John M. Baust

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

SummaryA new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. Hypo-Thermosol® (HTS), a hypothermic (4° C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbeccos Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME +5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (∼30%) and 5-d recovery (∼90%). Cells cryopreserved in CryoStor® CS 5, a HTS derivative containing 5% DMSO, yielded ∼75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME +5% DMSO, HTS, or CryoStor® CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor® CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor® CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use HTS (a dextran-based, intracellular-type solution), without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Experimental cryosurgery investigations in vivo

Cryosurgery is the use of freezing temperatures to elicit an ablative response in a targeted tissue. This review provides a global overview of experimentation in vivo which has been the basis of advancement of this widely applied therapeutic option. The cellular and tissue-related events that underlie the mechanisms of destruction, including direct cell injury (cryolysis), vascular stasis, apoptosis and necrosis, are described and are related to the optimal methods of technique of freezing to achieve efficacious therapy. In vivo experiments with major organs, including wound healing, the putative immunological response following thawing, and the use of cryoadjunctive strategies to enhance cancer cell sensitivity to freezing, are described.

Cryopreservation An emerging paradigm change

In 1949 Polge, Parks and Smith reported on the “chance” discovery of glycerol’s cryoprotective function during their efforts to preserve avian spermatozoa in the frozen state. In the following year, Smith extended these observations by successfully cryopreserving human red blood cells (RBCs) in glycerol. These two reports identified key elements that would play a crucial role in the evolution of the field of biopreservation including the need for a cryoprotective agent (CPA), the process by which cells could successfully be exposed to penetrating CPA and the manner of freezing and thawing. In 1959 Lovelock and Bishop first described the use of dimethyl sulfoxide as a CPA with its advantage of enhanced permeability versus glycerol for many cell types. In the following decades incremental advances were made focusing on changes in and study of the carrier media containing the CPAs as well as the mechanisms of cell cryoinjury and cryopreservation. Most notable was the Mazur et al. report in 1972 which put forth the “two-factor hypothesis” to describe the interrelationships between cooling rates and survival as influenced by either toxic “solution effects” experienced at sub-optimal slow cooling rates or lethal intracellular ice present at high cooling rates. In effect, these studies established a biophysical foundation upon which cryopreservation experimentation rested for nearly four decades. Other notable developments were discoveries by Fahy et al. and Rall and Fahy in the mid-1980s. This group reported on the novel vitrification strategy of cell preservation in which high concentrations (approaching 8 molar) of a cryoprotectant mixture could be titrated over a concentration gradient to create a medium that when “frozen” was devoid of ice even at liquid nitrogen storage temperatures. Beginning in 1998 a series of studies revealed that perturbations in the cell’s proteome and genome during and following the cryopreservation process would significantly impact survival. This effect was observed regardless of the cryopreservation protocol utilized, “optimized” or other.

Mechanisms of Cryoablation: Clinical Consequences on Malignant Tumors

While the destructive actions of a cryoablative freeze cycle are long recognized, more recent evidence has revealed a complex set of molecular responses that provides a path for optimization. The importance of optimization relates to the observation that the cryosurgical treatment of tumors yields success only equivalent to alternative therapies. This is also true of all existing therapies of cancer, which while applied with curative intent; provide only disease suppression for periods ranging from months to years. Recent research has led to an important new understanding of the nature of cancer, which has implications for primary therapies, including cryosurgical treatment. We now recognize that a cancer is a highly organized tissue dependent on other supporting cells for its establishment, growth and invasion. Further, cancer stem cells are now recognized as an origin of disease and prove resistant to many treatment modalities. Growth is dependent on endothelial cells essential to blood vessel formation, fibroblasts production of growth factors, and protective functions of cells of the immune system. This review discusses the biology of cancer, which has profound implications for the diverse therapies of the disease, including cryosurgery. We also describe the cryosurgical treatment of diverse cancers, citing results, types of adjunctive therapy intended to improve clinical outcomes, and comment briefly on other energy-based ablative therapies. With an expanded view of tumor complexity we identify those elements key to effective cryoablation and strategies designed to optimize cancer cell mortality with a consideration of the now recognized hallmarks of cancer.

Cryoablation of renal cancer: variables involved in freezing-induced cell death.

