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Title: Design of Experiment methodology applied for insulin and ascorbic acid concentrations in differentiation medium and calcium chloride concentration in alginate hydrogel for cultivated meat
Author: Noorul H. Ali, MS (bioengineering), Tufts University, Medford, MA 02155, United States
Correspondence: noorul.ali@tufts.edu
Abstract
Introduction
Methods
Results
Discussion
References
Appendix
Lipid storage is an important aspect of fat cells. Fat is necessary for cultured meat to resemble tradiBonal meat in texture and “mouth-feel”. Lipid storage is performed by adipocyte cells. Differentiation of cells into adipocytes is affected by insulin and ascorbic acid. Here, slightly posiBve effects of insulin and ascorbic acid on lipid storage are reported. Alginate hydrogels have demonstrated usage in industry as biomaterial scaffolds and food gels. Here, alginate hydrogel scaffolds are seeded with adipocytes and compared to ground beef in texture characteristics.
Fat is central to meat. Fat imbues muscle with the flavor and texture of meat. It renders while cooking, oxidizing to improve the texture of meat. It is significantly important for the meat’s overall flavor profile and palatability. In cultured meat, muscle and fat cells are layered on a scaffold to resemble meat derived from livestock (tradiBonal meat) in texture and structure. OpBmal amounts of lipids in fat cells, called adipocytes, are an important requirement of cultured meat. Lipid-encapsulaBng adipocyte cells form layers of fat on a scaffold. These cells are used downstream when layering muscle and fat cells into cultured meat. The accumulaBon of lipids in these adipocytes is thus of significant importance to the usability of cultured meat. OpBmizing media to maximize accumulaBon of lipids in adipocytes is an acBve area of research. The scaffold in which fat cells are arranged is also of prime importance. This scaffold must also have properBes that resemble tradiBonal fat in meat. Similar textural and structural properBes are necessary to give cultured meat a similar texture profile to tradiBonal meat. Design of experiment (DoE) is a methodology used to opBmize medium formulaBons. It has been previously used (cite) to opBmize medium formulaBons for the growth of cells. Cell growth is dependent on the medium cells are growing in. Different concentraBons of medium components affect cell growth differently. There may be interacBons between medium components as well. Design of experiment methodology varies concentraBons of medium components in a specific paUern to study the dependence of cell growth on various medium components and their interacBons. MulBple iteraBons of a medium are made. Each iteraBon differs in the concentraBon of certain medium components. Cell growth measurements are done for each medium formulaBon. These data are used to find opBmal concentraBons of medium components for highest cell growth. DoE methodology maps the dependence of each medium component to cell growth. InteracBons between medium components that posiBvely or negaBvely affect cell growth can be found using DoE. Here, we report usage of DoE methodology to find opBmal concentraBons of insulin and ascorbic acid for lipid accumulaBon in adipocyte cells. We varied the concentraBons of insulin and ascorbic acid in accumulaBon medium to find their role in accumulaBon of lipids in bovine stromal vascular cells. Alginate is a food-grade material that has been used to coat fruits and vegetables, as a microbial and viral protecBon product, and as a gelling, thickening, stabilizing or emulsifying agent [1]. Owing to its
