CN116555178A - Separation method and application of cryopreserved umbilical cord blood mononuclear cells - Google Patents
Separation method and application of cryopreserved umbilical cord blood mononuclear cells Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a separation method and application of cryopreserved umbilical cord blood mononuclear cells. According to the separation method provided by the invention, through optimization of recovery diluent and optimization of separation process, erythrocytes, platelets and granulocytes in the frozen umbilical blood are effectively removed, purity and activation rate of mononuclear cells obtained by separation of the frozen umbilical blood are realized, compared with the existing manual separation average level, the total cell yield is improved by 45%, and the recovery rate of CD34+ cells is 2.2 times of the existing level, so that the method is more beneficial to subsequent cell culture and separation research, and the risk of cell culture failure caused by cell clot and platelet residues is reduced. The separation method provided by the invention adopts raw materials or pharmaceutical excipients meeting pharmacopoeia requirements, and the single-core cell separation effect is stable, so that the frozen umbilical blood resource can be more effectively utilized, and the obtained single-core cell can more meet the subsequent research requirements.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a separation method and application of cryopreserved umbilical cord blood mononuclear cells.
Background
Umbilical cord blood mononuclear cells (Umbilical Cord Blood Mononuclear Cell, UCB-MNC) are cells with a single nucleus in umbilical cord blood, including lymphocytes and monocytes. The mononuclear cells of the cord blood contain abundant immune cells, are common raw materials in the field of immune research, and can be further purified and separated into a plurality of cell types, such as natural killer cells (natural killer cell, NK), T lymphocytes and B lymphocytes. In addition, mononuclear cells are one of the important starting cells for the isolation of hematopoietic stem cells (cd34+), mesenchymal stem cells, and the like. Efficient acquisition of high quality mononuclear cells is critical to the development of umbilical cord blood derived applications, as well as the sorting and purification of different cell types from the mononuclear cells.
The prior art is primarily directed to methods of mononuclear cell isolation from fresh cord blood.
Although there are differences between different clinical institutions and laboratories for specific isolation conditions of mononuclear cells, relatively well-defined sample processing methods and isolation conditions have been established. The basic principle of mononuclear separation is to purify and enrich mononuclear cells by using different sizes and specific gravities of cells in blood and density gradient centrifugation. The single-core specific gravity is around 1.070, whereas the specific gravity of erythrocytes and polynuclear leukocytes exceeds 1.080. The mononuclear cells can be obtained by density gradient centrifugation using a solution (layering solution) between the specific gravities. The most commonly used layering liquid is a solution with the mixed specific gravity of polysucrose and sodium diatrizoate of 1.070+/-0.001, and the common diatrizoate of China is replaced by diatrizoic acid meglumine (Ficoll). The single nucleus separating method for clinical and scientific research includes mainly density gradient lymphocyte separating liquid centrifuging process and hydroxyethyl starch precipitating process, or through hydroxyethyl starch, gelatin, methyl cellulose, etc. to precipitate red blood cell, and then density gradient centrifuging to separate single nucleus cell.
In practical clinical application, the frozen cord blood is mainly used as a raw material, and in the early research, related experimental research work is required to be carried out by taking the frozen cord blood as a starting material, but no complete mononuclear cell separation method aiming at the frozen cord blood as the starting material exists at present. Although some instruments can support isolation of mononuclear cells, manual isolation methods are necessary in view of the need for advanced scientific research. Besides the price of the instrument, the cost of consumable materials used for each separation is about thousand yuan, and the method has no universality for scientific research work. The mononuclear cells are obtained by separation through an instrument system, CD34+ cells are sorted, and the results of the existing literature show that the total cell yield obtained by the instrument has large fluctuation, wherein the average value of the recovery rate of the CD34+ cells is 24.2%, and the fluctuation of the results is large (4% -31%). Meanwhile, the purity of the mononuclear cells obtained by separation can have a great influence on the subsequent cell sorting.
