Method for cryopreservation of haemopoietic umbilical stem cells

FIELD: medicine.

SUBSTANCE: cryoprotector is opened in a laminar flow unit; a special syringe of asyringe pump is filled with a cryoprotector; that is followed by introducing a filter solution of the cryoprotector - 55% dimethylsulphoxide with 5% dextran 40 at temperature +4°C in a nucleated cell suspension with haemopoietic stem cells in a cryopackage with the concentrate and mixing mechanically in a mixing apparatus, transferring the system together with the cryoprotector flask into the laminar flow unit; the air is released from the cryopackage and portion of the suspension; the package is sealed and placed into a shrink bag; that is followed by programmed multi-stage freezing, the first stage of which keeping the mixture of the suspension with the stem cells and cryoprotector - a freezing sample - for 10 min at temperature +4°C; the second stage is cooled at a rate of 1°C/min to temperature -12°C; the thirst stage provides cooling at a rate of 20°C/min to temperature -60°C; at the fourth stage, the sample is heated at a rate of 10°C/min to temperature -18°C; at the fifth stage, the sample is cooled at a rate of 1°C/min to -60°C; at the end of the freezing program, the sample is cooled at a rate of 3°C/min to temperature -100°C; after freezing, the sample is placed into a quarantine dewar with liquid nitrogen until infection and bacteriological fungal contamination test results are obtained. After termination of the quarantine shelf life, the sample with haemopoietic stem cells are placed for long-term storage at temperature not exceeding -150°C, into the dewar with liquid nitrogen if observing negative test results. If the infection and bacterial and/or fungal contamination test results are positive, the sample with haemopoietic stem cells are transferred into the dewar with liquid nitrogen for infectious material for long-term storage.

EFFECT: invention enables increasing cell viability in the sample.

3 cl, 4 dwg

 

The invention relates to the field of medicine, namely to methods of preservation of hematopoietic stem cells.

The closest to the proposed is a method of cryopreservation of hematopoietic stem cells from umbilical cord blood [RF Patent №2416197, IPC A01N 1/02, A61K 35/14, A61K 35/44, 2011], including the introduction of a protecting solution of cryoprotectant 55% of dimethyl sulfoxide with 5% dextran 40 at a temperature of +4°C in a suspension of nucleated cells with hematopoietic stem cells (HSC) method: bags, mechanical agitation, air is released from the method: bags and suspension parts, sealing and placing it in a heat shrink package of software of the multi-stage freezing, then the sample is placed in quarantine Dewar with liquid nitrogen to determine the results of tests for infectious agents fungal and bacteriological contamination, after a quarantine period of storage, the sample is transferred to long-term storage at a temperature not exceeding -150°C in a Dewar with liquid nitrogen under the condition of negative test results, in the case of positive test results a sample of the hematopoietic stem cells are transferred into the Dewar for infectious material long-term storage.

Known for the reason that impede the achievement of the technical result provided by the proposed and�the acquisition, is to protect the sample from external influences not at all the stages of the method, the duration of the process of preparation for cryopreservation of HSC, relatively low viability after thawing of HSC.

The problem to be solved by the present invention is to reduce the probability of infection of hematopoietic stem cells of umbilical cord blood infection and bacterial and/or fungal contamination, reducing the length of training for long-term storage of cryopreserved hematopoietic stem cells of umbilical cord blood and cell viability of the hematopoietic stem cells of umbilical cord blood.

By carrying out the invention the problem is solved at the expense of achieving a technical result, which consists in improving the conditions of sterility, reduced time software multi-stage freeze and the increase in the number of viable hematopoietic stem cells of umbilical cord blood after a storage period.