biocompaBbility and nontoxicity, it has been used as a hydrogel and aerogel for wound dressings. Here, we use alginate hydrogel as a scaffold for fat cells. We perform texture profile analysis on hydrogel scaffolds with and without fat cells. We compare the mechanical properBes of our seeded scaffold with a tradiBonal meat control. Methods Media preparaBon for lipid accumulaBon: 25mL of inducBon medium was prepared with final concentraBons of 90% Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250µg insulin, 1 µM dexamethasone, 0.5mM isobutylmethylxanBne (IBMX), and 2 µM rosiglitazone. 9 accumulaBon medium formulaBons were prepared by varying concentraBons of insulin and ascorbic acid. ConcentraBons of both were obtained using design of experiment (4-factor, RSM central composite design) in JMP sogware (JMP StaBsBcal Discovery LLC). 100mL of base accumulaBon medium was prepared final concentraBons of 500 µg/mL Intralipid and 10 µM bioBn in DMEM. 9 media were formulated by dividing base accumulaBon medium equally adding varying amounts of insulin and ascorbic acid to each. The final concentraBons of insulin in media ranged from 0µg/mL to 8µg/mL, while ascorbic acid ranged from 0 to 120µM (Table 1). Media Ascorbic acid (µM) Insulin (µg/mL) M1 120 4 M2 0 0 M3 120 8 M4 240 4 M5 120 0 M6 0 4 M7 240 0 M8 240 8 M9 0 8 Table 1: Lipid accumulaBon media formulaBons Cell seeding and maintenance: Cryopreserved stromal vascular cells (SVCs) were thawed (37 ⁰C) and resuspended in growth medium (Gibco DMEM 1X + GlutaMAX) at 25,000 cells/mL. A 48-well plate was seeded with cells at a density of 7,500 cells/cm2. Growth medium was added to all wells Bll 90% confluency. InducBon medium was added once and respecBve accumulaBon media were added every 3 days for two weeks. A T-175 flask was seeded with SVCs at a density of 4,100 cells/cm2 and fed growth medium Bll 90% confluency. Cells in the T-175 flask were then fed inducBon medium once and accumulaBon medium twice. Oil Red O (ORO) staining and opBcal reading to test lipid accumulaBon: Fat cells in 48 well-plate were fixed in 4% formaldehyde (30 minutes). Wells were aspirated and rinsing thrice with 1X DPBS. 150µL of 1X propylene glycol was added to each well and removed. 150µL of
heated ORO soluBon (60 ⁰C) was added to each well (7 minutes) and removed. 150µL of 85% propylene glycol was added to each well (1 minute). Cells were washed with disBlled water . In a fume hood, 150 µL of 1X isopropanol was added to each well to elute the ORO stain. 100µL isopropanol supernatant from each well was transferred to a 96-well plate with two control wells containing 100µL isopropanol. Absorbance at 540nm was measured using a plate reader . Mean opBcal density measurements were used for each well because we had triplicated wells for each medium. The measurement was normalized by subtracBng the mean measurement of control isopropanol wells. Scaffold construcBon and seeding: Scaffold control was prepared with final concentraBons 10% calcium chloride and 1.8% alginate in water. The amounts of calcium chloride were varied from 10µL to 300µL in fixed alginate soluBons. Textures of formed hydrogels were observed under mulBple temperatures (4 ⁰C, 22 ⁰C, 37 ⁰C) and media condiBons (DPBS, standard growth medium, disBlled water) for 1 week. Hydrogel formed with 10% calcium chloride most resembled fat extracellular matrix (ECM) and was chosen for seeding. Fat cells were scraped and collected from T-175 flask. Seeded scaffold was prepared mixing 5% calcium chloride, 0.9% alginate and 0.9% scraped fat cells. Measurement of scaffold structural properBes: Texture p rofile analysis was performed on cont rol scaffold, seeded scaffold, and ground be ef. Analysis tested for hardness, adhesiveness, resilience, cohesion, springiness, gumminess and chewiness. A porBon of the sample was inserted into a texture profile analyzer (TA.XTPlus 100 Connect, Texture Technologies Corp. and Stable Micro Systems, Ltd.) to measure the mechanical properBes listed above for each sample. Results Insulin and ascorbic acid weakly promote lipid accumulaBon: PosiBve correlaBons are seen between both insulin and ascorbic acid concentraBons and lipid accumulaBon in adipocytes (Table 2). This suggests both posiBvely influence growth, though weakly as indicated by the low correlaBon scores. No correlaBon is observed between ascorbic acid and insulin concentraBons. We find no evidence of insulin and ascorbic acid concentraBons affecBng cell growth or interacBon between the two, evidenced by the null correlaBon score. Media with no insulin showed lower lipid accumulaBon (M2, M5). Only slightly lower lipid accumulaBon is observed in media without ascorbic acid and insulin (M2). Media without insulin (M2, M5) ranked lowest in lipid accumulaBon.
