Conceived and designed the experiments: NSS DN. Performed the experiments: NSS DN. Analyzed the data: NSS DN. Contributed reagents/materials/analysis tools: NSS DN MLY. Wrote the paper: NSS DN.
The authors have declared that no competing interests exist.
The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.
Hepatocytes constitute about 70% of the cellular population of the liver and play an indispensable role in over 500 metabolic, regulatory and immune functions
Various methodologies to maintain hepatocytes
Thus, influence of ECM on hepatocyte culture systems is important from two perspectives. From an application standpoint, development of scalable, simple, “in vivo” like and cost-effective
Variation in ECM compositions including addition of glycosaminoglycans and hepatic proteoglycans that promotes formation of gap junctions
Moreover, the developed
We have previously developed a system to synthesize mammalian preadipocyte cell secreted extracellular matrix proteins termed Adipogel in copius amounts. Adipogel has been shown to exhibit at least fibronectin [the most abundant ECM in the Space of Disse] and collagen IV that is present in the Space of Disse. We have also shown that Adipogel laden on top of collagen single gel hepatocyte cultures increased albumin differentiated function as compared to collagen double gel cultures
Numerous researchers have investigated the intermediary metabolism of hepatocytes, the primary functional cells of the liver
Dulbecco's Modified Eagle Medium [DMEM] containing 4.5 g/L glucose, fetal bovine serum [FBS], penicillin, streptomycin, and trypsin–EDTA were obtained from Invitrogen Life technologies [Carlsbad, CA]. Dexamethasone, isobutyl-methylxanthine, epidermal growth factor, insulin, glucagon, rosiglitazone and hydrocortisone were purchased from Sigma-Aldrich [St. Louis, MO]. 100 kDa cutoff protein centrifugal filters were purchased from Millipore Technologies [Billerica, MA].
Adipogel was generated from differentiating murine preadipocytes as described previously. Briefly, 3T3-L1 murine preadipocytes were cultured in T-175 cm2 flasks in Dulbecco's Modified Eagle's media supplemented with 10% FBS, 2% Penicillin and Streptomycin till the cells attained confluency. 48 hours post confluency, the cells were differentiated in culture media supplemented with 1 µM dexamethasone, 0.1 µM isobutyl-methylxanthine and 1 µM rosiglitazone for 2 days with media changes every two days. On the second day post-differentiation, cells were exposed to culture medium supplemented with 1 µM rosiglitazone for an additional 2 days. Media supernatant was collected on days 2 and 4 of differentiation and stored at 4°C prior to further processing.
In order to purify the extracellular matrix rich material, the differentiated preadipocyte conditioned media was centrifuged at 4000
Four different conditions were utilized for the metabolite measurements. Collagen single gel [CSG], collagen double gel [CDG] collagen-soluble Adipogel sandwich [CSG+solASG] and collagen-Adipogel sandwich cultures [CSGASG]. Secreted products were measured at the recovery stage, pre-stable stage and stable stage of culture. Urea and albumin synthesis was determined from day 3 to day 10 of culture.
