Review
Copyright ©The Author(s) 2023.
World J Hepatol. Feb 27, 2023; 15(2): 151-179
Published online Feb 27, 2023. doi: 10.4254/wjh.v15.i2.151
Table 1 Examples of non-liver decellularisation protocols
Organ
Species
Decellularisation technique
Recellularization
Significant outcome
Ref.
Heart
RatSDS + Triton X-100Neonatal cardio-myocytes(1) Maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology; (2) Macroscopic contractions were observed by day 4; and (3) By day 8, under physiological load and electrical stimulation, constructs could generate pump function in a modified working heart preparation.Ott et al[235]
PigFreeze and Thaw + hypotonic solution + trypsin/EDTA/NaN3 + Triton X-100/EDTA/NaN3 + deoxycholic acidChicken embryonic cardio-myocytesCardiac extracellular matrix supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro.Wainwright et al[236]
RatSDS vs POETE Not performedSDS decreased DNA and GAG and enriched the collagen content 10-fold.Bruyneel et al[237]
PigSDS vs Triton X-100 vs CHAPS vs OGPNot performed3% SDS as a detergent showed optimal decellularization.Ferng et al[238]
RatSDS + Triton X-100Induced cardiac progenitor cells(1) Optical mapping of recellularised scaffolds shows field-stimulated calcium transients that propagate across islands; and (2) Bipolar local stimulation demonstrated cell-cell coupling within scaffolds.Alexanian et al[239]
Kidney
RatSaline + SNP + Triton X-100, DNAse + SDSMurine pluripotent embryonic stem cells(1) Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures; and (2) Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation.Ross et al[240]
Monkey 1% SDS vs 1% Triton X-100Not performedSDS at 48C to be most effective in preserving the native architecture.Nakayama et al[241]
Pig0.5% SDS vs 0.25% SDS vs 1% Triton X-100 with 0.1% ammonium hydroxideNot performed0.5% SDS was the most effective detergent.Sullivan et al[242]
PigSDSNot performed(1) Kidney decellularized scaffolds implanted in Yorkshire pigs easily re-perfused, sustained blood pressure; (2) Scaffolds maintained renal ultrastructure; and (3) However, presence of inflammatory cells in the pericapsular region and complete thrombosis of the vascular tree were evident.Orlando et al[243]
Rat, pig, and humanSDSHUVECs + Rat Neonatal kidney cells (1) The resulting grafts produced rudimentary urine in vitro when perfused via their intrinsic vascular bed; and (2) Transplanted orthotopic grafts in rats, perfused by the recipient’s circulation, produced urine via the ureteral conduit in vivo.Song et al[244]
PigSonication + SDS + Triton X-100Not performed(1) Significant decrease in decellularization time with sonication; and (2) Sonicator power proved to have significant effect on the microarchitecture integrity of the scaffold.Manalastas et al[76]
Lung
RatHeparinized PBS + SDS + Triton X-100HUVECsOrthotopic Transplantation of grafts with 6 h of perfusion in vivo.Ott et al[245]
RatPBS + SNP + CHAPS + EDTA + BenzonaseRat neonatal lung epithelial + lung vascular endothelial cells (1) In vitro, the mechanical characteristics of the engineered lungs were like those of native lung tissue; and (2) In vivo gas exchange for short time intervals (45 to 120 min).Petersen et al[246]
MiceTriton X-100 + SDS + DNase Embryonic stem cells Demonstrated growth of foetal alveolar type II cells.Price et al[247]
RatHeparinized PBS + SDS + Triton X-100HUVECs + rat foetal lung cellsOrthotopic transplantation of grafts with 7 d of perfusion in vivo.Song et al[248]
Trachea
RabbitFreeze/thaw + Sonication + SDSNot performed(1) Orthoptic transplantation od decellularized scaffolds into segmental tracheal defects in rabbits; (2) Respiratory epithelium regeneration on the inner surface; and (3) Cartilaginous tubular structures could not maintain structural integrity.Hung et al[249]
PigFreeze and Thaw + Agitation/immersion + SDSNot performedSuccessful decellularization.Guimaraes et al[82]
RabbitsSonication + 1 % SDSNot performedOrthotopic transplantation of partially decellularized trachea with no immunosuppression treatment resulted in 2 mo of survival in two rabbits and one long-term survival (2 years) in one rabbit.Dang et al[71]
Nerve
Human Triton X-100 + SDS + EDTA + sonicationNot performedDetergent and sonication more effective than detergent only.Suss et al[74]
Small intestinal submucosa
PigSDS/Triton X-100/DNase vs Agitation and immersionNot performed SDS/Triton X-100 combination for decellularization proved superior.Syed et al[81]
Thyroid
RabbitSDS + immersion/agitationHTFCThe scaffolds exhibited good cytocompatibility, supported HTFCs growth, and proliferation.Weng et al[85]
Table 2 Summary of studies comparing different sterilization techniques used for decellularised scaffolds
Species
Organ
Sterilization technique
Outcome
Ref.
