DOI 10.1007/s10517-017-3692-3 Bulletin of Experimental Biology and Medicine, Vol. 162, No. 11, March, 2017
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METHODS Modification of Rat Lung Decellularization Protocol Based on Dynamic Conductometry of Working Solution E. V. Kuevda, E. A. Gubareva, I. S. Gumenyuk, A. S. Sotnichenko, I. V. Gilevich, R. Z. Nakokhov, T. V. Rusinova, T. G. Yudina, A. N. Red’ko, and S. N. Alekseenko Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 162, No. 11, pp. 665-667, November, 2016 Original article submitted June 17, 2016 We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA. Key Words: lung decellularization; biological carcasses; conductometry; sodium deoxycholate; rats Tissue engineering does not require organ donors and life-long immunosuppressive therapy and is considered as a potential alternative for transplantation [5-8]. Decellularization is the early stage of creation of tissue constructs implying elimination of tissue components with preservation of structural integrity and quantitative content of extracellular matrix (ECM) components [4,6,8,9]. Minimum modification of the biochemical content, morphological structure, and biomechanical properties of native organs is the main requirement to the scaffold quality [9,11-13]. Despite recent achievements of several research groups [1-3,8-10,12], no optimal protocol of lung decellularization meeting these requirements has been proposed until now. As detergents produce damaging effects on ECM, protocols based on recording of quantitative parameters as a criterion for termination of decellularization rather than visual changes of the organ color subjectively registered by the investigator have to be developed. Kuban State Medical University, Krasnodar, Russia. Address for correspondence:
[email protected]. E. V. Kuevda
This approach will help to reduce exposure to chemical agents and minimize ECM damage. Ion balance measured as relative resistivity of working solution during high-frequency current passage (conductometry) can be one of these parameters. By using this method, we can determine the moment of complete disintegration of cell membranes and terminate decellularization at this stage without additional damage to ECM proteins. Our aim was to perform comparative morphological analysis of rat lung samples decellularized under visual and conductometric control and to choose the protocol shortening the exposure to aggressive detergent (sodium deoxycholate) solution without impairing decellularization quality.
MATERIALS AND METHODS Experiments were performed on male Wistar rats weighting 220±50 g at the Laboratory of Fundamental Research in Regenerative Medicine, Kuban State Medical University. The study is conducted in accor-
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Bulletin of Experimental Biology and Medicine, Vol. 162, No. 11, March, 2017 METHODS
dance to the Rules for Investigations using Experimental Animals (Order No. 755 of the Ministry of Health of the USSR, August 12, 1972) and European Convention on the Protection of Vertebrate Animals used for Experiments or Other Scientific Purposes (Strasbourg, 1986), and was approved by the Local Ethic Committee (protocol No. 21/1). Explantation of the organocomplex was carried out after intraperitoneal administration of the lethal dose of barbiturates (150 mg/kg). The animals were randomized into the main (n=5) and reference (n=5) groups depending on the duration of sodium deoxycholate treatment. The control group (n=5) was created for estimation of decellularization efficiency. The procedure of organocomplex explantation was identical for all groups. The lungs of control group rats served as native control and were not treated with detergents. Organs were washed with phosphate buffer immediately after isolation. In animals of the treatment group, the pulmonary artery was cannuled at a distance of 2.5-3 cm from the orifice, fixed in a bioreactor (Harvard Apparatus), and perfused with detergent and enzyme solutions. Decellularization of the lung in rats of the reference group was started from perfusion with sterile phosphate buffer containing 1% penicillin– streptomycin and then successively with deionized water, 1% water solution of sodium deoxycholate, 0.1% solution of Triton-X, sterile phosphate buffer with 1% penicillin–streptomycin, and pig pancreatic DNase I in accordance to the modified protocol [1,2]. The exposure to sodium deoxycholate during decellularization of lungs from main group rats depended on changes in solution resistivity evaluated by a calibrated conductometer Hanna DiST WP 4 (0.01-19.99 m&/cm, error 2% of full scale). Resistivity of working solution was measured every 10 min after the start of the perfusion cycle (exposure to sodium deoxycholate) by plunging the conductometer for the working length up to reading stabilization within 15 sec. Working solution was vortexed immediately before measurements. The resistivity curves reflecting the time of attaining equilibrium plateau corresponding to the end of ion release from the damaged cells into the working solution with the detergent were constructed (Fig. 1). Perfusion with the detergent was terminated immediately after reaching the plateau of ion equilibrium. Native and decellularized samples were fixed in 10% neutral buffered formalin solution, dehydrated, and embedded into paraffin using Leica TP1020 tissue processor according to the standard method (EG1150H module device, Leica). For histological analysis, the paraffin sections (5 μ) were prepared using RM2235 rotation microtome (Leica) and mounted on highly adhesive slides, deparaffinized, hydrated, stained with
hematoxylin and eosin (Histolab) and with fluorophore DAPI (Sigma-Aldrich), and examined under an Olympus IX51 microscope. For verification lung decellularization, DNA content was measured quantitatively using Nano-Drop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.) and Dnease Blood and Tissue Kit (Qiagen) according to manufacturer’s protocol. Statistical analysis of the results was performed by Student’s t test using GraphPad Prism 6.04 software and presented as arithmetic mean (M)±standard error of mean (SEM). Differences were significant at p
