CN112458053A - Umbilical blood Treg cell in-vitro amplification method based on trophoblast cells and application - Google Patents

Umbilical blood Treg cell in-vitro amplification method based on trophoblast cells and application Download PDF

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CN112458053A
CN112458053A CN202011346010.2A CN202011346010A CN112458053A CN 112458053 A CN112458053 A CN 112458053A CN 202011346010 A CN202011346010 A CN 202011346010A CN 112458053 A CN112458053 A CN 112458053A
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陈勇军
刘少先
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Chengdu Yunce Medical Biotechnology Co ltd
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Abstract

The invention discloses an in-vitro expansion method and application of cord blood Treg cells based on trophoblast cells, and the specific technical method comprises the following steps: firstly, umbilical cord Wharton's jelly mesenchymal stem cells are used as trophoblast cells to induce the primary proliferation of Treg cells in cord blood mononuclear cells; then, separating by magnetic beads to obtain relatively pure Treg cells; and finally, stimulating the rapid expansion of the Treg cells by using the optimized expansion factor. The invention adopts human AB plasma, IL-2, rapamycin, RARA agonist and DNA methyltransferase inhibitor as optimized amplification factors, and can prepare more cord blood Treg cells with higher purity and stronger activity within 2 weeks. The cord blood is used as a raw material for expanding the Treg cells, can be prepared in batches, and can reduce the quality fluctuation of the Treg cells caused by individual difference of samples. The cord blood Treg cells have low immunogenicity, and can be used as universal cells for clinical research, such as autoimmune diseases, graft-versus-host diseases and the like.

