CN116396934B - Culture method of cord blood Treg cells - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention provides a culture method of cord blood Treg cells, which comprises the steps of firstly separating diluted cord blood by using a lymph separating liquid to obtain PBMC layer liquid, then further separating the PBMC layer liquid by using MACS buffer, CD25 MicroBeads II and LS column to obtain Treg cells, then sequentially using an ACTM culture medium and an EXPM culture medium to perform in-vitro expansion culture on the Treg cells, and combining IL-2, IL-10, TGF-beta and rapamycin to perform stimulated proliferation on the Treg cells, so that the number and cell activity of the Treg cells are effectively improved, and meanwhile, the culture success rate of the Treg cells is remarkably improved.
Description
Technical Field
The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to a culture method of cord blood Treg cells.
Background
Regulatory T cells (regulatory T cells, tregs) are an important T cell subset that plays an important role in regulating autoimmune responses, tissue damaging immunopathogenesis and maintaining the immune balance of the body. Cd4+cd25+ T cells are the most prominent population in the Tregs family, and were first found in 1995 to account for about 1% of normal human peripheral blood mononuclear cells. Experiments have shown that Treg cells have therapeutic effects on a variety of immune dysregulated diseases and have significant effects in animal models of graft versus host reactions, organ transplantation, autoimmune diseases, and the like.
Meanwhile, the research also finds that the MHCI antigen expression level of the cord blood-derived Treg cells is obviously reduced compared with that of the adult peripheral blood Treg cells, and proves that the cord blood-derived Treg cells can avoid immune rejection reaction of a transplantation receptor and can better play an immunosuppressive function in vivo. Although cord blood derived Treg cells have better therapeutic effects and higher levels than peripheral blood, a single fresh isolated Treg cell fraction still does not reach the number of clinical treatments.
In addition, the Treg cells from autologous blood are greatly influenced by factors such as age and physical conditions of a donor during in-vitro expansion culture, and a stable and effective expansion culture system does not exist during in-vitro culture of the Treg cells from autologous blood, so that the types of cytokines added by the conventional culture system are single, and the cell expansion effect is poor or even fails. Meanwhile, autologous blood-derived Treg cells cannot be used for allogeneic use, but can only be used for autologous reinfusion. In the prior art, no matter for Treg cells from autologous blood or cord blood, the biological activity of the expanded Treg cells is low, the success rate of cell expansion is low, and the cultured Treg cells cannot be applied in a variant way.
Disclosure of Invention
Based on the technical background, the inventor provides a culture method of cord blood Treg cells, which is characterized in that ACTM culture medium and EXPM culture medium are used for carrying out in-vitro expansion culture on the Treg cells for the first time, IL-2, IL-10, TGF-beta and rapamycin are added into the culture medium to stimulate proliferation of the Treg cells, so that the number of the cultured Treg cells is sufficient, the activity is high, the culture success rate of the Treg cells is remarkably improved, and meanwhile, the cultured Treg cells can be subjected to allogeneic or xenogeneic feedback.
The invention provides a culture method of cord blood Treg cells, which comprises the following steps:
step 1, diluting umbilical blood with normal saline to obtain umbilical blood diluent;
step 2, adding the cord blood dilution obtained in the step 1 into lymphocyte separation liquid, centrifuging, sucking out the upper layer liquid containing cord blood mononuclear cell layer, adding RPMI 1640 culture medium into the liquid containing cord blood mononuclear cell layer, re-suspending, centrifuging, and discarding supernatant;
step 3, adding MACS buffer and CD25 MicroBeads II (human CD25 magnetic beads) into the liquid containing the cord blood mononuclear cell layer after resuspension and centrifugation, uniformly mixing, placing in a dark place, adding MACS buffer and centrifuging, discarding supernatant, adding MACS buffer and resuspension, injecting into LS column (cell sorting column), adding MACS buffer and washing, centrifuging, discarding supernatant to obtain Treg cells;
step 4, placing Treg cells in a 48-well plate, and adding ACTM culture containing Human AB serum, rapamycin and IL-2Culturing with culture medium and magnetic beads until the number of cells reaches 4×10 5 Hole, re-suspending cell sediment, adding fetal calf serum and IL-2, inoculating in 48 hole plate for proliferation culture, and adding ACTM culture medium on 3 rd and 5 th days for 0 day;
and 5, adding an EXPM culture medium containing IL-2 on the 7 th day, and then supplementing the EXPM culture medium on the 9 th and 11 th days to obtain the Treg proliferation cells.
