RU2547426C1 - Method for cryopreservation of haemopoietic umbilical stem cells - Google Patents

Abstract

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

Description

The invention relates to medicine, namely to methods for preserving human hematopoietic stem cells.

Closest to the proposed is a method of cryopreservation of hematopoietic cord blood stem cells [RF Patent No. 2416197, IPC A01N 1/02, A61K 35/14, A61K 35/44, 2011], comprising the introduction of a cryoprotectant enclosing solution of 55% dimethyl sulfoxide with 5% dextran 40 at a temperature of + 4 ° C in a suspension of nucleated cells with hematopoietic stem cells (HSCs) in a cryopackage, mechanical mixing, air release from the cryopackage and part of the suspension, sealing and placing it in a heat-shrink bag, programmed multi-stage freezing, after four The sample is placed in quarantine dewar with liquid nitrogen until the test results for infectious agents and bacteriological fungal contamination are determined. After the quarantine shelf life, the sample is transferred to long-term storage at a temperature not exceeding -150 ° C in a dewar with liquid nitrogen, subject to negative results. testing, in case of positive test results, the sample with hematopoietic stem cells is transferred to the dewar for infectious material of long-term storage.

A known reason preventing the achievement of the technical result provided by the invention is the protection of the sample from external influences not at all stages of the method, the duration of the preparation of HSCs for cryopreservation, and the relatively low viability of HSCs after thawing.

The problem to which the present invention is directed is to reduce the likelihood of infection of hematopoietic cord blood stem cells with infection and bacterial and / or fungal contamination, reduce the duration of preparation for long-term storage of cryopreserved cord blood stem cells and preserve the viability of hematopoietic cord blood cells.

In the implementation of the invention, the task is solved by achieving a technical result, which consists in improving the sterility conditions, reducing the time of multi-stage program freezing and increasing the number of viable hematopoietic cord blood stem cells after a storage period.

The specified technical result is achieved by the method of cryopreservation of hematopoietic cord blood stem cells, which includes opening the cryoprotectant bottle in the laminar box, filling the cryoprotectant into a special syringe pump syringe, and introducing with the help of a syringe pump a cryoprotectant enclosing solution of 55% dimethyl sulfoxide 40 with 5% dextol ° C at a rate of 0.8 ml / min in a suspension of nucleated cells with hematopoietic stem cells in a cryopackage with simultaneous mechanical stirring using a device for stirring at + 4 ° C, stopping mechanical stirring immediately after the required amount of cryoprotectant is delivered to the cryopackage with concentrate, transferring the system together with the cryoprotectant bottle to the laminar box, letting air out of the cryopacket and part of the suspension, sealing and placing it in a heat-shrink bag , a multi-stage program freezing, in the first stage of which the suspension mixture with stem cells and a cryoprotectant - freezing sample - is kept for 10 min at a temperature of + 4 ° C, in the second stage I cool t at a rate of 1 ° C / min to a temperature of -12 ° C, in the third stage it is cooled at a speed of 20 ° C / min to a temperature of -60 ° C, at the fourth stage a sample is heated at a rate of 10 ° C / min to a temperature of -18 ° C, in the fifth stage, the sample is cooled at a rate of 1 ° C / min to -60 ° C and at the end of the freezing program, the sample is cooled at a rate of 3 ° C / min to a temperature of -100 ° C, after freezing, the sample is placed in quarantine dewar with liquid nitrogen , before determining the results of tests for infectious agents and bacteriological fungal contamination, after quarantine the shelf life, 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, provided that the test results are negative, in case of positive test results for infection and bacterial and / or fungal contamination, the sample with hematopoietic stem cells transferred to the dewar for infectious material of long storage.

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

The introduction of a cryoprotectant enclosing solution of 55% dimethyl sulfoxide with 5% dextran 40 at a temperature of + 4 ° C at a rate of 0.8 ml / min with a syringe pump into a suspension of nucleated cells with hematopoietic stem cells with simultaneous mechanical stirring allows uniform distribution of the cryoprotectant over the volume of leukocyte cell fractions. Due to this, the cryoprotectant solution penetrates almost all cells contained in this fraction, and the number of viable HSCs increases after thawing. The cryoprotectant injection rate of 0.8 ml / min is optimal for this method and is selected based on the diffusion rate of the cryoprotectant solution through the cell membrane. Mechanical stirring at + 4 ° C in the apparatus for mixing is stopped immediately after receipt of the required amount of cryoprotectant in a cryopack with concentrate. If mixing will last longer, then there is a likelihood of hemolysis.

After mechanical stirring, the system, together with the cryoprotectant bottle, is transferred to the laminar box, air is released from the cryopackage and part of the suspension, sealed and placed in a heat-shrink bag. Carrying out these operations in a laminar box reduces the likelihood of infection of hematopoietic stem cells of cord blood with infection and bacterial and / or fungal contamination.

