Cryoimmunology: Opportunities and challenges in biomedical science and practice.

Autologous and allogeneic cryoimmunological medicine is a brand new branch of biomedical science and clinical practice that examines the features and formation of the immune response to immunogenic properties of normal and malignant biological structures altered by ultralow temperature, as well as specific changes in the structural and functional characteristics of immune cells and tissues after cryopreservation. Cryogenic protein denaturation phenomenon provides important insights into the mechanisms underlying the damage to cryogenic lesions immediately after freeze-thawing sessions in bioscience and medicine applications. The newly formed cryocoagulated protein components (cryomodified protein components) are crucial in cryoimmunology from the perspective of the formation of immunological substances at ultralow temperatures. Dendritic cells and cryocell detritus (cryocell debris) formed in living biological tissue after exposure to ultralow temperature in vivo may be an indication of one of the essential mechanisms involved in the cryoimmunological response of living structures to the impact of ultralow temperature exposure. Hence, the formation of new autologous and allogeneic cryoinduced immunogenic substances is a novel concept in biomedical research globally. Accordingly, this review focuses on issues concerning the peculiarities of the interaction of the immune system with a dominant malignant neoplasm tissue after exposure to subzero temperatures, considering the original cryogenic technical approaches. We present an overview of the state-of-the-art methods of cryoimmunology, and their major developments, past and present. The need for the delineation of structural and functional characteristics of the biological substrates of the immune system after cryopreservation that can be used in adoptive cell therapy, especially in cancer patients, is emphasized.

Estimation of the stable frozen zone volume and the extent of contrast for a therapeutic substance.

BACKGROUND: In biomedical science and clinical practice, an estimation of the stable frozen zone volume and distribution of concentration fields of injected diagnostic and healing solutions in the tissues of living organisms is of great importance and does not currently have any mathematical solution aimed at its precise evaluation. OBJECTIVE: The aim of this research is the estimation of the stable frozen zone volume at ultra-low temperatures as well as the distribution of temperature areas and concentration fields of injected diagnostic and healing substances in vitro. The results can improve our understanding of the stable frozen zone volume and the extent of contrast for a therapeutic substance. MATERIALS AND METHODS: A cryogenic zone (ice ball) was generated at -180°C using liquid nitrogen without any difficulties in vitro. The effects of freeze-thaw processes using ultra-low temperature and the cryogenic response of a 1.5% gelatin solution in water (%g/v) kept at a constant temperature of 20°C and continuously stirred were mathematically analyzed. The stable frozen zone volume was illustrated in vitro and measured in terms of its length, depth and cryogenic margin using a standard medical ruler and Vernier caliper after a freezing period at -180°C, using liquid nitrogen to provide cooling and freezing of a small portion of this solution in the vessel at room temperature (20°C). Round-shaped cryoprobes with diameters of 15 mm and 50 mm were applied to create a frozen zone volume in vitro. A single cryoprobe was used per procedure. The sample exposure time was 3 min. After this time, the volume of the frozen region remains unchanged, which indicates that the equilibrium stationary state has been reached. The experimental design, cryogenic procedure and freeze-thaw processes of the hemisphere were described and illustrated in vitro item by item. The statistical analysis manifested significant differences that were found between the 50 mm and 15 mm cryoprobes with regards to the freezing diameter, depth, and cryogenic margin (P < 0.001). RESULTS: An illustrated analytical mathematical solution of equations determined the stable frozen zone volume and the radius of the sphere of the frozen medium in the equilibrium stationary state. The resulting assessment provided the basis for the creation of mini- and micro-cryoprobes as well as cryoneedles for local tissue freezing in living biological structures. A solution to the equations was obtained under the boundary conditions with a set stable temperature value on the boundary surface of the cryoprobe as well as at the surface well-away from it, where the temperature is equal to the stable temperature of the environment. For example, this solution gives that in the case of a hemispherical cryoprobe radius of 1 mm, the frozen zone volume was more than three orders of magnitude greater than the volume of the cryoprobe itself and was equal to approximately 4 cm3. The determination of the fractal dimension can consider the individual characteristics of the spread of the contrast medium or therapeutic substance(s) in living tissue. Based on fractal theory, our innovative mathematical formulas allow for the assessment of the effective distribution of contrast medium in living biological structures, specifically for tissues assessed for diagnostic purposes, and they enable the selection of an optimal treatment strategy in medical practice. CONCLUSION: A simple mathematical approach to solving the problems of assessing the stable frozen zone volume and distribution of temperature areas and concentration fields of injected diagnostic and healing substances in living biological structures, particularly living tissue in vitro, is presented in this study. The expressed quantitative mathematical formulas determine the stable stationary frozen zone volume and provide the basis for the creation of mini- and micro-cryoprobes. The application of fractal theory is proposed for assessing the distribution efficiency of contrast medium and therapeutic substance(s) in living biological structures for diagnostic purposes and for selecting a compassionate treatment strategy in medical professional practice.

