Preparation Of Soluble Liver Extracellular Matrix Bioink For Three Dimensional Bioprinting

Objective: Liver transplantation is the only definitive treatment for end-stage liver disease and its availability is restricted by organ donor shortages.The development of three-dimensional(3D)bioprint provides the prospect to address this limitation.Bioink encapsulates cells to assemble into hepatoid organs and generates cell-matrix interactions regulate cell functions through 3D bioprint.Therefore,the properties of bioink affect the entire biological functions of the organoids.Gelatin methacryloyl(Gel MA)is a natural material,which is commonly used to construct hepatoid organs with complex structures.However,Gel MA lacks the unique biochemical macromolecules of liver and cannot well simulate the natural growing niche of hepatocytes.Decellularized liver scaffold(DLS)retains the extracellular matrix(ECM)components.ECM is rich in bioactive substances that regulate cell biological behavior.Bioink derived from DLS can improve the biocompatibility of Gel MA.However,the process of preparing DLS into bioink have a destructive effect on ECM,it is necessary to establish a suitable and mild soluble ECM bioink protocol.Methods: 1% Triton X-100 and 0.1% sodium dodecyl sulfate(SDS)were employed to generate rat DLS,which was then detoxified,digested and lyophilized to prepare soluble ECM.Results: The results showed that the DLS was translucent,and the entire vascular structure was visualized after injection with trypan blue.Histological staining and scanning electron microscopy revealed that cell remnants were removed;DNA quantitative analysis showed that the residual DNA content of DLS was 32.98±6.68 ng/mg,which was significantly lower than that of normal liver(P < 0.05);Furthermore,soluble ECM retains collagen,glycosaminoglycans,hepatocyte growth factor,and basic growth factor.Live/dead staining and Alamarblue analysis results showed that the inclusion of soluble ECM in Gel MA hydrogel can significantly improve the cell viability of L-O2,and the higher the ECM content in the hydrogel,the better the cell viability of L-O2.Conclusions: These results demonstrated that 1% Triton and 0.1%SDS can completely remove cell components,while retaining vascular structure and ECM components.Furthermore,the soluble ECM obtained through washing,digestion and lyophilization retains the bioactive substances and can improved the biocompatibility of the biomaterial Gel MA.This reveals that we have successfully constructed a suitable protocol of liver ECM bioink,which can provide a basis for biomaterial research.