Description

Umbilical blood Treg cell in-vitro amplification method based on trophoblast cells and application
Technical Field
The invention relates to the technical field of biomedicine, in particular to an in-vitro amplification method of cord blood Treg cells based on trophoblast cells and application thereof.
Background
The Regulatory T cell (Treg) is CD4+Treg cells, an important cell subset in T cells, have considerable effects on the aspects of inducing organism immune tolerance, maintaining immune environment homeostasis, preventing autoimmune diseases and the like. The Shimon Sakaguchi team first identified CD25(IL-2 receptor alpha-chain, IL-2RA) as a surface marker for Treg cells in 1995, and researchers found that CD4 would be eliminated+CD25+Transplantation of lymphocytes from subpopulations into nude mice can cause various autoimmune diseases, and CD4 is imported+CD25+Treg cells can inhibit the occurrence of diseases. Meanwhile, the Treg cells also specifically express a transcription factor FoxP3, FoxP3 has an important regulation and control effect on development and functions of Tregs, and the gene mutation can cause development, differentiation disorder and dysfunction of the Treg cells. In addition, due to activated CD4 in humans+T cells also highly express CD25, and therefore, one also typically uses CD127 as a supplementary marker to more accurately identify Treg cells.
The immunomodulatory effects of Treg cells are achieved by direct intercellular contact and paracrine effects, the target cells of which are mainly Effector T cells (Teff) and Dendritic Cells (DCs). In addition, macrophages, B cells, NK cells, etc. are also included. The balance of the human immune system is maintained by Treg nuclear Teff cells, and once the balance between the Treg nuclear Teff cells is broken, various diseases can occur. The excessive Treg cell number is closely related to tumors, infection diseases and the like, and the insufficient Treg cell number can cause autoimmune diseases. Studies have shown that a reduction in the number of Treg cells or an abnormality in function occurs in a variety of autoimmune diseases. Intervention in the number and function of Treg cells may be a targeted target for the treatment of a variety of autoimmune and inflammatory diseases.
The number of Tregs in human body is very small, and the Treg cells in peripheral blood only account for CD4+5-10% of T lymphocytes, 100mL of peripheral blood can be separated and purified to 10%6An order of magnitude of Treg cells. The conventional amplification method needs a larger sample volume (250-400 mL of peripheral blood) and a longer amplification period (3-4 weeks). The unstable expression of FoxP3 is easy to occur in the amplification process, which results in the reduction of cell activity and is not favorable for clinical application. Meanwhile, the number and the function of the Treg cells in the body of the autoimmune disease patient are abnormal, so that the in-vitro expansion of the self-body Treg cells is not very facilitated.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for the in-vitro expansion of the Treg cells of the umbilical blood based on the trophoblast cells, which has high cell expansion multiple and strong inhibitory activity, and an application thereof.
The technical scheme adopted by the invention is as follows:
a method for in vitro expansion of cord blood Treg cells based on trophoblast cells comprises the following steps:
step 1: isolating mononuclear cells from cord blood;
step 2: adjusting the density of single nuclear cells to 0.5 × 10 using culture medium6～2×106cells/mL, inoculating into a culture bottle, and adding CD3/CD28 immunomagnetic beads, human AB plasma and IL-2;
inoculating umbilical cord Wharton's jelly mesenchymal stem cells, and counting single cell nuclei of umbilical cord blood: the number ratio of the umbilical cord Wharton's jelly mesenchymal stem cells is 2: 1-10: 1;
and step 3: on the second and fourth days, the culture medium is respectively supplemented, and human AB plasma and IL-2 are added;
and 4, step 4: on the sixth day, cells were collected and CD4 was isolated by magnetic bead sorting+CD25+CD127-Treg cells;
and 5: adjusting the Treg cells isolated in step 4 to 0.5X 10 using culture medium6～2×106cells/mL, inoculating into a culture bottle, and adding an optimized amplification factor and CD3/CD28 immunomagnetic beads;
step 6: on the eighth day, the tenth day and the twelfth day, the culture medium is respectively supplemented, and the optimized amplification factors are added;
and 7: collecting the cord blood Treg cells on the fourteenth day, and freezing and storing the cord blood Treg cells after performing quality detection;
the optimized amplification factors include: human AB plasma, IL-2, rapamycin, RARA agonists, DNA methyltransferase inhibitors.
Further, the addition amount of the human AB plasma and the IL-2 in the step 2 and the step 3 is as follows: the final concentration of the added solution is 5-10 vol% of human AB plasma, and the concentration of IL-2 is 300-1000 IU/ml.
Further, the final concentrations of the components after the optimized amplification factors are added in the steps 5 and 6 are as follows: 5-10 vol% of human AB plasma, 300-1000 IU/mL of IL-2, 10-100 nmol/L of rapamycin, 1-10 nmol/L of RARA agonist and 1-10 mu mol/L of DNA methyltransferase inhibitor.
Further, the ratio of the number of CD3/CD28 immunomagnetic beads to the number of cells in the step 2 and the step 5 is 1: 1-4: 1.
further, the freezing solution adopted in the freezing in the step 7 comprises the following components in percentage by volume: 50 vol% of culture medium, 40 vol% of human AB plasma and 10 vol% of dimethyl sulfoxide.
Further, the method for separating cord blood mononuclear cells in the step 1 comprises the following steps: cord blood was mixed with PBS buffer at 1: 2, then adding the mixture into separation liquid with the same volume, and separating the mononuclear cells by adopting a density gradient centrifugation method.
Further, the cell separation method in the step 4 is as follows: firstly, removing CD4 by magnetic bead negative sorting method-Cells, then enriched for CD25 by magnetic positive sorting+A cell.
Further, the expansion multiple of the cord blood Treg cells expanded in the step 7 is 1400-3800.
Cord blood Treg cell, wherein the cord blood Treg cell is CD4+CD25+High positive rateIn 90%, the positive rate of FoxP3 is more than 80%, the positive rate of HLA-DR is less than 5%, and the effective target ratio is 1: cord blood Treg cells to CD8 at 1+The inhibition rate of T cell proliferation is more than 80%.
An application of Treg cells in preparing the medicines for treating immune diseases, wherein the immune diseases are autoimmune diseases and graft-versus-host disease, and the autoimmune diseases are one of multiple sclerosis, systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, inflammatory bowel disease and psoriasis. .
The invention has the beneficial effects that:
(1) the umbilical cord Wharton's jelly mesenchymal stem cells are used as trophoblast cells, so that the differentiation and the amplification of Treg cells can be induced, the proportion of Treg cells in mononuclear cells is improved, and the subsequent magnetic bead sorting and rapid cell amplification are facilitated;