The mononuclear cell separation is carried out by fresh umbilical cord blood or frozen umbilical cord blood, the basic principles of the fresh umbilical cord blood and the frozen umbilical cord blood are the same, but the frozen umbilical cord blood cells are more fragile, and after resuscitating and in the separation process, cell coagulation and cell loss easily occur, so that the enrichment effect and the final cell state of the mononuclear cells are affected. The procedure and conditions for isolation of mononuclear cells from fresh cord blood are not applicable to isolation of cryopreserved cord blood mononuclear cells. Therefore, it is important to establish a method for separating the mononuclear cells of the frozen cord blood, and meanwhile, the separation effect and the cell state of the method should be clearly evaluated, so that the frozen cord blood can be ensured to obtain enough mononuclear cells with good state and good cell potential for subsequent research.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a separation method of frozen umbilical cord blood mononuclear cells, which has universality and is suitable for manual operation, and can be popularized and applied in experimental operation, so that mononuclear cells with good states and good cell potential can be obtained, and the requirements of experimental research are met.
In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:
in a first aspect, the invention provides a separation method of cryopreserved umbilical cord blood mononuclear cells, which sequentially comprises a cryopreserved umbilical cord blood resuscitation step, a density gradient centrifugation step and a mononuclear cell cleaning step;
the cryopreserved umbilical cord blood resuscitates at 37-40 ℃ and then sequentially goes through the steps of dilution, centrifugation, supernatant discarding and sieving to obtain a blood sample to be separated;
the dilution step uses a physiological saline solution containing 0.1% -5% (w/v) HSA and 1% (w/v) Dex 40;
the centrifugation condition of the step of centrifuging and discarding the supernatant is that the centrifugation is carried out for 15 min-20 min under the conditions of 4-8 ℃ and 400-500 g, and the rising speed is 1-9 and the falling speed is 1-5;
the sieving step is to obtain a cell suspension by re-suspending a precipitate obtained by centrifuging and discarding the supernatant and then passing through a 100 mu m cell sieve.
As a further technical scheme, the diluent is filtered through a 0.22 mu m filter membrane.
As a further technical scheme, the dilution step is that the recovered umbilical cord blood is stored at 18-25 ℃ after being diluted by the same volume of the diluent, and then diluted by 4-6 times, preferably 4 times;
preferably, the storage is carried out at 18 to 25℃for 3 to 7 minutes, more preferably 5 minutes.
As a further aspect, the volume of the pellet after resuspension is no more than twice the volume of the cryopreserved cord blood.
As a further technical scheme, the density gradient centrifugation step comprises the steps of diluting the cell heavy suspension to twice the volume of the frozen umbilical cord blood, adding the diluted cell heavy suspension to a lymphocyte separation liquid interface, and then carrying out density gradient centrifugation to separate blood samples to be separated;
the volume ratio of the cell re-suspension to the lymphocyte separation liquid after dilution is 2:1.
As a further technical scheme, the density gradient centrifugation is carried out for 30-40min under the conditions of 18-25 ℃ and 500-600 g, and the speed is increased by 1 and reduced by 1;
preferably, the centrifugation condition of the density gradient centrifugation is 18-25 ℃ and the centrifugation is carried out for 30min under 600g, and the speed is increased by 1 and reduced by 1.
As a further technical scheme, the mononuclear cell cleaning step is to add totipotent nucleic acid lyase into a mononuclear cell layer obtained after density gradient centrifugation, and after incubation, perform centrifugal cleaning at least twice by using physiological saline.
As a further technical scheme, the final concentration of the omnipotent nucleic acid lyase is 25-50U/ml in the incubation process.
As a further technical scheme, the dilution multiple of the twice centrifugal cleaning is 3-4 times, the centrifugal cleaning is carried out for 10-15 min under the condition of 400-500 g, the speed is increased by 4-9 and is reduced by 1-5, and the speed is preferably increased by 9 and is reduced by 3.
In a second aspect, the invention provides the use of the above separation method for the artificial separation of cryopreserved umbilical cord blood mononuclear cells.
