Method of cryopreservation of hematopoietic stem cells of umbilical blood

FIELD: agriculture.

SUBSTANCE: invention refers to medicine - transfusiology. Nuclear cells with hematopoietic stem cells (HSC) of cryoprotector - 55% of dimethyl sulfoxide solution with 5% dextran 40 arranged in a cryobag are added into a suspension. Mechanically mixed in a mixing device at +4°C, the cryobag is sealed and put into a shrink-wrap bag. Programmed freezing is carried out, at the first stage of which a sample is maintained for 10 min at +4°C, then cooled with the speed of 1°C/min down to the temperature of -12°C, then cooled down with the speed of 15°C/min down to the temperature of -60°C. After the sample thawing with the speed of 15°C/min down to the temperature of -18°C, it is cooled with the speed of 1°C/min down to -60°C . At the end of the freezing program the sample is cooled with the speed of 3°C/min down to -100°C. On completion of freezing the sample is put into a quarantine Dewar vessel with liquid nitrogen until results of tests for infection availability are ready. On completion of a quarantine period of storage, the sample is transferred for long-term storage at the temperature of at least -150°C into a Dewar vessel with liquid nitrogen provided the tests results are negative. In case the tests results are positive, the sample with HSC are transferred to a Dewar vessel for infection material for long-term storage.

EFFECT: invention makes it possible to increase HSC sterility during freezing, integrity and their viability.

 

Method of cryopreservation of hematopoietic stem cells in umbilical cord blood refers to medicine, in particular to the transfusion.

Cell technologies based on the use of stem cells develop around the world quite extensively. Medical centers in different countries are increasingly using autologous and allogeneic hematopoietic and mesenchymal stem cells in medical practice. Cord blood is one of the four main sources of stem cells. Transplantation of hematopoietic stem cells from umbilical cord blood is developing most rapidly. Only one American Register (National Marrow Donor Program) in 2008, 50,000 samples stored stem cells derived from umbilical cord blood. Medical research centers specifically examine and investigate the potential of optimal transport and cryopreservation of cells derived from umbilical cord blood. Theoretically, since the freezing stem cells and precursor cells are in liquid nitrogen at a temperature of -196°C and can remain in that state long enough. After 15 years of storage thawed hematopoietic stem cells (HSC) were recovered on average 84%. The safety functionality of these thawed precursor cells proved cast new eratively ability of hematopoietic stem cells. Cells CD34+CD38-isolated from thawed units of umbilical cord blood, after 15 years of freezing able to more than 250 - fold multiplication and moreover, engraftment occurs with the same frequency as the cells CD34+from freshly isolated cord blood. So, it was found that hematopoietic stem cells derived from umbilical cord blood, survive when transplanted in humans recipient.

Technology preservation of hematopoietic stem cells known methods of cryopreservation, for example, see p. the Russian Federation No. 2233589, A01N 1/02, date of publication of the patent 10.08.2004, "a Method for cryopreservation of hematopoietic stem cells", which involves adding a cell suspension of the cryoprotectant dimethyl sulfoxide (DMSO), a three-step freezing of the suspension and subsequent thawing. At the first stage, freezing to temperatures (-8,5)-(situated 10.5)°C with a speed of 0.7 to 1.2°C with the use of seeding (crystallization) at temperatures (for 3,5)-(-4)°C with a concentration of cryoprotectant 0,4-1,45 mol/l; at the second stage is cooled to temperatures (-30)-(-40)°C with a speed of 1.0 to 4.5°C; after the second stage thermal shutdown temperature (-30)-(-40)°C for 3-5 minutes, and at the third stage cool with a speed of 10-12°C/min to (-130)-(196)°C. Selection of the values of the temperatures and velocities of cooling like the Ana empirical laboratory. Hematopoietic stem cells obtained from different materials: cord blood, fetal liver, peripheral blood, bone marrow.

The disadvantages of this solution method of cryopreservation HSC are: method not technological, conducted in the laboratory at the stage of the experiment, because the tubes because of the small volume suitable only for laboratory studies, the concentration of cryoprotectant is DMSO through 3.12 percent to 11.3%, according to modern sources, the most effective concentration of DMSO solution to ensure the viability of stem cells is 10%, so 3,12 too little, and the range of concentrations too vague one cryoprotectant is DMSO is not enough, it is toxic, it also uses non-sterile DMSO solution, it should be diluted, but for clinical use cannot be increased from for contamination, gradation and the rate of cooling is inefficient, inadequate sterilization, because during freezing paid extra ice for crystallization (seeding), not specified as obtained cell suspension is and what it represents, the range of temperatures (-130)-(-196°C at the end point of the cooling increases the time of freezing.

