Cryosurgical technique: Assessment of the fundamental variables using human prostate cancer model systems q Daniel P. Klossner a,b , Anthony T. Robilotto a,b , Dominic M. Clarke a,b , Robert G. VanBuskirk a,b , John M. Baust a,b , Andrew A. Gage b,c , John G. Baust a, * a Institute of Biomedical Technology, Science 3 Suite 144, State University of New York, Binghamton, NY 13902, USA b Cell Preservation Services, Inc., Owego, NY 13827, USA c State University of New York, Buffalo, New York 14260, USA Received 6 June 2007; accepted 24 July 2007 Available online 15 August 2007 Abstract Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoab- lation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (5 to 40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlueä (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At 15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing tem- peratures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching 40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cryosurgery; Freezing; Prostate; Cancer; Freeze cycles Cryosurgery for the treatment of prostate cancer is an accepted procedure for both primary and salvage therapy, demonstrating long-term efficacy [4,21,27]. Present-day prostate cryosurgery is practiced with multiple cryoprobes, transrectal ultrasonography, and urethral warming cathe- ters, which support the management of the freezing process resulting in the attainment of targeted tumor temperatures, more efficient destruction of prostate tissue, and reduced morbidity [25,16,17,30]. Despite these advances, complete ablation of cancerous tissue sometimes fails due to techni- cal challenges, and rates of persistent disease after cryosur- gery substantiate the need for technique improvement, especially the management of freezing near the therapeutic margin [4,16,28,1,2]. The causes of persistent disease include selection of patients, disease understaging, limita- tions of imaging, and failure to freeze sufficiently. Of these factors, freezing techniques for attainment of targeted tem- peratures are suspected to contribute to significant thera- peutic error [31,5,6]. 0011-2240/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2007.07.003 q This study was funded in part by Galil Medical, Inc. (Plymouth Meeting, PA) and Cell Preservation Services, Inc. (Owego, NY). * Corresponding author. Address: Institute of Biomedical Technology, Department of Biology, Science 3 Suite 144, State University of New York, Binghamton, NY 13902, USA. Fax: +1 301 417 7077. E-mail address: JBaust@Binghamton.edu (J.G. Baust). www.elsevier.com/locate/ycryo Available online at www.sciencedirect.com Cryobiology 55 (2007) 189–199