Said technical result is achieved by a method of cryopreservation of hematopoietic stem cells of umbilical cord blood, including the opening of a bottle of cryoprotectant in a laminar box, filling the cryoprotectant in a special syringe syringe pump, the introduction of using syringe pump cladding RA�creators of cryoprotectant 55% of dimethyl sulfoxide with 5% dextran 40 at a temperature of +4°C at a rate of 0.8 ml/min in a suspension of nucleated cells with hematopoietic stem cells in the method: bags with simultaneous mechanical stirring in the apparatus for stirring at +4°C, the termination of mechanical agitation immediately after receipt of the required quantity of cryoprotectant in the method: bags with a concentrate transfer system together with a bottle of cryoprotectant in a laminar flow hood, air is released from the method: bags and suspension parts, sealing and placing it in a heat shrink package of software of the multi-stage freezing, at the first stage the mixture suspension with stem cells and the cryoprotectant is a sample of freeze - incubated for 10 min at +4°C, the second stage is cooled at a rate of 1°C/min to a temperature of -12°C, the third phase is cooled at a rate of 20°C/min to -60°C, in the fourth stage the sample is heated at a rate of 10°C/min to a temperature of -18°C, in the fifth phase, the sample was cooled at a rate of 1°C/min to -60°C and at the end of the program freeze the sample was cooled at a speed of 3°C/min to -100°C, after freezing, the sample is placed in quarantine Dewar with liquid nitrogen, until the determination of the results of tests for infectious agents fungal and bacteriological contamination, after a quarantine period of storage, the sample with hematopoietic stem cells is transferred to long-term storage at a temperature not exceeding -150°C in a Dewar with liquid nitrogen under the condition of negative test results, in the case �ood results of tests for infections and bacterial and/or fungal contamination of the sample with hematopoietic stem cells are transferred into the Dewar for infectious material long-term storage.

Opening the bottle of cryoprotectant and filling of cryoprotectant in a special syringe syringe pump is carried out in a laminar box in order to ensure the sterility of the process. This reduces the probability of infection of hematopoietic stem cells of umbilical cord blood infection and bacterial and/or fungal contamination.

Introduction using a syringe pump cladding solution of cryoprotectant 55% of dimethyl sulfoxide with 5% dextran 40 at a temperature of +4°C at a rate of 0.8 ml/min in a suspension of nucleated cells with hematopoietic stem cells with simultaneous mechanical mixing allows you to evenly distribute the cryoprotectant volume leukocyte cell fraction. Due to this, a solution of cryoprotectant penetrates into almost all cells that are contained in this fraction, and increases the number of viable HSC after thawing. The rate of introduction of cryoprotectant 0.8 ml/min is optimal for this method and selected based on the speed of diffusion of the cryoprotectant solution through the cell membrane. Mechanical stirring at +4°C in the apparatus for stirring is stopped immediately after the receipt of the required quantity of cryoprotectant in the method: bags with the concentrate. If mixing will last longer over time, it is likely obra�hardware hemolysis.

After mechanical mixing system together with a bottle of cryoprotectant is transferred to a laminar flow hood to let the air out of the method: bags and part of the suspension, sealed, and placed in a shrink package. These operations in a laminar box can reduce the probability of infection of hematopoietic stem cells of umbilical cord blood infection and bacterial and/or fungal contamination.

In the third stage, multistage software freeze the sample was cooled at a rate of 20°C/min to -60°C. by increasing the cooling rate is achieved by uniform cooling of the sample throughout the volume to the desired temperature.

At the fourth stage of multistage software freeze the sample is heated at a rate of 10°C/min to a temperature of -18°C. Due to a slower increase in temperature results in a seamless process of thawing due to the release of latent heat, which gives the possibility to overcome the recrystallization point and pay back the shock.

Thus, more rapid cooling in the third stage, and a slower heating in the fourth stage, in comparison with the prototype allows to avoid a sharp rise in temperature, thereby maintaining the viability of cells in the sample.

Examples of the implementation of the proposed method of cryopreservation of hematopoietic stem cell�OK umbilical cord blood are shown below.

In a laminar box, "Heat Scientific" Hera Safe KS 12 (Heraeus, USA) opened a bottle of cryoprotectant CryoSur-DEX 40, containing a solution of 55% of dimethyl sulfoxide with 5% dextran 40, and ran in a special syringe 20 ml syringe pump Pilot (Fresenius Vial, France).

The method: bags with a leukocyte concentrate was placed in a pretreatment apparatus CoolMix AS-210 (Biosafe, Switzerland), using a system of polymer backbones attached to a syringe pump. On the syringe pump Pilot exhibited the rate of injection of a solution of cryoprotectant equal to 0.8 ml/min When 5 ml of cryoprotectant entered method: bags with a concentrate pump Pilot gave the signal, immediately stopped stirring and transferred the system along with the bottle in a laminar flow hood.

