CN114703135A - In-vitro amplification method for cryopreserved umbilical cord blood Treg cells - Google Patents
In-vitro amplification method for cryopreserved umbilical cord blood Treg cells Download PDFInfo
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Abstract
The invention discloses an in-vitro expansion method of Treg cells of cryopreserved umbilical cord blood, which adopts cryopreserved umbilical cord blood, separation in CBMC and CD4+CD25+CD127lowOn the basis of Treg sorting, soluble carriers combined with anti-CD3, anti-CD28 and anti-CD2 are adopted to activate Treg cells, the activation effect of the Treg cells is enhanced, rapamycin is added at 0-3d, 5-7d and 12-15d respectively in the Treg culture process, the purity and the inhibitory activity of the Treg cells are obviously improved compared with the addition only in the activation process, and the expansion quantity of the Treg cells can be obviously improved compared with the addition in the whole culture process. The GMP-grade CS10 capable of being directly returned and the pansy cell preservation solution of the sansheng organisms are adopted, so that the vitality, the phenotype and the inhibitory activity of the Treg cells can be maintained.
Description
Technical Field
The invention relates to an in-vitro amplification method of umbilical cord blood, in particular to an in-vitro amplification method of cryopreserved Treg cells of umbilical cord blood.
Background
Regulatory T cells (tregs) are a subset of T lymphocytes with immunosuppressive effects, and are important regulators of the maintenance of immune tolerance in the body. CD4 in vivo+CD25+Treg cells account for CD4 only+5-10% of T cells account for only 1-2% of human peripheral blood mononuclear cells. In order to obtain sufficient quantities of cells for clinical use, large scale expansion of Treg cells in vitro is required. At present, the general method for obtaining Treg cells by in vitro expansion is to separate fresh umbilical cord blood or peripheral blood through lymphocyte separation liquid to obtain mononuclear cells, and then carry out magnetic sorting on the mononuclear cells to obtain CD4+CD25+And the cell activation and expansion process of the Treg cells is added with cytokine IL-2, anti-CD3/anti-CD28 magnetic beads or independent anti-CD3 and anti-CD28, and the Treg cell activation process or the whole culture process is added with rapamycin to inhibit the growth of toxic T lymphocytes. For the cryopreservation of the Treg cells, DMSO, FBS or human AB serum and culture medium mixed solution with different proportions are mostly adopted as cryopreservation solution to cryopreserve the Treg cells.
In the process of Treg culture activation, the activation effect is often poor by adopting the anti-CD3 and the anti-CD28, while the activation effect is better by adopting the magnetic beads of the anti-CD3/anti-CD28, but the magnetic bead residue has the problem of clinical safety and needs to be removed in clinical application. Most of the currently adopted frozen stock solutions of the Treg cells are of research and development grade, and the frozen Treg cells cannot be directly returned in vivo.