Sheep LiverCompared 6 different sterilization methods: (1) Freeze drying; (2) Ethylene oxide gas; (3) Gamma irradiation; (4) Gamma irradiation + Peracetic acid; (5) Gamma irradiation + Ethylene oxide gas; and (6) Gamma irradiation + Freeze drying(1) Peracetic acid or ethylene oxide + gamma irradiation was associated with the best outcome; and (2) Freeze drying and Gamma irradiation completely sterilized the liver, but also reduced the mechanical properties.Kajbafzadeh et al[96]
PorcineLiverCompared 3 different sterilization methods: (1) Peracetic acid; (2) Ethanol; and (3) Slightly acidic electrolyzed water (1) Ethanol caused a significant loss in collagen content; (2) The retained glycosaminoglycan content decreased in all treatments; and (3) Peracetic acid and slightly acidic electrolyzed water treatments achieved the highest efficiency of sterilization.Hussein et al[148]
MouseLungCompared 2 different sterilization methods: (1) Gamma irradiation; and (2) Peracetic acid(1) Irradiation produced significant structural distortion; and (2) Peracetic acid had less effect on the resulting architecture.Bonenfant et al[149]
PorcineTMJ Fibro-cartilage discCompared 3 different sterilization methods: (1) Peracetic acid; (2) Gamma irradiation; and (3) Ethylene oxide.(1) Gamma irradiation and Ethylene Oxide caused structural damage leading to inferior cell adhesions; and (2) Peracetic Acid caused minimal structural damage but also induced chemical modifications leading to better cell attachments.Matuska et al[146]
Porcine KidneyCompared 4 different sterilization methods: (1) 70% Ethanol; (2 0.2% Peracetic acid in 1 M NaCl; (3) 0.2% Peracetic acid in 4% Ethanol; and (4) Gamma irradiation(1) All four methods were successful in decontamination; (2) Gamma-irradiation was very damaging to collagen fibres and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells; and (3) 0.2% peracetic acid in 1 M NaCl was found to be the best method as it completely decontaminated the renal tissue and demonstrated to have preserved essential components of the ECM.Poornejad et al[139]
Porcine Liver Compared 2 different sterilization methods: (1) Hydrochloric acid; and (2) acetic acid.(1) ECM treated with Acetic acid showed higher initial attachment and albumin and urea production in HepG2/C3A cell cultures compared to Hydrochloric acid; and (2) Acetic acid preserved bioactive moieties compared to Hydrochloric acid.Coronado et al[97]
RabbitKidneyCompared 4 different sterilization methods: (1) Antibiotics (Penicillin G, Amphotericin B and Gentamicin; (2) Peracetic acid (0.5 %, 1% and 1.5 %); (3) Gamma irradiation 5 KG; and (4) 3 UV-irradiation 20-480 nm(1) UV-irradiation is not able to sterile; (2) Gamma irradiation resulted in reduced mechanical strength and altered microstructure; and (3) 0.5 % Peracetic acid was the most efficient method to completely decontaminate rabbit decellularized kidney while preserving the mechanical properties and main components of the matrix.Moradi et al[147]
Table 3 Liver decellularisation recellularisation studies
Species
Decellularisation method
Recellularisation cell type and route
Outcome
Ref.