Released the air out of the method: bags, squeezed out a small amount of blood from the package to fill up at a distance of 5 cm from the package; was fed up at this distance and did soldering at a distance of 1 cm from each other. Solder stations, leading to the tube-satellite.

The method: bags concentrate Packed in shrink bag, removed the air from the bag and heat-sealed with the help of a machine Shop Sealer FS-315 (Fuji Impulse Co. Ltd, EC) for sealing shrink package "CryoFlex", was placed on the refrigerant and transferred to programmatically freeze.

Packed in shrink bag, and the concentrate was placed in a cassette for programs�nogo freeze; set the camera on a tripod.

Control temperature sensor Sample software freezer Planer Kryo 560-16 (Planer pis, UK) placed in close proximity to the sample. Temperature control in the chamber was carried out by an additional sensor Chamber software freezer Planer Cryo 560/16 (Planer pis, UK). In the preparation of cryptococcal set schedule lined up for the set temperature of the chamber.

Also used the optional remote sensor monitoring system Scada Master, for temperature control and timely calibration of the camera.

The freezing of the concentrate was performed in a software freezer Planer Cryo 560/16 in the following stages: the first stage, a mixture of a slurry with stem cells and the cryoprotectant - sample freezing was kept 10 min at +4°C, the second stage is cooled at a rate of 1°C/min to a temperature of -12°C, the third phase was cooled at a rate of 20°C/min to -60°C, in the fourth stage the sample was heated at a rate of 10°C/min to a temperature of -18°C, at the fifth stage the sample was cooled at a rate of 1°C/min to -60°C and at the end of the program freeze the sample was cooled at a speed of 3°C/min to -100°C. the Data on the freezing procedure were recorded in the Protocol software freeze concentrate of stem cells, which is represented on f�G. 1. Protocol software freeze concentrate of stem cells according to the prior art shown in Fig.2.

The Protocol of cryopreservation of HSC by the proposed method for the duration of execution is 86 min (+1...3 min), and in the prototype 114 min (+1...2 min). Therefore, shortens the duration of training for long-term storage of cryopreserved hematopoietic stem cells of cord blood by the proposed method.

The results of all studies characterizing the cord blood sample and the resulting concentrate of stem cells, cord blood entered into the database NPO "Humanitrian" under a single identification number of the sample.

Concentrate stem cells from umbilical cord blood stored in a quarantine Dewar until then, until the results of tests for infectious agents fungal and bacteriological contamination (according to the order of Ministry of health of the Russian Federation No. 325 of 25 July 2003). After a quarantine period of storage the concentrate of cells of umbilical cord blood was transferred to a long-term storage at a temperature not exceeding -150°C in a Dewar with liquid nitrogen at negative test results (Anti-fflV-l and -2, HIV-lAg, Anti-HTLV-I and-II, Anti-HbcorAg, HBs-Ag, Anti-HCV). Data on withdrawal of the sample were recorded in the Protocol of seizure of the sample concentrate stem cells, information about changes in the localization� sample were recorded in the Protocol migration of stem cells concentrate, and also in the database NPO "Humanitrian" and in the "Journal of accounting and registration of the frozen samples of stem cells from umbilical cord blood".

Thawing of cryopreserved sample and washing of the concentrate of stem cells of umbilical cord blood from the cryoprotectant was performed by the standard technique as follows.

First, prepare the room, which made the procedure of defrosting and cleaning of the umbilical cord blood sample: included UV lamp for 15 min.

Consisted of a water bath in the network, set 37°C. the Warmed saline solution in a thermostat at 37°C for 15 min In a laminar box prepared wash buffer: saline solution containing 2.5% albumin human recombinant. Otbirali from the package to the final product, the entire anticoagulant.

Was removed the sample from the tanks, were recorded properly. Thawing of cryopreserved sample is conducted on software freezer Planer Kryo 560-16 as follows: maintained 5 minutes at a temperature of -160°C, the sample was heated at a speed of 3°C/min to -100°C, passed through the sample 5 min at a temperature of -100°C, the sample was heated at a rate of 1°C/min to -60°C, the sample was heated at a rate of 10°C/min to a temperature of -18°C, the sample was heated at a speed of 3°C/min to a temperature of -4°C, you�arrival sample 5 minutes at a temperature of -4°C, the sample was heated at a rate of 1°C/min to a temperature of 0°C, passed through the sample 5 min at 0°C. Subsequent thawing was performed in a water bath - thermostat medical TW-2.02.

