A Telese

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.

Development of human liver extracellular matrix hydrogel for three dimensional cell culture and cell transplantation

Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications.

Management of a rare cause of significant acute upper gastrointestinal bleeding: gastric lipoma resected by hybrid endoscopic submucosal dissection

Gastric lipomas are rare, benign, slowgrowing subepithelial tumors. Most lipomas remain asymptomatic and are detected incidentally at endoscopy. Large lipomas, however, may very rarely cause significant upper gastrointestinal (GI) bleeding and warrant removal [1]. For the management of large gastric lipomas, laparoscopic excision may be required, but endoscopic techniques such as endoscopic submucosal dissection (ESD) and unroofing have also been described [1, 2]. This endoscopic video case highlights this rare cause of upper GI bleeding and its definitive management by hybrid ESD. A 66-year-old man presented with melena, pallor, and fatigue. On admission, his hemoglobin level was 89g/L. He underwent an upper GI endoscopy during which a 3 cm ulcerated submucosal lesion was identified at the greater curvature (▶Fig. 1). An endoscopic clip and Hemospray (Cook Medical, Winston-Salem, North Carolina, USA) were applied, and hemostasis was achieved. Computed tomography imaging revealed an ovoid (fat-dense) lesion measuring 25×15mm, consistent with a lipoma (▶Fig. 2). Endoscopic ultrasound assessment of the lesion was subsequently performed, and showed the presence of a homogeneous and slightly hyperechoic lesion, 25×15mm, arising from the hyperechoic submucosal layer. A 22g needle was used to obtain core biopsies but unfortunately the sample was inadequate for diagnostic assessment. Endoscopic management of the lesion was agreed and the lesion was success-

A rare cause of small-bowel bleeding: haemorrhagic small-bowel lymphangioma diagnosed by antegrade double-balloon enteroscopy

Small-bowel lymphangiomata (SBLs) are benign and relatively uncommon tumors of the lymphatic system [1]. Although SBLs are usually clinically silent, they may rarely present with significant small-bowel bleeding, protein-losing enteropathy, and intussusception [2, 3]. A 54-year-old-man with a past medical history of stable chronic lymphocytic leukemia presented with transfusion-dependent obscure-overt gastrointestinal (GI) bleeding. Upper and lower GI endoscopies and small-bowel cross-sectional imagining were unremarkable. A smallbowel video capsule endoscopy (VCE) showed a white-speckled congested lesion, with active oozing (▶Fig. 1). The lesion was estimated to be about 2 cm in diameter and was deemed to be located within the jejunum. Antegrade double-balloon enteroscopy (DBE) was subsequently performed. The enteroscope was advanced to an estimated insertion depth of 240 cm post-pylorus, where the lesion seen at VCE was identified (▶Fig. 2, ▶Video1). This had a white–yellow appearance with overlying severely congested villi giving a “strawberry-like” mucosal pattern. The lesion was not deemed to be endoscopically resectable; multiple biopsies were taken and a reference tattoo was placed proximal to it. Histopathological exam confirmed a lymphangiomatous etiology without any evidence of dysplasia or malignancy (▶Fig. 3). Minimally invasive tattooguided laparoscopic resection has been planned. This case highlights the key, complementary role of small-bowel VCE and DBE for the diagnosis and minimally invasive management of clinically significant SBLs.