SCIENTIFIC ACTIVITY

• Investigation of the mechanisms of cryodamage, cryoprotection and reparation of biological systems of various level of organization under the effect of hypothermic and ultralow temperatures.
• Creation of efficient methods of artificial cryoprotection of different biological objects, development of this base of new cryopreservation technologies of cells, tissues, organs, food products and their application in medical practice and agriculture.
• Application of hypothermia, cryotherapy and cryopreserved biological objects in treatment of various diseases.
• Creation and production of experimental and industrial samples of various cryoapparatuses and equipment for needs of medicine and agriculture.

An important place in the scientific activity of the Institute is occupied by a physical complex (EPR, NMR, optic spectroscopy, microcalorimetry, UHV-dielectrometry) that allowed to reveal a phase state of intrace llular content, barrier properties of membranes for electrolytes and non-electrolytes, parameters of cell degradation and the character of their interaction with cryoprotectants. The possibility of cryoprotectant molecules to form the complexes with biocations and stabilize protein conformation was revealed, that was the hypothesis basis of biological systems cryoprotection by cryoprotectants from a damaging ef fect of freezing factors.Basing wide application of high-resolution NMR spectroscopy a non-destroying method of the investigation of macroergic compounds in a cell at various stages of its cryopreservation was elaborated. Further studies of the physical-chemical properties of cryoprotective solutions and cell suspensions in the range of low temperatures (from -196 °C to the devitrification point) made it possible to reveal a new mechanism of cell cryoinjury-a mechanoelectrical one, which consists in the fact that after changing the temperature of the sample at a crystal-glass interface there is observed cracking of the a substance and the newly formed surfaces become charged. Thus developed difference in potentials is capable of inducing ionization of the substance and electrostatic emission, which damages cells and subcellular structures.

One of the important tasks in the investigations was to study the mechanisms of the organism resistance during their cold adaptation under natural (poikilothermic, hibernating animals) and artificial conditions (homoiothermic animals, human being), which enabled to reveal the mechanisms of reversible restructuring of the system of genetic information and metabolic paths of cold-resistant organisms, capable of a long-term revival under extreme climatic conditions with the body temperature, close to 0°C and the state of hypometabolism. A study of these mechanisms of restructuring of metabolism and physiological functions in the organism of poikilothermic and hibernating animals provided for a chance to gain more knowledge on the functions of the "hibernation triggers" - new class of bioregulators, capable of inducing the state of hibernation in human being, thus opening up prospects for applying biomolecules of adaptogenes in medicine. The aim was to provide scientific grants for application of hypothermia and cryotherapy in medical practice on the basis of studying pathophysiological, biochemical, morphological and immunologic processes at the cellular, tissue, organ and organism levels.

The investigations on studying physical and chemical properties of membrane structures of cells and cytoskeletal proteins during their cooling, freezing and reparation are carried-out at the Institute. The investigations demonstrated an important role of lipids, cholesterol, enzymes, ion pumps in cold shock development and low temperature damages of cells. It was shown that the impairment of the barrier function of the cell plasma membranes during freeze-thawing is characterized by formation of micro- or macrodefects in them, the dimensions of which are dependent on the extent of tempera ture lowering, phase-structural transitions of lipids and the level of water freezing-out in the membrane. Of great importance was the observation that some types of the intercellular proteins, penetrating cryoprotectants and solutions of salts, which freeze in the form of hydrates, especially with acceleration of the rate of freezing, facilitate maintenance of the barrier functions of plasma membranes of the thawed cells. The investigations of the development and occurrence of osmotic and temperature stress of cells showed that the factors, which control the susceptibility of cells to cold shock, are the original volume and the difference in the osmotic pressure between the intracellular medium and the environment, and their injury relates to deformation of the elastic elements of the membrane cytoskeleton during its osmotic dehydration and impairment in the metastability of the cell. On the basis of the investigations conducted a new method of cryopreservation of red blood cells was developed basing on the application of "Propandiosakharol" multicomponent medium. In particular, application of non-penetrating cryoprotectants in combination with preliminary cold "adaptation" of cells at 0 °C, which is accompanied by a change in the structural state of the cytoskeletal proteins, made it possible to develop a washing-free method of cryopreservation of human red blood cells.

A mathematical model was plotted, which established interrelations between the shape and distribution of separate components in the cell, and which explained a physical cause of the event of separation of proteins and lipids in the lateral plane of the membrane during its deformation. The results of experiments confirmed that such structural rearrangements in the cell plasma membrane are accompanied by formation of micro- and macrodefects in it, and impairments in water and electrolyte exchange. On the basis of the conducted biophysical investigations and physical-mathematical calculations it was concluded on the development of injuries to membrane structures in a solid-phase matrix.