Compared with the prior art, the invention has the following beneficial effects:
according to the separation method provided by the invention, through optimization of recovery diluent and optimization of separation process, erythrocytes, platelets and granulocytes in the frozen umbilical blood are effectively removed, purity and activation rate of mononuclear cells obtained by separation of the frozen umbilical blood are realized, compared with the existing manual separation average level, the total cell yield is improved by 45%, and the recovery rate of CD34+ cells is 2.2 times of the existing level, so that the method is more beneficial to subsequent cell culture and separation research, and the risk of cell culture failure caused by cell clot and platelet residues is reduced.
The separation method provided by the invention adopts raw materials or pharmaceutical excipients meeting pharmacopoeia requirements, has stable single nuclear cell separation effect and higher purity, is more beneficial to subsequent cell culture and separation research, can more effectively utilize frozen cord blood resources, and enables the obtained single nuclear cells to more meet the subsequent research requirements.
In addition, the separation method provided by the invention can be suitable for a 50ml separation system and a 500ml system, and provides good reference value for daily scientific research, post production conversion and clinical application. Isolation of cryopreserved cord blood mononuclear cells is accomplished in a more time-efficient and economical manner.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram illustrating a technical flow of a separation method according to the present invention;
FIG. 2 shows the effect of different dilution factors on the isolation of mononuclear cells in example 1 of the present invention;
FIG. 3 shows the effect of different dilutions (different Dex40 concentrations) on the isolation of single nucleated cells from cryopreserved cord blood in example 2 of the present invention;
FIG. 4 shows the effect of different dilutions (different HSA concentrations) on the isolation of cryopreserved cord blood mononuclear cells in example 2 of the present invention;
FIG. 5 is a graph showing the effect of different density gradient centrifugation conditions on the isolation of mononuclear cells in example 3 of the present invention;
FIG. 6 shows the effect of the separation method of the present invention on separating mononuclear cells in a plurality of repeated separation batches;
FIG. 7 shows the results of a single nuclear cell separation multi-batch repeated CFU assay according to the separation method of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In a first aspect, the invention provides a separation method of cryopreserved umbilical cord blood mononuclear cells, which sequentially comprises a cryopreserved umbilical cord blood resuscitation step, a density gradient centrifugation step and a mononuclear cell cleaning step;
the cryopreserved umbilical cord blood resuscitates at 37-40 ℃ and then sequentially goes through the steps of dilution, centrifugation, supernatant discarding and sieving to obtain a blood sample to be separated;
the dilution step uses a physiological saline solution containing 0.1% -5% (w/v) HSA and 1% (w/v) Dex 40;
the centrifugation condition of the step of centrifuging and discarding the supernatant is that the centrifugation is carried out for 15 min-20 min under the conditions of 4-8 ℃ and 400-500 g, and the rising speed is 1-9 and the falling speed is 1-5;
the sieving step is to obtain a cell suspension by re-suspending a precipitate obtained by centrifuging and discarding the supernatant and then passing through a 100 mu m cell sieve.
In an alternative embodiment, the centrifugation conditions of the centrifugation supernatant removal step are 4-8 ℃ and 400g for 20min, and the rising speed is 9 and the falling speed is 3.
In an alternative embodiment, the diluent is filtered through a 0.22 μm filter.
In an alternative embodiment, the dilution step is that the umbilical cord blood after recovery is diluted by equal volume of the diluent, stored at 18-25 ℃, and then diluted by 4-6 times, preferably 4 times;
preferably, the storage is carried out at 18-25℃for 3-7 min, more preferably 5min.
In an alternative embodiment, the volume of sediment after resuspension is no more than twice the volume of the cryopreserved cord blood.
In an alternative embodiment, the density gradient centrifugation step comprises diluting the cell heavy suspension to twice the volume of the cryopreserved umbilical cord blood, adding the diluted cell heavy suspension to a lymphocyte separation liquid interface, and then performing density gradient centrifugation to separate the blood sample to be separated;
the volume ratio of the cell re-suspension to the lymphocyte separation liquid after dilution is 2:1.
In an alternative embodiment, the centrifugation conditions of the density gradient centrifugation are 18-25 ℃ and 500-600 g, the centrifugation is carried out for 30-40min, and the rising speed is 1 and the falling speed is 1;
preferably, the centrifugation condition of the density gradient centrifugation is 18-25 ℃ and the centrifugation is carried out for 30min under 600g, and the speed is increased by 1 and reduced by 1.
