Table 1.

Summary of protocols and main findings of studies in the liver bioengineering field.

References	Experimental model	Decellularization methods	Recellularization methods	Major contributions
Uygun et al., USA [8]	Female Lewis rats (150–200 g)	Protocol (i) Physical (ii) Chemical Quality assessments (i) Maintenance of ECM components and architecture: collagen and GAG content; immunostaining (ii) Acellularity/absence of nuclear content: histological evaluation and DAPI staining (iii) Maintenance of vasculature: Allura Red AC dye and scanning electron microscopy (SEM)	(i) Infusion of 50 × 106 of rat primary hepatocytes, through PV (ii) 40 × 106 microvascular endothelial cells through PV (iii) Biochemical analysis: albumin, urea, G6P, Ugt1a, and others	The first description of a protocol to generate a liver bioscaffold. Efficient recellularization was achieved supporting liver-specific function (albumin secretion, urea synthesis, and cytochrome P450) for a further transplantation
Shupe et al., USA [9]	Fisher 344 rats (not reported)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunohistochemical staining (ii) Acellularity/absence of nuclear content: DAPI staining (iii) Maintenance of vasculature: immunohistochemistry of laminin	(i) Infusion with WB344 rat liver progenitor cells through the IVC	A relatively simple method to decellularize a whole rat liver without removing the organ is presented
De Kock et al., Belgium [12]	Sprague-Dawley rats (250 g–300 g)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunostaining (ii) Acellularity/absence of nuclear content: SEM/hematoxylin and DAPI staining (iii) Biochemical parameters: VEGF immunostaining	Not performed	An effective and faster method of liver decellularization is presented
Soto-Gutierrez et al., USA [13]	Sprague-Dawley rats (250 g–300 g)	Protocol (i) Physical (ii) Chemical (iii) Enzymatic Quality assessments (i) Maintenance of ECM components: immunostaining and SEM (ii) Acellularity/absence of nuclear content: DNA quantification, H&E, and DAPI staining (iii) Biochemical parameters: bFGF/VEGF/HGF immunoassays (iv) Maintenance of vasculature: transmission electron microscopy (TEM), blue dye	(i) 10 to 50 × 106 primary mouse hepatocytes (ii) Three ways to seed cells: (a) direct parenchymal injection, (b) continuous perfusion, or (c) multistep infusion (iii) SEM showed hepatocyte engraftment (iv) Ki67 showed proliferating cells in recellularized liver (v) Functionality was measured by albumin production, ammonia metabolism, and CYP1A1/2 activity	Decellularization was accomplished in 48 h without the use of harsh detergents preserving 30-50% of growth factor content. It presents a perfusion technique (multistep infusion) resulting in ~90% grafting efficiency with maintenance of some liver functionalities
Bao et al., China [14]	Lewis rats (180–250 g)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: histological evaluation (ii) Acellularity/absence of nuclear content: SEM and DAPI staining	(i) 1 × 108 rat hepatocytes with spheroid formation (ii) Pretreatment with heparin (iii) Further transplantation	It is reported that the use of hepatocyte spheroids increases cell survival. Tissue-engineered liver can maintain hepatic functions up to 72 hours
Gessner et al., USA [16]	Wistar rats (250–300 g)	Protocol (i) Chemical (ii) Enzymatic Quality assessments (i) Maintenance of ECM components and architecture: SEM (ii) Acellularity/absence of nuclear content: SEM (iii) Maintenance of vasculature: ultrasound images/acoustic angiography	(i) 130 × 106 human hepatoblast-like cells, Hep3B cells	A nondestructive method, based on the analysis of ultrasound images, is presented in order to evaluate the microvascularization in the decellularized tissue. Recellularized matrix presented liver-specific functions
Mirmalek-Sani et al., USA [21]	Porcine (20-25 kg)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: SEM and immunostaining (ii) Acellularity/absence of nuclear content: histological evaluation (iii) Maintenance of vasculature: SEM	(i) Hepatoblastoma cells (HepG2) (ii) Total number of cells and infusion rate are not provided by the author	The protocol used for rats was adapted for application in a large animal model, with adequate preservation of essential ECM proteins for cell engraftment and function, as well as the vasculature required for nutrient distribution for whole-organ reseeding. Also showed nonimmunogenicity from decellularized matrices
Yagi et al., Japan [22]	Porcine (20-23 kg)	Protocol (i) Physical (ii) Chemical Quality assessments (i) Maintenance of ECM components and architecture: histological evaluation, immunostaining, and SEM (ii) Acellularity/absence of nuclear content: DAPI and DNA content quantification (iii) Maintenance of vasculature: vascular corrosion casting and digital radiography (iv) Biochemical parameters: immunohistochemical for HGF, bFGF, VEGF, and IGF-1	(i) 1 × 109 porcine hepatocytes were seeded by the multistep infusion method	The authors adapted protocols to be successfully applied in large-scale livers. Engraftment efficiency reported was approximately 74%
Kadota et al., Japan [15]	Lewis rats (200–500 g)	Protocol (i) Physical (ii) Chemical (iii) Enzymatic Quality assessments Not reported	(i) Coinfusion of primary hepatocytes and MSCs (ii) Different cell numbers tested: 3 × 108, 1 × 108 and 5 × 107, with 20% MSCs in different conditions	Authors suggest that MSCs act as supportive cells in this system improving the functionality of the protein production in the engineered tissue
