Bumsoo Han

Improved Cryosurgery by Use of Thermophysical and Inflammatory Adjuvants

In the present article, recent research efforts in our laboratory to improve cryosurgery by use of mechanistically derived adjuvants are reviewed. Our research has been focused on enhancing two freezing induced injury mechanisms - i) direct cell injury by use of thermophysical adjuvants, and ii) vascular injury by use of an inflammatory adjuvant. The thermophysical adjuvants are chemicals, usually salts, which can induce secondary crystallization, called eutectic solidification, in a cryolesion; thereby enhancing direct cell injury. The inflammatory adjuvant is a cytokine, tumor necrosis factor-alpha (TNF-α), which upregulates inflammation of microvasculature in tumors prior to freezing to promote vascular injury in the cryolesion. Even though the individual mechanism of injury enhancement within the cryolesion of each adjuvant requires further study, both adjuvants are envisioned to enlarge the complete killing zone so that the boundary of the cryolesion matches more closely with the edge of ice-ball. By bringing the edge of the cryolesion closer to the edge of iceball, the adjuvants hold promise for improvement of image guidance and outcome of cryosurgery.

Engineering Challenges in Tissue Preservation

Preserving native and engineered tissues for long periods without losing biological function is an important and challenging problem in biomedicine. Preservation is necessary to ensure an adequate ...

A Cryoinjury Model Using Engineered Tissue Equivalents for Cryosurgical Applications

Cryosurgery is emerging as a promising treatment modality for various cancers, but there are still challenges to be addressed to improve its efficacy. Two primary challenges are determining thermal injury thresholds for various types of cell/tissue, and understanding of the mechanisms of freezing induced cell/tissue injury within a cryolesion. To address these challenges, various model systems ranging from cell suspensions to three-dimensional in vivo tissues have been developed and used. However, these models are either oversimplifications of in vivo tissues or difficult to control and extract precise experimental conditions from. Therefore, a more readily controllable model system with tissue-like characteristics is needed. In this study, a cryoinjury model was developed using tissue engineering technology, and the capabilities of the model were demonstrated. Engineered tissue equivalents (TEs) were constructed by seeding and culturing cells in a type I collagen matrix. Two different cell lines were used in this study, AT-1 rat prostate tumor cells and LNCaP human prostate cancer cells. The constructed TEs underwent a freeze/thaw cycle imitating in vivo cryosurgery. Thermal conditions within TEs during freeze/thaw cycles were characterized, and the responses of TEs to these thermal conditions including freezing induced cellular injury and extracellular matrix damage were investigated at three different time points. The results illustrate the feasibility to establish thermal thresholds of cryoinjury for different cell/tissue types using the presently developed model, and its potential capabilities to study cell death mechanisms, cell proliferation or migration, and extracellular matrix structural damage after a freeze/thaw cycle.

Enhancement of cell and tissue destruction in cryosurgery by use of eutectic freezing

An in vitro study was performed to investigate a more effective method of destroying malignant tissue during cyrosurgery, which is based on eutectic crystallization. Eutectic formation is a solidification process through which water and solutes form a hydrate and can be recognized by a secondary heat release in differential scanning calorimetry (DSC). We investigated whether it is possible to induce eutectic crystallization by infusing concentrated salt solutions into cell suspension and tissue systems. These systems included AT-1 rat prostate tumor and normal rat liver tissues. In cell suspensions, the post-thaw viability significantly drops at or below the temperatures where eutectic crystallization occurred. When eutectic crystallization is induced in tissues, histological analysis shows significantly enhanced freezing injury. These results imply that this method may be of benefit in cryosurgical applications particularly at the edge of the iceball where tumor cell survival is in question. The possible advantages of inducing eutectic crystallization are i) enhancement of direct cell injury; ii) enlargement of effective cryosurgical cell/tissue destruction zone by selecting a salt with a high eutectic temperature; and iii) improvement of the efficacy of monitoring during cryosurgery.

Cryoinjury of MCF-7 Human Breast Cancer Cells and Inhibition of Post-Thaw Recovery Using TNF-α