In an alternative embodiment, the step of washing the mononuclear cells is to add a totipotent nucleic acid lyase to the mononuclear cell layer obtained after the density gradient centrifugation, and to perform at least two centrifugal washing steps using physiological saline after incubation.
In alternative embodiments, the final concentration of the totipotent nucleic acid lyase during incubation may be, for example, but not limited to, 25U/ml, 30U/ml, 35U/ml, 40U/ml, 45U/ml, or 50U/ml.
In an alternative embodiment, the dilution factor of the two centrifugal washes is 3-4 times, and the centrifugation is 10-15 min under 400-500 g conditions, with a speed up of 4-9 down 1-5, preferably a speed up of 9 down 3.
In a second aspect, the invention provides the use of the above separation method for the artificial separation of cryopreserved umbilical cord blood mononuclear cells.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
In a specific embodiment, the present invention provides a separation method as shown in fig. 1, specifically comprising the following steps:
(1) Saline injection containing 0.1% (w/v) HSA and 1% (w/v) Dex40 was prepared as a diluent, and the mixed diluent was filtered through a 0.22 μm filter into a 500ml collection bottle.
(2) Taking out the frozen umbilical blood from the liquid nitrogen tank, putting the frozen umbilical blood into a water bath kettle at 40 ℃ for thawing and resuscitating, taking out the frozen umbilical blood when the frozen umbilical blood is thawed to an ice-water mixture state, and enabling the overall resuscitating time to be no more than 2 minutes.
(3) The frozen cord blood was transferred to 250ml or 500ml centrifuge bottles, leaving a small blood sample for flow detection and cell counting. An equal volume of diluent was slowly added to the blood sample, and after 5 minutes at room temperature (18-25 ℃) a 4-fold dilution was performed, with an overall dilution of 8-fold. Putting the mixture into a centrifugal machine precooled to 4-8 ℃ for centrifugation, 400g,20min, and increasing the speed by 9 and reducing the speed by 3.
(4) After centrifugation, the supernatant was carefully aspirated, the cell pellet was resuspended in physiological saline, no more than twice the volume of the original blood sample, and the resuspended cell suspension was passed through a 100 μm cell sieve to remove the cell clot.
(5) The volume of blood sample was determined to be twice the volume of the starting blood sample and was slowly added to 15ml lymphocyte separation solution (Ficoll) by means of a pasteur pipette avoiding the destruction of the interface (resuspended sample volume: ficoll volume=2:1). To ensure that there is no vibration during centrifugation, granulocyte residual is reduced and balancing can be ensured by a balance. Centrifugation conditions: 18-25 ℃,600g,30min, 1 rise and 1 fall.
(6) Sucking mononuclear cell layer with Pasteur pipette after centrifugation, transferring to new 50ml tube, adding totipotent nucleic acid lyase (sample volume: totipotent nucleic acid lyase=10000:1, so that the final concentration of totipotent nucleic acid lyase is 50U/ml), incubating at room temperature for 5min, adding physiological saline for 3 times volume dilution, centrifuging again, 500g,10min, and increasing speed by 9 and decreasing speed by 3.
(7) The supernatant after centrifugation is sucked and removed, 6-8 ml of physiological saline is added to re-suspend the cell sediment, and centrifugation is carried out, 500g,10min, rising speed 9 and falling speed 3. The cell pellet obtained by centrifugation is the mononuclear cell (MNC) obtained by separation.
The present embodiment also provides a method for evaluating a mononuclear cell obtained by the above-described separation method, comprising: the recovery, activity, purity and potential of the obtained mononuclear cells were examined.