Drawings
Fig. 1 shows graphs of Treg cell proliferation on day 0, day 1, day 3, day 5, day 7, day 9, day 11, day 13, day 15, day 17, day 19 and day 21 of expansion culture in example 1;
figures 2 and 3 show positive staining photographs of anti-human CD45 and CD8 after PBMC injection and Treg expansion of example 1 injection, respectively.
Detailed Description
The features and advantages of the present invention will become more apparent and evident from the following detailed description of the invention.
The invention provides a culture method of cord blood Treg cells, which comprises the following steps:
step 1, diluting umbilical blood with normal saline to obtain umbilical blood diluent;
step 2, adding the cord blood dilution obtained in the step 1 into lymphocyte separation liquid, centrifuging, sucking out the upper layer liquid containing cord blood mononuclear cell layer, adding RPMI 1640 culture medium into the liquid containing cord blood mononuclear cell layer, re-suspending, centrifuging, and discarding supernatant;
step 3, adding MACS buffer and CD25 MicroBeads II (human CD25 magnetic beads) into the liquid containing the cord blood mononuclear cell layer after resuspension and centrifugation, uniformly mixing, placing in a dark place, adding MACS buffer and centrifuging, discarding supernatant, adding MACS buffer and resuspension, injecting into LS column (cell sorting column), adding MACS buffer and washing, centrifuging, discarding supernatant to obtain Treg cells;
step 4, placing Treg cells in a 48-well plate, and adding a cell line containing Human AB serum, rapamycin and ILACTM Medium and magnetic beads of-2 were cultured until the cell number reached 4X 10 5 Hole, re-suspending cell sediment, adding fetal calf serum and IL-2, inoculating in 48 hole plate for proliferation culture, and adding ACTM culture medium on 3 rd and 5 th days for 0 day;
and 5, adding an EXPM culture medium containing IL-2 on the 7 th day, and then supplementing the EXPM culture medium on the 9 th and 11 th days to obtain the Treg proliferation cells.
This step is specifically described below.
And 1, diluting the cord blood with normal saline to obtain cord blood diluent.
The volume ratio of the physiological saline to the cord blood is (0.8-1.5): 1, preferably 1:1. Evenly mixing to obtain the cord blood diluent.
And 2, adding the cord blood dilution obtained in the step 1 into the lymphocyte separation liquid, centrifuging, sucking out the upper layer liquid containing cord blood mononuclear cell layers, adding RPMI 1640 culture medium into the liquid containing cord blood mononuclear cell layers, re-suspending, centrifuging, and discarding the supernatant.
The volume ratio of the cord blood diluent to the lymphocyte separation liquid is (1.5-3): 1, preferably 2:1.
The cord blood dilution is preferably slowly transferred to the surface of the lymphocyte separation medium, so that a clear interface is formed between the cord blood dilution and the lymphocyte separation medium.
The centrifugation is preferably carried out in a centrifuge with a first centrifugation speed of 500 to 1000g, preferably 700 to 800g.
The first centrifugation time is 5 to 20 minutes, preferably 10 to 20 minutes.
When the upper layer contains liquid of mononuclear cell layer of cord blood (i.e. liquid of PBMC layer), care should be taken not to suck out the red blood cell layer together.
The second centrifugation speed is 300 to 800g, preferably 400 to 500g.
The second centrifugation time is 3 to 10 minutes, preferably 4 to 7 minutes. Ensuring the biological activity of Treg cells.
And 3, adding MACS buffer and CD25 MicroBeads II into the liquid containing the cord blood mononuclear cell layer after resuspension and centrifugation, uniformly mixing, placing in a dark place, adding MACS buffer again, centrifuging, discarding supernatant, adding MACS buffer for resuspension, injecting into an LS column, adding MACS buffer into the LS column, washing, centrifuging, discarding supernatant, and thus obtaining the Treg cells.
In the invention, the MACS buffer takes PBS (polybutylene succinate) as a diluent, and also contains 0.4-0.7% of BSA (N, O-bis (trimethylsilyl) acetamide) and 1-3 mmol/L of EDTA (ethylenediamine tetraacetic acid).
The amount of MACS buffer added for the first time was 1X 10 per Treg cell number 7 The individual Treg cells are supplemented with 70-100. Mu.L MACS buffer, preferably every 1X 10 7 mu.L of MACS buffer was added to each cell.