Figure 1: Optical density readings of eluted ORO stain. This reading is indicative of the quantity of lipids in cells. ORO is absorbed by lipids in cells and eluted from cells in the presence of isopropanol. Higher absorbance assay measurement indicates higher lipid accumulation. Readings are normalized by subtracting mean absorbance assay measurement of control well containing 1X isopropanol. Correlation matrix Lipid accumulation Ascorbic acid Insulin Lipid accumulation 1 0.3565 0.3565 Ascorbic acid 0.3565 1 0 Insulin 0.3565 0 1 Table 2: Correlation matrix between ascorbic acid concentration, insulin concentration, and lipid accumulation. Numbers represent correlation scores between mean optical density readings, insulin, and ascorbic acid concentrations. Correlation score is a metric of how strongly two variables are linked in multivariate analysis. No correlation is observed between insulin and ascorbic acid. Both are equally important to lipid accumulation according to the same correlation score obtained for both with respect to lipid accumulation. Seeded scaffold significantly differs from texture of ground beef: Control and seeded scaffolds (figure 9) differ significantly from ground beef (figures 2-8). Resilience and cohesion are relaBvely closer to ground beef control. Hardness, adhesiveness, cohesion, springiness, gumminess, and chewiness of both control and seeded scaffolds are significantly different from ground beef. These are mechanical properBes calculated by stress and strain measurements done by the texture analyzer . Control and seeded scaffolds are relaBvely similar in texture characterisBcs.
Figures 2-8: Texture profile comparisons between ground meat, alginate scaffolds seeded with and without cells (control) in terms of hardness, adhesiveness, resilience, cohesion, springiness, gumminess, and chewiness. Alginate control and seeded scaffolds differ significantly from control ground beef.
Figure 9: Unseeded alginate hydrogels in DPBS, water and no extra medium (storage)
Figure 10: Cells in T-175 flask in adipogenic accumulation medium. Globules are seen, which may be adipocytes.
Figure 11: Cells in M7 adipogenic accumulation medium. This well had relatively high lipid accumulation. Some globules can be seen.
Discussion Insulin and ascorbic acid generally promote lipid storage: We observed lowest lipid storage in cells accumulaBng lipids in two media formulaBons without insulin. Insulin is implicated in adipogenesis and lipid storage. Peroxisome proliferator-acBvated receptor γ (PPARγ) is a ligand-dependent transcripBon factor highly expressed in adipocytes and a master regulator of adipogenesis and lipid storage [2]. It upregulates adipocyte-specific proteins. Insulin is involved in cross-talk with PPARγ [3]. It sBmulates transcripBon factors involved in adipogenic differenBaBon. It also prevents lipolysis by inhibiBng hormone-sensiBve lipase. This seems to be one explanaBon for the observed lower lipid storage by cells in media without insulin. Lower adipogenic differenBaBon due to lack of insulin may be a cause for lower lipid content observed. For a future study, insulin may be varied along with cellular cycle arrest to observe its effects on differenBaBon, parBcularly the stage at which it is most effecBve at inducing adipogenic differenBaBon. Ascorbic acid is implicated in transcripBon of FABP4 which is directly coupled to PPARγ. Increased expression of FABP4 is linked to greater lipid accumulaBon [4-10]. Ascorbic acid increases adipogenic differenBaBon. Wells with high lipid storage contained ascorbic acid. 3D fat constructs were prepared using alginate hydrogels seeded with stromal vascular cells. Alginate hydrogels serve the funcBon of extracellular matrix (ECM) in which cells are embedded in tradiBonal meat Bssue. ECM consists of collagen and other materials. Alginate-derived hydrogels were selected for opBmal texture resembling livestock-derived fat Bssue and stability in different temperature and media. OpBmal hydrogel was seeded with lipid-containing adipocytes. Texture