × 10⁵ cells were seeded and cultured in vitro. A culture of adipose tissue-derived SVF (1 × 10⁵ cells) of bovine origin resulted in the formation of spindle-shaped, colony-forming cells within 4 days at passage 0 (Figure 1A,B). Starting with the culture with 1 × 10⁵ cells, the cumulative cell count reached approximately 3.0 × 10⁸ within a span of three weeks (Figure 1C). The assessment criteria for verifying MSCs include the expression of CD73, CD90, and CD105, coupled with the absence of CD45, as outlined in the ISCT guidelines. An attempt was made to assess the expression of MSC markers in bADSCs. As shown in Figure 1D, bADSCs showed a pronounced expression of CD73 (99%), CD90 (99%), and CD105 (99%), and was negative to CD45 (0.01%) (Figure S1). To investigate the cellular localization, immunofluorescence staining was conducted for CD73, CD90, and CD105, revealing strong positivity on the cellular surface of bADSCs, as shown in Figure 1E. This expression was consistently confirmed by Western blot analysis (Figure 1F).

The data suggest that bADSCs exhibit a spindle-shaped morphology and possess the capacity to repopulate and form colonies, while concurrently expressing specific markers associated with ADSCs. This observation meets the criteria necessary for classifying these cells as mesenchymal stem cells from adipose tissue. Moreover, bADSC culture in this setting exhibits considerable potential for generating abundant stem cells in vitro.

3.2. bADSC Is Capable of Differentiating into Fat Tissue with Diverse Fatty Acids

bADSC differentiation was induced on 6-well plate with a standard adipogenic cocktail for 21 days in vitro, as shown in Figure 2A. Lipid droplet formation commenced on the 4 days post adipogenic induction, (Figure S2), with a subsequent increase in droplet size. Multilocular lipid droplets developed, and the presence of intracellular lipid was confirmed through Oil Red O staining (Figure 2B). The quantification of the stained area revealed approximately a 4-fold higher absorbance compared to undifferentiated cells (Figure 2C,D). Adiponectin, an adipokine released by mature adipocytes, stands out as a distinctive marker of adipocyte. Studies have reported its capability to stimulate cell proliferation and the differentiation of pre-adipocytes into mature adipocytes. Quantification of adiponectin in the supernatant was conducted using ELISA at 21 days (Figure 2E). The control group exhibited negligible adiponectin production, whereas bADSC subjected to adipogenic induction showed a significant increase in adiponectin levels.

Next, the fatty acid composition of adipocytes derived from bADSCs was analyzed and compared with intact bovine adipose tissue (Figure 2F). The ratio of saturated fatty acids (SFA) to unsaturated fatty acids (UFA) in relation to total fats was found to be similar in both adipocytes and adipose tissue (Figure 2F and

Table 1. The analysis of the composition of the fatty acid. The fatty acid compositions in induced adipocytes and adipose tissue from bovine were compared, focusing on the differences between saturated and unsaturated fatty acids. bAdipocyte: bovine ADSC. N/D: not detected.

Common Name	Fame	Abbreviation	bAdipocyte	Fat Tissue
Saturated Fatty Acids
Stearic acid	Methyl stearate	C18:0	16%	9%
Palmitic acid	Methyl palmitate	C16:0	15%	24%
Eicosanoic Acid	Methyl arachidate	C20:0	2%	1%
Myristic acid	Methyl myristate	C14:0	2%	4%
Docosanoic acid	Methyl behenate	C22:0	2%	N/D
Lignoceric acid	Methyl lignocerate	C24:0	2%	N/D
Heptadecanoic Acid	Methyl heptadecanoate	C17:0	2%	1%
Pentadecanoic acid	Methyl pentadecanoate	C15:0	1%	1%
Unsaturated Fatty Acids
Oleic acid	cis-9-Oleic acid methyl ester	C18:1c	11%	20%
Linoelaidic acid	Methyl linolelaidate	C18:2n6t	9%	3%
Elaidic acid	trans-9-Elaidic acid methyl ester	C18:1t	8%	29%
Eicosatrienoic acid	cis-8,11,14-Eicosatrienoic acid methyl ester	C20:3n6	5%	1%
Docosahexaenoic acid	cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester	C22:6n3	4%	N/D
Palmitoleic acid	Methyl palmitoleate	C16:1	2%	4%
Eicosapentaenoic acid	cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester	C20:5n3	2%	N/D
Eicosenoic acid	Methyl cis-11-eicosenoate	C20:1n9	2%	1%
Eicosadienoic acid	cis-11,14-Eicosadienoic acid methyl ester	C20:2	1%	N/D
Erucic acid	Methyl erucate	C22:1n9	1%	N/D
Docosahexaenoic acid	cis-13,16-Docosadienoic acid methyl ester	C22:2	0%	0%
Myristoleic acid	Methyl myristoleate	C14:1	N/D	2%

). The content of oleic acid, which allows for good flavor, was high in both samples. In addition, the lower the SFA content, the better it is for health, because SFA is known to be a major cause of hyperlipidemia and hypercholesterolemia (Table 1).