In the third stage of the multi-stage program freezing, the sample is cooled at a rate of 20 ° C / min to a temperature of -60 ° C. By increasing the cooling rate, uniform cooling of the sample in the entire volume to the desired temperature is achieved.

In the fourth stage of the program multi-stage freezing, the sample is heated at a rate of 10 ° C / min to a temperature of -18 ° C. Due to the slower increase in temperature, a smooth thawing process occurs due to the release of latent heat, which makes it possible to bypass the recrystallization point and quench the thermal shock.

Thus, faster cooling in the third stage and slower heating in the fourth stage, compared with the prototype, can avoid a sharp jump in temperature, thereby maintaining the viability of the cells in the sample.

Examples of the proposed method for the cryopreservation of hematopoietic cord blood stem cells are given below.

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

A cryopackage with leukocyte concentrate was placed in a CoolMix AS-210 pre-preparation apparatus (Biosafe, Switzerland), and connected to a syringe pump using a polymer line system. A cryoprotectant solution injection rate of 0.8 ml / min was set on the Pilot syringe pump. When 5 ml of the cryoprotectant was delivered to the cryopackage with concentrate, the Pilot pump gave a signal, the stirring was stopped immediately and the system and the bottle were transferred to the laminar box.

Air was released from the cryopackage, a small amount of blood was squeezed out of the packet so that it filled the tube at a distance of 5 cm from the packet; soldered the tube at this distance and made soldering at a distance of 1 cm from each other. Soldered areas leading to a test tube satellite.

The cryopackage of the concentrate was packaged in a shrink bag, the air was removed from the bag and sealed using a Shop Sealer FS-315 apparatus (Fuji Impulse Co. Ltd, EC) to seal the CryoFlex shrink bag, placed on the refrigerant and transferred for program freezing.

The concentrate packed in a heat-shrink bag was placed in a cassette for program freezing; mounted in a camera on a tripod.

The Sample temperature control probe of the Planer Kryo 560-16 software freezer (Planer pis, UK) was placed in close proximity to the sample. The temperature in the chamber was controlled by an additional Chamber sensor of the Planer Cryo 560/16 software freezer (Planer pis, Great Britain). When compiling a cryoprotocol, a given schedule was built according to the temperature setting of the chamber.

We also used an additional remote sensor of the Master Scada monitoring system to monitor the temperature and timely calibration of the camera.

Concentrate was frozen in the Planer Cryo 560/16 program freezer according to the following steps: at the first stage, the suspension mixture with stem cells and cryoprotectant — the freezing sample was held for 10 min at + 4 ° C, and at the second stage it was cooled at a rate of 1 ° C / min to temperature -12 ° C, in the third stage it was cooled at a speed of 20 ° C / min to a temperature of -60 ° C, in the fourth stage the sample was heated at a speed of 10 ° C / min to a temperature of -18 ° C, in the fifth stage the sample was cooled at a speed 1 ° C / min to -60 ° C and at the end of the freezing program c was cooled at a rate of 3 ° C / min to a temperature of -100 ° C. Data on the freezing procedure were recorded in the protocol for program freezing of stem cell concentrate, which is shown in Fig. 1. The protocol for freezing a stem cell concentrate according to the prototype is shown in FIG.

The HSC cryopreservation protocol according to the proposed method for the execution time is 86 minutes (+ 1 ... 3 minutes), and in the prototype 114 minutes (+ 1 ... 2 minutes). Therefore, the duration of preparation for long-term storage of cryopreserved hematopoietic cord blood stem cells is reduced by the proposed method.

The results of all studies characterizing the cord blood sample and the obtained cord blood stem cell concentrate were entered into the database of Tyumenkriobank NGO under a single identification number of the sample.

Cord blood stem cell concentrate was stored in quarantine dewar until the results of tests for infectious agents and bacteriological fungal contamination were known (according to the order of the Ministry of Health of the Russian Federation No. 325 of July 25, 2003). After the expiration of the quarantine shelf life, the cord blood leukocyte concentrate was transferred to long-term storage at a temperature not exceeding -150 ° C in a dewar with liquid nitrogen with negative test results (Anti-fflV-l and -2, HIV-lAg, Anti-HTLV- I and -II, Anti-HbcorAg, HBs-Ag, Anti-HCV). The data on the withdrawal of the sample was recorded in the protocol for the withdrawal of the sample of stem cell concentrate, information on the change in the localization of the sample was recorded in the protocol for the transfer of stem cell concentrate, as well as in the database of Tyumenkriobank LLC and in the Journal of Registration and Registration of Frozen Cord Blood Stem Cells .

Thawing of the cryopreserved sample and washing of the cord blood stem cell concentrate from the cryoprotectant were carried out according to the generally accepted method as follows.

