Investigation of Lung Cancer Cell Response to Cryoablation and Adjunctive Gemcitabine-Based Cryo-Chemotherapy Using the A549 Cell Line.

Due to the rising annual incidence of lung cancer (LC), new treatment strategies are needed. While various options exist, many, if not all, remain suboptimal. Several studies have shown cryoablation to be a promising approach. Yet, a lack of basic information pertaining to LC response to freezing and requirement for percutaneous access has limited clinical use. In this study, we investigated the A549 lung carcinoma cell line response to freezing. The data show that a single 5 min freeze to -15 °C did not affect cell viability, whereas -20 °C and -25 °C result in a significant reduction in viability 1 day post freeze to <10%. These populations, however, were able to recover in culture. Application of a repeat (double) freeze resulted in complete cell death at -25 °C. Studies investigating the impact of adjunctive gemcitabine (75 nM) pretreatment in combination with freezing were then conducted. Exposure to gemcitabine alone resulted in minimal cell death. The combination of gemcitabine pretreatment and a -20 °C single freeze as well as combination treatment with a -15 °C repeat freeze both resulted in complete cell death. This suggests that gemcitabine pretreatment may be synergistically effective when combined with freezing. Studies into the modes of cell death associated with the increased cell death revealed the increased involvement of necroptosis in combination treatment. In summary, these results suggest that repeat freezing to -20 °C to -25 °C results in a high degree of LC destruction. Further, the data suggest that the combination of gemcitabine pretreatment and freezing resulted in a shift of the minimum lethal temperature for LC from -25 °C to -15 °C. These findings, in combination with previous reports, suggest that cryoablation alone or in combination with chemotherapy may provide an improved path for the treatment of LC.

The Assessment of a Novel Endoscopic Ultrasound-Compatible Cryocatheter to Ablate Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease that may be treated utilizing thermal therapies. Cryoablation is an effective, minimally invasive therapy that has been utilized for the treatment of various cancers, offering patients a quicker recovery and reduced side effects. Cryoablation has been utilized on a limited basis for the treatment of PDAC. With the recent reports on the success of cryoablation, there is a growing interest in the use of cryoablation as a standalone, minimally invasive procedure to treat PDAC. While offering a promising path, the application of cryoablation to PDAC is limited by current technologies. As such, there is a need for the development of new devices to support advanced treatment strategies for PDAC. To this end, this study investigated the performance of a new endoscopic ultrasound-compatible cryoablation catheter technology, FrostBite. We hypothesized that FrostBite would enable the rapid, effective, minimally invasive delivery of ultra-cold temperatures to target tissues, resulting in effective ablation via an endoscopic approach. Thermal properties and ablative efficacy were evaluated using a heat-loaded gel model, tissue-engineered models (TEMs), and an initial in vivo porcine study. Freeze protocols evaluated included single and repeat 3 and 5 min applications. Isotherm assessment revealed the generation of a 2.2 cm diameter frozen mass with the -20 °C isotherm reaching a diameter of 1.5 cm following a single 5 min freeze. TEM studies revealed the achievement of temperatures ≤ -20 °C at a diameter of 1.9 cm after a 5 min freeze. Fluorescent imaging conducted 24 h post-thaw demonstrated a uniformly shaped ellipsoidal ablative zone with a midline diameter of 2.5 cm, resulting in a total ablative volume of 6.9 cm3 after a single 5 min freeze. In vivo findings consistently demonstrated the generation of ablative areas measuring 2.03 cm × 3.2 cm. These studies demonstrate the potential of the FrostBite cryocatheter as an endoscopic ultrasound-based treatment option. The data suggest that FrostBite may provide for the rapid, effective, controllable freezing of cancerous pancreatic and liver tissues. This ablative power also offers the potential of improved safety margins via the minimally invasive nature of an endoscopic ultrasound-based approach or natural orifice transluminal endoscopic surgery (NOTES)-based approach. The results of this pre-clinical feasibility study show promise, affirming the need for further investigation into the potential of the FrostBite cryocatheter as an advanced, minimally invasive cryoablative technology.

Evaluation of a New Cystoscopic Cryocatheter and Method for the In Situ Destruction of Bladder Cancer: Preliminary In Vivo Study.