The detection of renal tumors has increased significantly over recent years resulting in a greater demand for novel, minimally invasive techniques. Cryoablation has emerged as a valuable treatment modality for the management of renal cancer. In an effort to detail the effects of freezing in renal cancer, the human renal cancer (RCC) cell line, 786-O, was evaluated in vitro. 786-O cells were exposed to a range of freezing temperatures from −5 to −40°C and compared to non-frozen controls. The data show that freezing to −5°C did not affect 786-O cell viability, while −10°C, −15°C, and −20°C results in a significant loss of viability (23, 70, and 91%, respectively). A complete loss of cell viability was evident at temperatures of −25°C and colder. Following this analysis, variables involved in the success of cryoablation were investigated. For each of the temperatures tested, extended freeze hold times and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared to a single cycle (62% vs. 22% at −10°C; 89% vs. 63% at −15°C, respectively). While these variables play an important part in the effective application of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. Apoptotic inhibition afforded 12% (−10°C), 25% (−15°C), and 11% (−20°C) protection following freezing. Using fluorescence microscopy analysis, the results demonstrated that apoptosis peaked at six hours post-thaw. Next, apoptotic initiating agents including 5-FU and resveratrol (RVT) applied prior to freezing exposure resulted in a significant increase in cell death compared to either application alone. Importantly, the combination of RVT and freezing was noticeably less effective when applied to normal renal cells. The results herein demonstrate the efficacy of freezing and describe a novel therapeutic model for the treatment of renal cancer that may distinguish between cancer and normal cells.

The pathophysiology of thermoablation: optimizing cryoablation.

Purpose of review To describe the response of prostate cancer to thermal therapies with an emphasis on cryoablative techniques. Recent findings Long-term follow-up studies demonstrate clearly the effectiveness of the use of modern cryoablative techniques in the management of prostate cancer. Recently published American Urology Association Best Practice Guidelines identify prostate cryoablation as both primary and salvage therapies. Recent findings demonstrate the effectiveness of −40°C exposure as lethal to prostate cancer genotypes following a double freeze-thaw encounter. In addition, the use of adjunctive agents to sensitize the cancer to freezing is reported. Summary Thermal therapeutic options, especially cryoablation, are of growing interest for the treatment of prostatic and renal cancers. The methods of application of cryoablative therapy and the mechanisms of cell death that are attendant to the freezing-thaw encounter are clearly understood. Research focused on the development of freeze sensitizing agents that work adjunctively is of central interest in furthering the efficacy of this therapy.

Issues Critical to the Successful Application of Cryosurgical Ablation of the Prostate

The techniques of present-day cryosurgery performed with multiprobe freezing apparatus and advanced imaging techniques yield predictable and encouraging results in the treatment of prostatic and renal cancers. Nevertheless, and not unique to cryosurgical treatment, the rates of persistent disease demonstrate the need for improvement in technique and emphasize the need for proper management of the therapeutic margin. The causes of persistent disease often relate to a range of factors including selection of patients, understanding of the extent of the tumor, limitations of the imaging techniques, and failure to freeze the tumor periphery in an efficacious manner. Of these diverse factors, the one most readily managed, but subject to therapeutic error, is the technique of freezing the tumor and appropriate margin to a lethal temperature [Baust, J. G., Gage, A. A. The Molecular Basis of Cryosurgery. BJU Int 95, 1187–1191 (2005)]. This article describes the recent experiments that examine the molecular basis of cryosurgery, clarifies the actions of the components of the freeze-thaw cycle, and defines the resultant effect on the cryogenic lesion from a clinical perspective. Further, this review addresses the important issue of management of the margin of the tumor through adjunctive therapy. Accordingly, a goal of this review is to identify the technical and future adjunctive therapeutic practices that should improve the efficacy of cryoablative techniques for the treatment of malignant lesions.

Gene Activation of the Apoptotic Caspase Cascade Following Cryogenic Storage

The improvement in preservation is now critical for support of the newly emerging fields of tissue engineering and regenerative medicine. The expanding number of complex biologics banked for therapeutic applications necessitates the development of new preservation technologies. Recent studies in biologic preservation have implicated apoptosis as a contributing factor to cell death. These studies have described the role and extent of apoptotic cell death following cryopreservation but have not addressed investigation into the signaling pathways responsible for program execution. Accordingly, we investigated the role of apoptotic gene activation of caspase3 at the genetic and protein levels following cryopreservation (culture media + dimethyl sulfoxide) in a human fibroblast cell model. Additionally, investigation into the extent of caspase-3 involvement in cell death following preservation in a newly developed cryopreservation medium was performed. In this study we report on the upregulation of transcripti...

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (−10°C) or TRAIL (500ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.

Cryoablative response of prostate cancer cells is influenced by androgen receptor expression

To investigate in prostate cancer cells the consequences of androgen‐insensitivity (AI) development on the cellular and molecular responses to freezing, as a challenge in prostate cancer treatment occurs when the androgen‐sensitive (AS) phenotype switches to an AI phenotype, the latter of which is often refractory to many therapies.