profile analysis was performed to compare the mechanical properBes of seeded and unseeded scaffolds with tradiBonal ground meat. Texture profile analysis (TPA) is a popular double compression test for determining the textural properBes of foods. It is occasionally used in other industries, such as pharmaceuBcals, gels, and personal care. During a TPA test samples are compressed twice using a texture analyzer to provide insight into how samples behave when chewed. The TPA test was ogen called the "two bite test" because the texture analyzer mimics the mouth's biBng acBon. Our scaffolds significantly differed in texture to ground beef. This was primarily because we selected a gel-like consistency in our manual selecBon of calcium chloride concentraBon in alginate soluBon. Alginate is a naturally derived polysaccharide. Calcium ions cause ionic crosslinking in alginate’s consBtuents by interacBng with carboxylic groups. This forms 3D units insoluble in water, or hydrogels. We selected a relaBvely lower calcium concentraBon in the hydrogel. This significantly impacted hardness, adhesiveness, gumminess, springiness, and chewiness metrics because we selected for a gel-like consistency. Unlike tradiBonal ground beef, the alginate hydrogel had higher cohesion and resilience. These metrics indicate how well a material regains its structure ager compression. Our gel-like hydrogel was much more fluid than ground beef hence had higher cohesion and resilience scores. Our scores for hardness, chewiness, gumminess, adhesiveness, and springiness were significantly different compared to ground beef because we selected for a gel-like consistency. Further work can be done to improve texture profile metrics by varying concentraBons of calcium chloride to form alginate hydrogels. Calcium chloride is linked to alginate gelaBon and its mechanical
properBes. A suitable concentraBon of calcium chloride that achieves similar texture profile metrics to ground beef may be useful as scaffold. References [1]: Gheorghita Puscaselu R, Lobiuc A, Dimian M, Covasa M. Alginate: From Food Industry to Biomedical ApplicaBons and Management of Metabolic Disorders. Polymers (Basel). 2020 Oct 20;12(10):2417. doi: 10.3390/polym12102417. PMID: 33092194; PMCID: PMC7589871. [2]: Ma X, Wang D, Zhao W, Xu L. Deciphering the Roles of PPARγ in Adipocytes via Dynamic Change of TranscripBon Complex. Front Endocrinol (Lausanne). 2018 Aug 21;9:473. doi: 10.3389/fendo.2018.00473. PMID: 30186237; PMCID: PMC6110914. [3]: Leonardini A, Laviola L, Perrini S, Natalicchio A, Giorgino F. Cross-Talk between PPARgamma and Insulin Signaling and ModulaBon of Insulin SensiBvity. PPAR Res. 2009;2009:818945. doi: 10.1155/2009/818945. Epub 2010 Feb 23. PMID: 20182551; PMCID: PMC2826877. [4]: Jurek, Sandra, Mansur A. Sandhu, Susanne Trappe, M. Carmen Bermúdez-Peña, MarBn Kolisek, Gerhard Sponder, and Jörg R. Aschenbach. “OpBmizing Adipogenic TransdifferenBaBon of Bovine Mesenchymal Stem Cells: A Prominent Role of Ascorbic Acid in FABP4 InducBon.” Adipocyte 9, no. 1 (2020): 35–50. doi:10.1080/21623945.2020.1720480. [5]: Cuaranta-Monroy I, Simandi Z, Kolostyak Z, et al. Highly effcient differenBaBon of embryonic stem cells into adipocytes by ascorbic acid. Stem Cell Res. 2014;13:88–97. [6]: Furuhashi M, Hotamisligil GS. FaUy acid-binding proteins: role in metabolic diseases and potenBal as drug targets. Nat Rev Drug Discov. 2008;7:489. [7]: Tontonoz P, Spiegelman BM. Fat and beyond: the diverse biology of PPARγ. Annu Rev Biochem. 2008;77:289–312. [8]: Garin-Shkolnik T, Rudich A, Hotamisligil GS, et al. FABP4 aUenuates PPARγ and adipogenesis and is inversely correlated with PPARγ in adipose Bssues. Diabetes. 2014;63:900–911. [9]: Ono M, Aratani Y , Kitagawa I, et al. Ascorbic acid phosphate sBmulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells. Exp Cell Res. 1990;187:309–314. [10]: Lee OH, Seo DH, Park CS, et al. Puerarin enhances adipocyte differenBaBon, adiponecBn expression, and anBoxidant response in 3T3-L1 cells. Biofactors. 2010;36:459–467.