After the blood became liquid in the air flow laminar box with a cloth dampened with 70% ethanol solution was carefully treated with a blood bag, and then through both ports of the package selected syringes (10 ml) concentrate all cells. Distributed concentrate equally in two conical centrifuge tubes (50 ml) and poured an equal volume of wash buffer. Centrifugation was performed at 400 g for 10 min at room temperature.

In a laminar box supernatant was removed and resuspendable precipitate the cells in wash buffer. Put the final product in a package in which through a sterile port was added by syringe the required amount of heparin to prevent blood coagulation.

Out of the package with the final product was collected and 0.5 ml of concentrate for analysis on the viability and number of cells by flow cytopfuorometry.

Cell viability was determined using the vital dye 7-amino-actinomycin D RUO (7AAD) (Beckman Coulter, USA), analyzing by flow cytofluorimetry Cytomics FC500 (Beckman Coulter, USA). Identification of the cells was performed by registering the two options: volume�mA and light scattering in the lateral direction (SS) and fluorescence intensity (7-AAD).

Fig.3 presents a histogram of the number of viable cells in the thawed sample as a result of the proposed method.

Fig.4 presents a histogram of the number of viable cells in the thawed sample as a result of the prototype method.

The histograms on the left of the vertical line (y value of 10°) registered live blood cells, and the right - dead. The data obtained showed that the application of the proposed method the number of viable hematopoietic stem cells of umbilical cord blood after storage, the sample was 87,24%, and according to the method prototype 79,01%.

Thus, the proposed method allows to reduce the probability of infection of hematopoietic stem cells of umbilical cord blood infection and bacterial and/or fungal contamination, to reduce the length of training for long-term storage of cryopreserved umbilical cord blood HSC and increase the number of viable hematopoietic stem cells of umbilical cord blood after a storage period.

1. Method of cryopreservation of hematopoietic stem cells of umbilical cord blood, including the opening of a bottle of cryoprotectant in a laminar box, filling the cryoprotectant in a special syringe syringe pump, the introduction of the protecting solution of cryoprotectant 55% �of metilsulfate with 5% dextran 40 at a temperature of +4°C in a suspension of nucleated cells with hematopoietic stem cells method: bags with the concentrate and simultaneous mechanical stirring apparatus for stirring, the transfer system together with a bottle of cryoprotectant in a laminar flow hood, air is released from the method: bags and suspension parts, sealing and placing it in a heat shrink package and the software implementation of the multi-stage freezing, at the first stage the mixture suspension with stem cells and the cryoprotectant - sample freezing incubated for 10 min at +4°C, the second stage is cooled at a rate of 1°C/min to a temperature of -12°C, the third phase is cooled at a rate of 20°C/min to -60°C, in the fourth stage the sample is heated at a rate of 10°C/min to a temperature of -18°C, in the fifth phase, the sample was cooled at a rate of 1°C/min to -60°C and at the end of the program freeze the sample was cooled at a speed of 3°C/min to -100°C, after freezing, the sample is placed in quarantine Dewar with liquid nitrogen, until the determination of the results of tests for infectious agents fungal and bacteriological contamination, at the expiration of the quarantine period of storage, the sample with hematopoietic stem cells is transferred to long-term storage at the temperature not exceeding -150°C in a Dewar with liquid nitrogen under the condition of negative test results, in the case of positive results of tests for infections and bacterial and/or fungal contamination of the sample with hematopoietic stem cells are transferred into �UAR with liquid nitrogen for infectious material long-term storage.

2. A method according to claim 1, wherein the cryoprotectant is introduced into the slurry with hematopoietic stem cells using a syringe pump at a speed of 0.8 ml/min.

3. A method according to claim 1, characterized in that the mechanical stirring is stopped immediately after the receipt of the required quantity of cryoprotectant in the method: bags with the concentrate.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncourology, and can be used in the non-surgical treatment of the patients suffering early stages of prostate malignant tumour. A method of treating prostate cancer with using prolonged prodrug of octreotide accompanying surgical or drug-induced castration involves a) measuring pre-therapeutic prostate-specific antigen; b) measuring pre-therapeutic blood plasma chromogranin A; c) selecting the patients having high blood chromogranin A of more than 3 nmol/l; d) conducting a therapy with prolonged prodrug of octreotide in a combination with dexamethasone in the selected patients; e) controlling the prostate-specific antigen variation every month and monitoring a decrease thereof in the patients by measuring it intra-therapeutically according to the stage d); the therapeutic efficacy according to the stage d) is evaluated by a therapeutic response, which is accompanied by maximum decrease of the prostate-specific antigen.

