Method for cryogenic preservation of haematopoietic umbilical cord stem cells

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to haematology. The cryogenic preservation of haematopoietic umbilical cord stem cells is carried out with a cryoprotectant solution - dimethylsulphoxide - added to a suspension of nuclear cells with the haematopoietic stem cells. That is followed by preparing for freezing by cooling the stem cell suspension in a cooling chamber to a temperature of +4°C. The multi-staged freezing of the stem cell suspension involves using a cryoprotectant solution that is 55% dimethylsulphoxide with 5% dextrane 40, which is added into a suspension of a leukocyte concentrate with the stem cells packed in a cryobag. That is followed by mixing mechanically in a mixing apparatus and added with a cryogenic preserving agent at a temperature of +4°C, de-aerating the cryobag with releasing a portion of the suspension, closing the bag, sealing it and placing into a shrink bag and freezing within several stages with computer assistance. At the first stage, the stem cell suspension mixed with the cryoprotectant solution (hereinafter referred to as a sample) is kept for 10 min at a temperature of +4°C, frozen at 1°C/min to a temperature of -12°C, cooled down at 15°C/min to a temperature of -60°C; after the sample is unfrozen at 10°C/min, it is cooled down at 1°C/min to -60°C; at the end of the freezing programme, the sample is cooled down at 3°C to -100°C. Upon completion of the freezing programme, the sample placed into a cryobox is placed into a quarantine vacuum flask containing liquid nitrogen to determine test results for the presence or absence of infectious agents and bacteriological and mycotic contamination. At the end of the quarantine period, the haematopoietic umbilical cord stem cell sample is transported into the vacuum flask containing liquid nitrogen for long storage at min -150°C with negative test results.

EFFECT: invention enables increasing the cells viability.

1 tbl, 1 dwg

 

The invention relates to medicine, in particular to Hematology, and can be used as a technology of cryopreservation of hematopoietic stem cells of umbilical cord blood.

The known method of cryopreservation of hematopoietic stem cells of umbilical cord blood new York Blood Center, which includes adding the cell suspension of the cryoprotectant dimethyl sulfoxide, a three-phase suspension freezing followed by thawing. At the initial stage of the freezing of the sample occurs at a rate of 10°C/min to a temperature of -3°C, further freezing to a temperature of -12°C and the subsequent freezing at the rate of 2°C/min to a temperature of -50°C/min, after which the sample is placed in liquid nitrogen [Technicalservicesbulletin 040-017 BioArchiveFreezingCurveCordBlood].

The known method of cryopreservation of hematopoietic stem cells of umbilical cord blood, is to add cladding solution of cryoprotectant dimethylsulfoxide in a suspension of nucleated cells with hematopoietic stem cells, preparation for freezing, comprising cooling the suspension with stem cells in the cooling chamber to a temperature of +4°C and multistage freezing mist with stem cells, characterized in that as a cryoprotectant using a solution of 55% of dimethyl sulfoxide with 5% dextran 40, which is made in�whole leukocyte concentrate with stem cells posted method: bags, mechanically stirred in an apparatus for mixing and add cryoconserved at a temperature of +4°C, let the air out of the method: bags and part of the suspension, close the package, sealed, and placed in a shrink package and implement software multistage 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, then cooled at a rate of 1°C/min to a temperature of -12°C, then cooled at a speed of 15°C/min to -60°C, after thawing the sample at a speed of 15°C/min to a temperature of -18°C 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 to -100°C, after freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of the tests on the presence or absence of infectious agents and bacteriological and fungal contamination after a quarantine period of storage, the sample with hematopoietic stem cells derived from umbilical cord blood, carry on for a long period of storage at a temperature of -150°C in a Dewar vessel with liquid nitrogen under the condition of negative test results, in the case of a put�individual test results for infections and bacterial and/or fungal contamination of the sample with hematopoietic stem cells are transferred into the Dewar infectious material long-term storage. Patent the invention 2416197, a class of the invention A01N 1/02, AK 35/14, AK 35/44.

The disadvantage of this method of cryopreservation of hematopoietic stem cells of umbilical cord blood is the poor viability of cells in the output.

The purpose of the invention is to increase the viability of stem cells.

This object is achieved in that the method of cryopreservation of hematopoietic stem cells of umbilical cord blood, is to add cladding solution of cryoprotectant dimethylsulfoxide in a suspension of nucleated cells with hematopoietic stem cells, preparation for freezing, comprising cooling the suspension with stem cells in the cooling chamber to a temperature of +4°C, and a multi-stage freezing mist with stem cells, and as a cryoprotectant using a solution of 55% of dimethyl sulfoxide with 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells placed method: bags, mechanically mixed in a machine for mixing and add cryoconserved at a temperature of +4°C, let the air out of the method: bags and part of the suspension, close the package, sealed, and placed in a shrink package and implement software multistage freezing, at the first stage the mixture suspension with stem cells and triprotic�the op - a sample of freeze - incubated for 10 min at +4°C, then cooled at a rate of 1°C/min to a temperature of -12°C, then cooled at a speed of 15°C/min to -60°C, after thawing of the sample it is 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° to a temperature of 100°C, after freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of the tests on the presence or absence of infectious agents, bacterial and fungal contamination, after a quarantine period of storage, the sample with hematopoietic stem cells derived from umbilical cord blood, transferred to long term storage at a temperature of -150°C in a Dewar vessel 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 the Dewar infectious material long-term storage, moreover, the thawing of the sample is carried out at a rate of 10°C/min.