The closest technical solution implementation cryopreservation of hematopoietic stem cells derived from povinnoi blood, is the manner specified in article authors: Abdulkadyrov K.M., Romanenko., Starkov N.N., SEL'tser AV Procurement and storage of umbilical cord blood". Material scientific-practical conference, St. Petersburg, 2000, http.www.gemabank.ru/pub/n1/html/. Method of cryopreservation of cord blood HSC is as follows. The cell suspension nucleated stem cells was obtained by sedimentation of red blood cells cord blood using gelatin with subsequent laundering. Then the cell suspension was poured into the vials for cryopreservation of 1.5 ml each, and cooled to a temperature of +4°C. to Prepare enclosing the solution - cryoprotector 10% DMSO and cooled in an ice bath to a temperature of +4°C. Freezing was carried out in the freezing apparatus according to the 3-way program. At the first stage of cell suspension nucleated cells from hematopoietic stem cells and cryoprotectant was cooled from a temperature of +4°C to -20°C at a rate of 1°C/min; the second stage is the reduction of temperature from -20°C to -40°C at a rate of 2°C/min, And the third stage was carried out by reducing the temperature from -40°C to -80°C with a rate of 4°C/min, followed by immersion tubes with cell suspension in liquid nitrogen (t=-196°C).

The disadvantages of this method of cryopreservation of hematopoietic stem cells in umbilical cord blood are the way to promyshlennogo the production unprocessed, ethnological, the tubes can only be used in laboratory studies, takes a lot of time the technological process in preparation for freezing, getting nucleated cells obtained by the laboratory, this time from gelatin as sedimentologica funds refused, in the deep cryogenic freezing there is no bypassing the point of crystallization, which increases the viability of stem cells, used as a cryoprotectant solution RPM1 in large scale production when receiving hematopoietic stem cells and cryogenic their freezing ineffective.

The technical result of the proposed solutions is to enhance the efficiency of cryopreservation with the use of modern industrial equipment, increasing sterility of hematopoietic stem cells during freezing, increase security and, consequently, the viability of HSC.

This result is achieved by a method for cryopreservation of hematopoietic stem cells in umbilical cord blood comprising adding fencing solution of cryoprotectant dimethyl sulfoxide in a concentration suspensions of nucleated cells from hematopoietic stem cells, preparation for freezing, including cooling mist with STV the new cells in the cooling chamber to a temperature of +4°C and multistage freezing mist with stem cells, at the same time as the cryoprotectant used solution 55% DMSO, 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells, placed in method : bags, mechanically stirred apparatus for mixing and adding cryoconserved Coolmix AS-210 at a temperature of +4°C, let the air out of the method : bags and part of the suspension concentrate, close the package, sealed and placed it in shrink package and implement software multistage freezing, at the first stage the mixture of the suspension concentrate with stem cells and cryoprotectant - sample freezing withstand 10' at a temperature of +4°C, then cooled at a rate of 1°C/min to a temperature of -12°C, then cooled with a speed of 15°C./min to a temperature of -60°C. after thawing the sample with a speed of 15°C/min to a temperature of -18°C, cool the sample at a rate of 1°C/min to -60°C and at the end of the program freezing the sample is cooled with a rate of -3°C to a temperature of -100°C. after completion of the program freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of tests for the presence or absence of infectious agents, bacterial and fungal contamination, after a quarantine period of storage of the sample with hematopoietic stem cells, p is obtained from umbilical cord blood, carry on prolonged storage at temperatures of at least

-150°C in the Dewar with liquid nitrogen, provided a negative test result, in the case of positive results of tests for infections, bacterial and/or fungal contamination of the sample with hematopoietic stem cells are transferred into the Dewar for infectious material long-term storage.

The invention consists in the combination of essential features, sufficient to achieve provided by the invention a technical result.

The essential features of the proposed method, coinciding with known characteristics are: a - add a fencing solution of cryoprotectant dimethyl sulfoxide in a concentration suspensions of nucleated cells from hematopoietic stem cells; B - preparation for freezing, including the cooling mist from stem cells in a cooling chamber to a temperature of +4°C; multistage freezing mist with stem cells.