The CD25 microblades II was 1X 10 per unit 7 The addition of 5 to 20. Mu.L, preferably 1X 10, per Treg cell 7 10-15 mu L of each Treg cell is added.
The temperature of the light-shielding place is 2-4 ℃, and the time of the light-shielding place is 3-5 min.
The addition amount of MACS buffer added for the second time is 1×10 7 1-5. Mu.L MACS buffer is added to each Treg cell, preferably every 1X 10 7 1-2. Mu.L MACS buffer was added to each Treg cell.
The addition amount of MACS buffer added for the third time is 1×10 8 400-700. Mu.L MACS buffer is added to each Treg cell, preferably every 1X 10 7 Each Treg cell was supplemented with 500. Mu.L of MACS buffer.
Step 4, placing Treg cells in a 48-well plate, adding ACTM culture medium containing Human AB serum, rapamycin and IL-2 and magnetic beads for culturing, when the cell number reaches 4×10 5 Cell pellet was resuspended, added with fetal bovine serum, IL-2, inoculated in 48 well plates for proliferation culture for 0 day on day 3 and day 5 with ACTM medium.
The ACTM culture medium contains 5-20wt% of Human AB serum, 50-150 nmol/L of rapamycin and 500-1000 IU/mL of IL-2.
Preferably, the ACTM culture medium contains 10-15wt% of Human AB serum, 80-120 nmol/L of rapamycin and 700-800 IU/mL of IL-2.
According to the invention, an ACTM culture medium is used for in-vitro expansion culture of Treg cells, wherein rapamycin can inhibit TOR complex of the cells, and a large number of experiments show that the adoption of the ACTM culture medium containing Human AB serum, rapamycin and IL-2 is beneficial to the improvement of the expansion number and the cell activity of the Treg cells.
The magnetic beads are magnetic beads coated by CD3/CD28, and can effectively stimulate the growth and proliferation of Treg cells.
The number ratio of the magnetic beads to the Treg cells is (2-5): 1, preferably 3:1.
The culture condition of Treg cells is 35-37 ℃, and CO 2 The content is 4-7%.
In step 4, the amount of IL-2 added for the second time is 400 to 2000U/mL, preferably 500 to 1500U/mL.
According to the preferred embodiment of the invention, IL-10, TGF-beta and rapamycin are added after the cell precipitation is resuspended, and the combined action of IL-2, IL-10, TGF-beta and rapamycin further stimulates the expansion of the Treg cells, so that the success rate of culturing the Treg cells is improved.
The addition amount of the IL-10 is 400 to 1700U/mL, preferably 500 to 1500U/mL.
The TGF-beta is added in an amount of 40 to 150ng/mL, preferably 50 to 100ng/mL.
The addition amount of the rapamycin is 50-150U/mL, preferably 90-120U/mL.
And 5, adding an EXPM culture medium containing IL-2 on the 7 th day, and then supplementing the EXPM culture medium on the 9 th and 11 th days to obtain the Treg proliferation cells.
The EXPM medium contains 500 to 1500U/mL IL-2, preferably 800 to 1200U/mL IL-2.
In the prior art, the in vitro culture of Treg cells is usually carried out by adopting a 1640 basal medium, and IL-2 is a commonly used cytokine for stimulating in vitro cell culture, but the successful expansion probability of cells is low. According to the invention, the ACTM culture medium and the EXPM culture medium are adopted, and the IL-2, the IL-10, the TGF-beta and the rapamycin are combined to stimulate and proliferate the cells, so that the proliferation quantity of the Treg cells is effectively increased, the activity and the culture success rate of the expanded and cultured Treg cells are obviously improved, and meanwhile, the Treg cells obtained through culture can be used for foreign bodies and also can be used for autologous transfusion.
Depending on the proliferation of the cells, the EXPM medium is supplemented on days 9, 11, 13, 15, 17, 19, 21.
The invention has the beneficial effects that:
(1) The invention creatively uses two types of culture mediums, combines IL-2, IL-10, TGF-beta, rapamycin and the like to stimulate and proliferate cells, and the obtained cells have sufficient quantity and high cell activity.