EFFECT: invention enables providing the higher therapeutic efficacy in the patients suffering prostate cancer at the different stages.

3 cl, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to balneology. A balneological agent for treating and preventing various diseases is prepared by the gradual and sequential mixing at room temperature of yellow clay, natural brine of Bolshoy Tambukan Lake, sage essence, dimethyl sulphoxide in certain relations.

EFFECT: agent possesses the more prominent therapeutic effect.

5 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: tissue-specific matrix is obtained by performing a perfusion washing and a decellularisation of a parenchymal organ. Herewith 60-120 minutes before the perfusion washing of the donor organ, measures to prevent intravascular blood cell aggregation and microcirculation disturbances are taken and involve the intramuscular or intravenous administration of a disaggregant or disaggregants (heparin and trental) into a donor. That is followed by an organ perfusion by administering into its vascular bed phosphate-buffered normal saline of the following composition: 138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, distilled water up to 1l and containing 1% serum albumin and 10-15% glycerol or 10-15% dimethyl sulphoxide with pH 7.4 in an amount equal to a double amount of the vascular bed of the organ at a perfusion pressure of 100-120 mm Hg in its arterial system. Thereafter, the decellularised organ is ground to a particle size of 0.5mm to 4mm; the ground fragments are portioned in an amount of 5-10g, and each portion is frozen by immersing into liquid nitrogen gas for 5-10 minutes. The frozen portions are re-ground to a particle size of no more than 600 mcm; then each portion is de-frozen by re-suspending in phosphate-buffered normal saline 30-70ml of the following composition containing 138 mM NaCl, 2.67 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, distilled water up to 1l , with pH 7.4, at a room temperature. The prepared suspension is cleaned from the particles of less than 200mcm; the prepared fraction is lyophilised and sterilised to prepare the target matrix samples.

EFFECT: using the invention enables providing the more complete decellularisation procedure ensured by preventing the disturbed microcirculation in the donor organ, providing higher density and uniformity of the re-cellularisation of the entire matrix and easier control thereof by increasing the matrix adhesive surface areas for the contact to inoculating cells as prepared in the form of powder.

4 cl, 5 ex, 9 dwg

FIELD: biotechnologies.

SUBSTANCE: invention relates to new heterogeneous ring compounds containing a pentatomic rings, condensed with other nuclei, only with one atom of oxygen as a heteroatom, namely to derivants of acetamid N-((1S)-1',2',3'-trimethoxy-6,7-dihydro-1H-benzo[5',6':5,4]cyclohepta-[3,2-f]benzofuran-1-il) with the general formula 1 , where R - substituent, R=Ph, pyridine-2-il, CH2OH, CH(CH3)OH, CH2CH2OH, CH2OAc, (CH2)8CO2Me or CH2N(CH2CH3)2, and also to their application as an active component of antitumoral medicinal preparation.

EFFECT: increase of activity with inhibition of proliferation of the tumour cells.

10 cl, 3 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: set task is solved by application of final slag, formed in production of ferrovanadium by alumino-silicothermic method as bactericidal material.

EFFECT: extension of raw material resources for bactericidal materials.

1 tbl

FIELD: chemistry.

SUBSTANCE: invention provides benzylidene furanone derivatives of (+)-usnic acid of formula 6-13 as anti-tumour agents. The compounds exhibit cytotoxic activity with respect to tumour cell lines CEM-13, U-937, MT-4.

EFFECT: high activity.

2 dwg, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention enables creating complex formulations of functional sports supplements of single foods with the specified concentrations of vitamins and mineral substances for sportsmen of various sports.

EFFECT: recovered physiological body saturation with vitamins and mineral substances on the basis of an algorithm for determining sportsmen's body saturation with these nutrients.

4 cl

FIELD: medicine.