Salient features of the process of cryopreservation of hematopoietic stem cells in umbilical cord blood are: G - as cryoprotectant use a solution of 55% DMSO, 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells, placed in method : bags; D - smesv method : bags mechanically stirred apparatus for mixing and adding cryoconserved Coolmix AS-210 at a temperature of +4°C, let the air out of the method : bags and part of the suspension concentrate, close the package, sealed and placed it in shrink pack; E - implement software freezing, at the first stage the mixture of the suspension concentrate with stem cells and cryoprotectant - sample freezing withstand 10' at a temperature of +4°C, then cooled at a rate of 1°C/min to a temperature of -12°C, then cooled with a speed of 15°C./min to a temperature of -60°C. after thawing the sample with a speed of 15°C/min to a temperature of -18°C, ohlazhaaut sample with the rate of 1°C/min to a temperature of -100°C; W - after the end of the program freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of tests for the presence or absence of infectious agents, bacterial and fungal contamination; And after a quarantine period of storage of the sample with hematopoietic stem cells derived from umbilical cord blood, transferred to long-term storage at a temperature of not less than -150°C in the Dewar with liquid nitrogen, provided a negative test result, in the case of positive results of tests for infections, bacterial and/or fungal contamination of the sample with hematopoietic stem cells are transferred into the Dewar for infectious material long-term storage.

the Method of cryopreservation of hematopoietic stem cells in umbilical cord blood is the following. Before freezing are preparing cell suspensions, which leukocyte concentrate (nucleated cells) with hematopoietic stem cells in umbilical cord blood obtained by separation scheme is made even more S100 (Switzerland) using sedimentologica tools hydroxyethylamine (BSE). To save the maximum number of HSC in the freezing phase crystallization using the protector, with non-load-bearing ability, dimethylsulfoxide. To reduce the toxicity of this solution is added a solution of dextran 40. As cryoprotectant solution taken 55% of dimethylsulfoxide with 5% dextran 40, which is injected into the cell suspension with hematopoietic stem cells, mechanically stirred apparatus for mixing CoolMix AS-210 (Switzerland) and cool it to a temperature of +4°C. after mixing of the method : bags with the contents of let the air out and part of the cell suspension, shut it, and sealed in several places with a tube going to it through 1 cm, creating satellites.

The method : bags with cell suspension, stem cells and cryoprotectant mark individual barcode with the specified content concentration, storage conditions, composition of the cryoprotectant, the date of cryosurgery and the name of the Bank, performing and preserving stem glue the key.

Then the method : bags with the contents Packed in special shrink package - "CryoFlex", (Nunc, Denmark)placed it with the method : bags in the cartridge for software freeze and place in software freezer - Planer Kryo 560-16 ("Planer Plc.", UK). Freezing is carried out under the special scheme for software-freezer Planer Kryo 560-16. The starting temperature in the freezer is set to +4°C and hold for 10 minutes. At the first stage of freezing leukocyte concentrate with stem cells and cryoprotectants, called the sample is cooled to a temperature of -12°C at 1°C/min In the second stage cooling, the sample is cooled down to -60°C with a speed of 15°C./min. In this period is the release of latent heat, which leads to short-term temperature increase - the defrosting process, to a temperature of -18°C at 15°C/min, This gives you the opportunity to bypass the point of crystallization to increase the viability of hematopoietic stem cells during freezing and after thawing (final thawing). At the third stage, after thawing the sample is cooled at a rate of 1°C/min to -60°C and at the end of the freezing process on the 4th stage, the sample is cooled with a rate of 3°C/min to a temperature of cooling to -100°C. Data on the procedure Zam is Rivonia recorded in the Protocol software freezing of the sample with stem cells. After freezing in the software the freezer method : bags with the sample placed in crimrose and placed last in quarantine Dewar with liquid nitrogen to determine the results of tests for the presence or absence of infectious agents, bacterial and fungal contamination. After a quarantine period of storage of the sample with stem cells is placed on long-term storage in a Dewar with liquid nitrogen at a temperature of not less than -150°C under 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 the stem cells are transferred into a special Dewar for infectious material long-term storage.

The results of all studies characterizing the sample harvested cell suspension with hematopoietic stem cells, cord blood, and all actions carried out with the sample, i.e. withdrawal of the sample, a change in the location of the sample included in the database enterprises LLC Pokrovsky Stem cell Bank" under a single identification number of the sample, as well as in the database Freezer Work.

