Copyright
©The Author(s) 2018.
World J Hepatol. Oct 27, 2018; 10(10): 719-730
Published online Oct 27, 2018. doi: 10.4254/wjh.v10.i10.719
Published online Oct 27, 2018. doi: 10.4254/wjh.v10.i10.719
Table 1 Composition of the different Krebs-Henseleit solution used
Components | KHB solution | KHA solution |
NaCl | 114 mmol/L | 114 mmol/L |
KH2PO4 | 1.2 mmol/L | 1.2 mmol/L |
KCl | 4.8 mmol/L | 4.8 mmol/L |
MgSO4 | 1.2 mmol/L | 1.2 mmol/L |
CaCl2 | 1.5 mmol/L | 1.5 mmol/L |
HEPES | 10 mmol/L | 10 mmol/L |
NaHCO3 | 25 mmol/L | 25 mmol/L |
Glucose | 25 mmol/L | 25 mmol/L |
Allopurinol | 1 mmol/L | 1 mmol/L |
Fructose | 5 mmol/L | 5 mmol/L |
Glycine | - | 3 mmol/L |
Adenosine | - | 10 µmol/L |
Ornithine | - | 6 mmol/L |
Sodium lactate | - | 10 mmol/L |
pH | 7.4 | 7.4 |
mOsm/kg H2O | 293 ± 6 (n = 10) | 328 ± 5 (n = 10) |
Table 2 Sequences of the primers employed
Primers | Fragment size | Genbank access number |
Cps1 5’-ATCTGAGGAAGGAGCTGTCT-3’ (sense) Cps1 5’-AAAACCACTTGTCAATGGAT-3’ (anti-sense) | 120 bp | NM_017072 |
Otc 5’-ATGACAGATGCAGTGTTAGC-3’ (sense) Otc 5’-CAGGATCTGGATAGGATGAT-3’ (anti-sense) | 120 bp | NM_013078 |
Actb 5’-CAACCTTCTTGCCAGCTCCTC-3’ (sense) Actb 5’-GACGAGCGCAGCGATATC-3’ (anti-sense) | 79 bp | NM_031144.2 |
Rn18S 5’-TAACCCGTTGAACCCCATT-3’ (sense) Rn18S 5’-CCATCCAATCGGTAGTAGCG-3’ (anti-sense) | 150 bp | X01117 |
Gapdh 5’-CCATCACCATCTTCCAGGAG-3’ (sense) Gapdh 5’-CCTGCTTCACCACCTTCTTG-3’ (anti-sense) | 576 bp | NM_017008.4 |
Table 3 Functional parameters of the cylindrical shaped bio-artificial liver1 (n = 6 independent runs)
Perfusion time | OsmB/OsmBC | Ht (%) | Hemolysis (%) | [Glucose]B/[Glucose]BC | [NH4+]B/[NH4+]BC | QNH4+ (μmol) |
0 min | 0.95 ± 0.05 | 43 ± 3 | 0.12 ± 0.06 | 0.12 ± 0.01 | 23.2 ± 1.2 | 31.1 ± 3.9 |
60 min | 0.99 ± 0.01 | 42 ± 6 | 0.30 ± 0.08 | 0.81 ± 0.03 | 1.4 ± 0.4 | 31.1 ± 4.2 |
120 min | 1.00 ± 0.01 | 38 ± 2 | 0.65 ± 0.10 | 0.94 ± 0.02 | 1.2 ± 0.2 | 31.3 ± 3.8 |
Table 4 Viability and functional parameters evaluated for liver microorgans in the cylindrical shaped bio-artificial liver and the Normothermic Reoxygenation System (n = 6 different liver microorgan preparations in independent bio-artificial liver runs/Normothermic Reoxygenation System incubations)
Table 5 Relative gene expression of Cps1 and Otc for liver microorgans in the cylindrical shaped bio-artificial liver and the Normothermic Reoxygenation System (n = 3 different liver microorgan preparations in independent bio-artificial liver runs/ Normothermic Reoxygenation System incubations)
Relative gene expression | Cps1 | Otc | |||||
Time (min) | 0 | 60 | 120 | 0 | 60 | 120 | |
Model | Cylindrical BAL | 1.05 ± 0.17 | 0.84 ± 0.09 | 0.63 ± 0.12 | 1.02 ± 0.26 | 0.87 ± 0.21 | 0.67 ± 0.20 |
NRS | 1.00 ± 0.24 | 0.96 ± 0.12 | 0.86 ± 0.10 | 1.00 ± 0.20 | 0.87 ± 0.16 | 0.82 ± 0.07 |
Table 6 Carbamyl phosphate synthetase I and ornithine transcarbamylase enzymatic activity in the cylindrical shaped bio-artificial liver and the Normothermic Reoxygenation System (n = 3 different liver microorgan preparations in independent bio-artificial liver runs/Normothermic Reoxygenation System incubations)
Enzymatic activity | CPSI (U/g wet tissue) | OTC (U/g wet tissue) | |||||
Time (min) | 0 | 60 | 120 | 0 | 60 | 120 | |
Model | Cylindrical BAL | 2.48 ± 0.62 | 2.72 ± 0.61 | 3.03 ± 0.86 | 241.7 ± 12.6 | 235.1 ± 11.9 | 222.0 ± 23.5 |
NRS | 2.47 ± 0.60 | 2.82 ± 0.76 | 3.12 ± 0.73 | 209.7 ± 33.2 | 251.8 ± 29.4 | 228.8 ± 32.8 |
Table 7 Ammonia detoxification capacity (μmol/g wet tissue) for fresh liver microorgans and hepatocyte suspensions used as biological component of the “flat bottom” and cylindrical bio-artificial livers (n = 6 different liver microorgan/hepatocyte preparations in independent runs)
Time (min) | Ammonia detoxification capacity (μmol/g wet tissue) | ||
Flat bottom BAL | Cylindrical BAL | ||
LMOs | LMOs | Hepatocytes | |
60 | 8.1 ± 1.2a | ND | 12.5 ± 1.8 |
120 | 13.2 ± 2.2 | ND | 18.6 ± 4.9 |
- Citation: Pizarro MD, Mamprin ME, Daurelio LD, Rodriguez JV, Mediavilla MG. Experimental bio-artificial liver: Importance of the architectural design on ammonia detoxification performance. World J Hepatol 2018; 10(10): 719-730
- URL: https://www.wjgnet.com/1948-5182/full/v10/i10/719.htm
- DOI: https://dx.doi.org/10.4254/wjh.v10.i10.719