Purpose: To assess the ability to deliver full-thickness bladder wall cryoablation through a cystoscopic approach using a new closed-loop 6F cryocatheter and thermal dose-controlled protocol. Materials and Methods: Evaluations were conducted using a chronic porcine model wherein 10 lesions/animal were created throughout the bladder (bladder wall, trigone region, ureteral orifice, and distal ureter). A 6F cryocatheter was passed through the working channel of a flexible cystoscope. Single 1- and 1.5-minute freeze protocols in a saline environment were evaluated and resultant lesion size was determined. A laparoscopic approach was utilized to observe the transmural extension of the ice propagation. Results: Studies demonstrated the generation of transmural lesions characterized by full-thickness histologic necrosis after freezing for 1.5 minutes regardless of tissue thickness (range 2-12 mm). All animals were found to have good overall health (maintained weight, appetite, mobility, and energy levels) throughout the recovery period. No significant deviations were noted in complete blood count and serum chemistry bloodwork except for elevated creatine kinase levels. Importantly, no fistulas or perforations were noted. Conclusions: The cryocatheter was able to rapidly and effectively freeze the bladder wall through a cystoscopic approach. The results showed the ability to consistently ablate an ∼1 cm diameter and up to 1.2 cm deep using a single 1.5-minute freeze protocol. Analysis of the ablation efficacy revealed ∼80% destruction within the frozen mass. Although further testing and refinement are needed, these studies demonstrate the potential of this new approach to provide a next-generation strategy for the treatment of bladder cancer.

An In Vitro Investigation into Cryoablation and Adjunctive Cryoablation/Chemotherapy Combination Therapy for the Treatment of Pancreatic Cancer Using the PANC-1 Cell Line.

As the incidence of pancreatic ductal adenocarcinoma (PDAC) continues to grow, so does the need for new strategies for treatment. One such area being evaluated is cryoablation. While promising, studies remain limited and questions surrounding basic dosing (minimal lethal temperature) coupled with technological issues associated with accessing PDAC tumors and tumor proximity to vasculature and bile ducts, among others, have limited the use of cryoablation. Additionally, as chemotherapy remains the first-line of attack for PDAC, there is limited information on the impact of combining freezing with chemotherapy. As such, this study investigated the in vitro response of a PDAC cell line to freezing, chemotherapy, and the combination of chemotherapy pre-treatment and freezing. PANC-1 cells and PANC-1 tumor models were exposed to cryoablation (freezing insult) and compared to non-frozen controls. Additionally, PANC-1 cells were exposed to varying sub-clinical doses of gemcitabine or oxaliplatin alone and in combination with freezing. The results show that freezing to -10 °C did not affect viability, whereas -15 °C and -20 °C resulted in a reduction in 1 day post-freeze viability to 85% and 20%, respectively, though both recovered to controls by day 7. A complete cell loss was found following a single freeze below -25 °C. The combination of 100 nM gemcitabine (1.1 mg/m2) pre-treatment and a single freeze at -15 °C resulted in near-complete cell death (<5% survival) over the 7-day assessment interval. The combination of 8.8 µM oxaliplatin (130 mg/m2) pre-treatment and a single -15 °C freeze resulted in a similar trend of increased PANC-1 cell death. In summary, these in vitro results suggest that freezing alone to temperatures in the range of -25 °C results in a high degree of PDAC destruction. Further, the data support a potential combinatorial chemo/cryo-therapeutic strategy for the treatment of PDAC. These results suggest that a reduction in chemotherapeutic dose may be possible when offered in combination with freezing for the treatment of PDAC.

Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model.

The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various "front end" strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.

Breast Cancer Cryoablation: Assessment of the Impact of Fundamental Procedural Variables in an In Vitro Human Breast Cancer Model.