The use of technical solutions "Method of cryopreservation of hematopoietic stem cells in cord blood compared with the prototype allows to improve the sterility of geopoetics the x stem cells during freezing to increase the safety of stem cells and their viability for cryoconservation due to the fact that as a cryoprotectant use a solution of 55% DMSO with 5% dextran 40, thereby reducing the toxicity of the tailings solution made in leukocyte suspension concentrate with hematopoietic stem cells with a thorough mechanical agitation, and also due to the fact that in multi-step freezing carry out thermal step - defrost, which allows you to bypass the point of crystallization of the sample freezing. When receiving leukocyte concentrate with stem cells using sedimentologica tools hydroxyethylamine (blood substitute) allows you to more accurately separate the leukocytes from erythrocytes and plasma. The use of modern license technical industrial equipment of foreign manufacture: USA, Switzerland, great Britain, Japan, Germany, Sweden, Denmark, etc. can improve the manufacturability of the process of cryopreservation, to spend less time, increase end up getting a higher quality end product. For example, the apparatus for mixing and adding cryoconserved - Coolmix AS-210 (Switzerland) allows you to evenly cool the stem cells in the process of adding cryoprotectant with simultaneous Khujand the implementation of continuous mixing leukocyte concentrate with stem cells method : bags at constant temperature and dropwise adding enclosing a solution of a mixture of DMSO with dextran 40. Method of cryopreservation of hematopoietic stem cells in umbilical cord blood was repeatedly carried out (more than 400 times) on the emerging enterprise LLC Pokrovsky stem cell Bank" and showed excellent results.

Method of cryopreservation of hematopoietic stem cells in umbilical cord blood, which consists in adding fencing solution of cryoprotectant dimethyl sulfoxide in a suspension of nucleated cells from hematopoietic stem cells, preparation for freezing, including the cooling mist from stem cells in a cooling chamber to a temperature of +4°C and multistage freezing mist with stem cells, characterized in that as a cryoprotectant use a solution of 55% DMSO, 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells, placed in method : bags, mechanically stirred apparatus for mixing and add cryoconserved at a temperature of +4°C, release the air from the method : bags and part of the suspension, close the package, sealed and placed it in shrink package and implement software multistage freezing, at the first stage the mixture is suspended from stem cells and cryoprotectant - sample freezing incubated 10 min at a temperature of +4°C, then cooled with what speed 1°C/min to a temperature of -12°C, then cooled at 15°C/min to a temperature of -60°C. after thawing the sample with a speed of 15°C/min to a temperature of -18°C is cooled sample at a rate of 1°C/min to -60°C and at the end of the program freezing the sample is cooled at 3°C to a temperature of -100°C. after completion of the program freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of tests for the presence or absence of infectious agents, bacterial and fungal contamination, after a quarantine period of storage of the sample with hematopoietic stem cells derived from umbilical cord blood, transferred to long-term storage at a temperature of not less than -150°C in the Dewar with liquid nitrogen, provided a negative test result, 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 the Dewar for infectious material long-term storage.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method related to field of medicine, namely to transfusiology. Method includes collecting umbilical cord blood (UCB) into container, weighing it with calculation of its volume, determining type of human leukocyte antigen for comparing hypocompatibility, presence or absence of infection, introduction into packet with UCB, anticoagulant and hydroxyethyl starch, mechanical mixing of packet content in various planes, incubation after mixing at room temperature with separation of erythrocyte mass (EM) by sedimentation, placement of centrifuge chamber in separator, connecting to it on symmetry axis main pipeline, the latter being connected via distribution unit via pipelines to sacks for collection of blood components and to packet with mixture of UCB, anticoagulant and hydroxyethyl starch. After that, chamber is filled with packet content, and mixture is subjected to separation, with its separation into EM, plasma and leukocyte concentrate with their distribution into corresponding sacks and into empty packet. After separation, viability and amount of SC are determined, they are checked on biological inoculation of sample of plasma and EM mixture, dose of plasma is tested for detection of antibodies, hemotransmissive infections, dose of EM is used to determine blood group and Rhesus factor.

EFFECT: method application makes it possible to increase quality and reliability of determining SM from various biological contaminations.

1 dwg

FIELD: medicine.

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FIELD: medicine.

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1 ex

FIELD: veterinary.

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1 ex

FIELD: veterinary.

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2 ex

FIELD: medicine.

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2 ex

FIELD: medicine.

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3 ex

FIELD: medicine.

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2 cl, 3 dwg, 4 tbl

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6 cl, 3 tbl, 4 ex

FIELD: medicine.

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5 cl, 11 ex, 1 tbl

FIELD: medicine.

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8 cl, 6 ex

FIELD: medicine.

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1 ex

FIELD: medicine.

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EFFECT: method application makes it possible to increase quality and reliability of determining SM from various biological contaminations.

1 dwg

FIELD: medicine.

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1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical industry, in particular, to balneology. Biologically active product of antler reindeer breeding for balneology, including ground fibrin of antler reindeer blood in native form, "Grapefruit cytrocept" and glycerine, taken in definite ratio.

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5 tbl, 1 ex

FIELD: medicine.

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3 cl, 3 ex, 2 tbl

FIELD: medicine.

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EFFECT: method increases treatment efficiency with reduction of terms of staying in hospital.

3 ex

FIELD: medicine.

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EFFECT: invention makes it possible to reduce preservation process toxicity.

11 cl, 6 ex, 1 tbl

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