3.3. Adipogenic Differentiation of bADSC on TVP Scaffold for Production of Cultivated Meat

The production of cultivated meat requires the integration of nutrients, such as fat or protein, with a scaffold to achieve structural similarity to intact tissue. TVP is utilized as a plant-based protein. In this study, an attempt was made to culture bADSCs on TVP coated with a gelatin solution. Given that the perfect adhesion to the TVP is impracticable, assessing the attachment rate of the total cells is imperative. To accomplish this, bADSCs were seeded onto TVP coated with gelatin and incubated for 2 days. TVP-unattached bADSCs would disperse in the culture medium and adhere on the bottom of the cultured flask based on our assumption. Thus, the cells in the supernatant and cultured flask were counted to identify the attachment rate. bADSCs adhered to the TVP were harvested by treating with trypsin-EDTA, and the cell count was determined. (Figure S3). Consequently, the attachment rate ranged approximately from 30% to 44%, with the remaining being unattached under these conditions. Subsequent experiments involved seeding bADSCs to maintain a final cell number of 2 × 10⁶ within the TVP. bADSC-seeded TVP was incubated for 2 days in growth medium before being transferred to (Figure 3A).

The differentiation potential of bADSCs was contrasted with that of cell-free TVP. Following the completion of the experimental treatment, the samples were subjected to heating to simulate the processing typically applied to edible tissue. When compared to cell-free TVP, the presence of bADSCs induced changes in both color and texture of TVP. This disparity was further pronounced after heating. Upon heating, TVP with bADSCs exhibited oil release (Figure 3B, red arrow), a phenomenon not observed in the cell-free counterpart. To confirm the adipogenic differentiation of bADSCs, TVP tissue was cryosectioned, and Oil Red O staining was carried out (Figure 3C). As shown in Figure 3C, the presence of lipid droplets within TVP was distinctly detected, confirming the adipogenic differentiation of bADSCs and the production of lipid droplets within the TVP matrix.

4. Discussion

Cultivated meat relies on the inherent capacity of stem cells to proliferate to achieve a sufficient cell number in vitro and to undergo specific differentiation in response to biological stimuli. Proliferation and differentiation potential of stem cells is obviously affected by their cellular activity. Numerous studies have endeavored to optimize the in vitro establishment of a mass culture system to obtain a substantial supply of healthy/highly proliferative stem cells. The differentiation process can commence once a stem cells is acquired. Taste, flavor, and texture comparable to conventional bovine tissue are essential traits to attain post-differentiation of stem cells in vitro. This underscores the importance of fat tissue, since the fat content is recognized as a critical determinant for meat flavor.

The objective of this study was to generate fat tissue from bADSCs within a plant-based scaffold. To achieve this goal, it was imperative to establish a stable culture system of bADSCs in vitro. We undertook the isolation and characterization of ADSCs from the subcutaneous fat of the bovine back. The culture of SVF revealed the presence of fibroblastic colony-forming cells, which demonstrated robust proliferation up to passage 8. Notably, approximately 2 g of fat tissue was used for cell isolation, resulting in a substantial yield of cells from the culture. The isolated cells displayed the characteristic spindle-shaped morphology and expressed CD73, CD90, and CD105, while lacking CD45. After confirmation of the presence of stem cell markers, bADSCs were induced to undergo adipocyte differentiation, as evidenced by the observation of lipid droplets. Comparison between bovine fat and adipocytes derived from bADSCs corroborated a similar composition of fatty acids. This suggests that in vitro adipogenesis of bADSCs can lead to the production of fat, potentially serving as a substitute for conventional meats. This finding implies the successful establishment of in vitro adipogenic induction in bovine ADSCs with characteristics comparable to intact tissue in terms of fatty acid.

In order to use cultured meat as a food source, it is essential to integrate fat tissue with other tissue components, such as proteins. bADSCs were plated on TVP as a scaffold and exposed to adipogenic stimuli for a period of 2 weeks. Adipogenesis progressed in the scaffold, and lipid droplets were observed in the TVP containing bADSCs. These results showed that bADSCs are capable of differentiating into adipocytes in a 3D culture system, and TVP stands out as a promising candidate scaffold for the development of hybrid cultured meat.

Cell-cultured meat presents a promising alternative to conventional meat production, with the potential to substantially reduce environmental impact, achieving reductions of 45% in energy consumption, 96% in greenhouse gas emissions, 99% in land use, and 96% in water usage [28]. In addition to its environmental benefits, this approach offers a shorter production timeline and enhanced efficiency in both resource utilization and scalability [28]. Advanced food technologies are increasingly being explored by the space industry to ensure a safe, sustainable food supply during long-duration missions in extreme environments [29]. Cell-culture meat technology could offer viable solutions to many of the current limitations faced in space food systems. Nevertheless, several technical hurdles must still be addressed to realize its full potential [28]. One limitation in the application of this study is the use of FBS, which increases production costs compared to conventional TVPs. Therefore, further research is needed to minimize or replace FBS with cost-effective, serum-free alternatives. In addition, the current manufacturing process is relatively complex and prone to contamination, making large-scale application challenging. Future studies should aim to simplify the production workflow to improve scalability and production efficiency.

This study employed Research Use Only (RUO) products to obtain in vitro grown adipocytes. The potential expansion of bADSCs-derived adipocytes is anticipated to be developed and produced with culture materials compatible with food. Availability of edible culture materials could expedite the development of cultured meat.