Disclosure of Invention
The invention aims to solve the technical problem of providing an in-vitro amplification method for cryopreserved umbilical cord blood Treg cells. The method can stably amplify the Treg cells with high purity and good functional activity.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: an in-vitro expansion method of cryopreserved umbilical cord blood Treg cells comprises the following steps:
(1) recovering cryopreserved umbilical cord blood, obtaining mononuclear cells from the thawed umbilical cord blood by a Ficoll separation method, and carrying out magnetic separation on the mononuclear cells to obtain CD4+CD25+CD127lowTreg cells; flow detection of CD4+CD25+Proportion of Treg cells, if CD4+CD25+If the proportion of Treg cells is more than 80%, carrying out the next experiment;
(2) activation and expansion of Treg cells, comprising the following steps:
treg cells were cultured in RPMI-1640 medium at (3-8). times.105Resuspending in RPMI-1640 medium supplemented with 5% -10% FBS, 200-1000IU/mL IL-2, 50-150nM rapamycin, 10-50. mu.L/mL CD3/CD28/CD2 soluble T cell activator at final concentration;
culturing to the 3 rd day, centrifuging and collecting cells for deactivation culture, wherein the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2 on the 3 rd day;
on day 5, centrifugally collecting cells and supplementing fresh culture medium, wherein the fresh culture medium RPMI-1640 contains 5% -10% FBS, 200-1000IU/mL IL-2 and 50-150nM rapamycin;
on day 7, the cells were collected by centrifugation and supplemented with fresh medium RPMI-1640 containing 5% -10% FBS, 200-1000IU/mL IL-2;
on the 10 th day of culture, fresh medium RPMI-1640 containing 5% -10% FBS and 200-1000IU/mL IL-2 was supplemented;
centrifuging and collecting cells for reactivation culture on day 12, wherein the RPMI-1640 culture medium on day 12 contains 5% -10% FBS, 200-1000IU/mL IL-2, 50-150nM rapamycin, and 10-50 μ L/mL CD3/CD28/CD2 soluble T cell activator;
centrifuging and collecting cells for deactivation culture at 15 days, wherein the RPMI-1640 culture medium contains 5-10% FBS and 200-1000IU/mL IL-2;
fresh culture medium is supplemented for 17 days and 19 days respectively, and the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2;
culturing for 21 days, collecting cells, and detecting Treg cell number, activity, purity, cell impurity residue and lymphocyte proliferation inhibiting activity, if 7-AAD-Cell proportion of more than 90%, CD4+CD25+FoxP3+The proportion of Treg cells is more than 80 percent, and the CD8+T cell content less than 5%, CD19+B cells are less than 0.5%, and the inhibitory activity is more than 70%, then the next step is carried out; if the standard is not met, discarding the batch of Treg cells;
(3) and (4) freezing and storing Treg cells: collecting Treg cells by centrifugation, counting, and freezing the cells at 5X 106Resuspending Treg cells at a density of 1 mL/mL, transferring the cell suspension into a cryopreservation tube, quickly putting the tube into a programmed cooling box, placing the tube into a refrigerator at minus 80 ℃, and transferring the tube into liquid nitrogen for storage for 24 hours.
According to the volume percentage, the cell frozen stock solution is 70 percent of compound electrolyte injection, 10 percent of DMSO, 10 percent of dextran 40 sodium chloride injection and 10 percent of human serum albumin intravenous infusion solution; wherein, the compound electrolyte injection comprises: every 500mL contains 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride and 0.15g of magnesium chloride; dextran 40 sodium chloride injection: every 500mL of the solution contains 30g of dextran 40 and 4.5g of sodium chloride; human serum albumin intravenous infusion solution: each 100mL of the injection contains 20g of human serum albumin, 16mM of N-acetyltryptophan sodium, 16mM of sodium caprylate, 140mM of sodium chloride and water for injection.
The cell freezing solution is CS10 or Panro cell preserving solution.
Resuscitating the cryopreserved umbilical cord blood in the step (1): 35mL of umbilical cord blood frozen in a 50mL freezing bag is placed in a constant-temperature water bath at 37-42 ℃ to be shaken left and right, and frozen objects in the freezing bag are continuously and lightly twisted by hands until the blood is completely melted.
Transferring the thawed umbilical cord blood to a 50mL centrifuge tube in the step (1), diluting by 6 times with physiological saline for injection, centrifuging at 600g and 4 ℃ for 30min, and collecting cell precipitates; resuspend the cells in 40mL of normal saline, mix the cell suspension with Ficoll at 1:1, centrifuge at 650g at 18 ℃ for 30min, collect the cord blood mononuclear cells, and wash 2 times with normal saline.