SUBSTANCE: agent for stimulating substantia Nissl synthesis in spinal cord motor neurons and spinal cord motor neuronal process growth, and a method for stimulating substantia Nissl synthesis in spinal cord motor neurons and spinal cord motor neuronal process growth. The agent represents a swine or foetal adrenal cortical alcohol extract which contains natural corticosteroids in the minor concentrations. The adrenal cortical alcohol extract of swine or foetal animal cells is prepared on the basis of organ preparations.

EFFECT: agent and method using the presented agent enable providing more effective stimulation of substantia Nissl synthesis in the spinal cord motor neurons which possesses an ability to neutralise toxic products and free radicals formed in process of acting, as well as ensuring higher growth of the processes of forming cell-to-cell communications, activating intracellular granular endoplasmic reticulum, increasing an energy level and antioxidant protection against aggressive radicals.

2 cl, 1 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to psychiatry, and may be used for treating and preventing disorders specified in a group consisting of depressive, affective and anxious disorders, particularly a major depressive disorder. For this purpose, a patient's therapy is added with a stage of administering a therapeutically effective amount of embryonated egg isolate.

EFFECT: group of inventions provides treating the above pathology, including by inhibiting glutamate and neurokinin 2 (NK2) receptors.

15 cl, 12 dwg, 11 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed are: a pharmaceutical combination for treatment of proliferative disease, which contains Hsp90 inhibitor ethamide 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid (AUY922) and mTOR inhibitor, representing everolimus (RAD001), a respective method of treating proliferative disease and a set for the same purpose.

EFFECT: synergic antiproliferative effect of the combination on cells of different types of cancer is demonstrated.

5 cl, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining 1,6-bis-(1,5,3-dithiazepan-3-yl)-2,5-disulphanylhexane of formula (1), possessing fungicidal activity against Botrytis cinerea and Rhizoctonia solani. Essence of method lies in interaction of mixture of 1,2-ethanedithiol and formaldehyde with water solution of ammonium salts NH4X (X=F, Br, OAc, NO3, 1/2SO4) with molar ratio HS(CH2)2SH:CH2O:NH4X=3:6:2 at room temperature (~20°C) and atmospheric pressure for 5-7 h.

EFFECT: output of 1,6-bis-(1,5,3-dithiazepan-3-yl)-2,5-disulphanylhexane of general formula (1) depending on applied ammonium salts (NH4X) constitutes 31-67%.

2 cl, 2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: paleontological objects with soft tissues are treated for 1-4 months with an embalming solution containing 40% formalin - 0.516%, crystalline phenol - 0.262%, glycerine - 79.077%, 96% alcohol - 18.576%, sodium chloride - 1.569%. The ratio of the embalming solution to the mass of the paleontological object must not be less than 3:1. Embalming is carried out at room temperature.

EFFECT: method of embalming paleontological objects with soft tissues provides prolonged preservation of soft tissues, minimises loss of soft tissues having different rotting stages, stops rotting processes, skin straightening, preserves natural colour thereof and natural morphometric parameters, which enables further use of the paleontological object for scientific purposes and for exhibition in dry form.

3 cl

FIELD: medicine.

SUBSTANCE: invention refers to physiology, cryobiology and medicine, namely to human blood nuclear cell preservation technique with the use of inert gas. The method involves cell saturation in Compoplast 300 plasticised container placed in a metal cryobarocontainer with inert gas, xenon at pressure 0.6 atm for 20min in the room temperature environment of 21±2°C; excessive gas is eliminated, and the biological object is removed from the metal container and frozen to -28°C in ethanol, placed into an electric freezer at -80°C. The bioobject is unfrozen in a water bath at 39±1.5°C for 1.5 min.

EFFECT: implementing the invention provides the reliable cryonic preservation of leukocytes in the inert gas medium and the high level of qualitative and morphological safety of leukocyte concentrate of human blood.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method of reducing impact of low-temperature jump on cryoprotectant solution is provided at the expense of remote processing of cryoprotectant solution with cells of living organisms by ultrasonic radiation with frequency 0.50-10 before its freezing.

EFFECT: reduction of low-temperature jump of cryoprotectant solutions, which makes it possible to eliminate overcooling effect, provide continuous and smooth character of freezing and increase integrity of defrosted cells after cryoconservation.

1 dwg

FIELD: medicine.