Female Lewis rats [Charles River Laboratories, Wilmington, MA] weighing 180 to 200 g [2 to 3 months old] were used as a hepatocyte source and were maintained in accordance with National Research Council guidelines. Experimental protocols were approved by the Subcommittee on Animal Care, Committee on Research, Massachusetts General Hospital (Protocol # 2005N000130). Using a modification of the two-step collagenase perfusion method
Type 1 collagen was prepared by extracting acid-soluble collagen from Lewis rat-tail tendons. To create a thin layer of collagen gel in 12-well tissue culture plates, 400 µl of an ice-cold mixture of 1 part of 10× concentrated DMEM and 9 parts of 1.25 mg/ml rat tail tendon type I collagen were evenly distributed over the bottom of each well. The plates were incubated at 37°C for 60 min to induce collagen gelation before cell seeding. Each well of the 12-well culture plates received 5×105 primary hepatocytes in suspension in 0.5 ml standard hepatocyte culture medium, which consisted of DMEM supplemented with 14 ng/ml glucagon, 7.5 µg/ml hydrocortisone, 0.5 U/ml insulin, 20 ng/ml EGF, 200 U/ml penicillin, 200 µg/ml streptomycin, and 10% FBS. Cultures were incubated in 90% air/10% CO2 at 37°C. Cells were rinsed with 1× PBS to remove non-adherent cells 4–6 hours after seeding. For the double collagen gel culture configuration, a second layer of 250 µl collagen was laden on top of the cells 48 hours post-seeding [
Albumin concentration in the collected medium samples was analyzed using a competitive enzyme-linked immunosorbent assay [ELISA]. Albumin protein and the antibody were purchased from MP Biomedicals. Urea concentration was determined via its specific reaction with diacetyl monoxime with a commercially available assay kit [Fisher Scientific, Pittsburgh, PA]. The absorbance was measured with a Thermomax microplate reader [Molecular Devices, Sunnyvale, CA]
The biochemical assays were performed at the recovery, pre-stable and stable stages of culture with media samples. Amino acids were fluorescently labeled using the AccQ-Tag system [Waters Co., Milford, MA], separated by high performance liquid chromatography [HPLC Model 2690, Waters Co.], and quantitated by a fluorescence detector [Model 474, Waters Co.]. Glucose and lactate levels were measured with commercially available kits [Sigma], the former based on the reaction of glucose catalyzed by glucose oxidase and the latter based on the conversion of lactate to pyruvate catalyzed by lactate oxidase. Acetoacetate and β-hydroxybutyrate were measured using a commercially available kit [Bioassay Systems].
MFA is a useful methodology to characterize the differential activation of metabolic pathways in hepatocyte cultures. Thus, based on a stoichiometric model for the metabolic reaction network prevalent in hepatocytes, intracellular reaction fluxes are estimated by mass balances around each intracellular metabolite and extracellular flux measurements. This gives the possibility to calculate intracellular metabolite fluxes, which are difficult to measure from relatively few measurements and to corroborate the metabolic network. The model used in this work was originally developed for perfused liver
The metabolic network is based on known stoichiometry of hepatic intermediary metabolism with consideration of carbon and nitrogen balances.
Albumin is a major protein product of hepatocytes and hence only this protein is considered.
The cellular uptake/secretion rates of metabolites are distinct from the intracellular fluxes of the corresponding metabolites. Thus, the intracellular and extracellular pools of substrates have been distinguished. Also, the mechanisms of active and passive transport have not been incorporated.
The metabolite pools are at pseudo steady-state with a single pool in the cell. The influx and efflux of metabolites into/from hepatocytes are calculated from the amount of metabolites remaining in the extracellular media after 24 h.
Following the above assumptions, the mathematical model consists of mass balances around 45 intracellular metabolites [
MetabolitePools |
Glucose-6Phosphate |
Ribulose-5Phosphate |
Ribose-5Phosphate |
Xylulose-5Phosphate |
Erythrose-4Phosphate |
Glyceraldehyde-3Phosphate |
Fructose-6Phosphate |
Fructose-1,6BisPhosphate |
PhosphoEnolPyruvate |
Pyruvate |
NADH |
FADH2 |
Acetyl-CoA |
Oxaloacetate |
Citrate |
2-oxo-gluterate |
Succinyl-CoA |
Fumarate |
Malate |
Ammonia |
Ornithine |
Citrulline |
Acetoacetyl-CoA |
Acetoacetate |
Alanine |
Cysteine |
Aspartate |
Glutamate |
Phenylalanine |
Glycine |
Histidine |
Isoleucine |
Lysine |
Leucine |
Methionine |
Asparagine |
Proline |
Glutamine |
Arginine |
Serine |
Threonine |
Valine |
Tyrosine |
CO2 |
Oxygen |
A total of 45 metabolites have been listed in the table.
Each data point represents the mean of two or three experiments [each with three biological replicates], and the error bars represent the standard error of the mean. Data was normalized to number of cells initially seeded and expressed as µmol/million cells/day.