Jiang et al., Taiwan [20]	Balb/c mice (10–30 g)	Protocol (i) Physical (ii) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunostaining and histological analyses (ii) Acellularity/absence of nuclear content: phase contrast microscopy, DNA content assay, and SEM	(i) 50 × 106 MSC undifferentiated or submitted to prior in vitro stimulation for hepatic differentiation (ii) Transplantation in chemical liver-injured mice	It provides evidence for increased hepatic differentiation of MSC in the decellularized scaffold when compared to 2D culture
Struecker et al., Germany [23]	Porcine (20-60 kg)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunostaining and GAG and collagen content quantification (ii) Acellularity/absence of nuclear content: DNA content quantification (iii) Maintenance of vasculature: computed tomography	Not performed	It presents a fast and effective method by inserting pressure gradients in the perfusion protocol, which improves the homogeneity of perfusion and the outcome of decellularization
Sabetkish et al., Iran [19]	Sprague-Dawley rat (250–300 g) and sheep (not reported)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: histological evaluation, immunostaining, tensile test, and collagen content (ii) Acellularity/absence of nuclear content: DAPI staining, SEM, and DNA content assay	(i) 18 × 106 GFP primary hepatocytes (ii) Histological evaluation, enzyme tests, and immunofluorescence were performed	By comparing different methods, the authors conclude that using Triton X for the decellularization method is more efficient in maintaining the ultrastructure and biomechanical properties of the tissue. Additionally, it shows that seeding the bioscaffold with cells from the same species is more efficient than xenotransplantation
Wang et al., China [25]	Bama miniature pigs (12-15 kg)	Protocol (i) Physical (ii) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunostaining, GAC, and collagen content quantification (ii) Acellularity/absence of nuclear content: SEM, DNA content quantification, and H&E staining	Not performed	The study evaluated different methods and defines SDS as the most efficient and fast agent
Mazza et al., England [26]	Human (lobes = 374 g–250 g) (whole liver = 1774 kg)	Protocol (i) Physical (ii) Chemical (iii) Enzymatic Quality assessments (i) Maintenance of ECM components and architecture: collagen and elastin quantification (ii) Acellularity/absence of nuclear content: DNA content quantification, histological evaluation, and DAPI staining	(i) 2 × 106 human hepatic cell lines were used: LX2 (hepatic stellate), HepG2, and Sk-Hep-1 cells (hepatocellular carcinoma)	The author was the first to adapt the protocol of decellularization and recellularization in human liver tissue
Maghsoudlou et al., England [17]	Sprague-Dawley rats (250 g–300 g)	Protocol (i) Chemical (ii) Enzymatic Quality assessments (i) Maintenance of ECM components and architecture: immunostaining (ii) Acellularity/absence of nuclear content: DNA content quantification, H&E staining, and SEM (iii) Maintenance of vasculature: Trypan blue perfusion	(i) 2 × 106 cells (HepG2—human hepatoma cell line)	The protocol of decellularization was optimized with the addition of EDTA to the detergent-enzymatic treatment, creating a denser and more compact matrix
Zhou et al., China [18]	Sprague-Dawley rats (300–350 g)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: immunostaining and GAG content assay (ii) Acellularity/absence of nuclear content: DNA content assay and SEM	(i) 2 × 107 cells (BRL rat liver cell line)	A method of liver decellularization by continuous perfusion of EDTA and Triton X-100/ammonium hydroxide followed by recellularization with hepatocytes and endothelial cells, showing reendothelization
Coronado et al., USA [24]	Porcine (not reported)	Protocol (i) Chemical Quality assessments (i) Maintenance of ECM components and architecture: protein content quantification (ii) Acellularity/absence of nuclear content: DNA content quantification, histological evaluation, and DAPI staining (iii) Biochemical parameters: HGF/FGF/EGF	(i) Primary porcine hepatocytes (ii) Porcine bioscaffold was lyophilized for cell seeding	Two different decellularization methods were performed in the porcine liver. The method using ammonia and acetic acid was the most efficient in the removal of genetic material. Hepatocytes presented better functionality when seeded in liver substrate (comparing to collagen I)
Mazza et al., England [27]	Human (not reported)	Protocol (i) Physical (ii) Chemical (iii) Enzymatic Quality assessments (i) Maintenance of ECM components and architecture: proteomic, histological, and immunostaining analyses (ii) Acellularity/absence of nuclear content: DNA quantification (iii) Maintenance of vasculature: SEM imaging, confocal autofluorescence microscopy, and chicken egg chorioallantoic membrane (CAM) assay	(i) 1.4-2 × 106 LX2, HepG2, HUVEC, and primary cells (hepatocytes and stellate cells)	Optimization of the previous work, the protocol consists in g-force oscillation and high shear stress to produce acellular liver cubes, using human liver tissue, followed by recellularization
Robertson et al., USA [10]	Sprague-Dawley rats (250–300 g)	Protocol (i) Chemical (ii) Enzymatic Quality assessments (i) Maintenance of ECM components and architecture: immunostaining and GAG content quantification (ii) Acellularity/absence of nuclear content: DNA content by Hoechst 33258 staining and SEM	(i) 1-20 × 106 rat liver cells or 20 × 106 human liver cells	Decellularization method using SDS+DNase was associated with the lowest amount of residual DNA and the highest retention of GAGs. Advances in the recellularization method by reseeding human liver cells in a rat bioscaffold and maintaining the bioengineered tissue for 28 days