Design of Experiment methodology applied for insulin and ascorbic acid concentrations in differentiation medium and calcium chloride concentration in alginate hydrogel for cultivated meat Noorul Hasan Ali, MS (bioengineering), Tufts University Abstract Lipid storage is an important aspect of fat cells. Fat is necessary for cultured meat to resemble tradiBonal meat in texture and “mouth-feel”. Lipid storage is performed by adipocyte cells. DifferenBaBon of cells into adipocytes is affected by insulin and ascorbic acid. Here, slightly posiBve effects of insulin and ascorbic acid on lipid storage are reported. Alginate hydrogels have demonstrated usage in industry as biomaterial scaffolds and food gels. Here, alginate hydrogel scaffolds are seeded with adipocytes and compared to ground beef in texture characterisBcs. IntroducBon Fat is central to meat. Fat imbues muscle with the flavor and texture of meat. It renders while cooking, oxidizing to improve the texture of meat. It is significantly important for the meat’s overall flavor profile and palatability. In cultured meat, muscle and fat cells are layered on a scaffold to resemble meat derived from livestock (tradiBonal meat) in texture and structure. OpBmal amounts of lipids in fat cells, called adipocytes, are an important requirement of cultured meat. Lipid-encapsulaBng adipocyte cells form layers of fat on a scaffold. These cells are used downstream when layering muscle and fat cells into cultured meat. The accumulaBon of lipids in these adipocytes is thus of significant importance to the usability of cultured meat. OpBmizing media to maximize accumulaBon of lipids in adipocytes is an acBve area of research. The scaffold in which fat cells are arranged is also of prime importance. This scaffold must also have properBes that resemble tradiBonal fat in meat. Similar textural and structural properBes are necessary to give cultured meat a similar texture profile to tradiBonal meat. Design of experiment (DoE) is a methodology used to opBmize medium formulaBons. It has been previously used (cite) to opBmize medium formulaBons for the growth of cells. Cell growth is dependent on the medium cells are growing in. Different concentraBons of medium components affect cell growth differently. There may be interacBons between medium components as well. Design of experiment methodology varies concentraBons of medium components in a specific paUern to study the dependence of cell growth on various medium components and their interacBons. MulBple iteraBons of a medium are made. Each iteraBon differs in the concentraBon of certain medium components. Cell growth measurements are done for each medium formulaBon. These data are used to find opBmal concentraBons of medium components for highest cell growth. DoE methodology maps the dependence of each medium component to cell growth. InteracBons between medium components that posiBvely or negaBvely affect cell growth can be found using DoE. Here, we report usage of DoE methodology to find opBmal concentraBons of insulin and ascorbic acid for lipid accumulaBon in adipocyte cells. We varied the concentraBons of insulin and ascorbic acid in accumulaBon medium to find their role in accumulaBon of lipids in bovine stromal vascular cells. Alginate is a food-grade material that has been used to coat fruits and vegetables, as a microbial and viral protecBon product, and as a gelling, thickening, stabilizing or emulsifying agent [1]. Owing to its
biocompaBbility and nontoxicity, it has been used as a hydrogel and aerogel for wound dressings. Here, we use alginate hydrogel as a scaffold for fat cells. We perform texture profile analysis on hydrogel scaffolds with and without fat cells. We compare the mechanical properBes of our seeded scaffold with a tradiBonal meat control. Methods Media preparaBon for lipid accumulaBon: 25mL of inducBon medium was prepared with final concentraBons of 90% Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250µg insulin, 1 µM dexamethasone, 0.5mM isobutylmethylxanBne (IBMX), and 2 µM rosiglitazone. 