Counting the obtained cord blood mononuclear cells in the step (1), and sorting the cord blood mononuclear cells with a Stemcell sorting buffer solution at 5X 107Resuspend cells at concentration/mL and pass through a 37 μm cell sieve; transferring the cell suspension to a round bottom flow type sample loading tube, adding 50 mu L of CD25 Positive Selection Cocktail antibody, mixing uniformly, and incubating at room temperature for 5 min; adding 30. mu.L of Releaseable RapidSpheresTMAnd 50. mu.L of CD4+Mixing T cell Enrichment Cocktail, and incubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing for 2-3 times, and then putting a magnetic pole to incubate for 10min at room temperature; pouring off the supernatant, removing the magnetic pole, adding a sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in the magnetic pole, incubating at room temperature for 5min, pouring off the supernatant, and repeating the process for 2 times; removing the magnetic pole, adding 1mL of sorting Buffer solution to resuspend the cells, adding 100 mu L of Release Buffer and mixing uniformly; add 50. mu.L of CD127highIncubating the Depletion Cocktail at room temperature for 5 min; add 10. mu.L of Dextran RapidSpheresTMIncubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in a magnetic pole, incubating for 5min, and collecting cell suspension; cells were harvested for use after centrifugation.
The cryopreserved umbilical cord blood Treg cells are obtained by the in-vitro amplification method of the cryopreserved umbilical cord blood Treg cells.
The invention has the beneficial effects that: the umbilical cord blood is cryopreserved, the umbilical cord blood is stored globally, resources are rich, and the umbilical cord blood can be prepared and stored in advance, so that autologous and allogeneic applications are met; isolation at CBMC and CD4+CD25+CD127lowOn the basis of Treg sorting, a soluble carrier (CD3/CD28/CD 2T cell activator) combined with anti-CD3, anti-CD28 and anti-CD2 is adopted to activate Treg cells, so that the activation effect of the Treg cells is enhanced, and the safety problem of magnetic beads for clinical application of the cells does not exist; rapamycin has the effects of inhibiting the growth of toxic T lymphocytes on one hand, and can enhance the inhibitory activity of Treg cells on the other hand. In the process of Treg culture, rapamycin is added in 0-3d, 5-7d and 12-15d respectively, compared with the rapamycin added only in the activation process, the purity and the inhibitory activity of Treg cells are obviously improved, and compared with the rapamycin added in the whole culture process, the Treg cell number can be obviously improved. Can maintain T by adopting GMP-grade directly-transfused CS10 and Panro cell preservation solution of TribiotaViability, phenotype and inhibitory activity of the reg cells.
Drawings
FIG. 1 is a cell morphology diagram of cord blood Treg cells at different stages of expansion in example 1 of the present invention.
Fig. 2 is a graph showing the results of the expansion fold of the Treg cells in the cord blood in example 1 and example 2 of the present invention.
FIG. 3A shows cord blood Treg cell CD4 in example 1 and example 2 of the present invention+CD25+Expression analysis result graph.
FIG. 3B shows the cord blood Treg cells FoxP3 in examples 1 and 2 of the present invention+Expression analysis result graph.
FIG. 4A shows CD19 in Treg cells of umbilical cord blood in examples 1 and 2 of the present invention+B cell residual analysis result chart.
FIG. 4B shows CD8 in Treg cells of cord blood in examples 1 and 2 of the present invention+T cell residual analysis result chart.
Fig. 5A is a graph showing the results of flow analysis of the inhibition of PBMC proliferation by cord blood Treg cells in example 1 of the present invention.
Fig. 5B is a graph showing the results of flow analysis of the inhibition of PBMC proliferation by cord blood Treg cells in example 2 of the present invention.
Fig. 5C is a graph of the statistical analysis difference of the inhibition of PBMC proliferation by cord blood Treg cells in example 1 and example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by a person skilled in the art without making any inventive step are within the scope of the present invention.