Mechanisms of cryoinjury and cryoprotection of reproductive cells (gametes, embryos, gonads, embryonic, fetal cells and tissues) is involved in the investigations of peculiar features of snap freezing of human and animal sexual cells, and of the role of low temperatures as a factor of "selection" during freezing of the cell suspension. There was developed a method of snap freezing of human sperm and embryos, which was subsequently patented in the USA, Great Britain, France, FRG, Japan. It turned out that ultrarapid freezing (10-12000 degrees/min) of reproductive cells effectively protects their functional integrity and, in particular, the state of the genome, plasma membranes, intracellular organelles and their acrosomal apparatus, which permits to successfully solve the problems of infertility of married couples. Currently a cryobank of donor sperm is functioning within Kharkov Center of Obstetrics and Gynecology, which houses more than 20 thousand doses of cryopreserved sperm. In this cryobank the ovarian tissue is also stored, the allotransplantation of which is an effective method of correction of the generative function in females during endocrine infertility.

A traditional trend of investigations in cryobiology of the reproductive systems is a study of the mechanisms of cryoinjury and cryoprotection of spermia of rare, extinct and industrial fish. For example, very important applied and experimental data were obtained on the nature of the factors, affecting the original cryoresistance of spermatozoa. An important role belongs to the studies of pathogenesis of cryodestruction and cryoprotection of embryonic, hemopoietic and fetal cells of the human liver . In particular, the functions of the genetic apparatus and system of energy pro duction of cells, their immunologic and biological properties was investigated in details. It turned out that central nerve system cells, embryonic progenitor cells of the erythroid growth after cryopreservation are well preserved and capable of restoring hemopoiesis in lethally irradiated animals, i.e. are capable of grafting and functioning in the recipient organism. Clinical trials testify to the fact that after transfusion of cryopreserved cells of the human embryonic liver to the patients with anemic and aplastic states of various genesis results in nearly a complete recovery of hemopoiesis. Embryonic and fetal tissues (pancreatic and other endocrine glands, embryos, placenta), varying in their origin, may be a source of obtaining biologically active substances, thus being the object of investigations for determining their clinical efficiency. The investigations of the mechanisms of resistance of the organisms during artificial cooling were reflected in the works on the investigations of neurophysiology of the processes, which control thermal regulation and cold adaptation of animals and human being. The conducted complex studies of the mechanisms of action of cooling on the metabolic and physiological processes, occurring in the tissues of the brain and in the cardiovascular system, made it possible to establish new rules in realization of the primary mechanisms of the neurohumoral response of the organism to cooling.

The investigations of profound anabiosis and anhydrobiosis are carried out. It was found that in biological objects, irrespective of the level of organization (immune globulins, viruses, phages, microbial cells and cell tissue culture, hybridomas, hemopoietic cells, etc.), after getting out of the state of profound anabiosis there are developed mainly non-lethal injuries of various components, which may be accompanied by reversible inhibition or stimulation of the functional activity. An important fact was the disclosure of the synthesis of stress-proteins by procaryotic cells at the stage of transfer to and getting out of the state of profound cold anabiosis. It turned out that the synthesis of these proteins is ind uced not only under the action of cooling, but also after exposure of biological systems to the action of hyperconcentrated solutions of salts during normothermia.

Creation and development of technological processes of cryopreservation of biomaterials, varying in their origin, is impossible without effective elaboration, synthesis and trial of cryoprotectants and cryopreservatives. Due to profound screening it was found, that active chemical cryoprotectants besides alcohols are concentrated mainly within a few classes of organic substances - polyols, amides, oxides. To develop and forecast new cryoprotective substances there was elaborated and introduced into practice a method of systemic investigation of the dependence of cryoprotective activity, cytotoxicity and total toxicity of compounds in homologic series of polyols and amides on their chemical structure and physical-chemical properties.There have been developed effective technological processes of cryopreservation of hemopoietic, immune competent cells, hybridomas, various strains of microorganisms on the basis of cryopreservatives, which should not be eliminated prior to administration into the organism.

The investigations in solving practical aims of the usage of low temperatures both to damage pathologically changed tissues and as the factor, stimulating processes of cryowound healing, are carried out at the Institute, this trend in cryomedicine found its application in combustiology. A study of a possible combined application of low temperatures with other physical factors (ultrasound, laser, plasma scalpel) allowed to elaborate new methods of atypical resection of the liver by an ultrasound scalpel following precoo ling of the part of the organ.