For extracellular metabolite measurements, the data-sets for each metabolite flux for each experimental condition were averaged from the sum of all replicates per experimental condition. The standard error of the mean was calculated from replicate data-set for each experimental condition. The mean and standard error of the mean of the extracellular metabolite measurements is quantitatively represented in the
RecoveryStage | |||||
Fluxno. | Metabolites | CSG | CDG | CSG+solASG | CSG+ASG |
1 | Glucose | −0.41±0.016 | −0.388±0.362 | −0.873±0.495 | −0.373±0.419 |
14 | Lactate | 0.582±0.092 |
0.743±0.061 | 1.274±0.555 | 0.971±0.759 |
23 | Urea | 1.952±0.051 |
1.479±0.123 | 1.828±0.442 | 1.533±0.312 |
45 | βhydroxybutyrate | −0.003±0.016 | −0.012±0.002 | 0.041±0.011 | 0.019±0.003 |
48 | Albumin | 0.0001±0 |
0.0002±0 | 0.0008±0.0004 |
0.0003±0.0002 |
49 | O2 | 0.916±0.128 | 1.169±0.163 | 2.005±0.28 | 1.528±0.213 |
50 | CO2 | 0.102±0.018 | 0.13±0.023 | 0.223±0.039 | 0.17±0.029 |
51 | Acetoacetate | 0.124±0.005 | 0.257±0.149 | 0.094±0.059 | 0.133±0.097 |
52 | Ornithine | −0.04±0.014 |
−0.067±0.015 | −0.031±0.027 |
−0.04±0.018 |
53 | Ammonia | −0.562±0.045 | −0.599±0.015 | −0.646±0.028 |
−0.601±0.006 |
55 | Cysteine | 0.016±0.067 | −0.04±0.02 | 0.059±0.056 |
0.053±0.052 |
56 | Aspartate | 0.017±0.021 | 0.005±0.005 | 0.006±0.012 | −0.002±0.01 |
57 | Glutamate | 0.027±0.075 | 0.02±0.014 | 0.042±0.027 | 0.032±0.011 |
58 | Phenylalanine | −0.049±0.035 | −0.052±0.015 | −0.126±0.041 |
−0.109±0.015 |
59 | Glycine | −0.163±0.075 | −0.167±0.01 | −0.209±0.026 |
−0.206±0.017 |
60 | Histidine | −0.256±0.121 | −0.176±0.04 | −0.511±0.179 |
−0.298±0.038 |
61 | Isoleucine | −0.025±0.025 | −0.03±0.016 | −0.092±0.042 |
−0.073±0.023 |
62 | Lysine | −0.116±0.083 | −0.159±0.059 | −0.285±0.097 |
−0.244±0.051 |
63 | Leucine | −0.03±0.031 | −0.036±0.019 | −0.135±0.087 |
−0.085±0.025 |
65 | Asparagine | −0.008±0 | −0.005±0 | 0±0 | −0.004±0 |
66 | Proline | −0.03±0.02 | −0.027±0.003 | −0.012±0.027 | −0.002±0.021 |
67 | Glutamine | 8.017±22.261 | 0.887±0.627 | 0.183±0.118 | 0.87±0.287 |
68 | Arginine | −0.021±0.071 | 0.139±0.227 | 0.183±0.118 |
−0.006±0.03 |
69 | Serine | −0.104±0.066 | −0.08±0.018 | −0.052±0.052 |
−0.13±0.004 |
70 | Threonine | −0.277±0.105 | −0.316±0.035 | −0.165±0.046 |
−0.375±0.023 |
71 | Valine | −0.018±0.031 | −0.025±0.027 | −0.373±0.018 |
−0.078±0.027 |
72 | Tyrosine | −0.169±0.007 |
−0.146±0.01 | −0.135±0.085 |
−0.185±0.009 |
Hepatocytes were cultured in four different configurations at a density of 500,000 cells/well in a 12 well plates for 10 days.