INTRODUCTION: Breast cancer is the most prominent form of cancer and the second leading cause of death in women behind lung cancer. The primary modes of treatment today include surgical excision (lumpectomy, mastectomy), radiation, chemoablation, anti-HER2/neu therapy, and/or hormone therapy. The severe side effects associated with these therapies suggest a minimally invasive therapy with fewer quality of life issues would be advantageous for treatment of this pervasive disease. Cryoablation has been used in the treatment of other cancers, including prostate, skin, and cervical, for decades and has been shown to be a successful minimally invasive therapeutic option. To this end, the use of cryotherapy for the treatment of breast cancer has increased over the last several years. Although successful, one of the challenges in cryoablation is management of cancer destruction in the periphery of the ice ball as the tissue within this outer margin may not experience ablative temperatures. In breast cancer, this is of concern due to the lobular nature of the tumors. As such, in this study, we investigated the level of cell death at various temperatures associated with the margin of a cryogenic lesion as well as the impact of repetitive freezing and thawing methods on overall efficacy. METHODS: Human breast cancer cells, MCF-7, were exposed to temperatures of -5°C, -10°C, -15°C, -20°C, or -25°C for 5-minute freeze intervals in a single or repeat freeze-thaw cycle. Samples were thawed with either passive or active warming for 5 or 10 minutes. Samples were assessed at 1, 2, and 3 days post-freeze to assess cell survival and recovery. In addition, the modes of cell death associated with freezing were assessed over the initial 24-hour post-thaw recovery period. RESULTS: Exposure of MCF-7 cells to -5°C and -10°C resulted in minimal cell death regardless of the freeze/thaw conditions. Freezing to a temperature of -25°C resulted in complete cell death 1 day post-thaw with no cell recovery in all freeze/thaw scenarios evaluated. Exposure to a single freeze event resulted in a gradual increase in cell death at -15°C and -20°C. Application of a repeat freeze-thaw cycle (dual 5-minute freeze) resulted in an increase in cell death with complete destruction at -20°C and near complete death at -15°C (day 1 survival: single -15°C freeze/thaw = 20%; repeated -15°C freeze/thaw = 4%). Analysis of thaw interval time (5 vs 10 minute) demonstrated that the shorter 5-minute thaw interval between freezes resulted in increased cell destruction. Furthermore, investigation of thaw rate (active vs passive thawing) demonstrated that active thawing resulted in increased cell survival thereby less effective ablation compared with passive thawing (eg, -15°C 5/10/5 procedure survival, passive thaw: 4% vs active thaw: 29%). CONCLUSIONS: In summary, these in vitro findings suggest that freezing to temperatures of 25°C results in a high degree of breast cancer cell destruction. Furthermore, the data demonstrate that the application of a repeat freeze procedure with a passive 5-minute or 10-minute thaw interval between freeze cycles increases the minimal lethal temperature to the -15°C to -20°C range. The data also demonstrate that the use of an active thawing procedure between freezes reduces ablation efficacy at temperatures associated with the iceball periphery. These findings may be important to improving future clinical applications of cryoablation for the treatment of breast cancer.

Investigation of Bladder Cancer Cell Response to Cryoablation and Adjunctive Cisplatin Based Cryo/Chemotherapy.

Due to a rising annual incidence of bladder cancer, there is a growing need for development of new strategies for treatment. In 2018, the World Cancer Research Fund and other groups reported that there were ~550,000 new cases worldwide of bladder cancer. It has been further estimated that >200,000 individuals die annually from bladder cancer worldwide. Various treatment options exist. However, many if not all remain suboptimal. While the preferred chemotherapeutic options have changed in the past few years there have been few advances in the bladder cancer medical device field. Cryoablation is now being evaluated as a new option for the treatment of bladder cancer. While several studies have shown cryoablation to be promising for the treatment of bladder cancer, a lack of basic information pertaining to dosing (minimal lethal temperature) necessary to destroy bladder cancer has limited its use as a primary therapeutic option. Concerns with bladder wall perforation and other side effects have also slowed adoption. In an effort to detail the effects of freezing on bladder cancer, two human bladder cancer cell lines, SCaBER and UMUC3, were evaluated in vitro. SCaBER, a basal subtype of muscle invasive bladder cancer, and UMUC3, an intermediate transitional cell carcinoma, are both difficult to treat but are reportedly responsive to most conventional treatments. SCaBER and UMUC3 cells were exposed to a range of freezing temperatures from -10 to -25°C and compared to non-frozen controls. The data show that a single 5 minute freeze to -10°C did not affect cell viability, whereas -15°C and -20°C results in a significant reduction in viability 1 day post freeze to <20%. These populations, however, were able to recover in culture. A complete loss of cell viability was found following a single freeze at -25°C. Application of a repeat (double) freeze resulted in complete cell death at -20°C. In addition to freezing alone, studies investigating the impact of adjunctive low dose (1 µM) cisplatin pre-treatment (30 minutes and 24 hours) in combination with freezing were conducted. The combination of 30 minute cisplatin pre-treatment and mild (-15°C) freezing resulted in complete cell death. This suggests that subclinical doses of cisplatin may be synergistically effective when combined with freezing. In summary, these in vitro results suggest that freezing to temperatures in the range of -20 to 25°C results in a high degree of bladder cancer cell destruction. Further, the data describe a potential combinatorial chemo/cryo therapeutic strategy for the treatment of bladder cancer.