The Institute has a standing reputation and maintains close contacts with the scientists of the Ukraine, CIS, and foreign cryobiological centers in the USA, UK, Germany, India, Czech Republic, Slovakia, Bulgaria, Belgium, Netherlands, Israel. The above makes it possible to realize complex programmes on cryobiology of the reproductive cells (sperm,oocytes,zigotes), preservation of cells, tissues, organs, and to do post-doc training of scientists. The important achievements of the Institute are the development and coordination of large complex programme on cryobiology. The activity of the workers of the Institute in various fields of cryobiology and cryomedicineinitiated maintenance of extensive links with higher educational establishments of the city of Kharkov and organization of scientific meetings. Starting from 1972, the Institute either independently or in combination with other establishments organized and held two conferences within CIS (1977, 1984), a mandatory Congress of cryobiologists of the Ukraine (1995), 3 schools for young scientists, 3 International Conferences on Cryobiology (1983, 1988, 1995). In 1999 two workshops and the UNESCO Chair on cryobiology meetings have been carried-out, as well as a range of close meetings with the scientists Germany,Japan, UK, Belgium. In 1995 a scientific Society for Cryobiology and Cryomedicine of the Ukraine was established on the initiative of scientists of the Institute. Its main aim is to protect social, economic, creative and other interests of the members, to contribute to the development of fundamental and applied problems of cry obiology and cryomedicine. The Society in its activity promotes to introduce scientific and research developments into a practice, to provide information exchange, to prepare scientific staff, to develop international cooperation. The Ukrainian Scientific Society of Cryobiology and Cryomedicine is voluntary independent public organization, has juridical personal rights. Society Chairman - Valentin Grischenko, Academician.

Since 1981 a Specialized Council (on defence of doctoral thesis) has been functioning with the Institute, which granted the degree of a Doctor of Sciences to 28 workers of the Institute, and the degree of a Candidate of Sciences(Ph.D.) - to 151 scientific workers. The scientific achievements of the workers of the Institute were reflected in 75 monographs and collections of scientific works, and more than 2,000 articles. The workers of the Institute were granted 63 patents and more than 200 author's certificates; 11 of them became winners of the State Prizes of the USSR and Ukraine (V.I.Grischenko, N.S.Pushkar, M.I.Shrago, T.N.Yurchenko, T.M.Sharlay, A.M.Belous, A.M.Vorotilin, V.A.Moiseyev, G.S.Lobyntseva, A.N.Goltsev, V.I.Lougovoy), 16 of them by Personal prizes of the National Academy of Sciences of the Ukraine, 6 were granted by the state prizes for young scientists.

In the process of setting, realization and extension of fundamental and applied researches in the field of cryobiology and cryomedicine in the Institute at the departments the scientific schools, that as a rule headed by famous scientists, professors, have been formed.

Scientific Schools

Professor Victor Moiseyev

Investigates physical and chemical mechanisms, laying in the base of cold impairments of systems, cold-resistance of biological objects and artificial methods of their cryoprotection. Damaging effect of thermomechanical tensions and electrical polarization, appearing in the course of cooling or heating of the samples in the temperature range from 0 to -196 C in a solid phase, was illustrated in artificial membranes and solutions of proteins, in cryopreserved erythrocytes and hepatocytes. By means of the methods of EPR-spectroscopy (NMR), a conformational dynamics of membrane bound proteins and protein-lipid complexes of oxidative-reductive reactions in cells under the effect of different physical and chemical factors ar e under study.

Professor Eugeny Gordienko

Studies the effect mechanisms of hypothermia and cryopreservation factors on human and animal gametes, gonads, embryos and embryonic cells with the aim to establish general biological reguliarities of both positive and negative low temperature effect on the mentioned bioobjects, develops new highly efficient methods of human fetoplacental tissue embryonic cells, embryos and gemetes cryopreservation. First in the Ukraine in 1991 in Kharkov in vitro concepted child (girl) was born due to the efforts of the deprtment scientific collabotators. To the present moment as the result of this technology application when treating infertility , more than 300 children were born in Kharkov. It has been demonstrated, that different by their nature human embryonic and fetoplacental tissues may serve as a transplantational material with a high spectrum of biological effect on human organism. These cells and tissues applica tion occured to be efficient when treating obstetrical pathology, infertility, climacterium, Diabetes mellitus and endocrine impairments, nerve and other deseases. Study of cryoprotection and cryodamage mechanisms of rare, undangered and marketable fish species is one of the important directions of the school work.