*
Pre-stableStage | |||||
Fluxno. | Metabolites | CSG | CDG | CSG+solASG | CSG+ASG |
1 | Glucose | 0.37±0.046 | 0.222±0.274 | −0.03±0.047 |
0.297±2.35 |
14 | Lactate | 0.393±0.359 | 0.001±0 | 0.883±0.196 | 0.637±0.546 |
23 | Urea | 0.97±0.06 |
1.852±0.13 | 1.869±0.135 | 1.555±0.486 |
45 | βhydroxybutyrate | −0.021±0.01 | −0.012±0.016 | 0.026±0.003 | −0.01±0.003 |
48 | Albumin | 0.0001±0 |
0±0 | 0.001±0.001 |
0.001±0 |
49 | O2 | 0.619±0.086 | 0.002±0.001 | 1.39±0.194 | 1.003±0.14 |
50 | CO2 | 0.062±0.009 | 0.0002±0.0001 | 0.139±0.02 | 0.1±0.014 |
51 | Acetoacetate | −0.054±0.014 | −0.011±0.025 | 0.054±0.013 |
−0.051±0.023 |
52 | Ornithine | −0.048±0.016 | −0.082±0.003 |
−0.023±0.013 | −0.066±0.01 |
53 | Ammonia | −0.394±0.135 | −0.638±0.008 |
−0.645±0.047 | −0.615±0.03 |
55 | Cysteine | −0.007±0.059 | −0.042±0.005 | 0.083±0.02 |
0.008±0.024 |
56 | Aspartate | 0.002±0.011 | 0.004±0.006 | 0±0.009 | 0.006±0.016 |
57 | Glutamate | 0.021±0.049 | 0.025±0.008 | 0.031±0.043 | 0.044±0.034 |
58 | Phenylalanine | −0.058±0.049 | −0.143±0.015 |
−0.164±0.012 |
−0.127±0.037 |
59 | Glycine | −0.192±0.03 | −0.22±0.018 | −0.205±0.035 | −0.179±0.046 |
60 | Histidine | −0.374±0.172 | −0.468±0.03 | −0.537±0.081 | −0.44±0.098 |
61 | Isoleucine | −0.07±0.058 | −0.13±0.116 | −0.118±0.064 | −0.073±0.053 |
62 | Lysine | −0.156±0.16 | −0.285±0.019 | −0.312±0.058 | −0.25±0.07 |
63 | Leucine | −0.074±0.065 | −0.087±0.018 | −0.138±0.065 | −0.086±0.058 |
65 | Asparagine | −0.007±0.002 | −0.002±0.003 |
−0.001±0.002 | −0.004±0 |
66 | Proline | −0.002±0.01 | −0.005±0.003 | −0.025±0.022 | −0.016±0.017 |
67 | Glutamine | 6.395±14.477 | 1.121±0.377 | 0.136±0.188 |
1.198±0.917 |
68 | Arginine | −0.021±0.039 | −0.007±0.021 | −0.062±0.052 | 0.017±0.078 |
69 | Serine | −0.073±0.101 | −0.152±0.01 | −0.181±0.026 | −0.142±0.032 |
70 | Threonine | −0.269±0.228 | −0.378±0.008 | −0.378±0.011 | −0.363±0.016 |
71 | Valine | −0.072±0.073 | −0.081±0.02 | −0.129±0.074 | −0.07±0.073 |
72 | Tyrosine | −0.095±0.068 | −0.122±0.024 | −0.166±0.041 | −0.107±0.068 |
Hepatocytes were cultured in four different configurations at a density of 500,000 cells/well in a 12 well plates for 10 days.
