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Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

ABSTRACT: A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development.

SUBMITTER: Rosines E

PROVIDER: S-EPMC2409245 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

Rosines E E Sampogna R V RV Johkura K K Vaughn D A DA Choi Y Y Sakurai H H Shah M M MM Nigam S K SK

Proceedings of the National Academy of Sciences of the United States of America 20071217 52

A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, foll ...[more]

PMID: 18087037

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Project description:OBJECTIVES:Subcutaneous implantations in small animal models are currently required for preclinical studies of acellular tissue to evaluate biocompatibility, including host recellularization and immunogenic reactivity. METHODS:Three rat subcutaneous implantation methods were evaluated in six Sprague Dawley rats. An acellular xenograft made from porcine pericardium was used as the tissue-scaffold. Three implantation methods were performed; 1) Suture method is where a tissue-scaffold was implanted by suturing its border to the external oblique muscle, 2) Control method is where a tissue-scaffold was implanted without any suturing or support, 3) Frame method is where a tissue-scaffold was attached to a circular frame composed of polycaprolactone (PCL) biomaterial and placed subcutaneously. After 1 and 4 weeks, tissue-scaffolds were explanted and evaluated by hematoxylin and eosin (H&E), Masson's trichrome,Picrosirius Red, transmission electron microscopy (TEM), immunohistochemistry, and mechanical testing. RESULTS:Macroscopically, tissue-scaffold degradation with the suture method and tissue-scaffold folding with the control method were observed after 4 weeks. In comparison, the frame method demonstrated intact tissue scaffolds after 4 weeks. H&E staining showed progressive cell repopulation over the course of the experiment in all groups with acute and chronic inflammation observed in suture and control methods throughout the duration of the study. Immunohistochemistry quantification of CD3, CD 31, CD 34, CD 163, and αSMA showed a statistically significant differences between the suture, control and frame methods (P < 0.05) at both time points. The average tensile strength was 4.03 ± 0.49, 7.45 ± 0.49 and 5.72 ± 1.34 (MPa) after 1 week and 0.55 ± 0.26, 0.12 ± 0.03 and 0.41 ± 0.32 (MPa) after 4 weeks in the suture, control, and frame methods; respectively. TEM analysis showed an increase in inflammatory cells in both suture and control methods following implantation. CONCLUSION:Rat subcutaneous implantation with the frame method was performed with success and ease. The surgical approach used for the frame technique was found to be the best methodology for in vivo evaluation of tissue engineered acellular scaffolds, where the frame method did not compromise mechanical strength, but it reduced inflammation significantly.

Project description:Functional cardiac tissue engineering holds promise as a candidate therapy for myocardial infarction and heart failure. Generation of "strong-contracting and fast-conducting" cardiac tissue patches capable of electromechanical coupling with host myocardium could allow efficient improvement of heart function without increased arrhythmogenic risks. Towards that goal, we engineered highly functional 1?cm?×?1?cm cardiac tissue patches made of neonatal rat ventricular cells which after 2 weeks of culture exhibited force of contraction of 18.0?±?1.4?mN, conduction velocity (CV) of 32.3?±?1.8?cm/s, and sustained chronic activation when paced at rates as high as 8.7?±?0.8?Hz. Patches transduced with genetically-encoded calcium indicator (GCaMP6) were implanted onto adult rat ventricles and after 4-6 weeks assessed for action potential conduction and electrical integration by two-camera optical mapping of GCaMP6-reported Ca2+ transients in the patch and RH237-reported action potentials in the recipient heart. Of the 13 implanted patches, 11 (85%) engrafted, maintained structural integrity, and conducted action potentials with average CVs and Ca2+ transient durations comparable to those before implantation. Despite preserved graft electrical properties, no anterograde or retrograde conduction could be induced between the patch and host cardiomyocytes, indicating lack of electrical integration. Electrical properties of the underlying myocardium were not changed by the engrafted patch. From immunostaining analyses, implanted patches were highly vascularized and expressed abundant electromechanical junctions, but remained separated from the epicardium by a non-myocyte layer. In summary, our studies demonstrate generation of highly functional cardiac tissue patches that can robustly engraft on the epicardial surface, vascularize, and maintain electrical function, but do not couple with host tissue. The lack of graft-host electrical integration is therefore a critical obstacle to development of efficient tissue engineering therapies for heart repair.

Dataset Information

Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

Publications

Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

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