The in-vitro amplification method of the cryopreserved umbilical cord blood Treg cells comprises the following steps:
(1) recovering cryopreserved umbilical cord blood, obtaining mononuclear cells from the thawed umbilical cord blood by a Ficoll separation method, and carrying out magnetic separation on the mononuclear cells to obtain CD4+CD25+CD127lowTreg cells; flow detection of CD4+CD25+Proportion of Treg cells, if CD4+CD25+If the proportion of the Treg cells is more than 80 percent, carrying out the next experiment;
(2) activation and expansion of Treg cells, comprising the following steps:
treg cells were cultured in RPMI-1640 medium at (3-8). times.105Resuspending in RPMI-1640 medium supplemented with 5% -10% FBS, 200 IU/mL IL-2, 50-150nM rapamycin, 10-50 μ L/mL CD3/CD28/CD2 soluble T cell activator;
culturing to the 3 rd day, centrifuging and collecting cells for deactivation culture, wherein the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2 on the 3 rd day;
on day 5, centrifugally collecting cells and supplementing fresh culture medium, wherein the fresh culture medium RPMI-1640 contains 5% -10% FBS, 200-1000IU/mL IL-2 and 50-150nM rapamycin;
on day 7, the cells were collected by centrifugation and supplemented with fresh medium RPMI-1640 containing 5% -10% FBS, 200-1000IU/mL IL-2;
on the 10 th day of culture, fresh medium RPMI-1640 containing 5% -10% FBS and 200-1000IU/mL IL-2 was supplemented;
centrifuging and collecting cells for reactivation culture on day 12, wherein the RPMI-1640 culture medium on day 12 contains 5% -10% FBS, 200-1000IU/mL IL-2, 50-150nM rapamycin, and 10-50 μ L/mL CD3/CD28/CD2 soluble T cell activator;
centrifuging and collecting cells on day 15, deactivating and culturing, wherein the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2;
fresh culture medium is supplemented for 17 days and 19 days respectively, and the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2;
culturing for 21 days, collecting cells, and detecting Treg cell number, activity, purity, cell impurity residue and lymphocyte proliferation inhibiting activity, if 7-AAD-Cell proportion of more than 90%, CD4+CD25+FoxP3+The proportion of Treg cells is more than 80 percent, and the CD8+T cell content less than 5%, CD19+B cellIf the content is less than 0.5 percent and the inhibitory activity is more than 70 percent, carrying out the next step; if the standard is not met, discarding the batch of Treg cells;
(3) and (4) freezing and storing Treg cells: collecting Treg cells by centrifugation, counting, and freezing the cells at 5X 106Resuspending Treg cells at a density of 1 mL/mL, transferring the cell suspension into a cryopreservation tube, quickly putting the tube into a programmed cooling box, placing the tube into a refrigerator at minus 80 ℃, and transferring the tube into liquid nitrogen for storage for 24 hours.
According to the volume percentage, the cell frozen stock solution is 70 percent of compound electrolyte injection, 10 percent of DMSO, 10 percent of dextran 40 sodium chloride injection and 10 percent of human serum albumin intravenous infusion solution; wherein, the compound electrolyte injection comprises: every 500mL contains 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride and 0.15g of magnesium chloride; dextran 40 sodium chloride injection: every 500mL of the solution contains 30g of dextran 40 and 4.5g of sodium chloride; human serum albumin intravenous infusion solution: each 100mL of the injection contains 20g of human serum albumin, 16mM of N-acetyltryptophan sodium, 16mM of sodium caprylate, 140mM of sodium chloride and water for injection.
The cell freezing solution is CS10 or Panro cell preserving solution.
Resuscitating the cryopreserved umbilical cord blood in the step (1): 35mL of umbilical cord blood frozen in a 50mL freezing bag is placed in a constant-temperature water bath at 37-42 ℃ to be shaken left and right, and frozen objects in the freezing bag are continuously and lightly twisted by hands until the blood is completely melted.
Transferring the thawed umbilical cord blood to a 50mL centrifuge tube in the step (1), diluting by 6 times with physiological saline for injection, centrifuging at 600g and 4 ℃ for 30min, and collecting cell precipitates; resuspend the cells in 40mL of physiological saline, mix the cell suspension with Ficoll at 1:1, centrifuge at 650g for 30min at 18 ℃, collect the cord blood mononuclear cells, and wash 2 times with physiological saline.