Cryoablation: physical and molecular basis with putative immunological consequences.

Cryoablation (CA) is unique as the singular energy deprivation therapy that impacts all cellular processes. CA is independent of cell cycle stage and degree of cellular stemness. Importantly, CA is typically applied as a non-repetitive (single session) treatment that does not support adaptative mutagenesis as do many repetitive therapies. CA is characterized by the launch of multiple forms of cell death including (a) ice-related physical damage, (b) initiation of cellular stress responses (kill switch activation) and launch of necrosis and apoptosis, (c) vascular stasis, and (d) likely activation of ablative immune responses. CA is not without limitation related to the thermal gradient formed between cryoprobe surface (∼-185°C) and the distal surface of the freeze zone (∼0°C) requiring freeze margin extension beyond the tumor boundary (up to ∼1 cm). This limitation is mitigated in part by commonly applied dual freeze thaw cycles and the use of freeze sensitizing adjuvants. This review will (1) identify the cascade of damaging effects of the freeze-thaw process, its physical and molecular-based relationships, (2) a likely immunological involvement (abscopic effect), and (3) explore the use of freeze-sensitizing adjuvants necessary to limit freezing beyond the tumor margin.

Assessment of a novel cryoablation device for the endovascular treatment of cardiac tachyarrhythmias.

OBJECTIVES: Cryoablation is an effective alternative treatment for cardiac arrhythmias offering shortened recovery and reduced side effects. As the use of cryoablation increases, the need for new devices and procedures has emerged. This has been driven by technological limitations including lengthy periods to generate a single lesion (3-5 min), uncertain transmurality, and differential efficacy. Furthermore, due to limited ablation capacity under high heat loads, cryo has had limited success in the treatment of ventricular arrhythmias. To this end, in this study we evaluated a new cryoablation catheter, ICEolate, for the targeted ablation of cardiac tissue. METHODS: Performance assessment included calorimetry, freeze zone isothermal distribution characterization and catheter ablation capacity in a submerged, circulating, heat-loaded ex vivo tissue model. A pilot in vivo study was also conducted to assess ablative capacity of the cryocatheter in a fully beating heart. RESULTS: Ex vivo studies demonstrated ice formation at the tip of a cryocatheter within 5 s and a tip temperature of ~-150°C within 10 s. The device repeatedly generated freeze zones of 2 cm × 3 cm in less than 2 min. Tissue model studies revealed the generation of a full thickness (5-10 mm) cryogenic lesion within 1 min with an opposite (transmural) surface temperature of <-60°C under a circulating 37°C heat load. Pilot in vivo studies demonstrated the delivery of an ablative "dose," producing a continuous full thickness transmural linear lesion in <60 s at both atrial and ventricular sites. CONCLUSION: These studies suggest that the supercritical nitrogen cryodevice and ICEolate cryocatheter may provide for rapid, effective, controllable freezing of targeted tissue. The ablative power, speed, and directional freeze characteristics also offer the potential of improved safety via a reduction in procedural time compared to current cryoablation devices. These technological developments may open new avenues for the application of cryo to treat other cardiac arrhythmogenic disorders.

Defeating Cancers' Adaptive Defensive Strategies Using Thermal Therapies: Examining Cancer's Therapeutic Resistance, Ablative, and Computational Modeling Strategies as a means for Improving Therapeutic Outcome.