9 accumulaBon medium formulaBons were prepared by varying concentraBons of insulin and ascorbic acid. ConcentraBons of both were obtained using design of experiment (4-factor, RSM central composite design) in JMP sogware (JMP StaBsBcal Discovery LLC). 100mL of base accumulaBon medium was prepared final concentraBons of 500 µg/mL Intralipid and 10 µM bioBn in DMEM. 9 media were formulated by dividing base accumulaBon medium equally adding varying amounts of insulin and ascorbic acid to each. The final concentraBons of insulin in media ranged from 0µg/mL to 8µg/mL, while ascorbic acid ranged from 0 to 120µM (Table 1). Media Ascorbic acid (µM) Insulin (µg/mL) M1 120 4 M2 0 0 M3 120 8 M4 240 4 M5 120 0 M6 0 4 M7 240 0 M8 240 8 M9 0 8 Table 1: Lipid accumulaBon media formulaBons Cell seeding and maintenance: Cryopreserved stromal vascular cells (SVCs) were thawed (37 ⁰C) and resuspended in growth medium (Gibco DMEM 1X + GlutaMAX) at 25,000 cells/mL. A 48-well plate was seeded with cells at a density of 7,500 cells/cm2. Growth medium was added to all wells Bll 90% confluency. InducBon medium was added once and respecBve accumulaBon media were added every 3 days for two weeks. A T-175 flask was seeded with SVCs at a density of 4,100 cells/cm2 and fed growth medium Bll 90% confluency. Cells in the T-175 flask were then fed inducBon medium once and accumulaBon medium twice. Oil Red O (ORO) staining and opBcal reading to test lipid accumulaBon: Fat cells in 48 well-plate were fixed in 4% formaldehyde (30 minutes). Wells were aspirated and rinsing thrice with 1X DPBS. 150µL of 1X propylene glycol was added to each well and removed. 150µL of
heated ORO soluBon (60 ⁰C) was added to each well (7 minutes) and removed. 150µL of 85% propylene glycol was added to each well (1 minute). Cells were washed with disBlled water . In a fume hood, 150 µL of 1X isopropanol was added to each well to elute the ORO stain. 100µL isopropanol supernatant from each well was transferred to a 96-well plate with two control wells containing 100µL isopropanol. Absorbance at 540nm was measured using a plate reader . Mean opBcal density measurements were used for each well because we had triplicated wells for each medium. The measurement was normalized by subtracBng the mean measurement of control isopropanol wells. Scaffold construcBon and seeding: Scaffold control was prepared with final concentraBons 10% calcium chloride and 1.8% alginate in water. The amounts of calcium chloride were varied from 10µL to 300µL in fixed alginate soluBons. Textures of formed hydrogels were observed under mulBple temperatures (4 ⁰C, 22 ⁰C, 37 ⁰C) and media condiBons (DPBS, standard growth medium, disBlled water) for 1 week. Hydrogel formed with 10% calcium chloride most resembled fat extracellular matrix (ECM) and was chosen for seeding. Fat cells were scraped and collected from T-175 flask. Seeded scaffold was prepared mixing 5% calcium chloride, 0.9% alginate and 0.9% scraped fat cells. Measurement of scaffold structural properBes: Texture p rofile analysis was performed on cont rol scaffold, seeded scaffold, and ground be ef. Analysis tested for hardness, adhesiveness, resilience, cohesion, springiness, gumminess and chewiness. A porBon of the sample was inserted into a texture profile analyzer (TA.XTPlus 100 Connect, Texture Technologies Corp. and Stable Micro Systems, Ltd.) to measure the mechanical properBes listed above for each sample. Results Insulin and ascorbic acid weakly promote lipid accumulaBon: PosiBve correlaBons are seen between both insulin and ascorbic acid concentraBons and lipid accumulaBon in adipocytes (Table 2). This suggests both posiBvely influence growth, though weakly as indicated by the low correlaBon scores. No correlaBon is observed between ascorbic acid and insulin concentraBons. We find no evidence of insulin and ascorbic acid concentraBons affecBng cell growth or interacBon between the two, evidenced by the null correlaBon score. Media with no insulin showed lower lipid accumulaBon (M2, M5). Only slightly lower lipid accumulaBon is observed in media without ascorbic acid and insulin (M2). Media without insulin (M2, M5) ranked lowest in lipid accumulaBon.