Counting the obtained cord blood mononuclear cells in the step (1), and sorting the cord blood mononuclear cells with a Stemcell sorting buffer solution at 5X 107Resuspend cells at concentration/mL and pass through a 37 μm cell sieve; transferring the cell suspension to a round bottom flow type sample loading tube, adding 50 mu L of CD25 Positive Selection Cocktail antibody, mixing uniformly, and incubating at room temperature for 5 min; add 30. mu.L of Releaseable RapidSpheresTMAnd 50. mu.L of CD4+T cell Enrichment Mixing and incubating Cocktail for 5min at room temperature; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing for 2-3 times, and then putting a magnetic pole to incubate for 10min at room temperature; pouring off the supernatant, removing the magnetic pole, adding a sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in the magnetic pole, incubating at room temperature for 5min, pouring off the supernatant, and repeating the process for 2 times; removing the magnetic pole, adding 1mL of sorting Buffer solution to resuspend the cells, adding 100 mu L of Release Buffer and mixing uniformly; add 50. mu.L of CD127highIncubating the Depletion Cocktail at room temperature for 5 min; add 10. mu.L of Dextran RapidSpheresTMIncubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in a magnetic pole, incubating for 5min, and collecting cell suspension; cells were harvested for use after centrifugation.
The cryopreserved umbilical cord blood Treg cells are obtained by the in-vitro amplification method of the cryopreserved umbilical cord blood Treg cells.
The CD3/CD28/CD2 soluble T cell activator used in the examples below was ImmunoCultTMHuman CD3/CD28/CD 2T cell activators, available from Stemcell Technologies Inc., are composed of soluble antibody complexes that bind and crosslink CD3, CD28 and CD2 cell surface ligands, providing the primary and costimulatory signals required for T cell activation.
Example 1
1) Resuscitating cryopreserved umbilical cord blood: about 35mL of umbilical cord blood frozen in a 50mL freezing bag is placed in a constant-temperature water bath at 37-42 ℃ to be shaken left and right, and frozen objects in the freezing bag are continuously and lightly twisted by hands until the blood is completely melted.
2) Separation of cord blood mononuclear cells: the thawed umbilical cord blood was transferred to a 50mL centrifuge tube, diluted 6-fold with physiological saline for injection, 600g, centrifuged at 4 ℃ for 30min, and the cell pellet was collected. Resuspend the cells in 40mL of normal saline, mix the cell suspension with Ficoll at 1:1, centrifuge at 650g at 18 ℃ for 30min, collect the cord blood mononuclear cells, and wash 2 times with normal saline.
3) Magnetic bead sorting of Treg cells: counting the cord blood mononuclear cells obtained in the step 2), and sorting the cord blood mononuclear cells with a Stemcell sorting buffer at 5X 107Resuspend cells at concentration/mL and pass through a 37 μm cell sieve; the cell suspension was transferred to a round-bottom flow-up tube, 50. mu.L of CD25 Positive Selection Cocktail antibody was added, and mixedAfter homogenizing, incubating for 5min at room temperature; adding 30. mu.L of Releaseable RapidSpheresTMAnd 50. mu.L of CD4+Mixing T cell Enrichment Cocktail, and incubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing for 2-3 times, and then putting a magnetic pole to incubate for 10min at room temperature; pouring off the supernatant, removing the magnetic pole, adding a sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in the magnetic pole, incubating at room temperature for 5min, pouring off the supernatant, and repeating the process for 2 times; removing the magnetic pole, adding 1mL of sorting Buffer solution to resuspend the cells, adding 100 mu L of Release Buffer and mixing uniformly; add 50. mu.L of CD127highIncubating the Depletion Cocktail at room temperature for 5 min; add 10. mu.L of Dextran RapidSpheresTMIncubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in a magnetic pole, incubating for 5min, and collecting cell suspension; cells were harvested for use after centrifugation.