After homogenization of the mononuclear cell suspension, small amounts of cells were taken for AO/PI counting (20. Mu.l). Purity assay was assessed by flow-through (leukocyte CD34/CD45 assay kit) of 0.5M cells. After 10. Mu.l of antibody was added to each tube of sample and incubated at room temperature for 20 minutes in the dark, 1ml of PBS was added and centrifuged at 300g for 5min. The supernatant was poured, 150. Mu.l of PBS was added thereto, and the mixture was subjected to on-line detection. The purity evaluation criteria was the proportion of mononuclear cells (i.e., the sum of the proportions of lymphocytes and monocytes) in leukocytes (CD 45 positive) before and after cord blood isolation by cryopreservation. The calculation formula of the mononuclear cell recovery rate is as follows:the formula combines raw cord blood with isolated mononuclear cells (stranglesThe total number of the barocytes and the monocytes) is compared, and granulocyte residual factors are included, so that the recovery rate of the mononuclear cells is reflected more strictly and accurately.
And carrying out apoptosis detection on the mononuclear cells, and taking 0.1M cells for apoptosis detection. 1ml of pre-chilled starting Buffer (Biolegend) or PBS was added for washing centrifugation, resuspended in 100. Mu.l of pre-chilled Binding Buffer (R & D), 5. Mu.l of Annexin V and 5. Mu.l of 7AAD (BD) were added, incubated at room temperature for 15 minutes in the absence of light, and 400. Mu.l of Binding Buffer were added for detection on-machine. Apoptosis rate (%) =necrotic or apoptotic cells% + early apoptotic cells%.
The stem differentiation potential of the mononuclear cells is detected through CFU (colony unit formation), and the purity and the cell state of CD34+ cells in the obtained mononuclear cells can be comprehensively reflected. CFU inoculation density is 20000 cells/ml, inoculated in 6-well plate, two duplicate wells per sample. Placing the mixture into a carbon dioxide incubator, culturing for 12-14 days, and counting.
Example 1
In this example, the influence of different dilution factors on the resuscitation effect in the resuscitation step was examined, and steps (1) to (7) in the above specific embodiment were repeated and performed in two groups, and in the first group, after standing at room temperature for 5 minutes in step (3), 2-fold dilution was performed, and total dilution was 4-fold. After 5 minutes at room temperature in step (3) of the second group, 4-fold dilution was performed, and the total dilution was 8-fold. After the separation of 8 umbilical cord blood samples under each group of dilution conditions, the ratio of the recovery rate of mononuclear cells, the cell activity rate and the purity of the blood samples obtained by the two groups of experiments are evaluated according to the evaluation method, and the results are shown in fig. 2, wherein the data in fig. 2 are 8 independent experiments (n=8) respectively completed under different dilution multiples, the frozen umbilical cord blood is subjected to single-core extraction after the dilution of different multiples, and the rest conditions are the same. Mononuclear cell recovery, cell viability and purity data are shown as mean±sem, P <0.05 was judged to have significant differences (multiplex T-test).
The data show that 8-fold dilution is significantly better than 4-fold dilution in recovery and purity of mononuclear cells. Meanwhile, in the actual operation process, the umbilical blood sample after 8-time dilution and recovery has more compact cell sediment, more obvious limit with the supernatant and convenient operation. The difference between different umbilical blood samples may affect the stability of the dilution effect, while 8-fold dilution can meet the requirement of saving the dilution liquid, has more universality for the different umbilical blood samples, and ensures good separation effect and cell state. Similar effects can be achieved with higher dilution factors, such as 10-fold or 12-fold, but too high dilution factors are not necessary and increase the risk of cell loss due to increased centrifuge volume.
Example 2
This example, repeating steps (1) - (7) of the above detailed description, compares the effect of different resuscitation dilutions on mononuclear cell separation effects:
first, steps (1) to (7) in the above embodiment are repeated, and after the same umbilical blood sample is resuscitated, the diluted solution is added to dilute the umbilical blood sample step by 8 times of volume, and then density gradient centrifugation is performed. Each set of experiments was repeated 3 times, and after the experiments were completed, the cell viability, the proportion of mononuclear cell recovery and the purity of mononuclear cells in blood samples obtained by the two sets of experiments were evaluated according to the evaluation method described above, and the results are shown in fig. 3 to 4. Wherein the dilution grouping is as follows, the remaining separation conditions are the same.
Group 1:
x% (w/v) Dex40+2.5% (w/v) HSA-NS (X is 0.2,1,5.3,10,20);
group 2:
x% (w/v) HSA+1% (w/v) Dex40-NS (X is 0.1,0.5,2.5,5,10).
