Experimental bio-artificial liver: Importance of the architectural design on ammonia detoxification performance

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)

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; mOsm/kg H₂O: Osmolality; KHB: KH-Base solution; KHA: KH-ammonia solution.

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

¹The cylindrical bio-artificial liver designed for liver microorgans functioning without any biological component. B: Blood; BC: Biological compartment; Ht: Hematocrit.

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)

Evaluated parameters		LDH release (%)			Oxygen Consumption (μmol/min·g wet tissue)
Time (min)		0	60	120	0	60	120
Model	Cylindrical BAL	1.9 ± 0.9	9.4 ± 3.0a	14.7 ± 3.1a	1.21 ± 0.24	1.15 ± 0.20	1.16 ± 0.21
	NRS	2.6 ± 0.1	8.3 ± 1.2a	15.5 ± 3.2a	1.13 ± 0.11	0.85 ± 0.15c	0.84 ± 0.15c

^aP < 0.05, different from other times;

^cP < 0.05, different from time 0. BAL: Bio-artificial liver; NRS: Normothermic Reoxygenation System; LDH: Lactate dehydrogenase.

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

BAL: Bio-artificial liver; NRS: Normothermic Reoxygenation System; Cps1: Carbamyl phosphate synthetase I; Otc: Ornithine transcarbamylase.

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

CPSI: Carbamyl phosphate synthetase I; OTC: Ornithine transcarbamylase; BAL: Bio-artificial liver; NRS: Normothermic Reoxygenation System.

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

^aP < 0.05, different from hepatocytes at time 60 min. 128 × 10⁶ hepatocytes = 1 g of liver. ND: Not detectable, means that the total ammonia mass remained equivalent to the 100% of initial dose at the indicated times; LMO: Liver microorgan; BAL: Bio-artificial liver.