Endothelial Cell Replacement of Human Veins, Modeling Vascular Repair and Endothelial Cell Chimerism.

Allogeneic transplant organs are potentially highly immunogenic. The endothelial cells (ECs) located within the vascular system serve as the primary interface between the recipient's immune system and the donor organ, playing a key role in the alloimmune response. In this study, we investigated the potential use of recipient-derived ECs in a vein recellularization model. In this study, human iliac veins underwent complete decellularization using a Triton X-100 protocol. We demonstrated the feasibility of re-endothelializing acellular blood vessels using either human umbilical cord vein endothelial cell or human venous-derived ECs, with this re- endothelialization being sustainable for up to 28 days in vitro. The re-endothelialized veins exhibited the restoration of vascular barrier function, along with the restoration of innate immunoregulatory capabilities, evident through the facilitation of monocytic cell transmigration and their polarization toward a macrophage phenotype following transendothelial extravasation. Finally, we explored whether recellularization with EC of a different donor could prevent antibody-mediated rejection. We demonstrated that in chimeric vessels, allogeneic EC became a target of the humoral anti-donor response after activation of the classical immune complement pathway whereas autologous EC were spared, emphasizing their potential utility before transplantation. In conclusion, our study demonstrates that replacement of EC in transplants could reduce the immunological challenges associated with allogeneic grafts.

Tumor decellularization reveals proteomic and mechanical characteristics of the extracellular matrix of primary liver cancer.

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.

Extracellular matrix drives tumor organoids toward desmoplastic matrix deposition and mesenchymal transition.

Patient-derived tumor organoids have been established as promising tools for in vitro modelling of multiple tumors, including cholangiocarcinoma (CCA). However, organoids are commonly cultured in basement membrane extract (BME) which does not recapitulate the intricacies of the extracellular matrix (ECM). We combined CCA organoids (CCAOs) with native tumor and liver scaffolds, obtained by decellularization, to effectuate a model to study the interaction between epithelial tumor cells and their surrounding ECM. Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin, including stiffness and presence of desmoplasia. The transcriptome of CCAOs in a tumor scaffold much more resembled that of patient-paired CCA tissue in vivo compared to CCAOs cultured in BME or liver scaffolds. This was accompanied by an increase in chemoresistance to clinically-relevant chemotherapeutics. CCAOs in decellularized scaffolds revealed environment-dependent proliferation dynamics, driven by the occurrence of epithelial-mesenchymal transition. Furthermore, CCAOs initiated an environment-specific desmoplastic reaction by increasing production of multiple collagen types. In conclusion, convergence of organoid-based models with native ECM scaffolds will lead to better understanding of the in vivo tumor environment. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) influences various facets of tumor behavior. Understanding the exact role of the ECM in controlling tumor cell fate is pertinent to understand tumor progression and develop novel therapeutics. This is particularly the case for cholangiocarcinoma (CCA), whereby the ECM displays a distinct tumor environment, characterized by desmoplasia. However, current models to study the interaction between epithelial tumor cells and the environment are lacking. We have developed a fully patient-derived model encompassing CCA organoids (CCAOs) and human decellularized tumor and tumor-free liver ECM. The tumor ECM induced recapitulation of various aspects of CCA, including migration dynamics, transcriptome and proteome profiles, and chemoresistance. Lastly, we uncover that epithelial tumor cells contribute to matrix deposition, and that this phenomenon is dependent on the level of desmoplasia already present.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Human branching cholangiocyte organoids recapitulate functional bile duct formation.

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Hydrogels derived from decellularized liver tissue support the growth and differentiation of cholangiocyte organoids.

Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.

Design by Nature: Emerging Applications of Native Liver Extracellular Matrix for Cholangiocyte Organoid-Based Regenerative Medicine.

Organoid technology holds great promise for regenerative medicine. Recent studies show feasibility for bile duct tissue repair in humans by successfully transplanting cholangiocyte organoids in liver grafts during perfusion. Large-scale expansion of cholangiocytes is essential for extending these regenerative medicine applications. Human cholangiocyte organoids have a high and stable proliferation capacity, making them an attractive source of cholangiocytes. Commercially available basement membrane extract (BME) is used to expand the organoids. BME allows the cells to self-organize into 3D structures and stimulates cell proliferation. However, the use of BME is limiting the clinical applications of the organoids. There is a need for alternative tissue-specific and clinically relevant culture substrates capable of supporting organoid proliferation. Hydrogels prepared from decellularized and solubilized native livers are an attractive alternative for BME. These hydrogels can be used for the culture and expansion of cholangiocyte organoids in a clinically relevant manner. Moreover, the liver-derived hydrogels retain tissue-specific aspects of the extracellular microenvironment. They are composed of a complex mixture of bioactive and biodegradable extracellular matrix (ECM) components and can support the growth of various hepatobiliary cells. In this review, we provide an overview of the clinical potential of native liver ECM-based hydrogels for applications with human cholangiocyte organoids. We discuss the current limitations of BME for the clinical applications of organoids and how native ECM hydrogels can potentially overcome these problems in an effort to unlock the full regenerative clinical potential of the organoids.