The data indicate that different concentrations of Dex40 dilutions have a greater effect on mononuclear cell isolation. Although the Dex40 solvent can protect cells and has an effect of settling red blood cells, too high a concentration of Dex40 solution causes cell loss, while too low a concentration cannot exert a good separation effect. Of the commonly used 2.5% HSA dilutions, the 1% Dex40 concentration was the best for the separation, significantly higher than the conventional 5.3% Dex40 solution (or 5% Dex40 concentration mentioned in other literature), with no significant difference in separation effect over the concentration range of 0.2% -1% (fig. 3: data shown as n=3, mean±sem, P <0.05 judged to be significant).
On this basis we compared dilutions containing different concentrations of HSA. The results show that HSA concentration has relatively little effect on the isolation of mononuclear cells, but that too high a concentration results in cell loss, with a higher recovery of mononuclear cells at a concentration of 0.1%. Unlike the prior art methods which required 2.5% HSA and resulted in low concentration of cell loss and apoptosis, the addition of 0.1% -5% human serum albumin to 1% Dex40-NS was demonstrated to be acceptable in this example (fig. 4: data shown as n=6, mean±sem, P <0.05 judged to be significantly different). Considering the cell yield and the cost of use, 1% Dex40-0.1% HSA-NS is the optimal diluent. If the addition of more human white blood cells is considered to have a protective effect on the cells for subsequent experiments, a maximum concentration of 5% HSA is recommended.
Example 3
This example, repeating steps (1) - (7) of the above detailed description, compares the effect of different resuscitation centrifugation conditions on mononuclear cell separation effects:
first, repeating steps (1) - (7) in the specific embodiment, recovering the same umbilical blood sample, adding the diluent, diluting step by step for 8 times of volume, and performing density gradient centrifugation. Each set of experiments was repeated 3 times, and after the end of the experiments, the cell viability, the proportion of mononuclear cell recovery and the purity of mononuclear cells in the blood samples obtained in the two sets of experiments were evaluated according to the evaluation method described above, and the results are shown in fig. 5. Wherein the density gradient centrifugation is grouped as follows, and the other separation conditions are the same.
Different centrifugation conditions were grouped as follows:
xg,30min, ramp up 1, ramp down 1 (x= 400,500,600,700,800).
The data show that the 400g density gradient centrifugation effect is significantly less than 600g, with increasing centrifugation speed, the single nuclei and cd34+ cell recovery rate increased, but too high centrifugation speed also resulted in cell loss, or the formation of clumps resulting in reduced recovery. Wherein the recovery of cd34+ cells is significantly lower than 600g at centrifugation speeds of 400g and 700 g. Taken together, 600g,30min are the optimal density gradient centrifugation conditions, 500g-600g are the acceptable centrifugation speed ranges (fig. 5: data shown as n=3, mean±sem, P <0.05 judged to have significant differences).
To sum up, to integrate the results of the cryopreservation umbilical cord blood resuscitator centrifugation in examples 1 to 3, in order to ensure the recovery rate of cells after resuscitation and reduce the damage to cells caused by resuscitation and centrifugation, 400g of the umbilical cord blood resuscitator centrifugation is centrifuged for 20min after 8 times of the umbilical cord blood resuscitator centrifugation is gradually diluted with 0.1% HSA-1% Dex40 physiological saline diluent, and the speed is increased by 9 and reduced by 3, so that the resuscitator is a mild resuscitator capable of ensuring the cell yield. Density gradient centrifugation at 600g for 30min at 1 up to 1 down to ensure the enrichment effect of mononuclear cells and maintain the separation condition of higher CD34+ cell content.