First, a room was prepared in which the procedure of thawing and washing of a cord blood sample was performed: UV lamps were turned on for 15 minutes.

Turned on the water bath in the network, set 37 ° C. Saline was heated in a thermostat at 37 ° C for 15 minutes. Wash buffer was prepared in a laminar box: physiological saline containing 2.5% human recombinant albumin. All anticoagulant was taken from the bag for the final product.

A sample was taken from the cryostorage, recorded appropriately. The cryopreserved sample was thawed on a Planer Kryo 560-16 program freezer as follows: it was held for 5 min at a temperature of -160 ° C, the sample was heated at a rate of 3 ° C / min to a temperature of -100 ° C, and the sample was kept for 5 min at a temperature of -100 ° C, heated the sample at a speed of 1 ° C / min to a temperature of -60 ° C, heated the sample at a speed of 10 ° C / min to a temperature of -18 ° C, heated the sample at a speed of 3 ° C / min to a temperature of -4 ° C, kept the sample for 5 min at a temperature of -4 ° C, heated the sample at a rate of 1 ° C / min to a temperature of 0 ° C, aged wali sample 5 minutes at 0 ° C. Subsequent thawing was carried out in a water bath - medical thermostat TW-2.02.

After the blood became liquid, in the air flow of the laminar box with a napkin moistened with a 70% solution of ethyl alcohol, the blood bag was thoroughly treated, after which the whole cell concentrate was taken through both ports of the bag with syringes (10 ml). The concentrate was distributed equally in two conical centrifuge tubes (50 ml) and poured with an equal volume of washing buffer. Centrifuged at 400 g for 10 min at room temperature.

In the laminar box, the supernatant was removed and the pellet resuspended with the cells in the wash buffer. The final product was placed in an empty bag into which the required amount of heparin was added via a sterile port with a syringe to prevent blood coagulation.

0.5 ml of the concentrate was taken from the package with the final product for analysis of cell viability and cell count by flow cytometry.

Cell viability was determined using the vital dye 7-amino-actinomycin D RUO (7AAD) (Beckman Coulter, USA), analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter, USA). Cell identification was performed by recording two parameters: lateral volume and light scattering (SS) and fluorescence intensity (7-AAD).

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

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

On the histograms to the left of the vertical line (at a value of 10 °), living blood cells are registered, and on the right - dead. The data obtained showed that as a result of the application of the proposed method, the number of viable hematopoietic cord blood stem cells after the storage period in the sample was 87.24%, and by the prototype method 79.01%.

Thus, the proposed method allows to reduce the likelihood of infection of hematopoietic cord blood stem cells with infection and bacterial and / or fungal contamination, shorten the preparation time for long-term storage of cryopreserved HSC cord blood and increase the number of viable hematopoietic cord blood stem cells after the storage period.

Claims (3)

1. A method of cryopreservation of hematopoietic cord blood stem cells, including opening a cryoprotectant vial in a laminar box, filling the cryoprotectant into a special syringe pump syringe, introducing a 55% dimethyl sulfoxide cryoprotectant enclosing solution with 5% dextran 40 at a temperature of + 4 ° C in suspension with cell suspension stem cells in a cryopackage with concentrate and simultaneous mechanical stirring in an apparatus for stirring, transferring the system together with a vial of cryoprotectant to la a mini-box, the release of air from the cryopackage and part of the suspension, sealing and placing it in a heat-shrink bag and the implementation of multi-stage program freezing, in the first stage of which the suspension mixture with stem cells and cryoprotectant is frozen for 10 minutes at a temperature of + 4 ° C, in the second the stage is cooled at a rate of 1 ° C / min to a temperature of -12 ° C, at the third stage it is cooled at a speed of 20 ° C / min to a temperature of -60 ° C, at the fourth stage a sample is heated at a rate of 10 ° C / min to a temperature of -18 ° C, in the fifth stage, cool sample at a rate of 1 ° C / min to -60 ° C and at the end of the freezing program, the sample is cooled at a rate of 3 ° C / min to a temperature of -100 ° C, after freezing, the sample is placed in a quarantine dewar with liquid nitrogen, until the test results are determined for infectious agents and bacteriological fungal contamination, after a quarantine shelf life 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, subject to negative results tested I, in the case of positive tests for infection and bacterial and / or fungal contamination of the sample with the hematopoietic stem cells transferred to a Dewar containing liquid nitrogen for long term storage of infective material. 2. The method according to p. 1, characterized in that the cryoprotectant is introduced into suspension with hematopoietic stem cells using a syringe pump at a rate of 0.8 ml / min. 3. The method according to p. 1, characterized in that the mechanical stirring is stopped immediately after receipt of the required amount of cryoprotectant in a cryopackage with concentrate.

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