Figure 1: Optical density readings of eluted ORO stain. This reading is indicative of the quantity of lipids in cells. ORO is absorbed by lipids in cells and eluted from cells in the presence of isopropanol. Higher absorbance assay measurement indicates higher lipid accumulation. Readings are normalized by subtracting mean absorbance assay measurement of control well containing 1X isopropanol. Correlation matrix Lipid accumulation Ascorbic acid Insulin Lipid accumulation 1 0.3565 0.3565 Ascorbic acid 0.3565 1 0 Insulin 0.3565 0 1 Table 2: Correlation matrix between ascorbic acid concentration, insulin concentration, and lipid accumulation. Numbers represent correlation scores between mean optical density readings, insulin, and ascorbic acid concentrations. Correlation score is a metric of how strongly two variables are linked in multivariate analysis. No correlation is observed between insulin and ascorbic acid. Both are equally important to lipid accumulation according to the same correlation score obtained for both with respect to lipid accumulation. Seeded scaffold significantly differs from texture of ground beef: Control and seeded scaffolds (figure 9) differ significantly from ground beef (figures 2-8). Resilience and cohesion are relaBvely closer to ground beef control. Hardness, adhesiveness, cohesion, springiness, gumminess, and chewiness of both control and seeded scaffolds are significantly different from ground beef. These are mechanical properBes calculated by stress and strain measurements done by the texture analyzer . Control and seeded scaffolds are relaBvely similar in texture characterisBcs.
Figures 2-8: Texture profile comparisons between ground meat, alginate scaffolds seeded with and without cells (control) in terms of hardness, adhesiveness, resilience, cohesion, springiness, gumminess, and chewiness. Alginate control and seeded scaffolds differ significantly from control ground beef.
Figure 9: Unseeded alginate hydrogels in DPBS, water and no extra medium (storage)
Figure 10: Cells in T-175 flask in adipogenic accumulation medium. Globules are seen, which may be adipocytes.
Figure 11: Cells in M7 adipogenic accumulation medium. This well had relatively high lipid accumulation. Some globules can be seen.
Discussion Insulin and ascorbic acid generally promote lipid storage: We observed lowest lipid storage in cells accumulaBng lipids in two media formulaBons without insulin. Insulin is implicated in adipogenesis and lipid storage. Peroxisome proliferator-acBvated receptor γ (PPARγ) is a ligand-dependent transcripBon factor highly expressed in adipocytes and a master regulator of adipogenesis and lipid storage [2]. It upregulates adipocyte-specific proteins. Insulin is involved in cross-talk with PPARγ [3]. It sBmulates transcripBon factors involved in adipogenic differenBaBon. It also prevents lipolysis by inhibiBng hormone-sensiBve lipase. This seems to be one explanaBon for the observed lower lipid storage by cells in media without insulin. Lower adipogenic differenBaBon due to lack of insulin may be a cause for lower lipid content observed. For a future study, insulin may be varied along with cellular cycle arrest to observe its effects on differenBaBon, parBcularly the stage at which it is most effecBve at inducing adipogenic differenBaBon. Ascorbic acid is implicated in transcripBon of FABP4 which is directly coupled to PPARγ. Increased expression of FABP4 is linked to greater lipid accumulaBon [4-10]. Ascorbic acid increases adipogenic differenBaBon. Wells with high lipid storage contained ascorbic acid. 3D fat constructs were prepared using alginate hydrogels seeded with stromal vascular cells. Alginate hydrogels serve the funcBon of extracellular matrix (ECM) in which cells are embedded in tradiBonal meat Bssue. ECM consists of collagen and other materials. Alginate-derived hydrogels were selected for opBmal texture resembling livestock-derived fat Bssue and stability in different temperature and media. OpBmal hydrogel was seeded with lipid-containing adipocytes. Texture