Improved recellularization of ex vivo vascular scaffolds using directed transport gradients to modulate ECM remodeling

The regeneration of functional, clinically viable, tissues from acellular ex vivo tissues has been problematic largely due to poor nutrient transport conditions that limit cell migration and integration. Compounding these issues are subcellular pore sizes that necessarily requires extracellular matrix (ECM) remodeling in order for cells to migrate and regenerate the tissue. The aim of the present work was to create a directed growth environment that allows cells to fully populate an ex vivo‐derived vascular scaffold and maintain viability over extended periods. Three different culture conditions using single (one nutrient source) or dual perfusion bioreactor systems (two nutrients sources) were designed to assess the effect of pressure and nutrient gradients under either low (50/30 mmHg) or high (120/80) relative pressure conditions. Human myofibroblasts were seeded to the ablumenal periphery of an ex vivo‐derived vascular scaffold using a collagen/hydrogel cell delivery system. After 30 days culture, total cell density was consistent between groups; however, significant variation was noted in cell distribution and construct mechanics as a result of differing perfusion conditions. The most aggressive transport gradient was developed by the single perfusion low‐pressure circuits and resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source). These investigations illustrate the influence of directed nutrient gradients where precisely controlled perfusion conditions significantly affects cell migration, distribution and function, resulting in pronounced effects on construct mechanics during early remodeling events. Biotechnol. Bioeng. 2013; 110: 2035–2045. © 2013 Wiley Periodicals, Inc.     

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO₂. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Future prospects for tissue engineered lung transplantation

The shortage of donor lungs for transplantation causes a significant number of patient deaths. The availability of laboratory engineered, functional organs would be a major advance in meeting the demand for organs for transplantation. The accumulation of information on biological scaffolds and an increased understanding of stem/progenitor cell behavior has led to the idea of generating transplantable organs by decellularizing an organ and recellularizing using appropriate cells. Recellularized solid organs can perform organ-specific functions for short periods of time, which indicates the potential for the clinical use of engineered solid organs in the future.

The present review provides an overview of progress and recent knowledge about decellularization and recellularization-based approaches for generating tissue engineered lungs. Methods to improve decellularization, maturation of recellularized lung, candidate species for transplantation and future prospects of lung bioengineering are also discussed.     

Future prospects for tissue engineered lung transplantation: Decellularization and recellularization-based whole lung regeneration

The shortage of donor lungs for transplantation causes a significant number of patient deaths. The availability of laboratory engineered, functional organs would be a major advance in meeting the demand for organs for transplantation. The accumulation of information on biological scaffolds and an increased understanding of stem/progenitor cell behavior has led to the idea of generating transplantable organs by decellularizing an organ and recellularizing using appropriate cells. Recellularized solid organs can perform organ-specific functions for short periods of time, which indicates the potential for the clinical use of engineered solid organs in the future.   The present review provides an overview of progress and recent knowledge about decellularization and recellularization-based approaches for generating tissue engineered lungs. Methods to improve decellularization, maturation of recellularized lung, candidate species for transplantation and future prospects of lung bioengineering are also discussed.