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Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential.

ABSTRACT: The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD). The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed as a potential alternative for generating functional tissues. Despite its growing interest, the optimal processing to achieve functional organ still remains unsolved. The biggest challenge remains is the time line for obtaining kidney. Keeping these facts in mind, we have assessed the effects of cryostorage (3 months) on renal tissue architecture and its potential for decellularization and recellularization in comparison to the freshly isolated kidneys. The light microscopy exploiting different microscopic stains as well as immuno-histochemistry and Scanning electron microscopy (SEM) demonstrated that ECM framework is well retained following kidney cryopreservation. The strength of these structures was reinforced by calculating mechanical stress which confirmed the similarity between the freshly isolated and cryopreserved tissue. The recellularization of these bio-scaffolds, with mesenchymal stem cells quickly repopulated the decellularized structures irrespective of the kidneys status, i.e. freshly isolated or the cryopreserved. The growth pattern employing mesenchymal stem cells demonstrated their equivalent recellularization potential. Based on these observations, it may be concluded that cryopreserved kidneys can be exploited as scaffolds for future development of functional organ.

SUBMITTER: Chani B

PROVIDER: S-EPMC5340383 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential.

Chani Baldeep B Puri Veena V Sobti Ranbir C RC Jha Vivekanand V Puri Sanjeev S

PloS one 20170307 3

The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD). The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed ...[more]

PMID: 28267813

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Project description:RationalePerfusion decellularization of cadaveric hearts removes cells and generates a cell-free extracellular matrix scaffold containing acellular vascular conduits, which are theoretically sufficient to perfuse and support tissue-engineered heart constructs. However, after transplantation, these acellular vascular conduits clot, even with anti-coagulation. Here, our objective was to create a less thrombogenic scaffold and improve recellularized-left ventricular contractility by re-lining vascular conduits of a decellularized rat heart with rat aortic endothelial cells (RAECs).Methods and resultsWe used three strategies to recellularize perfusion-decellularized rat heart vasculature with RAECs: retrograde aortic infusion, brachiocephalic artery (BA) infusion, or a combination of inferior vena cava (IVC) plus BA infusion. The re-endothelialized scaffolds were maintained under vascular flow in vitro for 7 days, and then cell morphology, location, and viability were examined. Thrombogenicity of the scaffold was assessed in vitro and in vivo. Both BA and IVC+BA cell delivery resulted in a whole heart distribution of RAECs that proliferated, retained an endothelial phenotype, and expressed endothelial nitric oxide synthase and von Willebrand factor. Infusing RAECs via the combination IVC+BA method increased scaffold cellularity and the number of vessels that were lined with endothelial cells; re-endothelialization by using BA or IVC+BA cell delivery significantly reduced in vitro thrombogenicity. In vivo, both acellular and re-endothelialized scaffolds recruited non-immune host cells into the organ parenchyma and vasculature. Finally, re-endothelialization before recellularization of the left ventricular wall with neonatal cardiac cells enhanced construct contractility.ConclusionsThis is the first study to re-endothelialize whole decellularized hearts throughout both arterial and venous beds and cavities by using arterial and venous delivery. The combination (IVC+BA) delivery strategy results in enhanced scaffold vessel re-endothelialization compared to single-route strategies. Re-endothelialization reduced scaffold thrombogencity and improved contractility of left ventricular-recellularized constructs. Thus, vessel and cavity re-endothelialization creates superior vascularized scaffolds for use in whole-organ recellularization applications.

Project description: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.

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Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential.

Publications

Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential.

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