4) Activation and expansion of Treg cells: treg cells were cultured in RPMI-1640 medium at 5X 105resuspend/mL, media supplemented with 10% FBS, 500IU/mL IL-2, 100nM rapamycin, 20. mu.L/mL CD3/CD28/CD2 soluble T cell activator; culturing to 3 days, centrifuging to collect cells, deactivating culture in RPMI-1640 medium containing 10% FBS and 500IU/mL IL-2; on day 5, cells were harvested by centrifugation and supplemented with fresh medium RPMI-1640 containing 10% FBS, 500IU/mL IL-2, 100nM rapamycin; on day 7, cells were harvested by centrifugation and supplemented with fresh medium RPMI-1640 containing 10% FBS, 500IU/mL IL-2; on the 10 th day of culture, fresh medium RPMI-1640 medium containing 10% FBS and 500IU/mL IL-2 was supplemented; centrifuging on day 12 to collect cells, and performing reactivation culture in RPMI-1640 medium containing 10% FBS, 500IU/mL IL-2, 100nM rapamycin, and 20 μ L/mL CD3/CD28/CD2 soluble T cell activator; centrifuging on day 15 to collect cells, deactivating and culturing, wherein the RPMI-1640 culture medium contains 10% FBS and 500IU/mL IL-2; supplementing fresh culture medium respectively for 17 days and 19 days, wherein the RPMI-1640 culture medium contains 10% FBS and 500IU/mL IL-2; and culturing for 21 days, harvesting cells, and detecting the quantity, the activity, the purity, the cell impurity residue and the lymphocyte proliferation inhibition activity of the Treg cells.
5) And (4) freezing and storing Treg cells: collecting Treg cells by centrifugation, counting, selecting three kinds of cell freezing stock solution at 5 × 106/mAnd (3) resuspending Treg cells at an L density, wherein the cryopreservation volume is 1mL, transferring the cell suspension into a cryopreservation tube, quickly putting the cryopreservation tube into a programmed cooling box, placing the box in a refrigerator at the temperature of-80 ℃, and transferring the cell suspension into liquid nitrogen for storage for 3 months after 24 hours. (frozen stock solution 1: 70% Compound electrolyte injection + 10% DMSO + 10% dextran 40 sodium chloride injection + 10% human serum Albumin intravenous infusion solution frozen stock solution 2: CS10(Stemcell Technology) frozen stock solution 3: Panro cell preservation solution (Nanjing Sansheng biology))
6) Recovering Treg cells: the cells are put in a water bath at 37 ℃ for fast thawing, transferred to a 15mL centrifuge tube, slowly added with 5mL compound electrolyte injection containing 0.5% HSA, and kept stand for 5min at room temperature. Reversing and mixing evenly, and taking 100 mu L of cell suspension for activity counting; the remaining cell suspension was centrifuged at 300g at 18 ℃ for 6min, the supernatant was discarded and resuspended in 2mL RPMI-1640 medium containing 10% FBS, 1mL for flow-type phenotypic analysis and 1mL for lymphocyte proliferation inhibition experiments.
The results show that 20. mu.L/mL of CD3/CD28/CD2 soluble T cell activator, 500IU/mL of IL-2 and 100nM of rapamycin in the example can obviously induce the high-purity Treg cell proliferation. The cell activity of the cord blood Treg cells on day 21 is 96.8%, and the cell activity is CD4+CD25+The positive rate is 97.8 percent, and the FoxP3+The positive rate is 94.8%, CD8+Less than 2.5% T cells, CD19+B cells less than 0.1%; the expansion multiple of the cells is 1195 times; the activity of inhibiting PBMC proliferation is 84.5%.