SUBSTANCE: what is presented for embalming of dead bodies for the purpose of organ harvesting and transplantation drills is a method, wherein tissues and organs are saturated with embalming solutions; the dead body is immersed into water at t 40°C for 120-160 min; cannulas are inserted into a femoral artery, and a blood flow is washed with warm normal saline with 1-2% sodium citrate added; the femoral artery is sealed, and a solution containing formalin and glycerol is inserted into the artery.

EFFECT: keeping the internal organ structure by increasing the preserving properties of the solution and providing the small vessel filling.

FIELD: chemistry.

SUBSTANCE: invention relates to a method of lyophilisation of a composition, which contains purified antithrombin III (AT III) and a crystallised substance, selected from alanine, mannitol, glycine or NaCl. The claimed method includes freezing the composition at a temperature from -52°C to -60°C for 6-15 hours, annealing the composition at -30°C for 1 hour, re-freezing the composition at a temperature from -52°C to -60°C for 2-15 hours at keeping the product temperature between -48°C and -52.7°C for 4-10 before lyophilisation and drying the composition with obtaining a lyophilised cake. The invention also relates to a pharmaceutical set, which contains the said lyophilised cake and a liquid reagent.

EFFECT: invention provides obtaining the lyophilised composition, containing AT III, which preserves its activity and stability.

14 cl, 24 dwg, 5 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula

,

where X=S, SO or SO2, and one of radicals R1 and R2 is a hydrogen atom and the other has a value given in the claim, which are used for depigmentation of the skin and/or hair and/or body hair and for skin disinfection, to cosmetic application of the disclosed compounds, and to compounds of formula (I), wherein R1 and R2 can also be a hydrogen atom at the same time. The invention also relates to a cosmetic composition and a pharmaceutical composition based on the disclosed compounds and to methods of producing said compounds.

EFFECT: improved properties.

19 cl, 2 tbl, 30 ex

FIELD: medicine.

SUBSTANCE: preserving the myocardium in transplantation involves perfusion and storage of the isolated heart with using a Custodiol solution at a temperature of 2-4°C. The heart tissue culture into the donor precedes a 10-12-hour infusion of Levosimendan 10 mcg/kg of body weight, while the perfusion and storage of the isolated organ is ensured by adding Levosimendan into the Custodiol solution at 10 mcg of the preparation per 1 litre of the solution. The surgical stage of the heart transplantation after the recipient's aorta de-clamping and the blood flow recovery is followed by introducing Levosimendan 12.5 mg into the continuing bypass circuit and infusing it for 1 hour.

EFFECT: invention aims at preventing the ischemic and re-perfusion myocardial injuries, providing the adequate function of the donor's heart in the recipient's body.

2 ex

FIELD: medicine.

SUBSTANCE: composition contains recombinant interferon specified in a group: recombinant interferon alpha, recombinant interferon beta, recombinant interferon gamma, antibiotics, antiseptics, hypromellose and water in the following proportions, g per 1 ml of the composition: recombinant interferon, IU - 100-1,000,000; antibiotics, g - 0.00001-0.5; antiseptics, g - 0.00001-0.5; hypromellose, g - 0.00001-0.5; water - the rest up to 1 ml. The above composition can contain broad-spectrum antibiotics specified in a group: amoxicillin, ampicillin, oxacillin, methicillin, cephalexin in an amount of 0.00001-0.5 g per 1 ml of the composition. The composition contains antiseptics specified in a group: lysozyme, biclotymol, fusafungine, ambazon, benzyldimethyl-myristoylamino-propylammonium, co-trimoxazole, amylmetacresol, dichlorobenzyl alcohol, cetylpyridinium chloride, benzoxonium chloride in an amount of 0.00001-0.5 g per 1 ml of the composition.

EFFECT: invention enables providing higher therapeutic efficacy of the composition.

3 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine. After fixing tissues and dissection, an anatomic preparation is immersed into an intermediate solution - acetone for 4 weeks, cooled to -20°C, placed into a sealed metal chamber filled with a composition containing a medical siloxane polymer, a cross-linking agent and a catalyst. The composition is diluted in acetone in the concentration of 50-75%. An excessive pressure of 500 mm Hg is generated in the chamber; the preparation is cooled to a temperature of -8°C. The composition fills the intercellular spaces of the preparation throughout 2-3 weeks; thereafter the preparation is cured in a thermostat at a temperature of 38°C for 4 hours.

EFFECT: invention enables providing higher quality of the anatomic preparations.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

Up!