Sustained in vivo perfusion of a re-endothelialized tissue engineered kidney graft in a human-scale animal model.

Introduction: Despite progress in whole-organ decellularization and recellularization, maintaining long-term perfusion in vivo remains a hurdle to realizing clinical translation of bioengineered kidney grafts. The objectives for the present study were to define a threshold glucose consumption rate (GCR) that could be used to predict in vivo graft hemocompatibility and utilize this threshold to assess the in vivo performance of clinically relevant decellularized porcine kidney grafts recellularized with human umbilical vein endothelial cells (HUVECs). Materials and methods: Twenty-two porcine kidneys were decellularized and 19 were re-endothelialized using HUVECs. Functional revascularization of control decellularized (n = 3) and re-endothelialized porcine kidneys (n = 16) was tested using an ex vivo porcine blood flow model to define an appropriate metabolic glucose consumption rate (GCR) threshold above which would sustain patent blood flow. Re-endothelialized grafts (n = 9) were then transplanted into immunosuppressed pigs with perfusion measured using angiography post-implant and on days 3 and 7 with 3 native kidneys used as controls. Patent recellularized kidney grafts underwent histological analysis following explant. Results: The glucose consumption rate of recellularized kidney grafts reached a peak of 39.9 ± 9.7 mg/h at 21 ± 5 days, at which point the grafts were determined to have sufficient histological vascular coverage with endothelial cells. Based on these results, a minimum glucose consumption rate threshold of 20 mg/h was set. The revascularized kidneys had a mean perfusion percentage of 87.7% ± 10.3%, 80.9% ± 33.1%, and 68.5% ± 38.6% post-reperfusion on Days 0, 3 and 7, respectively. The 3 native kidneys had a mean post-perfusion percentage of 98.4% ± 1.6%. These results were not statistically significant. Conclusion: This study is the first to demonstrate that human-scale bioengineered porcine kidney grafts developed via perfusion decellularization and subsequent re-endothelialization using HUVEC can maintain patency with consistent blood flow for up to 7 days in vivo. These results lay the foundation for future research to produce human-scale recellularized kidney grafts for transplantation.

Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds.

Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.

The consequence of biologic graft processing on blood interface biocompatibility and mechanics.

Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.

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.

Physiologically Modeled Pulse Dynamics to Improve Function in In Vitro-Endothelialized Small-Diameter Vascular Grafts.

The lack of a functional endothelium on small-diameter vascular grafts leads to intimal hyperplasia and thrombotic occlusion. Shear stress conditioning through controlled hydrodynamics within in vitro perfusion bioreactors has shown promise as a mechanism to drive endothelial cell (EC) phenotype from an activated, pro-inflammatory wound state toward a quiescent functional state that inhibits responses that lead to occlusive failure. As part of an overall design strategy to engineer functional vascular grafts, we present a novel two-phase shear conditioning approach to improve graft endothelialization. Axial rotation was first used to seed uniform EC monolayers onto the lumenal surface of decellularized scaffolds derived from the human umbilical vein. Using computer-controlled perfusion circuits, a flow-ramping paradigm was applied to adapt endothelia to arterial levels of fluid shear stress and pressure without graft denudation. The effects of constant pulse frequencies (CF) on EC quiescence were then compared with pulse frequencies modeled from temporal fluctuations in blood flow observed in vivo, termed physiologically modeled pulse dynamics (PMPD). Constructs exposed to PMPD for 72 h expressed a more functional transcriptional profile, lower metabolic activity (39.8% ± 8.4% vs. 62.5% ± 11.5% reduction, p = 0.012), and higher nitric oxide production (80.42 ± 23.93 vs. 48.75 ± 6.93 nmol/10(5) cells, p = 0.028) than those exposed to CF. By manipulating in vitro flow conditions to mimic natural physiology, endothelialized vascular grafts can be stimulated to express a nonactivated phenotype that would better inhibit peripheral cell adhesion and smooth muscle cell hyperplasia, conditions that typically lead to occlusive failure. Development of robust, functional endothelia on vascular grafts by modulation of environmental conditions within perfusion bioreactors may ultimately improve clinical outcomes in vascular bypass grafting.

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization.

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

New strategies in kidney regeneration and tissue engineering.

PURPOSE OF REVIEW: The severe shortage of suitable donor kidneys limits organ transplantation to a small fraction of patients suffering from end-stage renal failure. Engineering autologous kidney grafts on-demand would potentially alleviate this shortage, thereby reducing healthcare costs, improving quality of life, and increasing longevity for patients suffering from renal failure. RECENT FINDINGS: Over the past 2 years, several studies have demonstrated that structurally intact extracellular matrix (ECM) scaffolds can be derived from human or animal kidneys through decellularization, a process in which detergent or enzyme solutions are perfused through the renal vasculature to remove the native cells. The future clinical paradigm would be to repopulate these decellularized kidney matrices with patient-derived renal stem cells to regenerate a functional kidney graft. Recent research aiming toward this goal has focused on the optimization of decellularization protocols, design of bioreactor systems to seed cells into appropriate compartments of the renal ECM to nurture their growth to restore kidney function, and differentiation of pluripotent stem cells (PSCs) into renal progenitor lineages. SUMMARY: New research efforts utilizing bio-mimetic perfusion bioreactor systems to repopulate decellularized kidney scaffolds, coupled with the differentiation of PSCs into renal progenitor cell populations, indicate substantial progress toward the ultimate goal of building a functional kidney graft on-demand.

In vitro method for real-time, direct observation of cell-vascular graft interactions under simulated blood flow.