Example 2
The method for inducing and expanding the Treg cells in the umbilical cord blood is basically the same as the specific steps in the example 1, except that in the step 4, the Treg cells are cultured in RPMI-1640 medium at a speed of 3X 105Density resuspension at/mL, concentration of activated amplification factor added: the FBS concentration is 5%, the IL-2 concentration is 1000IU/mL, the rapamycin concentration is 50nM, and the CD3/CD28/CD2 soluble T cell activator is 10 uL/mL. The results show that the phenotype of the Treg cells in the embodiment is similar to that in the embodiment 1, the cell viability of the Treg cells in the umbilical cord blood at the 21 st day is 95.6%, and the cell viability is CD4+CD25+The positive rate is 96.9 percent, and the FoxP3+The positive rate is 89.1%, CD8+Less than 5.0% T cells, CD19+B cells less than 0.1%; the expansion multiple of the cells is 816 times;the activity of inhibiting PBMC proliferation is 63.8%.
The results are shown in FIGS. 1-5C.
In summary, the disclosure of the present invention is not limited to the above-mentioned embodiments, and persons skilled in the art can easily set forth other embodiments within the technical teaching of the present invention, but such embodiments are included in the scope of the present invention.
Claims (8)
1. An in-vitro amplification method for cryopreserved umbilical cord blood Treg cells is characterized by comprising the following steps:
(1) recovering cryopreserved umbilical cord blood, obtaining mononuclear cells from the thawed umbilical cord blood by a Ficoll separation method, and carrying out magnetic separation on the mononuclear cells to obtain CD4+CD25+CD127lowTreg cells; flow detection of CD4+CD25+Proportion of Treg cells, if CD4+CD25+If the proportion of the Treg cells is more than 80 percent, carrying out the next experiment;
(2) activation and expansion of Treg cells, comprising the following steps:
treg cells were cultured in RPMI-1640 medium at (3-8). times.105Resuspending in RPMI-1640 medium supplemented with 5% -10% FBS, 200 IU/mL IL-2, 50-150nM rapamycin, 10-50 μ L/mL CD3/CD28/CD2 soluble T cell activator;
culturing to the 3 rd day, centrifuging and collecting cells for deactivation culture, wherein the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2 on the 3 rd day;
on day 5, centrifugally collecting cells and supplementing fresh culture medium, wherein the fresh culture medium RPMI-1640 contains 5% -10% FBS, 200-1000IU/mL IL-2 and 50-150nM rapamycin;
on day 7, the cells were collected by centrifugation and supplemented with fresh medium RPMI-1640 containing 5% -10% FBS, 200-1000IU/mL IL-2;
on the 10 th day of culture, fresh medium RPMI-1640 containing 5% -10% FBS and 200-1000IU/mL IL-2 was supplemented;
centrifuging and collecting cells for reactivation culture on day 12, wherein the RPMI-1640 culture medium on day 12 contains 5% -10% FBS, 200-1000IU/mL IL-2, 50-150nM rapamycin, and 10-50 μ L/mL CD3/CD28/CD2 soluble T cell activator;
centrifuging and collecting cells on day 15, deactivating and culturing, wherein the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2;
fresh culture medium is supplemented for 17 days and 19 days respectively, and the RPMI-1640 culture medium contains 5% -10% FBS and 200-1000IU/mL IL-2;
culturing for 21 days, collecting cells, and detecting Treg cell number, activity, purity, cell impurity residue and lymphocyte proliferation inhibiting activity, if 7-AAD-Cell proportion of more than 90%, CD4+CD25+FoxP3+The proportion of Treg cells is more than 80 percent, and the CD8+T cell content less than 5%, CD19+B cells are less than 0.5%, and the inhibitory activity is more than 70%, then the next step is carried out; if the standard is not met, discarding the batch of Treg cells;
(3) and (4) freezing and storing Treg cells: collecting Treg cells by centrifugation, counting, and freezing the cells at 5X 106Resuspending Treg cells at a density of 1 mL/mL, transferring the cell suspension into a cryopreservation tube, quickly putting the tube into a programmed cooling box, placing the tube into a refrigerator at minus 80 ℃, and transferring the tube into liquid nitrogen for storage for 24 hours.