The technical method can be suitable for a 50ml small-volume centrifugation system or a 500ml centrifugation system of whole umbilical blood, the separation effect of multiple umbilical blood batches shows that the average value of the total cell yield is 31.8 percent (16% -47%), the average value of the recovery rate of CD34+ cells is 54.3 percent (29% -100%), the average value of the recovery rate of pure mononuclear cells is 41.2 percent (20% -76%) (fig. 6, n=10, mean+ -SEM), and the CFU of the mononuclear cells has good differentiation potential, which shows that the separated cells can have good differentiation potential, and are one of important indexes for comprehensively reflecting the purity and the cell state of CD34+ (fig. 7, n=6, mean+ -SEM; CB is the umbilical blood frozen before separation, MNC is the mononuclear cells after separation, and BFU-E, CFU-GM and CFU-GEMM respectively represent the erythroid explosive colony, the granulosa colony and the mixed colony formation condition of the mononuclear cells. Compared with the existing manual separation method, the total cell yield of the separation method of the technology is improved by 45%, the recovery rate of CD34+ cells is 2.2 times of the existing level, and the separation level of the separation method of the technology is equal to or better than the existing instrument separation level. The reagent raw materials used in the separation process all meet the GMP production requirement, meet the Chinese pharmacopoeia standard or belong to pharmaceutical excipients, can provide high-quality initial cell materials for research, and have good practical clinical application prospect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The separation method of the cryopreserved umbilical cord blood mononuclear cells is characterized by sequentially comprising a cryopreserved umbilical cord blood resuscitation step, a density gradient centrifugation step and a mononuclear cell cleaning step;
the cryopreserved umbilical cord blood resuscitates at 37-40 ℃ and then sequentially goes through the steps of dilution, centrifugation, supernatant discarding and sieving to obtain a blood sample to be separated;
the dilution step uses a physiological saline solution containing 0.1% (w/v) to 5% (w/v) HSA and 1% (w/v) Dex 40;
the centrifugation condition of the step of centrifuging and discarding the supernatant is that the centrifugation is carried out for 15 min-20 min under the conditions of 4-8 ℃ and 400-500 g, and the rising speed is 1-9 and the falling speed is 1-5;
the sieving step is to obtain a cell suspension by re-suspending a precipitate obtained by centrifuging and discarding the supernatant and then passing through a 100 mu m cell sieve.
2. The separation method according to claim 1, wherein the dilution is filtered through a 0.22 μm filter.
3. The separation method according to claim 1, wherein the dilution step is performed by diluting the resuscitated cord blood with a diluent in equal volume, preserving at 18-25 ℃, and then diluting again 4-6 times, preferably 4 times;
preferably, the storage is carried out at 18 to 25℃for 3 to 7 minutes, more preferably 5 minutes.
4. The method of claim 1, wherein the volume of sediment after resuspension is no more than twice the volume of the cryopreserved cord blood.
5. The separation method according to claim 1, wherein the density gradient centrifugation step comprises diluting the cell suspension to twice the volume of the cryopreserved umbilical cord blood, adding the diluted cell suspension to a lymphocyte separation liquid interface, and performing density gradient centrifugation to separate the blood sample to be separated;
the volume ratio of the cell re-suspension to the lymphocyte separation liquid after dilution is 2:1.
6. The separation method according to claim 5, wherein the centrifugation conditions of the density gradient centrifugation are 18 to 25 ℃ and 500 to 600g, centrifugation is carried out for 30 to 40 minutes at a speed of 1 to 1;
preferably, the centrifugation condition of the density gradient centrifugation is 18-25 ℃ and the centrifugation is carried out for 30min under 600g, and the speed is increased by 1 and reduced by 1.
7. The method according to claim 1, wherein the step of washing the mononuclear cells comprises adding a totipotent nucleic acid lyase to a mononuclear cell layer obtained by density gradient centrifugation, incubating the mixture, and washing the mixture at least twice with physiological saline.
8. The method of claim 7, wherein the final concentration of the omnipotent nucleic acid lyase during incubation is 25-50U/ml.
9. The separation method according to claim 7, wherein the dilution ratio of the two centrifugal washes is 3 to 4 times, the centrifugation is carried out for 10 to 15 minutes under 400 to 500g conditions, and the rising speed is 4 to 9 and the falling speed is 1 to 5, preferably the rising speed is 9 and the falling speed is 3.
10. Use of the separation method according to any one of claims 1 to 9 for the artificial separation of cryopreserved umbilical cord blood mononuclear cells.
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