Data on a novel liver bioscaffold (rDLS) generated from regenerative liver with activated extracellular matrix for functional liver regeneration

The data presented in this article are related to the original research article entitled “A novel bioscaffold with naturally-occurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation” (Yang et al., in press) [1]. This article describes a decellularized liver scaffold (DLS) that derived from partial hepatectomy liver (rDLS) which supported primary hepatocyte survival and promoted blood patency, as compared with a conventional scaffold that generated from naïve liver (nDLS). Analysis by immunochemistry and scanning electron microscope (SEM) showed that the vessel network and extracellular matrix (ECM) components were similar to the nDLS. The rDLS could prevent blood clotting after transplanted it in vivo, identified by immunofluorescence staining for the integrin (αIIb, α4) expression and liver transplantation models (mice, pigs) a formed well-blood petency liver lobules. These data indicate that the novel scaffold (rDLS) with naturally-occurring “activated ECM” that may be useful for the implantation in vivo of a bioengineered organoid that is able to exert function long term without clotting in future clinic.

The data presented in this article are related to the original research article entitled "A novel bioscaffold with naturallyoccurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation" (Yang et al., in press) [1]. This article describes a decellularized liver scaffold (DLS) that derived from partial hepatectomy liver (rDLS) which supported primary hepatocyte survival and promoted blood patency, as compared with a conventional scaffold that generated from naïve liver (nDLS). Analysis by immunochemistry and scanning electron microscope (SEM) showed that the vessel network and extracellular matrix (ECM) components were similar to the nDLS. The rDLS could prevent blood clotting after transplanted it in vivo, identified by immunofluorescence staining for the integrin (αIIb, α4) expression and liver transplantation models (mice, pigs) a formed well-blood petency liver lobules. These data indicate that the novel scaffold (rDLS) with

Value of the data
The data demonstrate for the first time that by using a novel approach to generate a decellularized liver scaffold (DLS) from regenerative liver (rDLS) which is different from the scaffold that generated from naïve livers (nDLS). The nDLS has been widely studies in the field.
The data providing here for the first time support the rDLS with "active ECM environment" for preventing thrombosis in vivo, and the idea opens a new window for other researchers promising step toward to engineer functional organoids.

Data
Naïve decellularized liver scaffold (nDLS)-based tissue engineering has been impaired by the lack of a suitable extracellular matrix (ECM) to provide "active micro-environmental" support [2]. Generation of the novel decellularized liver scaffold (rDLS) with "active micro-environmental" is shown in Fig. 1. We anesthetized mice with pentobarbital sodium salt and performed approximately 30-45% partial hepatectomy. The liver was perfused with detergent solution after three days [3] and the extracellular matrix was evaluated by SEM, DNA content, and immunofluorescence staining for ECM components (Fig. 2). The data indicate that rDLS has similar structure and ECM proteins compared to the nDLS. To address the anti-clotting effect in vivo, the rDLS was grafted into animals by portal-renal arterialized auxiliary heterotopic (mice) (Fig. 3) and inorthotropic (pig) (Fig. 4) liver transplantation. At this stage, the rDLS had lower expression of integrins (αIIb, α4) (Fig. 5) and higher blood patency than those of nDLS (Fig. 4). These are the first report on the anti-thrombosis of the rDLS. In addition, the rDLS/seed cell complex showed the better liver lobule formation after transplantation as compare to nDLS/seed cell complex in vivo (Fig. 6).

Generation of rDLS
The C57BL/6 mouse strain and pigs (China, Chongqing) was used in this study. A surgical method and perfusion procedure [4] was used for generation of rDLS. The Committee for the experiments involving animals care, handling and surgical procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of the Third Military Medical University, Chongqing, China (Protocol no. SYXK-PLA-20120031).

DNA content measurement
DNA content was quantified as previously described [5]. In brief, the rDLS was digested with proteinase K (Invitrogen Inc., Carlsbad, CA, USA) at 37°C and centrifuged at 2980g for 30 min. Supernatants were purified and centrifuged at 9000g for 30 min. Aqueous layers were removed and added to 3 M sodium acetate solution. Samples were treated with ethanol at À 20°C for at least 8 h to precipitate the DNA quantified using the Picogreen DNA assay (Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer's instructions. DNA fragments were separated by electrophoresis on a 3% low melting point agarose gel with ethidium bromide at 60 V for 1 h and visualized under ultraviolet light (BioRad, Hercules, CA, USA).

Scanning electron microscopy
RDLS samples were assessed by scanning electron microscopy as previously described [6]. Briefly, rDLS samples were sectioned into small pieces (8 mm 3 ), fixed in 4% glutaraldehyde stained with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 h. Then, sections were washed in PBS and dehydrated using a graded series of alcohol. Critical drying was performed in absolute ethanol. Each sample was mounted on an aluminum stub and sputter-coated with a 7-nm layer of gold (Cressington 108 sputter coater, Cressington Scientific Instruments Ltd., Watford, UK) before examination using a JEM 6335F field emission scanning electron microscope (JEOL, Tokyo, Japan).

Immunofluorescence staining for platelet aggregation
The fixed DLS samples were embedded in flash-frozen in liquid nitrogen and sectioned to 5 μm. Sections were blocked with 10% fetal bovine serum for 30 min and primary antibodies (αIIb, α4, 1:100-500) were applied overnight at 4°C. after secondary antibody, sections were mounted using fluorescence mounting medium (Dako, Glostrup, Denmark). Fluorescence signals were observed under a fluorescence microscope (SMZ25/SMZ18; Nikon, Tokyo, Japan). Analysis was performed using Image J software (National Institutes of Health, Bethesda, MI, USA).

Surgical mouse and pig model for thrombosis measurement
Mice were anesthetized and opened along the midline to expose the right kidney. After clamping the right branches of the renal artery and vein, a nephrectomy was performed. Once the right kidney was removed, the DLS was placed into the nephrectomy site. The portal vein and inferior vena cava of the host were introduced into the DLS graft's portal vein, and another of the same size was introduced and secured in the inferior vena cave. An end-to-end anastomosis was made between the cuff of the recipient's left renal artery branch and the scaffold's portal vein. The connection of the recipient's inferior vena cava to that of the scaffold was performed using end-to-side anastomosis and Any blood lost during surgery was replaced with lactated Ringer's solution.