2. The in vitro expansion method of the cryopreserved umbilical cord blood Treg cells according to claim 1, wherein the cell cryopreserved solution is 70% compound electrolyte injection + 10% DMSO + 10% dextran 40 sodium chloride injection + 10% human serum albumin intravenous infusion solution in volume percentage; wherein, the compound electrolyte injection comprises: every 500mL contains 2.63g of sodium chloride, 2.51g of sodium gluconate, 1.84g of sodium acetate, 0.185g of potassium chloride and 0.15g of magnesium chloride; dextran 40 sodium chloride injection: every 500mL of the solution contains 30g of dextran 40 and 4.5g of sodium chloride; human serum albumin intravenous infusion solution: each 100mL of the injection contains 20g of human serum albumin, 16mM of N-sodium acetyltryptophanate, 16mM of sodium caprylate, 140mM of sodium chloride and water for injection.
3. The in vitro expansion method of the cryopreserved umbilical cord blood Treg cells according to claim 1, wherein the cell cryopreservation solution is CS 10.
4. The in vitro expansion method of the cryopreserved umbilical cord blood Treg cells according to claim 1, wherein the cell cryopreservation solution is a Pano cell preservation solution.
5. The in vitro expansion method of the cryopreserved umbilical cord blood Treg cells according to claim 1, wherein the resuscitation of the cryopreserved umbilical cord blood in the step (1): 35mL of umbilical cord blood frozen in a 50mL freezing bag is placed in a constant-temperature water bath at 37-42 ℃ to be shaken left and right, and frozen objects in the freezing bag are continuously and lightly twisted by hands until the blood is completely melted.
6. The in vitro expansion method of the cryopreserved umbilical cord blood Treg cells according to claim 5, wherein in the step (1), the thawed umbilical cord blood is transferred to a 50mL centrifuge tube, diluted 6 times with physiological saline for injection, and centrifuged at 600g and 4 ℃ for 30min to collect cell precipitates; resuspend the cells in 40mL of normal saline, mix the cell suspension with Ficoll at 1:1, centrifuge at 650g at 18 ℃ for 30min, collect the cord blood mononuclear cells, and wash 2 times with normal saline.
7. The method for in vitro expansion of cryopreserved umbilical cord blood Treg cells according to claim 6, wherein in the step (1), the umbilical cord blood mononuclear cells obtained are counted, and 5 x 10 by using a Stemcell sorting buffer7Resuspend cells at concentration/mL and pass through a 37 μm cell sieve; transferring the cell suspension to a round bottom flow type sample loading tube, adding 50 mu L of CD25 Positive Selection Cocktail antibody, mixing uniformly, and incubating at room temperature for 5 min; adding 30. mu.L of Releaseable RapidSpheresTMAnd 50. mu.L of CD4+Mixing T cell Enrichment Cocktail, and incubating at room temperature for 5 min; adding sorting buffer solution to 2.5mL, blowing, beating and mixing uniformly for 2-3 times, and then putting the mixture into a magnetic pole to incubate for 10min at room temperature; pouring off the supernatant, removing the magnetic pole, adding a sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in the magnetic pole, incubating at room temperature for 5min, pouring off the supernatant, and repeating the process for 2 times; removing the magnetic pole, adding 1mL of sorting Buffer solution to resuspend the cells, adding 100 mu L of Release Buffer, and mixing uniformly; add 50. mu.L of CD127highIncubating the Depletion Cocktail at room temperature for 5 min; add 10. mu.L of Dextran RapidSpheresTMIncubating at room temperature for 5 min; adding the sorting buffer solution to 2.5mL, blowing, uniformly mixing, placing in a magnetic pole, incubating for 5min, collecting cell suspension, and centrifuging to obtain cells for later use.
8. The cryopreserved umbilical cord blood Treg cells obtained by the in vitro expansion method for the cryopreserved umbilical cord blood Treg cells according to any one of claims 1 to 7.
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