BACKGROUND: Diverse thermal ablative therapies are currently in use for the treatment of cancer. Commonly applied with the intent to cure, these ablative therapies are providing promising success rates similar to and often exceeding "gold standard" approaches. Cancer-curing prospects may be enhanced by deeper understanding of thermal effects on cancer cells and the hosting tissue, including the molecular mechanisms of cancer cell mutations, which enable resistance to therapy. Furthermore, thermal ablative therapies may benefit from recent developments in computer hardware and computation tools for planning, monitoring, visualization, and education. METHODS: Recent discoveries in cancer cell resistance to destruction by apoptosis, autophagy, and necrosis are now providing an understanding of the strategies used by cancer cells to avoid destruction by immunologic surveillance. Further, these discoveries are now providing insight into the success of the diverse types of ablative therapies utilized in the clinical arena today and into how they directly and indirectly overcome many of the cancers' defensive strategies. Additionally, the manner in which minimally invasive thermal therapy is enabled by imaging, which facilitates anatomical features reconstruction, insertion guidance of thermal probes, and strategic placement of thermal sensors, plays a critical role in the delivery of effective ablative treatment. RESULTS: The thermal techniques discussed include radiofrequency, microwave, high-intensity focused ultrasound, laser, and cryosurgery. Also discussed is the development of thermal adjunctive therapies-the combination of drug and thermal treatments-which provide new and more effective combinatorial physical and molecular-based approaches for treating various cancers. Finally, advanced computational and planning tools are also discussed. CONCLUSION: This review lays out the various molecular adaptive mechanisms-the hallmarks of cancer-responsible for therapeutic resistance, on one hand, and how various ablative therapies, including both heating- and freezing-based strategies, overcome many of cancer's defenses, on the other hand, thereby enhancing the potential for curative approaches for various cancers.

Dose Escalation of Vitamin D3 Yields Similar Cryosurgical Outcome to Single Dose Exposure in a Prostate Cancer Model.

Vitamin D3 (VD3) is an effective adjunctive agent, enhancing the destructive effects of freezing in prostate cancer cryoablation studies. We investigated whether dose escalation of VD3 over several weeks, to model the increase in physiological VD3 levels if an oral supplement were prescribed, would be as or more effective than a single treatment 1 to 2 days prior to freezing. PC-3 cells in log phase growth to model aggressive, highly metabolically active prostate cancer were exposed to a gradually increasing dose of VD3 to a final dose of 80 nM over a 4-week period, maintained for 2 weeks at 80 nM, and then exposed to mild sublethal freezing temperatures. Results demonstrate that both acute 24-hour exposure to 80 nM VD3 and dose escalation resulted in enhanced cell death following freezing at -15°C or colder, with no significant differences between the 2 exposure regimes. Apoptotic analysis within the initial 24-hour period postfreeze revealed that VD3 treatment induced both caspase 8- and 9-mediated cell death, most notably in caspase 8 at 8-hour postfreeze. These results indicate that both the intrinsic and extrinsic apoptotic pathways are involved in VD3 sensitization prior to freezing. Additionally, both acute and gradual dose escalation regimes of VD3 exposure increase prostate cancer cell sensitivity to mild freezing. Importantly, this study expands upon previous reports and suggests that the combination of VD3 and freezing may offer an effective treatment for both slow growth and highly aggressive prostate cancers.

Best practices for cryopreserving, thawing, recovering, and assessing cells.

Long-term storage of cell stocks insures that cells are available for use whenever needed. Cryopreservation of cells is the method of choice for preservation of important or rare cell stocks. There are several factors to consider when establishing a protocol for freezing, thawing, and recovery of cells after storage. These parameters may include cell concentration, cryoprotectant choice and concentration, and thawing rate among others. Further, the assessment of cell viability and/or function prior to and following cryopreservation is imperative in order to accurately determine downstream utility as well for optimizing the cryopreservation process. This chapter is designed to provide guidance and insight into developing robust and successful protocols for preserving cells that will preserve cell stocks and provide optimal cell yield and viability.

Best practices in cell culture: an overview.

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

The promise of organ and tissue preservation to transform medicine.

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

Assessment of Cryosurgical Device Performance Using a 3D Tissue-Engineered Cancer Model.