Female Lewis ratsSDS + Triton X-100(1) Primary rat hepatocytes via the Portal vein; and (2) Rat cardiac microvascular endothelial cells via portal vein(1) Demonstrated Successful decellularization/Recellularization with cell viability and function; (2) Demonstrated the feasibility of transplanting these recellularised liver grafts in vivo with minimal ischemic damage; and (3) The recellularised graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro.Uygun et al[86]
Fisher 344 ratsTriton X-100 + SDSRat liver progenitor cell line WB344 through the inferior vena cava(1) Perfusion with 0.1% SDS for 1 hour completely cleared all DNA; and (2) Supplementation of all perfusion solutions with antibiotics/antimycotics prevented microbial growth, and the IDL could be stored at 4°C for several weeks.Shupe et al[156]
Male Sprague Dawley ratsTrypsin + EGTA + Triton X-100Primary mice hepatocytes via: (1) Direct parenchymal injection; (2) Continuous perfusion via the portal vein; and (3) Multistep infusion via the portal veinSystematic comparison of three different reseeding methods showed that a multistep strategy provides the greatest seeding efficiency and the presence of functional hepatocytes.Soto-Gutierrez et al[164]
Male Lewis ratsSDS + Triton X-100Primary rat hepatocytes via the portal vein (from spheroid culture)(1) Layer-by-layer heparin deposition was used to avoid thrombosis, followed by repopulation of hepatocytes, and successfully implanted as a TEL into the portal system; (2) Treatment of extended hepatectomized rats with a TEL improved liver function and prolonged survival; mean lifespan was extended from 16 to 72 h; and (3) At 72 h post operation, the TEL sustained functional and viable hepatocytes.Bao et al[174]
FerretDistilled water + Triton X-100 + ammonium hydroxideHuman foetal liver cells + human umbilical vein endothelial cells co-infusion via the portal veinDemonstrated delivery of cells to different compartments of the liver tissue via different pathways EC delivered through the vena cava selectively seeded larger and smaller blood vessels up to the pericentral area of the liver lobule and cells seeded through the portal vein reached predominantly the periportal area of the liver lobule.Baptista et al[90]
Adult male Sprague–Dawley ratsSDS or Triton X-100 + sodium hydroxidePrimary rat hepatocytes via the portal veinDecellularised scaffolds constructed by perfusion of Triton X-100 were of superior quality and can provide a more effective and ideal scaffold for tissue engineering and regenerative medicine.Ren et al[161]
PorcineSDS + DNasePorcine hepatocytes via the portal VEINDemonstrated a protocol to decellularise rapidly a full-size porcine liver with small detergent volumes within 24 h.Bühler et al[153]
HumanDistilled water + SDS + Triton X-100Human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2) via suspensionDecellularised human liver cubic scaffolds were repopulated for up to 21 d using human cell lines with excellent viability, motility and proliferation and remodelling of the extracellular matrix.Mazza et al[154]
PigletTriton X-100 + ammonium hydroxideMurine endothelial cells (MS1) with combination of static and perfusion techniques (via the portal vein)(1) Developed an effective method for re-establishing the vascular network within decellularised liver scaffolds by conjugating anti-endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells; (2) This procedure resulted in uniform endothelial attachment throughout the liver vasculature extending to the capillary bed of the liver scaffold and greatly reduced platelet adhesion upon blood perfusion in vitro; and (3) The reendothelialized livers, when transplanted to recipient pigs, were able to withstand physiological blood flow and maintained for up to 24 hKo et al[89]
PorcineSDS + Triton X-100Rat primary hepatocytes and human umbilical vein endothelial cells (cells cultured in scaffolds, but not in a perfusion circuit)(1) The heparinized scaffolds showed improved anticoagulation and cytocompatibility compared to the control scaffold both in vitro and in vivo test; and (2) The layer-by-layer technique showed that heparinisation did not interfere with hepatocyte or endothelial cell repopulation.Bao et al[176]