(2) The culture method is not only limited to expanding the Treg cells, but also can obviously improve the culture success rate and the biological activity of the Treg cells, the expansion quantity of the Treg cells can reach more than 800 times stably, and the activity rate is basically maintained to be more than 98%;
(3) The Treg cells obtained by culture in the culture method can be subjected to allogenetic or even xenogenic feedback, and are safe and effective;
(4) The culture method is simple to operate, can realize the large-scale expansion of the cord blood-derived Treg cells, and develop new ideas for further research and clinical application of the Treg cells.
Examples
The invention is further illustrated by the following specific examples, which are intended to be illustrative of the invention and are not intended to limit the scope of the invention.
The cytokines, the culture medium and the like adopted in the embodiment of the invention are all commercial products.
Example 1
(1) The cord blood in the anticoagulation tube was transferred to a 50mL centrifuge tube with a disposable pipette, and 2mL of peripheral blood was drawn with a Pasteur pipette and dispensed into two EP (epoxy) tubes, one for bacterial and fungal detection and one for sample retention. Adding an equivalent amount of physiological saline into a centrifuge tube containing cord blood by a disposable pipette for dilution, wherein the volume ratio of the physiological saline to the cord blood is 1:1, and obtaining cord blood diluent.
(2) A new 50-mL centrifuge tube is additionally taken, 12.5ml of lymphocyte separation liquid is added, and the umbilical cord blood dilution liquid is slowly transferred to the surface of the lymphocyte separation liquid, so that a clear interface is formed between the umbilical cord blood dilution liquid and the lymphocyte separation liquid, and the volume ratio of the umbilical cord blood dilution liquid to the lymphocyte separation liquid is 2:1. The tube was transferred to a centrifuge and centrifuged at 800g for 15 min. After centrifugation, the supernatant liquid containing the cord blood mononuclear cells (PBMC) layer was aspirated, transferred to another 50mL centrifuge tube, taking care not to aspirate the red blood cell layer together. PBMCs were resuspended to 40 mL with disposable pipette plus RPMI 1640 medium, centrifuged at 500g for 5min and the supernatant discarded after centrifugation.
(3) Every 1X 10 according to the number of cells 7 Each cell was added with 90. Mu.L of MACS buffer (MACS buffer in which PBS was used as a diluent, and 0.5% BSA (N, O-bis (trimethylsilyl) acetamide) and 2 mmol/L EDTA (ethylenediamine tetraacetic acid)) and 10. Mu.L of CD25 microblads II, thoroughly mixed, and left at 4℃for 5min in the absence of light. Then according to every 1×10 7 Adding 1~2 mL MACS buffer to each cell, centrifuging, discarding supernatant, and every 1×10 8 Individual cells were resuspended with 500 μl MACS buffer. The cell suspension was poured into a rinsed LS column (cell sorting column), the column was washed 3 times with 3 mL MACS buffer, and finally the Treg cells were washed off with 5mL MACS buffer, centrifuged, and the supernatant was discarded to give Treg cells, which were counted.
(4) Treg cells were cultured in 48-well round bottom plates and ACTM medium (available from Beijing daceae Biotechnology Co., ltd.) containing 10wt% Human AB serum, 100nmol/L rapamycin and 800 IU/mL IL-2 was added, and CD3/CD28 coated magnetic beads were added at a ratio of cell:magnetic bead=1:3 to stimulate cell growth. At 37℃with 5% CO 2 Culturing under conditions until the number of cells reaches 4×10 5 Cells were passaged and passage time and fold expansion were recorded. Cell pellet was then resuspended in ACTM medium, 1mL of fetal bovine serum was added, and 1000U/mL IL-2, 1000U/mL IL-10, 100ng/mL TGF- β, 100U/mL rapamycin were added based on the amount of medium added and inoculated into 48 well plates for a time period of day 0. ACT containing 1000U/mL IL-2, 1000U/mL IL-10, 100 ng/mLTGF-beta, 100U/mL rapamycin was supplemented on days 3 and 5M medium.
(5) EXPM medium (available from Biotechnology Inc. of Beijing) containing 1000U/mL IL-2 was supplemented on day 7 and cells were transferred to a 1L cell culture bag. Simultaneously, 350mL of EXPM medium containing 1000U/mL of IL-2 was added on days 9 and 11, and the culture was performed until 21 days, with the addition of the liquid according to the proliferation of cells.
