A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc(+)HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc(+)HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models

Whole Organ Tissue Vascularization: Engineering the Tree to Develop the Fruits

Tissue engineering aims to regenerate and recapitulate a tissue or organ that has lost its function. So far successful clinical translation has been limited to hollow organs in which rudimental vascularization can be achieved by inserting the graft into flaps of the omentum or muscle fascia. This technique used to stimulate vascularization of the graft takes advantage of angiogenesis from existing vascular networks. Vascularization of the engineered graft is a fundamental requirement in the process of engineering more complex organs, as it is crucial for the efficient delivery of nutrients and oxygen following in-vivo implantation. To achieve vascularization of the organ many different techniques have been investigated and exploited. The most promising results have been obtained by seeding endothelial cells directly into decellularized scaffolds, taking advantage of the channels remaining from the pre-existing vascular network. Currently, the main hurdle we need to overcome is achieving a fully functional vascular endothelium, stable over a long time period of time, which is engineered using a cell source that is clinically suitable and can generate, in vitro, a yield of cells suitable for the engineering of human sized organs. This review will give an overview of the approaches that have recently been investigated to address the issue of vascularization in the field of tissue engineering of whole organs, and will highlight the current caveats and hurdles that should be addressed in the future

Whole Organ Tissue Vascularization: Engineering the Tree to Develop the Fruits

Tissue engineering aims to regenerate and recapitulate a tissue or organ that has lost its function. So far successful clinical translation has been limited to hollow organs in which rudimental vascularization can be achieved by inserting the graft into flaps of the omentum or muscle fascia. This technique used to stimulate vascularization of the graft takes advantage of angiogenesis from existing vascular networks. Vascularization of the engineered graft is a fundamental requirement in the process of engineering more complex organs, as it is crucial for the efficient delivery of nutrients and oxygen following in-vivo implantation. To achieve vascularization of the organ many different techniques have been investigated and exploited. The most promising results have been obtained by seeding endothelial cells directly into decellularized scaffolds, taking advantage of the channels remaining from the pre-existing vascular network. Currently, the main hurdle we need to overcome is achieving a fully functional vascular endothelium, stable over a long time period of time, which is engineered using a cell source that is clinically suitable and can generate, in vitro, a yield of cells suitable for the engineering of human sized organs. This review will give an overview of the approaches that have recently been investigated to address the issue of vascularization in the field of tissue engineering of whole organs, and will highlight the current caveats and hurdles that should be addressed in the future

ECM from decellularised tissues as an additive for polysaccharidic hybrid gels

The use of decellularised tissues represents a valid and emerging alternative over traditional synthetic scaffolds, which have limited ability to mimic the sophisticated tissue specificity1. Within the tissue engineering context, gels composed by decellularised tissues have been produced through enzymatic digestion followed by basic pH treatment2. Nevertheless, low viscosity, stability and reproducibility often limit their applicative potential. Herein, ECM, obtained from porcine blood vessels, was imbedded within alginate gels and compared to both alginate and alginate/gelatin gels aiming to process decellularised tissues in diverse physical forms and therefore broaden their application. Porcine blood vessels were decellularised1 and gels were further obtained adapting the procedure previously described2. Gels containing ECM or gelatin (8 mg/ml) and different concentrations of alginate (2-20 mg/ml) were produced by internal gelation (CaCO3 2,8% w/v, D- (+)-gluconic acid δ-lactone 0,5% w/v). The alginate samples were obtained preserving the final polymer concentration (10,13,18, 28 mg/ml). Rheological characterization was performed by time, frequency and temperature sweep analyses3. Stability tests were conducted using cell culture medium (complete DMEM medium) from 3 hours up to 7 days. Additionally, preliminary biological characterization was assessed through DNA content after seeding EA.hy 926 for 1 day. ECM-loaded alginate gels (AlgECM) samples were successfully obtained for all the concentration tested. All the samples could be removed from a mould while retaining the shape. The storage and loss moduli of all the tested alginate concentrations were frequency-independent, with the storage modulus higher than the loss modulus, therefore exhibiting gel behaviour. Higher final polymer concentration resulted in gels with higher complex viscosity. Overall, AlgECM samples showed higher values of both storage and loss moduli and higher stability in the medium comparing with unloaded alginate gels. Samples obtained with gelatin could not be produced at polymer concentrations lower than 18 mg/ml. The AlgECM samples remained stable in cell culture medium; samples with the lowest concentration of alginate (2 mg/ml of alginate and 8 mg/ml w/v of ECM) degraded after 7 days. A first biological characterization indicated an increased number of cells for AlgECM gels compared to alginate and alginate/gelatin samples. A novel gel composed of alginate and native vascular decellularised ECM is here proposed. AlgECM gels able to combine the properties of its components. Alginate improved ECM gels reproducibility and allowed the tailoring of gels rheological properties through the variation of alginate concentration. The use of ECM should promote the creation of a tissue-specific material, able to enhance cell growth and proliferation. However, a wider biological characterization should be conducted to test the ECM influence. References 1. Cells Tissues Organs 200:363–373, 2015. 2. Biomaterials 29:1630-1637, 2008. 3. Carbohyd. Polym. 103:339–347, 2014

Terminal sterilization of equine-derived decellularized tendons for clinical use

In the last few years, the demand for tissue substitutes has increased and decellularized matrices has been widely proposed in the medical field to restore severe damages thanks to high biocompatibility and biomechanical properties similar to the native tissues. However, biological grafts represent a potential source of contamination and disease transmission; thus, there is the need to achieve acceptable levels of sterility. Several sterilization methods have been investigated with no consensus on the outcomes in terms of minimizing structural damages and preserving functional features of the decellularized matrix for transplantation in humans. With the aim of making decellularized tendons safe for clinical use, we evaluated the cytocompatibility, and biochemical, structural and biomechanical variations of decellularized equine tendons sterilized with peracetic acid or β-irradiation and differently wet- or dry- stored at 4 °C or â\u88\u92 80 °C, respectively. Considering that both sterilization and long-term storage are crucial steps that could not be avoided, our results pointed at ionizing β-rays as terminal sterilization method for decellularized grafts followed by frozen dry storage. Indeed, this approach can maintain the integrity of collagen-based structures and can avoid biomechanical changes, thus making xenogeneic decellularized tendons a promising candidate for clinical use

Whole rat stomach decellularisation using a detergent-enzymatic protocol

BACKGROUND: Conditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol. METHODS: Stomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications. RESULTS: The detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced. CONCLUSION: Gastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions

Arterial Decellularized Scaffolds Produced Using an Innovative Automatic System

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for alpha-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation. (C) 2015 S. Karger AG, Base

Arterial Decellularized Scaffolds Produced Using an Innovative Automatic System

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for alpha-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation. (C) 2015 S. Karger AG, Base