The method is implemented as follows.

Method of cryopreservation of hematopoietic stem cells of umbilical cord blood, which is added in�and enclosing solution of the cryoprotectant dimethyl sulfoxide in a suspension of nucleated cells with hematopoietic stem cells, preparation for freezing comprising cooling the suspension with stem cells in the cooling chamber to a temperature of +4°C, and a multi-stage freezing mist with stem cells, and as a cryoprotectant using a solution of 55% of dimethyl sulfoxide with 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells placed method: bags, mechanically stirred in an apparatus for mixing and add cryoconserved at a temperature of +4°C, let the air out of the method: bags and part of the suspension, close the package, sealed, and placed in a shrink package and implement software multistage 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, then cooled at a rate of 1°C 7 min to a temperature of -12°C, then cooled at a speed of 15°C/min to -60°C, after thawing of the sample it is 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 to a temperature of 100°C, after freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of the tests on the presence or absence of infectious agents, bacterial and fungal� contamination, after a quarantine period of storage, the sample with hematopoietic stem cells derived from umbilical cord blood, transferred to long term storage at a temperature of -150°C in a Dewar vessel 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 the Dewar infectious material long-term storage, and thawing of the sample is carried out at a rate of 10°C/min.

Here is an example of practical realization of the proposed method.

The following table 1 Indicators concentrate umbilical cord blood in the allocation of hematopoietic stem cells" shows the quantitative changes of indicators concentrate umbilical cord blood in the allocation of hematopoietic stem cells during two different methods of cryopreservation. The difference of the considered methods of cryopreservation of samples is a predetermined speed, programmable defrost the freezer Chuo 560-16 ("PlanerPlc". UK). In the first method the rate of thawing was 15°C/min, in the second - 10°C/min. From the table it is seen that the cryopreservation of our proposed method, the cell viability significantly increased � average 4.46% compared to the previously used method of cryopreservation of hematopoietic stem cells of umbilical cord blood.

The study included 85 samples of concentrate leukocyte fraction pupovinoy blood. Before freezing the allocation of leukocyte fractions were carried out on the unit SepaxSlOO (Biosafe, Switzerland). The Method: Bags (Pail. USA) with the concentrate and the cryoprotectant (DMSO solution 10%) were frozen Packed in shrink pack (Thermogenesis, USA). The first method of freezing of the concentrate was performed in a programmable freezer, Shuo 560-16 (Planer. UK) according to the following scheme (Hubeletal.. 2003 [6], in the modification Pokrovsky Bank of stem cells (Patent No. 2416197. The invention priority from December 11, 2009 )):

1) the starting temperature of 4°C maintained for 10 minutes.

2) the sample is cooled at a rate of 1°C/min to -12°C;

3) the sample is cooled at a speed of 15°C/min to -60°C:

4) the sample is heated at a speed of 15°C/min to -18 C;

5) the sample is cooled at a rate of 1°C/min to 60°C;

6) the sample is cooled at a rate of 3°C/min to -100°C.

The second method was carried out according to the following program:

1) the starting temperature of 4°C and held for 10 minutes.

2) the sample is cooled at a speed of -1°C/min to -12 C;

3) the sample is cooled at a speed of 20 C/min to 60 seconds.

4) the sample is heated at a rate of 10°C/min to -18 C;

5) the sample is cooled at a speed of -1°C/min to -60°C;

6) the sample is cooled with a speed� -3°C/min to -100°C.

After thawing, all samples were processed (washed from the cryoprotectant is DMSO solution 10% (Pall, UK)) and conducted a study of quality indicators.

In all samples was determined by the number of nucleated cells, the absolute number of CD34+/CD45+cells and viability in concentrate leukocyte fractions before freezing, after thawing and washing of the cells from the cryoprotectant.

After thawing and washing from the cryoprotectant concentrate leukocyte suspensions were counting the number of nucleated cells using a Hematology analyzer "ACTdiff 2" BeckmanCulter ("BeckmanCulter, USA), and the number and viability of CD34/45+cells on flow cytometer tomixF500 (BeckmanCoulter).

Statistical analysis was performed by parametric statistical methods. The results are presented as M±m, where M is the arithmetic mean, m - standard error of the mean. Reliable results were considered at p<0,05. Statistical analysis of the data was performed using the software package Microsoft Office Excel 2003 for Windows 2003 version 1.0, IBM® SPSS® Statistics version 19.0.

The graph of the dependence of the temperature of the sample of hematopoietic stem cells of umbilical cord blood from time cryosurgery" predstavliava freeze concentrate umbilical cord CRO�I. The abscissa axis is set to the duration of the freeze, the ordinate is the temperature of a sample of hematopoietic stem cells of umbilical cord blood.

The allocation of leukocyte fraction with hematopoietic stem cells from the umbilical cord blood sample is carried out by " automatic separation apparatus SepaxS100 (Biosafe, Switzerland) using sedimentologica means of hydroxyethyl starch (HES). With the aim of preserving the maximum number of hematopoietic stem cells during freezing in phase crystallization using protector with non-load-bearing ability, dimethyl sulfoxide. It pre-added to the dextran solution to reduce the toxicity of the solution. The cryoprotectant - razor 55% of dimethylsulfoxide with 5% dextran 40 is injected into the cell suspension with hematopoietic stem cells. Further mechanical stirring and cooling to a temperature of 4°C is produced on apparatus for mixing CollMixAS-210 (Switzerland). At the end of the mixing process of the method: bags with contents release the air and a portion of cell suspension, shut it, and sealed in several places with a tube that goes to it, in the apparatus for sealing shrink package "CryoFlex" (Nunc, Denmark), thereby creating a so-called "satellites". As a method: bags system is used to collect and store �povinnoi blood MacoPharma (France).

The method: bags with cell suspensions, stem cells and cryoprotectant (sample) tags customized barcode with the specified concentration of the content, storage condition, the composition of the cryoprotectant, date krishnarajendra and the name of a Bank, implementing and preserving stem cells.

After the method: bags with contents packaged in special heat-shrinkable package - "CryoFlex" (Nunc, Denmark), placed it with the method: bags in the cassette for program freezing and placed in a programmable freezer - PlanerKryo 560-16 ("PlanerPlc.", UK). The freezing is performed in a special program, a given programmable freezer. The initial temperature in the freezer is set equal to +4°C and hold for 10 minutes. At the first stage of freezing leukocyte concentrate with hematopoietic stem cells and the cryoprotective agent is cooled at a rate of 1°C to a temperature of -12°C. In the second stage cooling, the sample was cooled at a speed of 15°C to a temperature of -60°C. to compensate For the release of latent heat, leading to transient increase in temperature, the defrosting process occurs at a rate of 10°C/min to a temperature of -18°C. This process gives greater opportunity to bypass the point of crystallization by reducing the temperature gradient to increase the viability of gemap�ethical stem cells in the process of freezing and after thawing. In the third stage after thawing, the sample is cooled at a rate of 1°C/min to -60°C, at the end of the freezing process, in stage 4, the sample is cooled at a speed of 3°C/min to -100°C. the Data on the freezing procedure recorded in the Protocol software freeze sample with hematopoietic stem cells. After freezing in a programmable freezer the method: bags with a leukocyte concentrate hematopoietic stem cells and the cryoprotective agent is placed in crimrose with subsequent placement in a quarantine Dewar with liquid nitrogen. The sample is in a quarantine Dewar to provide test results on the presence or absence of infectious agents, bacteriological and fungal contamination. After a quarantine period of storage and provided negative test results frozen sample is placed on long-term storage in the Dewar vessel with liquid nitrogen at temperatures below -150°C.

The results of all studies characterizing the sample prepared cell suspension with hematopoietic stem cells of umbilical cord blood and solution of cryoprotectant, all the actions performed with the sample, such as sample withdrawal, a change in the location of the sample, contribute to the database PR�enterprises "LTD Pokrovsky Bank of Stem cells" and the database FreezerWork under a single identification number of the sample.

Method of cryopreservation of hematopoietic stem cells of umbilical cord blood, is to add cladding solution of cryoprotectant is dimethylsulfoxide in a suspension of nucleated cells with hematopoietic stem cells, preparation for freezing, comprising cooling the suspension with stem cells in the cooling chamber to a temperature of +4°C, and a multi-stage freezing mist with stem cells, and as a cryoprotectant using a solution of 55% of dimethyl sulfoxide with 5% dextran 40, which is made in leukocyte suspension concentrate with stem cells placed method: bags, then mechanically stirred in an apparatus for mixing and add cryoconserved at a temperature of +4°C, let the air out of the method: bags and part of the suspension, close the package, sealed, put it in a shrink package and implement software multistage freezing, at the first stage the mixture suspension with stem cells and the cryoprotective agent (the sample) is incubated for 10 min at +4°C, then cooled at a rate of 1°C/min to a temperature of -12°C, then cooled at a speed of 15°C/min to -60°C, after thawing of the sample it is cooled at a rate of 1°C/min to -60°C and at the end of the program freeze the sample was cooled with a speed�þ 3°C to -100°C, after freezing the sample, placed in crimrose, placed in quarantine Dewar with liquid nitrogen to determine the results of the tests on the presence or absence of infectious agents and bacteriological and fungal contamination after a quarantine period of storage, the sample with hematopoietic stem cells derived from umbilical cord blood, transferred to long term storage at a temperature of -150°C in a Dewar vessel with liquid nitrogen under the condition of negative test results, characterized in that the thawing of the sample is carried out at a rate of 10°C/min.



 

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