Cryoinjury of MCF-7 human breast cancer cells and its enhancement using tumor necrosis factor-alpha (TNF-α) as an adjuvant, were investigated. Through a series of experiments in a two level factorial design critical parameters affecting cryotherapy responses were identified. The cryoinjury was investigated by quantifying the effects of four freeze/thaw (F/T) parameters, selected to be within the expected range for a cryosurgical iceball. Thermal parameters considered were cooling rate (5 and 50 °C/min), end temperature (−20 and −80 °C), hold time (0 and 10 min), and thawing rate (20 and 100 °C/min). After exposing the cells to the selected F/T conditions, survival was assessed and statistically analyzed to determine the effect of each parameter and their interactions. A statistical analysis shows that the end temperature and hold time were the two most significant parameters in the range studied. This suggests that proper control of these two parameters is important to achieve desired cryodestruction of MCF-7 cells. Enhancement of cryoinjury by TNF-α was also investigated in a tissue equivalent cryoinjury model in which a cryosurgical iceball is formed. MCF-7 cells cultured in a collagen matrix underwent a controlled F/T with or without TNF-α pre-treatment at 100 ng/ml for 24 hours. Post-thaw viability of MCF-7 cells was assessed at three hours, and at one and three days after freezing. Although the TNF-α treatment alone induced neither apoptotic nor necrotic cell death, the combination of TNF-α pre-treatment and freezing enhanced the immediate cryoinjury of MCF-7 cells, and significantly impaired the post-thaw recovery. Without TNF-α treatment, MCF-7 cell cultures were repopulated, reaching approximately 80% survival at day 3 even after severe cryoinjury (≤ 20% survival) at three hours. In contrast, this repopulation was significantly inhibited by TNF-α pre-treatment, in which case the viability of the frozen region remained below 40% at day 3. The effects of TNF-α on the cryoinjury of MCF-7 cells suggest that TNF-α may serve as a potent adjuvant to cryosurgery of breast cancer.

Phase change behavior of biomedically relevant solutions

Understanding the phase change behavior of biomedically relevant solutions is critical to the development of cryopreservation and cryosurgery protocols. In the present study, the phase change characteristics, particularly thermodynamically non-equilibrium phenomena including supercooled ice nucleation and eutectic formation, of various compositions and concentrations of biological solutions with/without glycerol as a cryoprotective agent (CPA) were investigated using a differential scanning calorimeter (DSC) and cryomicroscopy. The eutectic transitions (crystallization and melting) were observed in not only water-NaCl binary solutions, but also phosphate buffered saline (PBS) solutions. The eutectic transitions in both water-NaCl and PBS solutions were depressed during freezing far below their thermodynamic equilibrium eutectic temperatures. Contrary to freezing, eutectic melting starts very close to the thermodynamic equilibrium eutectic temperature during thawing. However, the eutectic transitions disappeared when a small amount of glycerol (∼0.1M) was added.Copyright

Effect of thermal properties on heat transfer in cryopreservation and cryosurgery

Biological materials in both cryopreservation and cryosurgery are composed of various chemicals and experience a wide range of temperature change. Therefore, their thermal properties including specific heat, latent heat (including water/ice and eutectic phase change) and thermal conductivity are expected to change significantly during freezing/thawing. The effects of thermal properties on heat transfer in cryopreservation/cryosurgery were studied experimentally and numerically. Thermal properties of various biological aqueous solutions were measured over a wide temperature range (−150~30°C). To estimate the effect of thermal property changes on the heat transfer, numerical simulations of both cryopreservation (cooled from outside) and cryosurgery (cooled from inside) geometries were performed with constant and temperature-dependent properties. The results show that the constant-property case significantly under-predicts the heat transfer over the temperature-dependent-property case regardless of the geometry.Copyright

Effects of a Cryoprotective Agent on Thermal Properties of Solutions at Subzero Temperatures

The study focuses on evaluating the effects of adding a cryoprotective agent (CPA) on the thermal properties of solutions. Preliminary results for Phosphate Buffered Saline (PBS) with glycerol at subzero temperatures are reported. A Differential Scanning Calorimeter (DSC) was used to measure the specific heat and to observe latent heat release. Cryomicroscopy was performed using a cryostage to control the cooling and heating rate of the sample and visually observe microscopic changes. The specific heat data obtained from the DSC is also used for predicting bulk thermal conductivity by a transient heat transfer model using a thermistor. Thermal properties measurement results show opposing trends prior to and after devitrification and melting events with respect to glycerol concentration.Copyright

Characterization of Heating, Movement and Visualization of Magnetic Nanoparticles for Biomedical Applications

Magnetic nanoparticles can be used for a variety of biomedical applications. They can be used in the targeted delivery of therapeutic agents, as contrast agents in MR imaging and in the hyperthermic treatment of cancers. Previous studies using these particles have not dealt with a quantitative characterization of movement and heating of these particles in biological environments. In the present study, the thermal characteristics of magnetic nanoparticles in water and collagen were investigated. In other studies, the movement of these particles in collagen in a known magnetic field was studied; infra-red (IR) imaging was used to visualize these particles in vitro. The results show that the amount of temperature rise increases with the concentration of nanoparticles regardless of the microenvironments. However, the amount of heating in collagen is significantly less than water at the same nanoparticle concentration. IR imaging can be used to visualize these particles in vitro over a wide range of concentrations of these nanoparticles.Copyright