decellularization-recellularization-methodology-for-human-saphenous

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

University of Gothenburg

Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity.

Characterization and engraftment of long-term serum-free human fetal liver cell cultures.

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for tissue-specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells (P <0.001). The presence of antibodies was associated with female gender (P < 0.001), systolic hypertension (P < 0.01), and elevated TNF-α (P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.

Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.

One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH.

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes.

Extrahepatic portal vein obstruction can have severe health consequences. Variceal bleeding associated with this disorder causes upper gastrointestinal bleeding, leading to substantial morbidity and mortality. We report the clinical transplantation of a deceased donor iliac vein graft repopulated with recipient autologous stem cells in a patient with extrahepatic portal vein obstruction.

Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.

Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.

Isolation and characterization of human primary enterocytes from small intestine using a novel method.

We aimed to produce an acellular human tissue scaffold with a view to test the possibility of recellularization with bone marrow stem cells to produce a tissue-engineered small intestine (TESI). Human small-bowel specimens (n = 5) were obtained from cadaveric organ donors and treated sequentially with 6% dimethyl sulfoxide in hypotonic buffer, 1% Triton X-100, and DNase. Each small intestine (SI) piece (6 cm) was recellularized with EPCAM+ and CD133+ allogeneic bone marrow stem cells. Histological and molecular analysis demonstrated that after decellularization, all cellular components and nuclear material were removed. Our analysis also showed that the decellularized human SI tissue retained its histoarchitecture with intact villi and major structural proteins. Protein films of common extracellular matrix constituents (collagen I, laminin, and fibronectin) were found in abundance. Furthermore, several residual angiogenic factors were found in the decellularized SI. Following recellularization, we found viable mucin-positive goblet cells, CK18+ epithelial cells in villi adjacent to a muscularis mucosa with α-actin+ smooth muscle cells, and a high repopulation of blood vessels with CD31+ endothelial cells. Our results show that in the future, such a TESI would be ideal for clinical purposes, because it can be derived from the recipient's own immunocompatible bone marrow cells, thus avoiding the use of immunosuppression.

Recellularization of acellular human small intestine using bone marrow stem cells.

Cell-based therapies involving tissue engineering represent interesting and potentially important strategies for the treatment of patients with various disorders. In this study, using a detergent-enzymatic method, we prepared an intact three-dimensional scaffold of an extracellular matrix derived from a human cadaver donor trachea, which we repopulated with autologous stem cells and implanted into a 76-year-old patient with tracheal stenosis including the lower part of the larynx. Although the graft provided the patient with an open airway, a week after the surgery, the mucous membrane of the graft was covered by a 1-2 mm thick fungal infection, which was treated with local and systemic antifungal therapy. The airway lumen was postoperatively controlled by fiber endoscopy and found stable and sufficient. However, after 23 days, the patient died due to cardiac arrest but with a patent, open, and stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft during autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of α-sma-positive muscle cells, serous glands, and nerve fibers with S-100-positive nerve cells in the submucosa and intact chondrocytes in the cartilage. Our findings suggest that although autologous stem cells-engineered tracheal matrices may represent a tool for clinical tracheal replacement, further preclinical studies are required for generating functional airway grafts and long-term effects of such grafts.

Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report.

Hepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method.

TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy.

Scaffold characteristics are decisive for repopulating the acellular tissue with cells. A method to produce such a scaffold from intact organ requires a customized decellularization protocol. Here, we have decellularized whole, intact porcine hearts by serial perfusion and agitation of hypotonic solution, an ionic detergent (4% sodium deoxycholate), and a nonionic detergent (1% Triton X-100). The resultant matrix was characterized for its degree of decellularization, morphological and functional integrity. The protocol used resulted in extensive decellularization of the cardiac tissue, but the cytoskeletal elements (contractile apparatus) of cardiomyocytes remained largely unaffected by the procedure although their membranous organelles were completely absent. Further, several residual angiogenic growth factors were found to be present in the decellularized tissue. 

An alternative approach to decellularize whole porcine heart.

Vascular diseases are increasing health problems affecting > 25 million individuals in westernized societies. Such patients could benefit from transplantation of tissue-engineered vascular grafts using autologous cells. One challenge that has limited this development is the need for cell isolation, and risks associated with ex vivo expanded stem cells. Here we demonstrate a novel approach to generate transplantable vascular grafts using decellularized allogeneic vascular scaffolds, repopulated with peripheral whole blood (PWB) in vitro in a bioreactor. Circulating, VEGFR-2 +/CD45 + and a smaller fraction of VEGFR-2 +/CD14 + cells contributed to repopulation of the graft. SEM micrographs showed flat cells on the luminal surface of the grafts consistent with endothelial cells. For clinical validation, two autologous PWB tissue-engineered vein conduits were prepared and successfully used for by-pass procedures in two pediatric patients. These results provide a proof of principle for the generation of transplantable vascular grafts using a simple autologous blood sample, making it clinically feasible globally. 

In Vivo Application of Tissue-Engineered Veins Using Autologous Peripheral Whole Blood: A Proof of Concept Study.

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Significantly Accelerated Wound Healing of Full-Thickness Skin Using a Novel Composite Gel of Porcine Acellular Dermal Matrix and Human Peripheral Blood Cells.

The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.

Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture.

Despite progress in the field of decellularization and recellularization, the outcome for pancreas has not been adequate. This might be due to the challenging dual nature of pancreas with both endocrine and exocrine tissues. We aimed to develop a novel and efficient cold-perfusion method for decellularization of porcine pancreas and recellularize acellular scaffolds with human fetal pancreatic stem cells. Decellularization of whole porcine pancreas at 4°C with sodium deoxycholate, Triton X-100 and DNase efficiently removed cellular material, while preserving the extracellular matrix structure. Furthermore, recellularization of acellular pieces with human fetal pancreatic stem cells for 14 days showed attached and proliferating cells. Both endocrine (C-peptide and PDX1) and exocrine (glucagon and α-amylase) markers were expressed in recellularized tissues. Thus, cold-perfusion can successfully decellularize porcine pancreas, which when recellularized with human fetal pancreatic stem cells shows relevant endocrine and exocrine phenotypes. Decellularized pancreas is a promising biomaterial and might translate to clinical relevance for treatment of diabetes.

Cold-perfusion decellularization of whole-organ porcine pancreas supports human fetal pancreatic cell attachment and expression of endocrine and exocrine markers.

Suchitra Sumitran-Holgersson

Department of Surgery,
Institute of Clinical Sciences,
Sahlgrenska Academy,
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy

University of Gothenburg

Decellularization and Recellularization Methodology for Human Saphenous Veins

Suchitra Sumitran-Holgersson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Surgery,Institute of Clinical Sciences,Sahlgrenska Academy,Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy

University of Gothenburg

Decellularization and Recellularization Methodology for Human Saphenous Veins

Department of Surgery,
Institute of Clinical Sciences,
Sahlgrenska Academy,
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy