CN110074093B - Programmed cooling and freezing method for human umbilical cord blood hematopoietic stem cells - Google Patents
Programmed cooling and freezing method for human umbilical cord blood hematopoietic stem cells Download PDFInfo
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Abstract
The invention discloses a programmed cooling and freezing method of human umbilical cord blood hematopoietic stem cells, which comprises the steps of pretreating the human umbilical cord blood hematopoietic stem cells obtained after separation, and cooling to 4 ℃ to obtain a sample; the samples were transferred to a programmed cooling chamber and operated as follows: 1) waiting for 3-4min at 4 ℃; 2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; 3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; 4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; 5) cooling at a speed of 2 ℃/min until the temperature of the sample is-40 ℃; 6) cooling at a speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen. The invention has the advantages that through reasonably setting the cooling rate and the cooling control point, on one hand, the freezing process is shortened, and the program cooling and freezing cost is reduced; on the other hand, the cells can stably pass through the phase change point, the cells are prevented from being damaged in the freezing storage process, and the cell survival rate of the recovered cells is improved.
Description
The technical field is as follows:
the invention relates to a programmed cell cooling and freezing method, in particular to a programmed cooling and freezing method for human umbilical cord blood hematopoietic stem cells.
Background art:
human umbilical cord blood is blood in umbilical cord and blood vessel near fetus of placenta at birth of newborn, and human umbilical cord blood stem cells contained in the human umbilical cord blood stem cells are often used for transplantation treatment of various blood system diseases and immune system diseases, including malignant tumor of blood system, hematopoietic failure of bone marrow, congenital metabolic diseases, congenital immunodeficiency diseases, autoimmune diseases, certain solid tumors, respiratory diseases, cardiovascular diseases, endocrine diseases, digestive diseases and the like. Thus, cord blood has become an important source of hematopoietic stem cells.
The number of human umbilical cord blood hematopoietic stem cells is a direct influence factor for determining the transplantation efficiency, and the human umbilical cord blood hematopoietic stem cells are particularly precious because the human umbilical cord blood hematopoietic stem cells cannot be massively expanded in vitro like other stem cells (such as mesenchymal stem cells). Cord blood hematopoietic stem cells are generally collected and processed, then sent to a cord blood bank for cryopreservation and wait for reuse, and the number of the umbilical cord blood hematopoietic stem cells after cryopreservation can directly influence the later clinical transplantation efficiency, so the cryopreservation and recovery process of the umbilical cord blood hematopoietic stem cells has higher requirements than other types of stem cells.
The successful cryopreservation of the biological sample means that the cells still keep alive and have certain biological activity after being frozen and recovered. Successful cryopreservation of biological samples depends on the rate of freezing, precise temperature control and reasonable temperature gradient (+50 ℃ to-80 ℃). When the temperature is reduced to-80 ℃, most biological samples can be transferred to liquid nitrogen for long-term storage. In the process of cell cryopreservation, a liquid phase is converted into a solid phase, cell metabolism is stopped, water is lost, the concentration of salt and metabolites in cells is changed, osmotic pressure imbalance is further caused, cells are broken to die, and the survival rate of the cryopreserved cells is directly influenced. Meanwhile, the freezing rate is too fast or too slow, which easily causes the loss of water in cells or the formation of large ice crystals, and is not favorable for the survival of the cells.
At present, methods for cryopreservation of human umbilical cord blood hematopoietic stem cells are roughly classified into a non-programmed freezing method and a programmed freezing method. The non-program controlled cooling freezing method is to freeze and store hematopoietic stem cells in liquid nitrogen at-196 ℃ directly without process-controlled cooling, and the greatest defect of the freezing storage method is that a large amount of ice crystals are generated in the cells in the process of rapidly reducing the temperature, and the ice crystals support cell membranes to cause cell death; meanwhile, along with the prolonging of the preservation time, the recovery rate of the hematopoietic stem cells is seriously reduced, the preservation time is not more than two years, the preservation effect is easily influenced by the working state of a low-temperature refrigerator, and the successful resuscitation of the cryopreserved human umbilical cord blood hematopoietic stem cells cannot be ensured. The program-controlled cooling freezing method can effectively ensure the survival rate of human umbilical cord blood hematopoietic stem cells with limited quantity by using a program-controlled cooling instrument, selecting proper freezing stock solution, preparing proper probes and setting proper temperature increasing and decreasing speed; however, the total time consumption is long, about 1 hour and 10 minutes, and the consumption of liquid nitrogen is large due to long time consumption in the freezing process, so that the cost is high; meanwhile, the method has low cell recovery rate and cell survival rate and cannot meet the use requirement of the human umbilical cord blood hematopoietic stem cells.
The invention content is as follows:
the invention aims to provide a method for freezing the human umbilical cord blood hematopoietic stem cells by program cooling, which has the advantages of reasonable design of a cooling program, greatly shortened time consumption in the freezing storage process, reduced cost of freezing the stem cells by program cooling, and greatly improved recovery rate and cell survival rate of the umbilical cord blood hematopoietic stem cells after recovery.
The invention is implemented by the following technical scheme: the programmed cooling and freezing method of the human umbilical cord blood hematopoietic stem cells comprises the steps of pretreating the human umbilical cord blood hematopoietic stem cells obtained after separation, and cooling to 4 ℃ to obtain a sample; the sample was transferred to a programmed cooling chamber and operated as follows: 1) waiting for 3-4min at 4 ℃; 2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; 3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; 4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; 5) cooling at the speed of 2 ℃/min until the temperature of the sample is-40 ℃; 6) and cooling at the speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen to enable the sample to be completely submerged by the liquid nitrogen.
Further, the pretreatment specifically comprises the step of adding a cryoprotectant to the human umbilical cord blood hematopoietic stem cells, wherein the volume ratio of the human umbilical cord blood hematopoietic stem cells to the cryoprotectant is 4-5: 1, fully mixing the human umbilical cord blood hematopoietic stem cells with the cryoprotectant, placing the mixture in ice water, and standing for 10min to 4 ℃ to obtain the sample.
In the process, the cryoprotectant is Cryo-Sure DEX 40 produced by origen and is a commercially available reagent consumable.
The invention has the advantages that: by reasonably setting the cooling rate and the cooling control point, on one hand, the relationship between the phase change point of the human umbilical cord blood hematopoietic stem cells and the cooling control point is concerned, so that the cooling control point approaches to the phase change point of the human umbilical cord blood hematopoietic stem cells, on the basis, the temperature compensation effect is generated by rapid cooling and intracellular water crystallization heat release, then the box body is rapidly heated, the human umbilical cord blood hematopoietic stem cells can stably pass through the phase change point in the cryopreservation process, the human umbilical cord blood hematopoietic stem cells are prevented from being damaged in the cryopreservation process, the cell viability of the recovered human umbilical cord blood hematopoietic stem cells is improved, and the storage time of the human umbilical cord blood hematopoietic stem cells can be further prolonged; on the other hand, the freezing process is shortened, the time consumption of the freezing process is controlled within 55min, the use amount of liquid nitrogen in the program-controlled cooling process is reduced, and the program cooling and freezing cost of the stem cells is reduced.
Description of the drawings:
in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow cytogram of CD34+ cell rate before cryopreservation in example 3.
FIG. 2 is a flow cytogram of CD34+ cell rate after recovery in example 3.
Fig. 3 is a temperature programmed graph of example 3.
FIG. 4 is a partial enlarged view of the frozen phase transition point temperature curve of example 3.
FIG. 5 is a flow cytogram of CD34+ cell rate before cryopreservation in example 4.
FIG. 6 is a flow cytogram of CD34+ cell rate after freeze resuscitation of example 4.
FIG. 7 is a flow cytogram of CD34+ cell rate before cryopreservation in example 5.
FIG. 8 is a flow cytogram of CD34+ cell rate after freeze resuscitation of example 5.
Fig. 9 is a temperature programmed graph of example 5 freezing.
FIG. 10 is a partially enlarged view of the frozen phase transition point temperature curve of example 5.
The specific implementation mode is as follows:
the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
The samples were transferred to a programmed cooling chamber and operated as follows:
1) waiting for 3min at 4 ℃; the temperature of the program cooling box and the temperature of the probe are close to 4 ℃ at the same time, and preparation is made for the next cooling;
2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; the method aims to gradually reduce the temperature to the phase transition point of the human umbilical cord blood hematopoietic stem cells, so that the temperature reduction control point is closer to the phase transition point of the human umbilical cord blood hematopoietic stem cells, the process of cell crystallization and heat release can be controlled more accurately, the cell temperature reduction curve is more stable, the temperature of a sample is reduced by no more than 5 ℃ in the subsequent rapid temperature reduction process, the temperature change degree is reduced, the cells can be better protected, and the freezing survival rate of the cells is improved;
3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; the purpose is to prepare for the next temperature compensation; in the process, the temperature of the sample is reduced from minus 5 ℃ in the last step to minus 10 ℃, the temperature of the sample is reduced by no more than 5 ℃, and the influence on the activity of the cells is small;
4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; the human umbilical cord blood hematopoietic stem cells in the programmed cooling box continuously release heat, after the cooling rate is reduced, the temperature of the box body of the programmed cooling box is increased from-54 ℃ to-17 ℃, the programmed cooling instrument can exhaust a large amount of air through the fan, the temperature rise of nitrogen in the box body is reduced, so that after the heat release of the human umbilical cord blood hematopoietic stem cells is completed, the temperature of a sample cannot be rapidly reduced, and the temperature of the sample is slowly reduced all the time, thereby avoiding the damage of phase transition points to the human umbilical cord blood hematopoietic stem cells, rapidly increasing the temperature, utilizing the temperature compensation effect, enabling the human umbilical cord blood hematopoietic stem cells to stably pass through the phase transition points, improving the recovery rate and the cell survival rate of the recovered umbilical cord blood hematopoietic stem cells, and further prolonging the preservation time of the umbilical cord blood hematopoietic stem cells;
5) cooling at a speed of 2 ℃/min until the temperature of the sample is-40 ℃; on the basis of not influencing the survival rate of human umbilical cord blood hematopoietic stem cells, the cryopreservation process is shortened, and the time consumed by cryopreservation is reduced;
6) cooling at a speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen; the aim is to rapidly reduce the temperature to-80 ℃ and transfer the liquid nitrogen to the liquid nitrogen for long-term storage.
In the embodiment, the freezing time can be controlled within 55min, and compared with the existing program control cooling freezing method, the freezing time is effectively shortened, the liquid nitrogen consumption can be reduced, and the cost is reduced; meanwhile, in the implementation, the relationship between the phase change point of the human umbilical cord blood hematopoietic stem cells and the temperature reduction control point is focused, so that the temperature reduction control point approaches to the phase change point of the human umbilical cord blood hematopoietic stem cells, on the basis, the temperature compensation effect is generated through rapid temperature reduction and intracellular water crystallization heat release, then the box body is rapidly heated, the human umbilical cord blood hematopoietic stem cells can stably pass through the phase change point in the cryopreservation process, the human umbilical cord blood hematopoietic stem cells are prevented from being damaged in the cryopreservation process, the cell viability of the recovered human umbilical cord blood hematopoietic stem cells is improved, and the storage time of the human umbilical cord blood hematopoietic stem cells can be prolonged.
Example 2:
The samples were transferred to a programmed cooling chamber and operated as follows:
1) waiting for 3.5min at 4 ℃; the temperature of the program cooling box and the temperature of the probe are close to 4 ℃ at the same time, and preparation is made for the next cooling;
2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; the method aims to gradually reduce the temperature to the phase transition point of the human umbilical cord blood hematopoietic stem cells, so that the temperature reduction control point is closer to the phase transition point of the human umbilical cord blood hematopoietic stem cells, the process of cell crystallization and heat release can be controlled more accurately, the cell temperature reduction curve is more stable, the temperature of a sample is reduced by no more than 5 ℃ in the subsequent rapid temperature reduction process, the temperature change degree is reduced, the cells can be better protected, and the freezing survival rate of the cells is improved;
3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; the purpose is to prepare for the next temperature compensation; in the process, the temperature of the sample is reduced from-5 ℃ in the last step to-10 ℃, the temperature of the sample is reduced by no more than 5 ℃, the temperature change of the sample is small, and the influence on the activity of cells is small;
4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; the human umbilical cord blood hematopoietic stem cells in the programmed cooling box continuously release heat, after the cooling rate is reduced, the temperature of the box body of the programmed cooling box is increased from-54 ℃ to-17 ℃, the programmed cooling instrument can exhaust a large amount of air through the fan, the temperature rise of nitrogen in the box body is reduced, so that after the heat release of the human umbilical cord blood hematopoietic stem cells is completed, the temperature of a sample cannot be rapidly reduced, and the temperature of the sample is slowly reduced all the time, thereby avoiding the damage of phase transition points to the human umbilical cord blood hematopoietic stem cells, rapidly increasing the temperature, utilizing the temperature compensation effect, enabling the human umbilical cord blood hematopoietic stem cells to stably pass through the phase transition points, improving the recovery rate and the cell survival rate of the recovered umbilical cord blood hematopoietic stem cells, and further prolonging the preservation time of the umbilical cord blood hematopoietic stem cells;
5) cooling at a speed of 2 ℃/min until the temperature of the sample is-40 ℃; on the basis of not influencing the survival rate of human umbilical cord blood hematopoietic stem cells, the cryopreservation process is shortened, and the time consumed by cryopreservation is reduced;
6) cooling at a speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen; the aim is to rapidly reduce the temperature to-80 ℃ and transfer the liquid nitrogen to the liquid nitrogen for long-term storage.
In the embodiment, the freezing time can be controlled within 55min, and compared with the existing program control cooling freezing method, the freezing time is effectively shortened, the liquid nitrogen consumption can be reduced, and the cost is reduced; meanwhile, in the implementation, the relationship between the phase change point of the human umbilical cord blood hematopoietic stem cells and the temperature reduction control point is focused, so that the temperature reduction control point approaches to the phase change point of the human umbilical cord blood hematopoietic stem cells, on the basis, the temperature compensation effect is generated through rapid temperature reduction and intracellular water crystallization heat release, then the box body is rapidly heated, the human umbilical cord blood hematopoietic stem cells can stably pass through the phase change point in the cryopreservation process, the human umbilical cord blood hematopoietic stem cells are prevented from being damaged in the cryopreservation process, the cell viability of the recovered human umbilical cord blood hematopoietic stem cells is improved, and the storage time of the human umbilical cord blood hematopoietic stem cells can be prolonged.
Example 3:
The samples were transferred to a programmed cooling chamber and operated as follows:
1) waiting for 4min at 4 ℃; the temperature of the program cooling box and the temperature of the probe are close to 4 ℃ at the same time, and preparation is made for the next cooling;
2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; the method aims to gradually reduce the temperature to the phase transition point of the human umbilical cord blood hematopoietic stem cells, so that the temperature reduction control point is closer to the phase transition point of the human umbilical cord blood hematopoietic stem cells, the process of cell crystallization and heat release can be controlled more accurately, the cell temperature reduction curve is more stable, the temperature of a sample is reduced by no more than 5 ℃ in the subsequent rapid temperature reduction process, the temperature change degree is reduced, the cells can be better protected, and the freezing survival rate of the cells is improved;
3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; the purpose is to prepare for the next temperature compensation; in the process, the temperature of the sample is reduced from-5 ℃ in the last step to-10 ℃, the temperature of the sample is reduced by no more than 5 ℃, the temperature change of the sample is small, and the influence on the activity of cells is small;
4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; the human umbilical cord blood hematopoietic stem cells in the programmed cooling box continuously release heat, after the cooling rate is reduced, the temperature of the box body of the programmed cooling box is increased from-54 ℃ to-17 ℃, the programmed cooling instrument can exhaust a large amount of air through the fan, the temperature rise of nitrogen in the box body is reduced, so that after the heat release of the human umbilical cord blood hematopoietic stem cells is completed, the temperature of a sample cannot be rapidly reduced, and the temperature of the sample is slowly reduced all the time, thereby avoiding the damage of phase transition points to the human umbilical cord blood hematopoietic stem cells, rapidly increasing the temperature, utilizing the temperature compensation effect, enabling the human umbilical cord blood hematopoietic stem cells to stably pass through the phase transition points, improving the recovery rate and the cell survival rate of the recovered umbilical cord blood hematopoietic stem cells, and further prolonging the preservation time of the umbilical cord blood hematopoietic stem cells;
5) cooling at a speed of 2 ℃/min until the temperature of the sample is-40 ℃; on the basis of not influencing the survival rate of human umbilical cord blood hematopoietic stem cells, the cryopreservation process is shortened, and the time consumed by cryopreservation is reduced;
6) cooling at a speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen; the aim is to rapidly reduce the temperature to-80 ℃ and transfer the liquid nitrogen to the liquid nitrogen for long-term storage.
In the embodiment, the freezing time can be controlled within 55min, and compared with the existing program control cooling freezing method, the freezing time is effectively shortened, the liquid nitrogen consumption can be reduced, and the cost is reduced; meanwhile, in the implementation, the relationship between the phase change point of the human umbilical cord blood hematopoietic stem cells and the temperature reduction control point is focused, so that the temperature reduction control point approaches to the phase change point of the human umbilical cord blood hematopoietic stem cells, on the basis, the temperature compensation effect is generated through rapid temperature reduction and intracellular water crystallization heat release, then the box body is rapidly heated, the human umbilical cord blood hematopoietic stem cells can stably pass through the phase change point in the cryopreservation process, the human umbilical cord blood hematopoietic stem cells are prevented from being damaged in the cryopreservation process, the cell viability of the recovered human umbilical cord blood hematopoietic stem cells is improved, and the storage time of the human umbilical cord blood hematopoietic stem cells can be prolonged.
Example 4:
embodiment 4 is a non-programmed cooling freezing method for human umbilical cord blood hematopoietic stem cells in the prior art, and the pretreatment is performed on the human umbilical cord blood hematopoietic stem cells obtained after separation, specifically, a cryoprotectant is added to the human umbilical cord blood hematopoietic stem cells, and the volume ratio of the human umbilical cord blood hematopoietic stem cells to the cryoprotectant is 5: 1, fully mixing human umbilical cord blood hematopoietic stem cells with a cryoprotectant, placing the mixture in ice water, standing for 10min, and cooling to 4 ℃ to obtain a sample; samples were directly frozen and stored in liquid nitrogen at-196 ℃.
Example 5:
embodiment 5 is a prior art human umbilical cord blood hematopoietic stem cell prior programmed cooling freezing method, which is to pre-treat human umbilical cord blood hematopoietic stem cells obtained after separation, wherein the pre-treatment is to add a cryoprotectant into the human umbilical cord blood hematopoietic stem cells, the addition volume ratio of the human umbilical cord blood hematopoietic stem cells to the cryoprotectant is 5: 1, and the human umbilical cord blood hematopoietic stem cells and the cryoprotectant are fully mixed and placed in ice water for standing for 10min and cooled to 4 ℃ to obtain a sample, and the specific process comprises the following steps of waiting for 3-4min at ① 4 ℃, cooling 1 ℃ at ② min until the sample temperature is-4 ℃, cooling 30 ℃ at ③ min until the box temperature is-60 ℃, cooling 17 ℃ at ④ min until the box temperature is-20 ℃, cooling 3 ℃ at ⑤ min until the sample temperature is-10 ℃, cooling 2 ℃ at ⑥ min until the sample temperature is-40 ℃, cooling 10 ℃ at ⑦ min until the sample temperature is-80 ℃, and transferring the sample temperature to liquid nitrogen.
Example 6:
examples 1, 2 and 3 are methods for performing temperature programmed freezing of human umbilical cord blood hematopoietic stem cells according to the present invention, example 4 is a conventional method for performing non-temperature programmed freezing of human umbilical cord blood hematopoietic stem cells, and example 5 is a conventional method for performing temperature programmed freezing of human umbilical cord blood hematopoietic stem cells. The human umbilical cord blood hematopoietic stem cells of the same lot were subjected to cryopreservation treatment by the methods of examples 1, 2, 3, 4 and 5, and were numbered as test groups 1, 2, 3, 4 and 5, respectively.
Comparison of liquid nitrogen usage and liquid nitrogen cost
The human umbilical cord blood hematopoietic stem cells of the same batch are subjected to cryopreservation treatment by adopting the methods of the test groups 1, 2, 3, 4 and 5, the usage amount and the usage cost of liquid nitrogen in the processes of the test groups 1, 2, 3, 4 and 5 are detected, the price of the liquid nitrogen is calculated according to the market price of 6 yuan/L, and the specific detection results are shown in table 1.
TABLE 1 comparison of liquid nitrogen usage and freezing cost
| | Test group | 1 | |
|
Test group 4 | Test group 5 |
| Liquid nitrogen usage amount (L times) | 29.4 | 29.7 | 30 | 0 | 40 | |
| Cost of liquid nitrogen (Yuan/Shi) | 176.4 | 178.2 | 180 | 0 | 200 |
As can be seen from table 1, compared with the test group 4 (i.e., the existing non-programmed freezing method) and the test group 5 (i.e., the existing programmed freezing method), the test groups 1, 2, and 3 have low liquid nitrogen usage, so that the liquid nitrogen usage cost is reduced, and finally the program freezing cost of the human umbilical cord blood hematopoietic stem cells is reduced.
Secondly, comparing the counting statistical results of the nucleated cells of the human umbilical cord blood hematopoietic stem cells before freezing and after freezing recovery
The human umbilical cord blood hematopoietic stem cells of the same batch were subjected to cryopreservation by the methods of the test groups 1, 2, 3, 4, and 5, and nucleated cell count statistics were performed on the human umbilical cord blood hematopoietic stem cells of the test groups 1, 2, 3, 4, and 5 before freezing and after freezing recovery, respectively, wherein the results of the nucleated cell count statistics before freezing are shown in table 2, and the results of the nucleated cell count statistics after freezing recovery are shown in table 3.
TABLE 2 statistics of nucleated cell counts before cryopreservation of five groups of cells
TABLE 3 statistics of nucleated cell counts after five groups of cells were thawed
The human umbilical cord blood hematopoietic stem cells of test groups 1, 2, 3, 4 and 5 before and after freezing and thawing were counted by a nucleated cell counting method, wherein the cell viability rate (number of surviving cells/total number of cells) is × 100%, which is used for representing the ratio of the number of cells with biological activity to the total number of cells, the smaller the difference between the cell viability rate before freezing and the cell viability rate after freezing and thawing indicates the lower the damage degree of the freezing process to the cell activity, and the recovery rate of nucleated cells (number of nucleated cells after freezing and thawing/number of nucleated cells before freezing) is × 100%, which is used for representing the damage degree of the freezing process to the cell structure, and the higher the recovery rate of nucleated cells indicates the lower the damage degree of the freezing process to the cell structure.
As is clear from Table 2, the cell viability rates of the respective test groups were the same before the freezing treatment.
As can be seen from table 3, after freeze-thawing, the total number of nucleated cells in each test group decreased, but as can be seen from the recovery rate of nucleated cells, the degree of damage to the cell structure by the test groups 1, 2, and 3 (i.e., the programmed temperature-lowering freezing method of the present invention) was lower than that by the test group 4 (i.e., the conventional non-programmed temperature-lowering freezing method) and the test group 5 (i.e., the conventional programmed temperature-lowering freezing method), which is more favorable for the use of the human umbilical cord blood hematopoietic stem cells after freeze-thawing and the extension of the storage time.
As can be seen from the comparison of Table 2 and Table 3, the cell viability of the test groups 1, 2 and 3 (i.e., the programmed cooling and freezing method of the present invention) is not changed, which indicates that the programmed cooling and freezing treatment of the human umbilical cord blood hematopoietic stem cells by the method of the present invention can effectively maintain the cell activity of the human umbilical cord blood hematopoietic stem cells, and further can prolong the preservation time of the umbilical cord blood hematopoietic stem cells; the cell viability of the test group 4 (i.e., the existing non-programmed cooling freezing method) and the cell viability of the test group 5 (i.e., the existing programmed cooling freezing method) are both reduced, which indicates that the cell viability of the human umbilical cord blood hematopoietic stem cells can be destroyed by performing programmed cooling freezing treatment on the human umbilical cord blood hematopoietic stem cells by using the existing non-programmed cooling freezing method and the existing programmed cooling freezing method, and the use of the human umbilical cord blood hematopoietic stem cells after freezing recovery is not facilitated.
Thirdly, comparing the counting results of the flow cytometry method before freezing and after freezing recovery of the human umbilical cord blood hematopoietic stem cells
The human umbilical cord blood hematopoietic stem cells of the same lot were subjected to cryopreservation by the methods of test groups 3, 4, and 5, and the human umbilical cord blood hematopoietic stem cells of test groups 3, 4, and 5 before freezing and after freezing recovery were counted by flow cytometry, respectively, the CD34+ cell rate flow cytograms of test group 3 before freezing and after freezing recovery are shown in fig. 1 and 2, the CD34+ cell rate flow cytograms of test group 4 before freezing and after freezing recovery are shown in fig. 5 and 6, and the CD34+ cell rate flow cytograms of test group 5 before freezing and after freezing recovery are shown in fig. 7 and 8. The data of the flow cytograms of the CD34+ cell rates before freezing and after freezing recovery in the test groups 3, 4 and 5 were counted, and the results are shown in Table 4.
TABLE 4 statistical results of flow cytometry before and after freezing recovery of three groups of cells
| Group of | |
Test group 4 | Test group 5 |
| CD34+ cell Rate (%) > before freezing | 0.32 | 0.28 | 0.58 |
| CD34+ cell Rate (%) | 0.300 | 0.16 | 0.45 |
| Recovery of CD34+ cells (%) | 93.7 | 57.1 | 77.6 |
Counting the human umbilical cord blood hematopoietic stem cells of the test groups 3, 4 and 5 before freezing and after freezing recovery by adopting a flow cytometry method, wherein the CD34+ cells before freezing indicate the proportion of CD34+ cells in the human umbilical cord blood hematopoietic stem cells before freezing, the CD34+ cells after freezing indicate the proportion of CD34+ cells in the human umbilical cord blood hematopoietic stem cells after freezing, the CD34+ cell recovery rate indicates the damage degree of the freezing process to the cell structure, and the higher the CD34+ cell recovery rate indicates the lower the damage degree of the freezing process to the cell structure.
As can be seen from table 4, the recovery rates of CD34+ cells in test group 3 (i.e., the temperature-programmed freezing method of the present invention) were lower than those in test group 4 (i.e., the conventional non-temperature-programmed freezing method) and test group 5 (i.e., the conventional temperature-programmed freezing method), and the damage to the cell structure was more favorable for the use of the human umbilical cord blood hematopoietic stem cells after freezing and thawing, which is consistent with the above-mentioned statistics of the nucleated cell count.
Example 7:
example 3 is a method for performing temperature programmed freezing on human umbilical cord blood hematopoietic stem cells according to the present invention, and example 5 is a conventional method for performing temperature programmed freezing on human umbilical cord blood hematopoietic stem cells. The human umbilical cord blood hematopoietic stem cells of the same lot were subjected to cryopreservation treatment by the methods of examples 3 and 5, and the samples were numbered as test groups 3 and 5, respectively, and the programmed freezing and cooling processes of the test groups 3 and 5 were monitored, respectively. The temperature programmed curve of test group 3 is shown in fig. 3, and a partial enlarged view thereof is shown in fig. 4; the temperature programmed curve of test group 5 is shown in fig. 9, and a partial enlarged view thereof is shown in fig. 10;
as can be seen from a comparison of fig. 3 and fig. 9, the phase transition point temperature change of the test group 3 (i.e., the temperature programmed freezing method of the present invention) is significantly smaller than that of the test group 5 (i.e., the curve sag is smaller) in the test group 3 as compared with the test group 5 (i.e., the temperature programmed freezing method of the present invention), and the result can be seen more clearly in fig. 4 and fig. 10; therefore, the test group 3 (namely the programmed cooling and freezing method of the invention) can slow down the damage of the phase change point to the human umbilical cord blood hematopoietic stem cells, and the temperature compensation effect is generated through rapid cooling and intracellular water crystallization heat release, and then the rapid temperature rise of the box body enables the human umbilical cord blood hematopoietic stem cells to stably pass through the phase change point in the freezing storage process, thereby avoiding the human umbilical cord blood hematopoietic stem cells from being damaged in the freezing storage process, improving the cell viability of the recovered human umbilical cord blood hematopoietic stem cells, and further prolonging the storage time of the human umbilical cord blood hematopoietic stem cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (2)
1. The programmed cooling and freezing method for the human umbilical cord blood hematopoietic stem cells is characterized in that the human umbilical cord blood hematopoietic stem cells obtained after separation are pretreated and cooled to 4 ℃ to obtain a sample; the sample was transferred to a programmed cooling chamber and operated as follows: 1) waiting for 3-4min at 4 ℃; 2) cooling at a speed of 1 ℃/min until the temperature of the sample is-5 ℃; 3) cooling at a speed of 21 ℃/min until the temperature of the program cooling box is-54 ℃; 4) cooling at a speed of 17 ℃/min until the temperature of the program cooling box is-17 ℃; 5) cooling at the speed of 2 ℃/min until the temperature of the sample is-40 ℃; 6) and cooling at the speed of 10 ℃/min until the temperature of the sample is-80 ℃, and transferring into liquid nitrogen to enable the sample to be completely submerged by the liquid nitrogen.
2. The programmed cooling and freezing method for human umbilical cord blood hematopoietic stem cells according to claim 1, wherein the pretreatment comprises adding a cryoprotectant to the human umbilical cord blood hematopoietic stem cells, and the volume ratio of the human umbilical cord blood hematopoietic stem cells to the cryoprotectant is 4-5: 1, fully mixing the human umbilical cord blood hematopoietic stem cells with the cryoprotectant, placing the mixture in ice water, and standing for 10min to 4 ℃ to obtain the sample.
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