*
StableStage | |||||
Fluxno. | Metabolites | CSG | CDG | CSG+solASG | CSG+ASG |
1 | Glucose | 0.072±0.016 |
−0.001±0.04 | 0.073±0.043 |
0.072±0.0358 |
14 | Lactate | 0.071±0.022 | 0.068±0.015 | 0.095±0.013 |
0.103±0.039 |
23 | Urea | 1.074±0.77 |
1.946±0.155 | 1.847±0.253 | 1.703±0.299 |
45 | βhydroxybutyrate | −0.021±0.005 | −0.021±0.005 | 0.073±0.022 | 0.073±0.022 |
48 | Albumin | 0±0 |
0.0007±0.0001 | 0.0017±0.0002 |
0.0008±0.0002 |
49 | O2 | 0.112±0.016 | 0.107±0.015 | 0.149±0.021 | 0.162±0.023 |
50 | CO2 | 0.013±0.003 | 0.012±0.002 | 0.017±0.003 | 0.018±0.003 |
51 | Acetoacetate | 0.071±0.009 | 0.071±0.009 | −0.024±0.014 | −0.024±0.014 |
52 | Ornithine | −0.052±0.016 |
−0.082±0.003 | −0.025±0.01 |
−0.066±0.011 |
53 | Ammonia | 0.346±0.545 | −0.005±0.006 | −0.045±0.007 | 0.005±0.011 |
55 | Cysteine | 0.017±0.052 | 0.042±0.005 | −0.088±0.018 | −0.008±0.027 |
56 | Aspartate | 0.005±0.002 | 0.003±0.001 | 0.001±0.001 | 0.003±0.001 |
57 | Glutamate | 0.048±0.022 |
0.142±0.006 | 0.085±0.01 | 0.092±0.021 |
58 | Phenylalanine | −0.001±0.072 |
−0.198±0.011 | −0.252±0.01 | −0.167±0.012 |
59 | Glycine | 0.032±0.083 | −0.035±0.014 | −0.092±0.015 |
−0.006±0.037 |
60 | Histidine | −0.034±0.019 |
−0.091±0.001 | −0.092±0.001 | −0.086±0 |
61 | Isoleucine | 0.037±0.109 |
0.172±0.017 | −0.133±0.036 |
−0.019±0.057 |
62 | Lysine | 0.038±0.073 | −0.003±0.015 | −0.096±0.013 |
−0.041±0.017 |
63 | Leucine | 0.021±0.098 |
0.134±0.017 | −0.164±0.032 |
−0.045±0.051 |
65 | Asparagine | 0.006±0.003 |
0±0 | −0.003±0.002 |
0.004±0 |
66 | Proline | 0.325±0.343 | 0.021±0.006 | 0.069±0.014 |
0.063±0.006 |
67 | Glutamine | 13.714±3.82 |
6.444±0.746 | 0.125±0.518 |
2.496±1.908 |
68 | Arginine | 0.001±0.002 |
0.008±0.001 | −0.003±0.002 |
−0.001±0.002 |
69 | Serine | 0.027±0.035 |
−0.038±0.004 | −0.061±0.005 |
−0.03±0.003 |
70 | Threonine | −0.046±0.047 |
−0.188±0.002 | −0.411±0.617 | −0.73±0.94 |
71 | Valine | 0.051±0.108 |
0.203±0.014 | −0.222±0.35 |
0.007±0.057 |
72 | Tyrosine | 0.002±0.072 |
0.104±0.02 | −0.116±0.026 |
0.03±0.047 |
Hepatocytes were cultured in four different configurations at a density of 500,000 cells/well in a 12 well plates for 10 days.
*
The MFA framework was applied to each biological experimental data-set of extracellular metabolite measurements in µmol/million cells/day input units. The output of the MFA for each biological experimental data-set was averaged using the sum over all computationally derived replicates for the unknown [non-measured] fluxes. The standard error of the mean was calculated from the computationally derived replicates for the unknown [non-measured] fluxes.
The summary statistics were calculated using t-test by comparing data from all experiments from one experimental condition for (e.g.: CSG+solASG) vs. data from all experiments from another experimental condition (for e.g.: CDG). This statistical method was used for extracellular metabolite measurements and computed intracellular fluxes while comparing a specific metabolite flux between different experimental conditions. Statistical significance was determined using the Student's t-test for unpaired data. Differences were considered significant when the probability was less than or equal to 0.05.
We have previously performed a preliminary characterization of the extracellular matrix components derived as a basement membrane extract from preadipocytes during the differentiation process
The preadipocytes are cultured in T-175 cm2 flasks in Dulbecco's Modified Eagle's media supplemented with 10% FBS, 2% Penicillin and Streptomycin till the cells attain confluency. 48 hours post confluency, the cells are differentiated in culture media supplemented with 1 µM dexamethasone, 0.1 µM isobutyl-methylxanthine and 1 µM rosiglitazone for 2 days with media changes every two days. On day 2, the differentiation medium is supplemented with rosiglitazone only.