The investigations on revealing the mechanisms of cell and subcellular structure adaptation and damage mechanisms under cooling (+4° C) and a deep freezing (-196° C) effect are performed. The interaction mechanisms of the systems of bioenergetics, ion transport, xenobiotics metabolism and lipid exchange are studied in a cellular level. The possibility of a directed regulation of cell function by means of metabolically active compounds is under study. Theoretical and applied problems of liver cell cold storage in vivo and in vitro are developed in the department. Elaborated in the department new methods of hepatocytes non-enzymatic isolation and preservation permitted to extend the application possibilities of liver isolated cells in a medical practice, as well as a test-model for studying phar mokinetics and the mechanisms of toxic and medicinal preparation effect. Directions of liver cell clinical application - "artificial liver" development for extracorporal joining up to a patient and hepatocyte transplantation, are developed in the department. Fetal liver cells, that are better survived, proliferate, produce the substances, stimulating regeneration of a recipint's damaged liver, possess low immunogenity, are preferable. The importance of the given direction of the work is al so reinforced by the fact, that embryonic and fetal liver contains stem polypotent cells-precursors of a whole number of cell types in an organism.

Professor Anatoly Goltsev

Carries out the investigations on studying the fundamental and applied aspects of immunology, pathophysiology. In particular, the application possibilities of native and cryopreserved products of fetoplacental complex (PFPC) as the immune reactivity modulators of mammalian organism are studied. The realization mechanisms of PFPC immune modulating activity are studied, one estimates the degree of its manifestation depending on the terms of fetus development, the character of pregnancy course etc. The elaboration of the methods of PFPC preservation, providing the integrity of functional activity of biological substrates, possessing an immune modulating activity is an importan link of investigations. The works are performed on cytokines-immune modulators identification in native and cryopreserved PFPC. Such investigations will give the possibility to decode the mechanismof PFPC effect on lymphohemopoietic and immunocompetent sphere of an organism and apply them in a clinical practice.

Doctor of Biological Sciences Tatyana Bondarenko

R&D works are conducted in two directions. The first one is related to the development of the methods of cell cryopreservation, as well as endocrinic tissues of organs in order to create the bank of samples for this material xenotransplantation. The second direction concerns the problem of cellular homeostasis cryopreservation. One of the methodical approaches to the studying of intracellular processes is the application of biologically active compounds, affecting the membrane permeability of cells. Bee venom, mellitin and its synthetic analogue, PI4A peptide are used as biologically active substances.

Studies the effect of temperature and osmotic conditions of the medium on barrier function of biomembranes, their structure and state of membrane-associated cytoskeleton. The role of changes in medium temperature and osmotic behaviour as the factors, causing the formation and stabilization of transmembrane pores, is investigated. The efficiency of cation and non-ionic amphiphates as the agents, providing the locking of osmo- and thermoinduced pores, is analysed. A great value is given to the study of the changes in membrane proteins and cytoskeleton ones at diiferent cell modification variants, including vesiculation. The correlation between the sensibilization mechanisms of cells to temperature and osmotic shock and the changes of ions transport, including anionic transport, has been established.

Over 25 years in Cryobiology and experimental Cryomedicine. Pioneer of Electron Microscopy investigations in the institute. Mammalian and human cell and tissue properties (light and electron microscopy levels) at low temperatures have been developed. Over 100 publications and 4 monographs. Imaging, software development, data modeling, analysis. Design and implementation of new algorithms, methods and equipment. Holder over 22 patents. In the department new technical means for conducting an electron microscopic investigations, original automated device for cell and tissue preparation, analysis have been developed. Studies structural and functional state of mammalian epithelium and parenchymatous cells under cooling effect. Types of cell and tissue damages under low temperature effect are summarized.

Investigates the responses of biological structures of different organizational level on cooling of various degree and the effect of cryoprotective substances. Studies main regularities of the development of morphological changes, providing structural and physiological homeostasis of cells when decreasing the temperature in vivo and in vitro. The study of reactive changes in organs, tissues, cells and subcellular structures in response to cryopreserved fetoplacental tissue transplantation and determination of a character and intensity level of structural rearrangements in organs-targets is conducted.

Studies the processes, occurring in an organism, organs and tissues after local cold effects with the aim of creation new treating methods and approval existing ones. In this case the possibilities of cold usage to destroy a pathological focus are determined and it has been shown, that dosed cooling may cause manifested stimulating effect on regenerative processes in an acute and chronic inflammation focus.