Cryo-Assisted Resection En Bloc, and Cryoablation In Situ, of Primary Breast Cancer Coupled With Intraoperative Ultrasound-Guided Tracer Injection: A Preliminary Clinical Study.

The aim of the study was to perform cryosurgery on a primary breast tumor, coupled with simultaneous peritumoral and intratumoral tracer injection of a blue dye, to evaluate lymphatic mapping. We explored the ability of our strategy to prevent tumor cells, but not that of injected tracers, to migrate to the lymphovascular drainage during conventional resection of frozen breast malignancies. Seventeen patients aged 51 (14) years (mean [standard deviation]), presenting primary breast cancer with stage I to IV, were randomly selected and treated in The Rudolfinerhaus Private Clinic in Vienna, Austria, and included in this preliminary clinical study. Under intraoperative ultrasound, 14 patients underwent curative cryo-assisted tumor resection en bloc, coupled with peritumoral tracer injection, which consisted of complete tumor freezing and concomitant peritumor injection with a blue dye, before resection and sentinel lymph node dissection (group A). Group B consists of 3 patients previously refused any standard therapy and had palliative tumor cryoablation in situ combined with intratumoral tracer injection. The intraoperative ultrasound facilitated needle positioning and dye injection timing. In group A, the frozen site extruded the dye that was distributed through the unfrozen tumor, the breast tissue, and the resection cavity for 12 patients. One to 4 lymph nodes were stained for 10 of 14 patients. The resection margin was evaluable. Our intraoperative ultrasound-guided performance revealed the injection and migration of a blue dye during the frozen resection en bloc and cryoablation in situ of primary breast tumors. Sentinel lymph node mapping, pathological determination of the tumor, and resection margins were achievable. The study paves the way for intraoperative cryo-assisted therapeutic strategies for breast cancer.

Clinical Practice Guidelines for Cryosurgery of Pancreatic Cancer: A Consensus Statement From the China Cooperative Group of Cryosurgery on Pancreatic Cancer, International Society of Cryosurgery, and Asian Society of Cryosurgery.

Pancreatic cancer (PC), one of the most lethal malignancies, accounts for 8% to 10% of digestive system cancers, and the incidence is increasing. Surgery, chemotherapy, and radiotherapy have been the main treatment methods but are not very effective. Cryosurgery was first used in 1984 for treatment of locally advanced PC and has since become a considerable treatment for most cases of unresectable PC. During the past decade, cryosurgery has been applied in some hospitals in China, and the newly developed technique of computed tomography- and/or ultrasound-guided percutaneous cryosurgery has shown better results than chemotherapy in cases of unresectable locally advanced PC, with the 1-year survival rate reported to be more than 50%. To develop standardized criteria for the application of cryosurgery in PC, the International Society of Cryosurgery and Asian Society of Cryosurgery assembled experts from Austria, Japan, and China to discuss treatment methods and arrive at a consensus on the indications, contraindications, and preferred techniques of PC cryosurgery.

Experimental study on pulmonary cryoablation in a porcine model of normal lungs.

Objective of this study is to analyze the range of necrosis after using different freezing times and freeze-thaw cycles during percutaneous cryosurgery, in order to create a suggestion for optimizing the technique for lung cryoablation. Six healthy pigs were given a CT scan and histological investigation after percutaneous cryosurgery on both lungs. Three cryoprobes were inserted into both the left and right lungs of each pig, respectively. Cryoablation was performed with two cycles of an active 10-minute freezing using argon in the left lung, each freeze followed by an active 5-minute thaw using helium. In contrast to the left lung cryoablation, the right lungs underwent 3 cycles of freeze/thaw, the first and second cycles consisted of an active 5-minute freezing followed by an active 5-minute thaw, and the third cycle of 10-minute freezing and an active 5-minute thaw. The CT imaging change of an ice ball was continuously observed. The lung tissues were taken 4 hours after cryosurgery on day 3 and on day 7, respectively, for pathological observation. One pig presented acute symptoms including bradycardia and hypothermia 30 minutes after cryosurgery, and died 4 hours after the freezing, and the other 5 pigs experienced a weak condition for 4-6 hours and then exhibited relatively normal behavior and regularly took food. The freezing area (ice ball) on CT imaging during the cryoablation grew gradually in relation to the increase over time, and along with the increase in the number of cycles. The size of the cryolesion on the lung samples became larger than the ice ball during cryosurgery, regardless of whether 2 or 3 freeze-thaw cycles were performed. The area of necrosis histologically gradually increased for the time being. Percutaneous cryosurgery on the lung can achieve complete ablation of targeted tissue. Three freeze-thaw cycles are recommended, and the range of cryoablation may not be mandatory "1 cm safe border" during cryosurgery in order to avoid harming the organ and tissue which is close to the cancer. Correct use of the technique is especially important to treat the lung neoplasms, especially the malignant tumors, which are close to the heart and large vessels.