During the differentiation process, cell exposed media is collected and processed further for generation of cell derived extracellular matrix. We have previously identified a highly viscoelastic material on days 2 and 4 of adipocyte differentiation resembling extracellular matrix components secreted by preadipocytes to maintain adipose tissue cell-cell contact, morphological induction of adipocytes and functional and gene expression indicative of mature adipocyte lineage. In order to purify the extracellular matrix-rich material, the cell exposed media is centrifuged at 4000
Routine culture of primary hepatocytes is difficult and cumbersome due to their ability to develop compromised function. We have developed a primary hepatocyte culture system that supersedes the traditional methodology of maintaining hepatocyte function and polarity in collagen double gel sandwich systems. Hepatocytes when cultured on single collagen gel with a soluble matrix of Adipogel in the culture media showed comparable urea secretion rates [
[A] Urea and [B] Albumin Secretion rate of Hepatocytes cultured in five different configurations at a density of 500,000 cells/well in a 12 well plate; CSG corresponds to culture on single collagen gel; CDG corresponds to culture in collagen double gel sandwich configuration; CSG+solASG corresponds to hepatocytes cultured on collagen single gel with soluble Adipogel in the media; CSG+ASG corresponds to culture on collagen single gel with Adipogel overlaid on top. Adipogel was utilized at a 1∶5 ratio with culture media and media was changed on days 0, 1,2,5,7 and 9. While the urea secretion rates are similar for the CDG [positive control] and the CSG+solASG conditions, the albumin secretion rate is significantly higher for the CSG+solASG condition as compared to CDG cultures.
We have performed a metabolic analysis of the different culture configurations viz. the collagen single gel [CSG], collagen double gel [CDG], collagen-Adipogel sandwich [CSG+ASG] and collagen single gel+soluble Adipogel [CSG+solASG]. To elucidate the effect of Adipogel on hepatocyte metabolism, we have compared the CDG to the CSG+solASG conditions as described below. We have chosen day 4 [recovery stage], day 7 [pre-stable stage] and day 10 [stable stage] of analysis to investigate the delayed effect [day4, day7] as well as the immediate effect [day10] of Adipogel supplementation on hepatic metabolism. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation
As shown in
Pathways | FluxNo. | RecoveryStage | Pre-stableStage | StableStage |
CSG+solASG | CSG+solASG | CSG+solASG | ||
Glucose Uptake | 1 | Base | Decrease significantly | Increase significantly |
PPP | 2,3,5 | Base | Decrease significantly | Same |
PPP | 4,6 | Base | Decrease significantly | Decrease significantly |
Gluconeogenesis | 7,8 | Base | Increase significantly | Same |
Gluconeogenesis | 9 | Base | Same | Increase significantly |
Glycerol Uptake | 10 | Base | Decrease significantly | Decrease significantly |
Gluconeogenesis | 11–14 | Base | Same | Decrease significantly |
TCA | 15–18 | Base | Decrease significantly | Same |
Pyruvate Synthesis | 24,26 | Base | Same | Increase significantly |
Amino Acid Metabolism | 25,27,28 | Base | Same | Decrease significantly |
Amino Acid Metabolism | 30,31,33,34 | Base | Same | Decrease significantly |
Amino Acid Metabolism | 36,37 | Base | Same | Increase significantly |
Amino Acid Metabolism | 38,40 | Base | Same | Decrease significantly |
Lipid Metabolism | 42–44 | Base | Same | Increase significantly |
Amino acid Uptake | 53–55 | Base | Same | Increase significantly |
Amino acid Uptake | 56 | Base | Same | Decrease significantly |
Amino acid Uptake | 57 | Base | Same | Decrease significantly |
Amino acid Uptake | 58 | Base | Decrease significantly | Increase significantly |
Amino acid Uptake | 59–64 | Base | Same | Increase significantly |
Amino acid Uptake | 65 | Base | Same | Decrease significantly |
Amino acid Uptake | 66 | Base | Same | Decrease significantly |
Amino acid Uptake | 68–72 | Base | Same | Increase significantly |
Hepatocytes were cultured on collagen single gel at a density of 500,000 cells/well in a 12 well plate. Adipogel was utilized at a 1∶5 ratio with culture media and media was changed on days 0, 1,2,5,7 and 9 of culture. Metabolic Flux Analysis was performed on [A] recovery stage, [B] pre-stable stage and [C] stable stage of culture.