profile analysis was performed to compare the mechanical properBes of seeded and unseeded scaffolds with tradiBonal ground meat. Texture profile analysis (TPA) is a popular double compression test for determining the textural properBes of foods. It is occasionally used in other industries, such as pharmaceuBcals, gels, and personal care. During a TPA test samples are compressed twice using a texture analyzer to provide insight into how samples behave when chewed. The TPA test was ogen called the "two bite test" because the texture analyzer mimics the mouth's biBng acBon. Our scaffolds significantly differed in texture to ground beef. This was primarily because we selected a gel-like consistency in our manual selecBon of calcium chloride concentraBon in alginate soluBon. Alginate is a naturally derived polysaccharide. Calcium ions cause ionic crosslinking in alginate’s consBtuents by interacBng with carboxylic groups. This forms 3D units insoluble in water, or hydrogels. We selected a relaBvely lower calcium concentraBon in the hydrogel. This significantly impacted hardness, adhesiveness, gumminess, springiness, and chewiness metrics because we selected for a gel-like consistency. Unlike tradiBonal ground beef, the alginate hydrogel had higher cohesion and resilience. These metrics indicate how well a material regains its structure ager compression. Our gel-like hydrogel was much more fluid than ground beef hence had higher cohesion and resilience scores. Our scores for hardness, chewiness, gumminess, adhesiveness, and springiness were significantly different compared to ground beef because we selected for a gel-like consistency. Further work can be done to improve texture profile metrics by varying concentraBons of calcium chloride to form alginate hydrogels. Calcium chloride is linked to alginate gelaBon and its mechanical
properBes. A suitable concentraBon of calcium chloride that achieves similar texture profile metrics to ground beef may be useful as scaffold. References [1]: Gheorghita Puscaselu R, Lobiuc A, Dimian M, Covasa M. Alginate: From Food Industry to Biomedical ApplicaBons and Management of Metabolic Disorders. Polymers (Basel). 2020 Oct 20;12(10):2417. doi: 10.3390/polym12102417. PMID: 33092194; PMCID: PMC7589871. [2]: Ma X, Wang D, Zhao W, Xu L. Deciphering the Roles of PPARγ in Adipocytes via Dynamic Change of TranscripBon Complex. Front Endocrinol (Lausanne). 2018 Aug 21;9:473. doi: 10.3389/fendo.2018.00473. PMID: 30186237; PMCID: PMC6110914. [3]: Leonardini A, Laviola L, Perrini S, Natalicchio A, Giorgino F. Cross-Talk between PPARgamma and Insulin Signaling and ModulaBon of Insulin SensiBvity. PPAR Res. 2009;2009:818945. doi: 10.1155/2009/818945. Epub 2010 Feb 23. PMID: 20182551; PMCID: PMC2826877. [4]: Jurek, Sandra, Mansur A. Sandhu, Susanne Trappe, M. Carmen Bermúdez-Peña, MarBn Kolisek, Gerhard Sponder, and Jörg R. Aschenbach. “OpBmizing Adipogenic TransdifferenBaBon of Bovine Mesenchymal Stem Cells: A Prominent Role of Ascorbic Acid in FABP4 InducBon.” Adipocyte 9, no. 1 (2020): 35–50. doi:10.1080/21623945.2020.1720480. [5]: Cuaranta-Monroy I, Simandi Z, Kolostyak Z, et al. Highly effcient differenBaBon of embryonic stem cells into adipocytes by ascorbic acid. Stem Cell Res. 2014;13:88–97. [6]: Furuhashi M, Hotamisligil GS. FaUy acid-binding proteins: role in metabolic diseases and potenBal as drug targets. Nat Rev Drug Discov. 2008;7:489. [7]: Tontonoz P, Spiegelman BM. Fat and beyond: the diverse biology of PPARγ. Annu Rev Biochem. 2008;77:289–312. [8]: Garin-Shkolnik T, Rudich A, Hotamisligil GS, et al. FABP4 aUenuates PPARγ and adipogenesis and is inversely correlated with PPARγ in adipose Bssues. Diabetes. 2014;63:900–911. [9]: Ono M, Aratani Y , Kitagawa I, et al. Ascorbic acid phosphate sBmulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells. Exp Cell Res. 1990;187:309–314. [10]: Lee OH, Seo DH, Park CS, et al. Puerarin enhances adipocyte differenBaBon, adiponecBn expression, and anBoxidant response in 3T3-L1 cells. Biofactors. 2010;36:459–467.