Living matter: the "lunar eclipse" phenomena.

The present investigations describe a unique phenomenon, namely the phenomenon of the "lunar eclipse", which has been observed and discovered by the author in living substance during the freeze-thawing processes in vivo using temperatures of various intensities and its cryosurgical response in animal experiment. Similar phenomena author has observed in nature, namely the total lunar eclipse and total solar eclipse. In this experimental study 76 animals (mongrel dogs) were investigated. A disc cryogenic probe was placed on the pancreas after the laparotomy. For cryosurgical exposure a temperature range of -40 degrees C, -80 degrees C, -120 degrees C and -180 degrees C was selected in contact with pancreas parenchyma. The freeze-thaw cycle was monitored by intraoperative ultrasound before, during and after cryosurgery. Each cryolesion was observed for one hour after thawing intraoperatively. Immediately after freezing, during the thawing process, the snow-white pancreas parenchyma, frozen hard to an ice block and resembling a full moon with a sharp demarcation line, gradually assumed a ruby-red shade and a hemispherical shape as it grew in size depend on reconstruction vascular circulation from the periphery to the center. This snow-white cryogenic lesion dissolved in the same manner in all animal tissues. The "lunar eclipse" phenomenon contributes to a fundamental understanding of the mechanisms of biological tissue damage during low temperature exposure in cryoscience and cryomedicine. Properties of the pancreas parenchyma response during the phenomenon of the "lunar eclipse" provide important insights into the mechanisms of damage and the formation of cryogenic lesion immediately after thawing in cryosurgery. Vascular changes and circulatory stagnation are commonly considered to be the main mechanism of biological tissue injury during low temperature exposure. The phenomenon of the "lunar eclipse" suggests that cryosurgery is the first surgical technique to use anti-angiogenesis with an immediately following cryoaponecrosis and cryoapoptosis in the treatment of malignant tumor. Both the "lunar eclipse" in vivo as well as the similar phenomena, namely the total moon and total solar lunar eclipses, are is part of living nature.

Cryosurgery: early ultrastructural changes in liver tissue in vivo.

BACKGROUND: Experimental observations with regard to freezing in vitro cell lines and fluid systems led to the application of low temperatures to in vivo biological systems. For the first time, this report describes the cryosurgical response of liver parenchyma and the early ultrastructural cellular changes in liver tissue, i.e., cryosurgery, in vivo. MATERIALS AND METHODS: Forty-eight animals were used for the experiment. The dogs were divided into four groups. In group A, the liver tissue was frozen to -80 degrees C and in group B, to -180 degrees C. Temperatures of -80 degrees C and -180 degrees C in contact with liver tissue was selected for cryosurgical exposure. For transmission electron microscopy, the specimens were taken immediately and 1 h after the finishing of the freeze-thaw cycles intraoperatively. Further, the next specimens were taken in 24 h, this time also intraoperatively. RESULTS: The electronic microscopic analysis showed that, after local cryodestruction at temperatures of -80 degrees C and -180 degrees C, similar processes occurred within the liver tissue in the early postcryosurgical phase-immediately and 1 h after cryosurgical session. The hepatocytes in the center of the cryozone changed upon thawing. Ultrastructural changes in the hepatic cells, where the first signs of dystrophic processes had been noticed, were increased. CONCLUSIONS: Our new insights prove on the cell level that suddenly and progressively damaged liver cells in the postcryosurgical zone lead to aseptic cryoaponecrosis and then to aseptic cryoapoptosis of vital normal tissue. The vascular capillary changes and circulatory stagnation demonstrate together with cryoaponecrosis and cryoapoptosis the anti-angiogenesis mechanisms, which are some of the main mechanisms of biological tissue injury following the low temperature exposure.