In the development of engineered vascular grafts, assessing the material's interactive properties with peripheral blood cells and its capacity to endothelialize are important for predicting in vivo graft behavior. Current in vitro techniques used for characterizing cell adhesion at the surface of engineered scaffolds under flow only facilitate a terminal quantification of cell/surface interactions. Here, we present the design of an innovative flow chamber for real-time analysis of blood-biomaterial interactions under controllable hemodynamic conditions. Decellularized human umbilical veins (dHUV) were used as model vascular allografts to characterize platelet, leukocyte, and endothelial cell (EC) adhesion dynamics. Confluent EC monolayers adhered to the lumenal surface of the grafting material were flow conditioned to resist arterial shear stress levels (up to 24 dynes/cm(2)) over a 48 h period, and shown to maintain viability over the 1 week assessment period. The basement membrane was imaged while whole blood/neutrophil suspensions were perfused across the HUV surface to quantify cell accumulation. This novel method facilitates live visualization of dynamic events, including cell adhesion, migration, and morphological adaptation at the blood-graft interface on opaque materials, and it can be used for preliminary assessment of clinically relevant biomaterials before implantation.

Adaptation of endothelial cells to physiologically-modeled, variable shear stress.

Endothelial cell (EC) function is mediated by variable hemodynamic shear stress patterns at the vascular wall, where complex shear stress profiles directly correlate with blood flow conditions that vary temporally based on metabolic demand. The interactions of these more complex and variable shear fields with EC have not been represented in hemodynamic flow models. We hypothesized that EC exposed to pulsatile shear stress that changes in magnitude and duration, modeled directly from real-time physiological variations in heart rate, would elicit phenotypic changes as relevant to their critical roles in thrombosis, hemostasis, and inflammation. Here we designed a physiological flow (PF) model based on short-term temporal changes in blood flow observed in vivo and compared it to static culture and steady flow (SF) at a fixed pulse frequency of 1.3 Hz. Results show significant changes in gene regulation as a function of temporally variable flow, indicating a reduced wound phenotype more representative of quiescence. EC cultured under PF exhibited significantly higher endothelial nitric oxide synthase (eNOS) activity (PF: 176.0±11.9 nmol/10(5) EC; SF: 115.0±12.5 nmol/10(5) EC, p = 0.002) and lower TNF-a-induced HL-60 leukocyte adhesion (PF: 37±6 HL-60 cells/mm(2); SF: 111±18 HL-60/mm(2), p = 0.003) than cells cultured under SF which is consistent with a more quiescent anti-inflammatory and anti-thrombotic phenotype. In vitro models have become increasingly adept at mimicking natural physiology and in doing so have clarified the importance of both chemical and physical cues that drive cell function. These data illustrate that the variability in metabolic demand and subsequent changes in perfusion resulting in constantly variable shear stress plays a key role in EC function that has not previously been described.

Preimplantation processing of ex vivo-derived vascular biomaterials: effects on peripheral cell adhesion.

The use of ex vivo-derived scaffolds as vascular conduits has shown to be a clinically valid approach to repair or bypass occluded vessels. Implantation of allogeneic tissue grafts requires careful processing to lower immunogenicity and prevent bacterial infection. However, the mechanical/chemical treatments used to prepare biological scaffolds can result in significant alterations to the native structure and surface chemistry, which can affect in vivo performance. Of particular importance for vascular grafts are binding interactions between the implanted biomaterial and host cells from the circulation and adjacent vasculature. Here we present a comparison of four strategies used to decellularize allogeneic human umbilical vein (HUV) scaffolds: ethanol/acetone, sodium chloride, sodium dodecyl sulfate (SDS), or Triton X-100. Scanning electron microscopy revealed that all four techniques achieved removal of native cells from both the lumenal and ablumenal surfaces of HUV grafts. Platelets and promyelocytic HL-60 cells showed preferential binding on the more loosely structured ablumenal surface, although low surface coverage was observed overall by peripheral blood cells. Vascular endothelial cell adhesion was highest on HUV decellularized using ethanol/acetone, and significantly higher than on SDS-processed grafts (p = 0.016). Primary cells showed high viability on the lumenal surface regardless of decellularization technique (over 95% in all cases). These results demonstrate the critical effects of various chemical processing strategies on the adhesive properties of ex vivo-derived vascular grafts. Careful application-specific consideration is warranted when selecting a processing strategy that minimizes innate responses (e.g. thrombosis, inflammation) that are often deleterious to graft survival.

Lymphangiogenesis-independent resolution of experimental edema.

Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis, and excess VEGF-C has been shown to be ameliorative for edema produced by lymphatic obstruction in experimental models. However, it has recently been shown that edema can resolve in the mouse tail even in the complete absence of capillary lymphangiogenesis when distal lymph fluid crosses the regenerating wound site interstitially. This finding has raised questions about the action of VEGF-C/VEGF receptor (VEGFR) signaling during the resolution of experimental edema. Here, the roles of VEGFR-2 and VEGFR-3 signaling in edema resolution were explored. It was found that edema resolved following neutralization of either VEGFR-2 or VEGFR-3 in the mouse tail skin, which inhibited lymphangiogenesis. Neutralization of either VEGFR-2 or VEGFR-3 reduced angiogenesis at the site of obstruction at day 10 (9.2 +/- 1.2% and 11.5 +/- 1.0% blood capillary coverage, respectively) relative to controls (14.3 +/- 1.5% blood capillary coverage). Combined VEGFR-2/-3 neutralization more strongly inhibited angiogenesis (6.9 +/- 1.5% blood capillary coverage), leading to a reduced wound repair of the lymphatic obstruction and extended edema in the tail skin. In contrast, improved tissue repair of the obstruction site increased edema resolution. Macrophages in the swollen tissue were excluded as contributing factors in the VEGFR-dependent extended edema. These results support a role for VEGFR-2/-3-combined signaling in the resolution of experimental edema that is lymphangiogenesis independent.