As the clinical use of cryoablation for the treatment of cancer has increased, so too has the need for knowledge on the dynamic environment within the frozen mass created by a cryoprobe. While a number of factors exist, an understanding of the iceball size, critical isotherm distribution/penetration, and the resultant lethal zone created by a cryoprobe are critical for clinical application. To this end, cryoprobe performance is typically characterized based on the iceball size and temperature penetration in phantom gel models. Although informative, these models do not provide information as to the impact of heat input from surrounding tissue nor give any information on the ablative zone created. As such, we evaluated the use of a tissue-engineered tumor model (TEM) to assess cryoprobe performance including iceball size, real-time thermal profile distribution, and resultant ablative zone. Studies were conducted using an Endocare V-probe cryoprobe, with a 10/5/10 double freeze-thaw protocol using prostate and renal cancer TEMs. The data demonstrate the generation of a 33- to 38-cm3 frozen mass with the V-Probe cryoprobe following the double freeze of which ∼12.7 and 6.5 cm3 was at or below -20°C and -40°C, respectively. Analysis of ablation zone using fluorescence microscopy 24 hours postthaw demonstrated that the internal ∼40% of the frozen mass was completely ablated, whereas in the periphery of the iceball (outer 1 cm region), a gradient of partial to minimal destruction was observed. These findings correlated well with clinical reports on renal and prostate cancer cryoablation. Overall, this study demonstrates that TEMs provide an effective model for a more complete characterization of cryoablation device performance. The data demonstrate that while the overall iceball size generated in the TEM was consistent with published reports from phantom models, the integration of an external heat load, circulation, and cellular components more closely reflect an in vivo setting and the impact of penetration of the critical (-20°C and -40°C) isotherms into the tissue. This is important as it is well appreciated in clinical practice that the heat load of a tissue, cryoprobe proximity to vasculature, and so on, can impact outcome. The TEM model provides a means of characterizing the impact on ablative dose delivery allowing for a better understanding of probe performance and potential impact on ablative outcome.

Integrating Molecular Control to Improve Cryopreservation Outcome.

Cryopreservation (CP) is a critical component in enabling on-demand access to biological material (macromolecules, cells, and tissues), yet CP has evolved little over the last several decades. Today's CP processes often yield a suboptimal "product," which has slowed progression in areas such as cell therapy and stem cell research. Recent discoveries focusing on molecular control and buffering of cell stress responses to CP as well as the development of new devices that improve sample freezing and thawing are now providing a path to improve sample quality. Numerous reports now identify the problems associated with CP-induced delayed-onset cell death (CIDOCD), with a focus on the mitigation of cell stress responses through freeze media formulation to optimize survival during "cold chain" sample management. Importantly, these new approaches that manage cell stress response are now providing a basis for advancements in cell therapy development. Herein, we provide an overview of the molecular stress responses of cells to CP, including the impact of hypothermic and cell death continuums and the strategies for improving preservation outcome.

Characterization of Pancreatic Cancer Cell Thermal Response to Heat Ablation or Cryoablation.

One of the most lethal carcinomas is pancreatic cancer. As standard treatment using chemotherapy and radiation has shown limited success, thermal regimens (cryotherapy or heat ablation) are emerging as viable alternatives. Although promising, our understanding of pancreatic cancer response to thermal ablation remains limited. In this study, we investigated the thermal responses of 2 pancreatic cancer cell lines in an effort to identify the minimum lethal temperature needed for complete cell death to provide guidance for in vivo applications. PANC-1 and BxPC-3 were frozen (-10°C to -25°C) or heated (45°C-50°C) in single and repeated exposure regimes. Posttreatment survival and recovery were analyzed using alamarBlue assay over a 7-day interval. Modes of cell death were assessed using fluorescence microscopy (calcein acetoxymethyl ester/propidium iodide) and flow cytometry (YO-PRO-1/propidium iodide). Freezing to -10°C resulted in minimal cell death. Exposure to -15°C had a mild impact on PANC-1 survival (93%), whereas BxPC-3 was more severely damaged (33%). Exposure to -20°C caused a significant reduction in viability (PANC-1 = 23%; BxPC-3 = 2%) whereas -25°C yielded complete death. Double freezing exposure was more effective than single exposure. Repeat exposure to -15°C resulted in complete death of BxPC-3, whereas -20°C severely impacted PANC-1 (7%). Heating to 45°C resulted in minimum cell death. Exposure to 48°C yielded a slight increase in cell loss (PANC-1 = 85%; BxPC-3 = 98%). Exposure to 50°C caused a significant decline (PANC-1 = 70%; BxPC-3 = 9%) with continued deterioration to 0%. Double heating to 45°C resulted in similar effects observed in single exposures, whereas repeated 48°C resulted in significant increases in cell death (PANC-1 = 68%; BxPC-3 = 29%). In conclusion, we observed that pancreatic cancer cells were completely destroyed at temperatures <-25°C or >50°C using single thermal exposures. Repeated exposures resulted in increased cell death at less extreme temperatures. Our data suggest that thermal ablation strategies (heat or cryoablation) may represent a viable technique for the treatment of pancreatic cancer.

