Table 1. Perfusion protocol.

	Steps and Reagents
Days	1st	2nd	3rd	4th	5th
−1	Thaw liver overnight at 4 °C
0	dH2O	0.025% Trypsin-EDTA
1	dH2O	0.01% SDS	0.1% SDS	1% SDS
2	dH2O	0.025% Trypsin-EDTA	1% SDS
3	dH2O	3% TX100
4	dH2O	3% TX100
5	dH2O	3% TX100
6	dH2O	3% TX100	1X PBS
7	dH2O	0.025% Trypsin-EDTA
8	dH2O	1% SDS
9	dH2O	1% SDS
10	dH2O	1% SDS
11	dH2O	1% SDS
12	dH2O	1% SDS
13	dH2O	PBS/AA 5%	3% TX100
14	dH2O	PBS/AA 5%	dH2O	0.1%PAA/4% EtOH	1X PBS sterile

The decellularization of the whole human liver was achieved by repeating three times the procedure shown in the Table (the freezing/thawing step was performed just once).

Abbreviations: dH2O (distilled water), TX100 (Triton X100), SDS (sodium dodecyl sulfate), A-A 5% (Antibiotic and Antimycotic), PAA (paracetic acid) and EtOH (ethanol).