A proof of concept study on real-time LiMAx CYP1A2 liver function assessment of donor grafts during normothermic machine perfusion.

No single reliable parameter exists to assess liver graft function of extended criteria donors during ex-vivo normothermic machine perfusion (NMP). The liver maximum capacity (LiMAx) test is a clinically validated cytochromal breath test, measuring liver function based on 13CO2 production. As an innovative concept, we aimed to integrate the LiMAx breath test with NMP to assess organ function. Eleven human livers were perfused using NMP. After one hour of stabilization, LiMAx testing was performed. Injury markers (ALT, AST, miR-122, FMN, and Suzuki-score) and lactate clearance were measured and related to LiMAx values. LiMAx values ranged between 111 and 1838 µg/kg/h, and performing consecutive LiMAx tests during longer NMP was feasible. No correlation was found between LiMAx value and miR-122 and FMN levels in the perfusate. However, a significant inverse correlation was found between LiMAx value and histological injury (Suzuki-score, R = - 0.874, P < 0.001), AST (R = - 0.812, P = 0.004) and ALT (R = - 0.687, P = 0.028). Furthermore, a significant correlation was found with lactate clearance (R = 0.683, P = 0.043). We demonstrate, as proof of principle, that liver function during NMP can be quantified using the LiMAx test, illustrating a positive correlation with traditional injury markers. This new breath-test application separates livers with adequate cytochromal liver function from inadequate ones and may support decision-making in the safe utilization of extended criteria donor grafts.

Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro.

The well-established 3D organoid culture method enabled efficient expansion of cholangiocyte-like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal-invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD- and EHBD-tissue. We demonstrate successful organoid-initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional-specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP-derived BCOs and gallbladder-derived BCOs expressing gallbladder-specific genes. Moreover, BCOs have limited hepatocyte-fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid-initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte-like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid-initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine.

Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.

Biliary disorders can lead to life-threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue-specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte-markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.

Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion.

Human whole-liver perfusion-decellularization is an emerging technique for producing bio-scaffolds for tissue engineering purposes. The native liver extracellular matrix (ECM) provides a superior microenvironment for hepatic cells in terms of adhesion, survival and function. However, current decellularization protocols show a high degree of variation in duration. More robust and effective protocols are required, before human decellularized liver ECM can be considered for tissue engineering applications. The aim of this study is to apply pressure-controlled perfusion and test the efficacy of two different detergents in porcine and human livers. To test this, porcine livers were decellularized using two different protocols; a triton-x-100 (Tx100)-only protocol (N = 3) and a protocol in which Tx100 was combined with SDS (N = 3) while maintaining constant pressure of 120 mm Hg. Human livers (N = 3) with different characteristics (age, weight and fat content) discarded for transplantation were decellularized using an adapted version of the Tx-100-only protocol. Decellularization efficacy was determined by histology and analysis of DNA and RNA content. Furthermore, the preservation of ECM components was assessed. After completing the perfusion cycles with detergents the porcine livers from both protocols were completely white and transparent in color. After additional washing steps with water and DNase, the livers were completely decellularized, as no DNA or cell remnants could be detected. The Tx100-only protocol retained 1.5 times more collagen and 2.5 times more sGAG than the livers decellularized with Tx100 + SDS. The Tx100-only protocol was subsequently adapted for decellularizing whole-organ human livers. The human livers decellularized with pressure-controlled perfusion became off-white in color and semi-transparent within 20 h. Livers decellularized without pressure-controlled perfusion took 64-96 h to completely decellularize, but did not become white or transparent. The addition of pressure-controlled flow did remove all cells and double stranded DNA, but did not damage the ultra-structure of the ECM as was analyzed by histology and scanning electron microscopy. In addition, collagens and sGAG were maintained with the decellularized ECM. In conclusion, we established effective, robust and fast decellularization protocols for both porcine and human livers. With this protocol the duration of decellularization for whole-organ human livers has been shortened considerably. The increased pressure and flow did not damage the ECM, as major ECM components remained intact.

Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds.

Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.

From organoids to organs: Bioengineering liver grafts from hepatic stem cells and matrix.

Due to the complex function and structure of the liver, resourceful solutions for treating end-stage liver disease are required. Currently, liver transplantation is the only curative therapeutic option. However, due to a worldwide donor shortage, researchers have been looking in other fields for alternative sources of transplantable liver tissue. Recent advances in our understanding of liver physiology, stem cell and matrix biology, have accelerated tissue engineering research. Most notable is the discovery of a culture system to grow liver-like organoids from human hepatic stem cells. The extensive expansion capacity of these stem cells has contributed greatly to the availability of hepatocyte-like cells for tissue engineering. In addition, new techniques are explored to obtain biological liver scaffolds from full size donor organs. This review summarizes these state-of-art techniques which may lay the groundwork towards re-creating transplantable tissue from autologous or allogenic stem cells in the coming decade.