Abstract
Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.
Highlights
Decellularized tissues have provided an option for engineering tissue both for transplantation and for disease modeling
There were no macroscopic differences observed between the rat livers pre-treated with EDTA and those treated with detergent enzymatic treatment (DET) only (Fig 1A and 1B)
EDTA-DET treatment was more efficient in removing cellular proteins as the nuclear protein Sub1 and the cytoplasmic protein Tim13 could be identified in the DET sample (Fig 1E and 1F)
Summary
Decellularized tissues have provided an option for engineering tissue both for transplantation and for disease modeling. Ideal scaffolds should have architectural and mechanical characteristics allowing migration and proliferation of introduced cells, a defined biodegradation profile, and a minimal immune response [1,2] For complex organs, such as the liver, scaffold choice is limited to decellularized materials, wherein cell removal from the whole-organ yields a three-dimensional extracellular matrix (ECM) [1,3]. Current decellularization protocols cause substantial harm to the ECM and may render the vasculature too porous for successful transplantation. This is effectively shown in vascular casting images in rat livers decellularized by 1% SDS and 1% TX100 that demonstrate destruction of the blood vessel network [9]
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