Experimental example
Experimental example 1 cell number and viability detection
Cell number and viability assays were performed on Treg cell suspensions from example 1 on day 0, day 1, day 3, day 5, day 7, day 9, day 11, day 13, day 15, day 17, day 19 and day 21, respectively, 10 μl of each Treg cell suspension was added to 10 μl of AO/PI dye (nucleic acid dye acridine orange/propidium iodide), mixed well, and added to a cell counting plate, and cell viability was measured by a cellmeter Auto 2000. The test results are shown in table 1 and fig. 1.
TABLE 1
As can be seen from Table 1 and FIG. 1, treg cells grew slowly in vitro during the first 3 days, starting from day 5 and entering the exponential growth phase, starting from the original 1X 10 cells 6 cells started to proliferate, and the cells started to enter the plateau phase on day 17, and proliferated slowly until the cell number was 8.45X10 8 cells, at day 21, treg cells stably expanded to 882 times the number of primordial cells. The Treg cell viability was maintained substantially above 98% throughout the cell culture.
Experimental example 2 cell flow assay
Respectively taking 2×10 6 Example 1 of cells Treg cells isolated with MACS buffer and expanded cultured Treg cell suspensions were washed twice with PBS containing 5% FBS (fetal bovine serum), resuspended with PBS containing 10% FBS, and the expanded cultured Treg cell suspensions were used as sample groups, and the Treg cells isolated with MACS buffer were used as control groups, and the flow rate was increased to two2.5Ul of antibodies CD4, CD25, CD45RA and CD45RO are added into the tube, the mixture is fully and uniformly mixed, and incubated for 30min in dark, washed twice with PBS containing 10% FBS, resuspended with 1640 basal medium, and the CD4 is detected by an up-flow instrument + CD25 + Cell content of CD45RA, CD45 RO. The test results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the isolated Treg cells were detected after magnetic bead sorting, CD4 + CD25 + The purity of (C) is about 91%, and the purity of (C) is about 91% for CD45RA + About 72% of the expression of CD45RO + The expression of (2) is about 36%. The Treg cells obtained by the amplification culture method of the invention are used for preparing CD4 + CD25 + The purity expression of (C) is increased to more than 96%, and the purity expression of (C) is improved to CD45RA + The expression of (C) was reduced to 0.09%, for CD45RO + The expression of (2) was increased to 93% or more. The increase in expression of Treg cell functional molecules indicates that the expanded Treg cells have stronger functions.
Experimental example 3 stimulation test
Peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) are extracted, hydroxyfluorescein diacetate succinimidyl ester (5, 6-carboxyfluorescein diacetate succinimidyl ester, CFSE) is marked as effector cells, the Treg cells separated by MACS buffer and the expanded and cultured Treg cells in the example 1 are irradiated by X-ray sublethal dose 30 Gy, the peripheral blood mononuclear cells and the Treg cells separated by MACS buffer are mixed and cultured according to the proportion of 1:1, the peripheral blood mononuclear cells and the expanded and cultured Treg cells are mixed and cultured according to the proportion of 1:1, 10:1 and 20:1 respectively, and the proliferation of the effector cells is detected by flow cytometry after 7 d. The results are shown in Table 3.
TABLE 3 Table 3
As can be seen from table 3, the inhibition ability of the Treg cells isolated was that the proliferation efficiency was only about 0.59% even at an effector cell concentration ratio of 1:1, and the proliferation of effector cells could not be effectively inhibited. The inhibition rate of the Treg cells after the expansion culture is about 89% when the ratio of the Treg cells to the effector cells is 1:1, and the inhibition rate of the Treg cells after the expansion culture is about 75% and 30% when the ratio of the effector cells to the Treg cells after the expansion culture is 10:1 and 20:1, so that the Treg cells after the expansion culture have the capability of inhibiting proliferation of the effector cells, and have a great inhibition effect on proliferation of the effector cells.
Experimental example 4 establishment of GVHD model (graft versus host disease)
Extraction of PBMC isolated from example 1, resuspended to 1X 10 5 Mu L, 1X 10 7 Is intravenously injected into NOD-Prkdcsccid IL2rgnull (NPG) mice from the tail of the mice, and is used as a control group; the experimental group adopts 1×10 7 Example 1 expanded cultured Treg cells were injected intravenously from the tail of mice into NOD-Prkdcscid IL2rgnull (NPG) mice. Mice were continuously observed for changes in body weight, hair, morphology and mobility after injection, and after the control mice developed graft-versus-host disease (GVHD), the spleen and liver of the control mice and the experimental mice were taken at the corresponding time points for immunohistochemical analysis, dewaxed, hydrated, and then developed with anti-human CD8 and CD45 antibodies, and human CD8 and CD45 positive cells were detected. Positive staining photographs of the control and experimental groups are shown in fig. 2 and 3, respectively.