Cryopreservation: Evolution of Molecular Based Strategies.

Cryopreservation (CP) is an enabling process providing for on-demand access to biological material (cells and tissues) which serve as a starting, intermediate or even final product. While a critical tool, CP protocols, approaches and technologies have evolved little over the last several decades. A lack of conversion of discoveries from the CP sciences into mainstream utilization has resulted in a bottleneck in technological progression in areas such as stem cell research and cell therapy. While the adoption has been slow, discoveries including molecular control and buffering of cell stress response to CP as well as the development of new devices for improved sample freezing and thawing are providing for improved CP from both the processing and sample quality perspectives. Numerous studies have described the impact, mechanisms and points of control of cryopreservation-induced delayed-onset cell death (CIDOCD). In an effort to limit CIDOCD, efforts have focused on CP agent and freeze media formulation to provide a solution path and have yielded improvements in survival over traditional approaches. Importantly, each of these areas, new technologies and cell stress modulation, both individually and in combination, are now providing a new foundation to accelerate new research, technology and product development for which CP serves as an integral component. This chapter provides an overview of the molecular stress responses of cells to cryopreservation, the impact of the hypothermic and cell death continuums and the targeted modulation of common and/or cell specific responses to CP in providing a path to improving cell quality.

Investigation of the Impact of Cell Cycle Stage on Freeze Response Sensitivity of Androgen-Insensitive Prostate Cancer.

BACKGROUND: Cryoablation, an effective means of ablating cancer, is often used in conjunction with adjuvants that target cancer cells in a specific cell cycle stage to increase treatment efficacy. The objective of this study was to investigate the impact of cell cycle stage on cancer freeze response as well as investigate the potential cellular kinetic effect of calcitriol, the active metabolic of vitamin D3, when used as a cryosensitizing adjuvant in order to maximize prostate cancer cell death. METHODS: Cell cycle distribution of PC-3 cells was analyzed via flow cytometry to compare gap 1, synthesis, and gap 2/mitosis phase subpopulations pre- and postfreeze as well as changes elicited by calcitriol pretreatment. Distinct gap 1, synthesis, and gap 2/mitosis phase populations were obtained through fluorescence-activated cell sorting and synthesis phase thymidine synchronization. Posttreatment viability was assessed using alamarBlue and fluorescence microscopy to assess live, apoptotic, and necrotic subpopulations. RESULTS: A small but statistically significant increase in synthesis phase and decrease in gap 2/mitosis phase populations was noted at 6 hours postfreeze in asynchronous samples. Synchronization in synthesis phase yielded an increase in cell death when combined with freezing to both -15°C and -20°C. Calcitriol pretreatment increased the gap 1 phase population by 20% and a synergistic decrease in viability following freezing. However, gap 1-sorted populations combined with calcitriol treatment did not exhibit this synergistic effect. Fluorescence microscopy of fluorescence-activated cell sorting-sorted cells revealed necrosis as the predominant form of cell death in all phases, though apoptosis did play a role. CONCLUSION: Although initial results suggested a potential sensitivity, PC-3 cells exposed to freezing as sorted populations did not reveal significant differences in cell death. As such, the data from this study suggest that there is no difference in cell cycle stage sensitivity to freezing injury.