Optimal decellularization of rat hearts and diaphragms and morphological evaluation

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Abstract

Tissue engineering involves the design, evaluation, modification and maintenance of living cells or tissues embedded in biological (natural) or artificial scaffolds. Biological scaffolds need to be decellularized to become completely non-immunogenic while preserving the tissue structure and extracellular matrix. The primary goals of the present paper were to investigate and optimize different decellularization protocols of rats heart and diaphragm and establish the most reliable technique (paraffin vs. cryosections) to evaluate the morphology of the decellularized tissues. Hearts and diaphragm were decellularized with detergent-enzymatic based protocols, including deoxycholate and DNAse. Compared to published decellularization protocols, our was able to reduce exposure time (for heart: up to 3 hours — deoxycholate and 1 hour — DNAse; for diaphragm: up to 6 hours — deoxycholate and 2 hour — DNAse) of detergents and length (up to 24 hours). Results of morfological studies showed the absence of cells and preservation of the extracellular matrix. DNA quantification showed that about 81% and 74% of heart and diaphragm nuclear material, respectively, was removed by the decellularization process. Our results suggest that the investigated decellularization protocol was superior to others in removing DNA content and preserving the ECM of rats hearts and diaphragms.

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About the authors

E. A. Gubareva

International Research, Clinical and Education Center of Regenerative Medicine, Kuban State Medical University, Krasnodar, Russia

A. S Sotnichenko

International Research, Clinical and Education Center of Regenerative Medicine, Kuban State Medical University, Krasnodar, Russia

I. V Gilevich

International Research, Clinical and Education Center of Regenerative Medicine, Kuban State Medical University, Krasnodar, Russia

P. Macchiarini

International Research, Clinical and Education Center of Regenerative Medicine, Kuban State Medical University, Krasnodar, Russia; Advanced Center for Translational Regenerative Medicine (ACTREM), Karolinska Institutet, Stockholm, Sweden

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