In fig. 2 and 3, brown color indicates positive staining, and in fig. 2, it can be seen that positive staining occurs for both anti-human CD45 and CD8 antibodies after injection of PBMC cells, and in fig. 3, positive staining occurs for anti-human CD45 after injection of Treg cells of example 1, but no positive staining occurs for CD8 antibodies, and in vivo experiments in mice indicate that expansion of cultured Treg cells by the method of the present invention does not cause graft versus host reaction in the recipient.
The invention has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.
Claims (6)
1. A method of culturing umbilical cord blood Treg cells, the method comprising the steps of:
step 1, diluting umbilical blood with normal saline to obtain umbilical blood diluent;
step 2, adding the cord blood dilution obtained in the step 1 into lymphocyte separation liquid, centrifuging, sucking out the upper layer liquid containing cord blood mononuclear cell layer, adding RPMI 1640 culture medium into the liquid containing cord blood mononuclear cell layer, re-suspending, centrifuging, and discarding supernatant;
step 3, adding MACS buffer and CD25 MicroBeads II into the liquid containing the cord blood mononuclear cell layer after resuspension and centrifugation, uniformly mixing, placing in a dark place, adding MACS buffer again, centrifuging, discarding supernatant, adding MACS buffer for resuspension, injecting into an LS column, adding MACS buffer into the LS column, washing, centrifuging, discarding supernatant, and obtaining Treg cells;
step 4, placing Treg cells in a 48-well plate, adding ACTM culture medium containing Human AB serum, rapamycin and IL-2 and magnetic beads for culturing, when the cell number reaches 4×10 5 Hole, re-suspending cell sediment, adding fetal calf serum and IL-2, inoculating in 48 hole plate for proliferation culture, adding ACTM culture medium on day 3 and day 5 for 0 day;
the ACTM culture medium contains 10wt% of Human AB serum, 100nmol/L rapamycin and 800 IU/mL IL-2;
after cell pellet was resuspended, 1mL of fetal bovine serum, 1000U/mL IL-2, 1000U/mL IL-10, 100ng/mL TGF-beta, 100U/mL rapamycin was added;
ACTM medium containing 1000U/mL IL-2, 1000U/mL IL-10, 100 ng/mLTGF-beta, 100U/mL rapamycin was supplemented on days 3 and 5;
step 5, adding an EXPM culture medium containing IL-2 on the 7 th day, and then supplementing the EXPM culture medium on the 9 th and 11 th days to obtain Treg proliferation cells;
the EXPM medium contains 1000U/mL IL-2, and 350mL EXPM medium containing 1000U/mL IL-2 is supplemented on days 9 and 11.
2. The method according to claim 1, wherein in step 1,
the volume ratio of the normal saline to the cord blood is (0.8-1.5): 1.
3. the culture method according to claim 1, wherein in step 2,
the volume ratio of the cord blood diluent to the lymphocyte separation liquid is (1.5-3): 1.
4. the culture method according to claim 1, wherein in step 3,
the addition amount of MACS buffer added for the first time is 1×10 7 Adding 70-100 mu L of MACS buffer into each Treg cell;
the CD25 microblades II was 1X 10 per unit 7 5-20 mu L of each Treg cell is added.
5. The culture method according to claim 1, wherein in step 3,
the addition amount of MACS buffer added for the second time is 1×10 7 Adding 1-5 mu L of MACS buffer into each Treg cell;
the addition amount of MACS buffer added for the third time is 1×10 8 400-700. Mu.L MACS buffer was added to each Treg cell.
6. The method according to claim 1, wherein in step 4,
the magnetic beads are magnetic beads coated by CD3/CD28, and the number ratio of the magnetic beads to the Treg cells is (2-5): 1.
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| CN114703135A (en) * | 2022-05-09 | 2022-07-05 | 协和干细胞基因工程有限公司 | In-vitro amplification method for cryopreserved umbilical cord blood Treg cells |
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| CN114703135A (en) * | 2022-05-09 | 2022-07-05 | 协和干细胞基因工程有限公司 | In-vitro amplification method for cryopreserved umbilical cord blood Treg cells |
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