Porcine SDSHuman EA.hy926 endothelial cells and HepG2 hepatic carcinoma cells via the portal vein(1) The study demonstrated, exposing scaffold to heparin-gelatin mixture improved endothelial cell ability to migrate and cover vessel discs, perhaps by exploiting gelatin’s multiple integrin binding sites which facilitate endothelial cell binding; and (2) Scaffolds repopulated with Hep G2 hepatocytes and endothelial cells after heparin gelatin coating showed improved ex vivo blood perfusion, in comparison to uncoated scaffolds.Hussein et al[87]
Male Lewis ratsTrypsin + EGTA + Triton X-100Primary rat hepatocytes via the bile duct and the portal veinThe study results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.Ogiso et al[173]
Male Lewis ratsTrypsin + EGTA + Triton X-100(1) Primary rat hepatocytes via the Bile duct; and (2) LSECs via the portal vein(1) Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone; (2) LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity; and (3) Successfully achieved continuous blood flow into the vascularized liver graft by extracorporeal perfusion for at least 8 hoursKojima et al[172]
Female Lewis ratsSDS + Triton X-100Human EA.hy926 endothelial cells via the portal vein(1) Coupled the cell-binding domain REDV to the vasculature of decellularised rat livers; and (2) REDV coupling increased cell attachment, spreading and proliferation of endothelial cells within the scaffold resulting in uniform endothelial lining of the vasculature, and a reduction in platelet adhesion and activationDevalliere et al[88]
Female Lewis ratSDS(1) Rat cholangiocytes via the common bile duct; and (2) Rat hepatocytes via the portal vein(1) Demonstrated for the first time, whole liver grafts co-populated with hepatocytes and cholangiocyte; (2) Cholangiocytes formed duct-like structures, with the viable hepatocyte mass residing in the parenchymal space, in an arrangement highly comparable to the native tissue; and (3) Both albumin and urea assay results confirmed hepatocyte functionality and the gene expression analysis of cholangiocytes in recellularised liver grafts indicated viability and sustained gene expression of functional proteins.Chen et al[177]
Adult Sprague–Dawley ratsTriton X-100 + NH4OHRat sinusoidal endothelial cells were perfused via the Portal vein in either RPMI media or in 5% gelatin hydrogel solution(1) Used immortalized endothelial cells to repopulate decellularised rat liver scaffolds; (2) Gelatin hydrogels-based perfusion significantly increased the number of cells that were retained in the scaffolds; and (3) The Doppler ultrasound detected active blood flows within the re-endothelialised liver scaffolds 8 d post heterotopic transplantation.Meng et al[190]
Male Lewis ratsTrypsin/EGTA solution + Triton X-100/EGTAHuman induced pluripotent stem cells derived hepatocyte-like cells via bile duct(1) The first study to generate a recellularised liver model with human hepatic function using human induced pluripotent stem cells; and (2) This result suggested that the BD was an appropriate recellularization pathway regardless of the hepatocyte type.Minami et al[250]
PorcineSDS + Triton X-100Human umbilical vein endothelial cells via the superior vena cava followed by via the portal veinDecellularised whole porcine livers revascularized with human umbilical endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleen has been removed sustained perfusion for up to 20 d.Shaheen et al[191]
PorcineTriton X-100 + SDS(1) Human umbilical vein endothelial cells via the vena cava and the portal vein; and (2) Porcine hepatocytes via the bile duct(1) Co-seeded primary porcine hepatocytes after human umbilical vein endothelial cell reendothelialization; and (2) Repopulated scaffolds were implanted heterotopically in a pig model and produced improved biochemical function in an acute liver failure model.Anderson et al[175]
Female Sprague-Dawley ratsSDS + DNaseHuman umbilical vein endothelial cells via the Portal vein(1) Used aptamers (short, single-stranded DNA or RNA molecules that selectively bind to specific targets) with CD31 specificity; and (2) Aptamer coated scaffolds showed higher endothelial cell coverage, enabled perfusion with blood for 2 h with reduced platelet adhesion ex vivo, and restored liver function in a hepatic fibrosis rat model.Kim et al[192]