At the recovery stage and pre-stable stage, cysteine synthesis [v55] is significantly higher for CSG+solASG vs. CDG condition. At the recovery stage, glutamine [v67] and arginine synthesis rate [v68] is also significantly higher for CSG+solASG vs. CDG condition. At the stable stage, serine [v69], glycine [v59], isoleucine [v61], lysine [v62], leucine [v63], methionine [v64], arginine [v68], valine [v71] and tyrosine [v72] uptake rates are significantly higher for CSG+solASG condition as compared to CDG condition. Thus, we can correlate increased amino acid uptake rates and changes in glucose metabolism to hepatocellular function at the stable stage of culture.
Using a previously developed hepatic metabolic network comprising of 72 reactions and 27 extracellular metabolite measurements
We also performed a comparison of the CSG+solASG condition at different time points of the differentiation process. A comparison of the recovery stage and pre-stable stage cultures revealed significant increase in PPP and decrease in TCA cycle fluxes for the CSG+solASG condition with no significant difference in albumin synthesis rate [
Primary hepatocytes are cultured
Particularly, there is some literature evidence for the effects of ECM proteins on enhanced albumin synthesis of hepatocytes
While hepatic function is enhanced by utilizing the above mentioned culture systems, the albumin synthetic functional capacity of the cells is not maximal. In this study, Adipogel, a natural cell secreted extracellular matrix, can be added in soluble media format to hepatocyte cultures [
In this context, we have performed a metabolic analysis of the different culture configurations viz. the collagen single gel [CSG], collagen double gel [CDG], collagen-Adipogel sandwich [CSG+ASG] and the collagen single gel+soluble Adipogel [CSG+solASG]. The analysis shows that the key amino acids upregulated for the CSG+solASG conditions are serine [v69], glycine [v59], isoleucine [v61], lysine [v62], leucine [v63], methionine [v64], arginine [v68], valine [v71] and tyrosine [v72] uptake rates as compared to the CDG condition at the stable stage of culture.
There is also an increased albumin synthesis rate for the CSG+solASG condition as compared to the CDG condition on day 10 of culture. The albumin flux is negligible and the amino acid fluxes contribute very little to albumin synthesis rates from a stoichiometric balance as also previously described using a similar metabolic network model
On the other hand, at the recovery stage and pre-stable stage of analysis, soluble Adipogel does not reveal increases in amino acid uptake rate as compared to the CDG condition probably due to a delayed response to Adipogel supplementation 48 hours before analysis. However, the albumin synthesis rate is significantly higher for the stable stage as compared to the recovery stage of hepatocyte cultures in CSG+solASG condition. Thus, a quantitative estimate of intracellular metabolic pathways can identify key pathways implicated in Adipogel supplementation of hepatocyte cultures and its effect on cell function.
We have previously developed metabolic flux models of the perfused rat liver
Arrows indicate direction of reaction assumed in the model. Numbers refer to reaction numbers listed in
For the CSG+solASG condition, differential effects on hepatic metabolism were observed on different days of analysis. As shown in
[A] Recovery stage [B] Pre-stable stage and [C] Stable stage of culture. Hepatocytes cultured in five different configurations at a density of 500,000 cells/well in a 12 well plate; CSG corresponds to culture on single collagen gel; CDG corresponds to culture in collagen double gel sandwich configuration; CSG+solASG corresponds to hepatocytes cultured on collagen single gel with soluble Adipogel in the media; CSG+ASG corresponds to culture on collagen single gel with Adipogel overlaid on top;. Adipogel was utilized at a 1∶5 ratio with culture media and media was changed on days 0, 1,2,5,7 and 9. Metabolic Flux Analysis was performed on [A] recovery stage [B] pre-stable stage and [C] stable stage of culture. MFA results for CDG vs. CSG+solASG conditions are represented in the figures.
In general, the increase in transamination reaction fluxes with increased amino acid uptake rates might imply channeling of the amino acids towards albumin protein synthesis. Thus, the addition of these amino acids at early stages of the culture period in conjunction with Adipogel could potentially improve function significantly. Overall, enhanced albumin synthesis in the presence of Adipogel may be due to selective alterations in intracellular fluxes associated with these pathways that can be utilized for improving function.
Previous work referring to the development of a novel preadipocyte cell differentiation system has been utilized in this work for the generation of a natural basement membrane extract termed Adipogel with a unique extracellular matrix and growth factor composition
We have shown that Adipogel can be utilized for augmenting hepatocyte differentiated function
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