Establishing a procedure for dimethyl sulfoxide removal from cardiovascular allografts: a quantitative study
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ORIGINAL PAPER
Establishing a procedure for dimethyl sulfoxide removal from cardiovascular allografts: a quantitative study Claudio Gatto • Luca Dainese • Marina Buzzi • Adriana Terzi • Anna Guarino • Pasquale Paolo Pagliaro Gianluca Polvani • Jana D’Amato Tothova
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Received: 19 April 2012 / Accepted: 14 July 2012 / Published online: 27 July 2012 Ó Springer Science+Business Media B.V. 2012
Abstract The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium (BASE). Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min of continuous
Claudio Gatto, Luca Dainese and Marina Buzzi contributed equally to the work. C. Gatto J. D’Amato Tothova (&) AL.CHI.MI.A.S.r.l, Research and Development, Viale Austria 14, 35020 Ponte San Nicolo`, PD, Italy e-mail: [email protected] L. Dainese A. Guarino G. Polvani Cardiovascular Tissue Bank of Lombardia—Centro Cardiologico Monzino, Via Parea 4, 20138 Milan, Italy M. Buzzi A. Terzi P. P. Pagliaro Cardiovascular Tissue Bank of Emilia Romagna—Policlinco S. Orsola-Malpighi, Via Massarenti 9, 40138 Bologna, Italy
washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content. Keywords Cardiovascular allografts Cryopreservation Dimethyl sulfoxide removal Rinsing procedure HPLC Tissue transplantation CRYO.ON BASE Validation
Introduction The preservation of structural integrity is of paramount importance for the success of cryopreserved allograft transplantations. The amphipathic solvent Me2SO is extensively used for the cryopreservation of a wide array of biological samples, both for clinical and research purposes. Me2SO penetrates tissues and binds water molecules, thereby preventing the formation of ice crystals that cause cell and membrane damage and disrupt tissue architecture (Pegg 2007). Most freezing media used in tissue processing, therefore, contain Me2SO which ensures optimal tissue recovery after thawing. A serious drawback of these methods is the toxicity caused to the host by the local and systemic absorption of Me2SO from the graft (Fahy 2010; Ruiz-Delgado et al. 2009).
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