Carries out the investigations on the search and organic synthesis of new cryoprotective substances, their chemical structure modification in the study of physical and chemical characteristics, toxico-pharmacological activity and cryoprotective activity for chemical compounds, biostructures of different organizational levels, important for cryoprotection. The investigations, directed to a profound study of cryoprotectant's effect on physiological parameters of animals organism and objects of a plant origin. Thus the possibility of oligomers of polyethylenoxide series with a differ ent molecular weight to decrease plasm and blood dynamic viscosity at normothermia and hypothermia of homoiotermal animals was established. PEO-400 and PEO-1500 normalizing effect on an aqueous-saline balance at brain edema-swelling, caused by cranio-cerebral trauma of traumatic and cold origin was shown, the possibility of the usage of efficient compositions, containing cryoprotectants and biologically active compounds to increase resistance of homoiothermal animals to a deep cooling is studied. The possibility of PEO-400 and PEO-1500 positive effect on the changes of brain bioelectric activity indices, as well as lipid peroxidation and antioxidative enzymic systems in different organs and tissues at animals' deep cooling has been shown. On the base of these data the original medical preparation "Cryopolyethylenoxide-400, 15%-solution for injections", that was successfully tested in profile clinics was proposed and recommended for introduction in medical practice as efficient osmo-diuretic agent .

Develops its investigations in the following directions:natural and artificial hypothermia, neuropathological processes and the methods of their correction and the effect of electromagnetic fields on biological objects. A central and peripheric mammalians' nerve system is the main object of investigations. The methods of the study of secretion and neuromediators capture and other biologically active substances, hematoencephalic barrier permeability for different substances, neurochemical and electrophysiological investigation methods of brain activity are used to achieve these aims. The investigations conducted on the effect of artificial cooling on an organism gave the possibility to estimate in a new way and interpret the mechanisms of cooling effect, optimize the indices of depth and duration of cold effect during different physiological functions impairments.

Investigates the mechanisms of natural and artificial cell adaptation to low temperatures. Cold-resistant insects, avoiding freezing and human erythrocytes are the objects under study. The investigations of molecular mechanisms by means of temperature and osmotic cell stabilization under the effect of non-penetrating cryoprotectants and low temperatures allowed to reveal the characteristic of spectrine-actine complex proteins to give an increased erythrocyte stability to freezing effect with their preexposition at 0-4C in the polyethylenoxide solutions with m.m. 1500. The obtained experimental data allowed to substantiate the notion about the determining role of cytoskeletal proteins and processes, regulating their structural and functional state in the mechanisms of temperature and osmotic cell stabilization.

Professor Alla Tsutsayeva

Studies the problem of investigation the induction mechanisms of non-lethal damages in biological objects of different organizational levels, depending on their initial state and their transition regimens into a state of deep cold anabiosis (cryopreservation) and unhydrobiosis (lyophilization), the methods of their returning to normothermia conditions. Studies the character of the change in immune biological properties of cryopresereved transplants of the tissue cells used at molecular, cellular and tissue levels. Investigates the peculiarities of interaction between cr yopreserved transplants and a recipient's organism. Studies the character and mechanisms of CNS interaction and immune system depending on the cooling degree, induction mechanisms, as well as treatment of immune deficient states. Cryopreservation technolologies of cord blood, bone marrow, lymphocytes, used for the treatment of hemodepressive state, not requiring washing-out are developed. Developments of technologies for cryopreservation of hybrodomas and cellular cultures are cond ucted.

Two scientific theoretical journals are published at the Institute:

1. "Problems of Cryobiology" founded by National Academy of Sciences of the Ukraine; Department of Molecular Biology, Biochemistry, Experimental and Clinical Physiology; Institute for Problems of Cryobi ology & Cryomedicine. The Journal is included into the list of scientific publications, approved by Higher Certifying Commission of the Ukraine, where the results of candidate's and doctor's theses can be presented. Founded in 1985 under the title "Kriobiologiya", since 1991 - "Problems of Cryobiology". Volume - 7,2 quires. Texts are given in Russian and English. Subscription to the journal can be accomplished through Ukrainian Post State Enterprise "Ukrpochta" according to an export catalogue or via the Journal Editorial Office. Index 70474. Registration No 0233-7673. Quarterly published. Journal's Responsible Secretary - Evgeny Popov, tel: (0572) 721039

Published the under the aegis of Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of the Ukraine and Kharkov Scientific Medical Society. The Journal comprises the papers, concerning fundamental and applied problems of medicine.