(2) the invention adopts human AB plasma, IL-2, rapamycin, RARA agonist, DNA methyl transaminase inhibitor as the optimized amplification factor, can maintain the stable expression of FoxP3, and enhance the activity of cord blood Treg cells;
(3) according to the invention, the cord blood is used as a raw material for Treg cell amplification, so that the mass preparation can be realized, and the quality fluctuation of Treg cells caused by individual difference of samples can be reduced;
(4) the Treg cells of the cord blood obtained by the invention have low immunogenicity, and can be used as universal cells for clinical research.
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FIG. 1 is a schematic flow chart of the method of the present invention.
FIG. 2 is a graph showing the results of an analysis of the induction of Treg cell proliferation in mononuclear cells using trophoblasts in example 1 of the present invention.
FIG. 3 shows the results of the cell morphology detection before and after the expansion of the cord blood Treg cells in example 1 of the present invention.
FIG. 4 shows the results of the phenotypic analysis of the cells at different time points after expansion of the cord blood Treg cells in example 1 of the present invention.
FIG. 5 shows the result of the purity test of Treg cells from cord blood in example 1; a is the analysis result of detecting the expression of CD8 and CD19 by flow cytometry; b is the analysis result of detecting the expression of CD3 and CD56 by flow cytometry.
FIG. 6 shows the results of the inhibition experiment of the cord blood Treg cells on lymphocyte proliferation in example 1 of the present invention; a is cord blood Treg cell suppression CD8+Flow analysis of T cell proliferation; b is cord blood Treg cell suppression CD8 with different proportions+Results of an assay for T cell proliferation; c is cord blood Treg cell suppression CD4+Flow analysis of T cell proliferation; d is cord blood Treg cell suppression CD4 with different ratios+Results of T cell proliferation assay.
FIG. 7 shows the results of the fold expansion analysis of the cord blood Treg cells in example 1 and comparative example 1 of the present invention.
FIG. 8 shows the results of phenotypic analysis of cord blood Treg cells in example 1 and comparative example 1 of the present invention.
FIG. 9 shows the results of the fold expansion analysis of the cord blood Treg cells in example 1 and comparative example 2 of the present invention.
FIG. 10 shows the results of phenotypic analysis of cord blood Treg cells in example 1 and comparative example 2 of the present invention.
FIG. 11 is the results of the analysis of the expression of HLA-DR and CD45RA in cord blood Treg and peripheral blood Treg cells in example 1 and comparative example 3 of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The reagents involved in the invention are as follows: lymphoprepTMLymphocyte isolates were purchased from stem cell; CD4+CD25+CD127-Cell sorting kits were purchased from Milteny Biotec; X-VIVO 15 medium was purchased from Lonza; human AB serum was purchased from Fisher Scientific; CD3/CD28 immunomagnetic beads were purchased from Thermo Fisher; IL-2 was purchased from Jiangsu Jinsili pharmaceutical industry; rapamycin was purchased from Milteny Biotec; RARA agonist (AM580) and DNA methyltransferase inhibitor (5-Azacytidine) were purchased from STEMCELL; CD3, CD4, CD8, CD19, CD25, CD25, CD45RA, CD56, FoxP3 and HLA-DR flow detection antibodies, fixation/rupture buffer and fluorescent dye CFSE were purchased from Biolegend; dimethyl sulfoxide (DMSO) was purchased from Santa Cruz.
A method for in vitro expansion of cord blood Treg cells based on trophoblast cells comprises the following steps:
step 1: isolating mononuclear cells from cord blood; the separation method of the cord blood mononuclear cells comprises the following steps: cord blood was mixed with PBS buffer at 1: 2, then adding the mixture into separation liquid with the same volume, and separating the mononuclear cells by adopting a density gradient centrifugation method.
Step 2: adjusting the cord blood mononuclear cells to 0.5 × 10 with the culture medium6～2×106cells/mL, inoculating into a culture bottle, and adding CD3/CD28 immunomagnetic beads, human AB plasma and IL-2;
taking the cord blood mononuclear cells separated in the step 1, suspending the cord blood mononuclear cells by using X-VIVO 15 culture medium basis weight, and carrying out concentration according to a certain density of 0.5 multiplied by 106～2×106cells/mL were inoculated into T175 flasks, and CD3/CD28 immunomagnetic beads were added, along with human AB plasma and IL-2. Wherein the ratio of the CD3/CD28 immunomagnetic beads to the number of cells is 1: 1-4: 1; after the addition, the human AB plasma is 5-10 vol%, and the final concentration of IL-2 is 300-1000 IU/mL.
Resuscitating 3 rd generation umbilical cord Wharton's jelly mesenchymal stem cells as trophoblast cells according to the single nucleus cells of the umbilical cord blood: number ratio of trophoblast cells 2: 1-10: 1 were inoculated into the same T175 flask.
And step 3: on the next and fourth days, respectively adding X-VIVO 15 culture medium in an amount required to maintain cell density of 0.5 × 106～2×106cells/mL; and adding human AB plasma and IL-2, wherein the added human AB plasma is 5-10 vol%, and the final concentration of IL-2 is 300-1000 IU/mL.
And 4, step 4: on the sixth day, cells were collected and CD4 was isolated by magnetic bead sorting+CD25+CD127-Treg cells; specifically, a two-step sorting method, in which CD4 is first separated by a magnetic bead negative sorting method+T cells, followed by isolation of CD25 using magnetic bead positive sorting+A cell.
And 5: adjusting the Treg cells separated in the step 4 to 0.5X 10 by adopting X-VIVO 15 culture medium6～2×106cells/mL, inoculated into T175 flask, addedCD3/CD28 immunomagnetic beads and optimized amplification factors are added. The number ratio of CD3/CD28 immunomagnetic beads to Treg cells is 1: 1-4: 1. the concentration of each component after adding the optimized amplification factor is as follows: 5-10 vol% of human AB plasma, 300-1000 IU/mL of IL-2, 10-100 nmol/L of rapamycin, 1-10 nmol/L of RARA agonist and 1-10 mu mol/L of DNA methyltransferase inhibitor.
Step 6: on the eighth, tenth and twelfth days, the culture medium X-VIVO 15 was added to maintain the cell density at 0.5X 106～2×106cells/mL; and adding optimized amplification factors: human AB plasma, IL-2, rapamycin, RARA agonists, DNA methyltransferase inhibitors; the final concentration is respectively 5-10 vol% of human AB plasma, the concentration of IL-2 is 300-1000 IU/mL, the concentration of rapamycin is 10-100 nmol/L, the concentration of RARA agonist is 1-10 nmol/L, and the concentration of DNA methyltransferase inhibitor is 1-10 mu mol/L.
And 7: and collecting the cord blood Treg cells on the fourteenth day, and freezing and storing the cord blood Treg cells in liquid nitrogen by using a frozen stock solution after quality detection. And (3) freezing and storing the qualified product, wherein the freezing and storing liquid comprises the following components in percentage by volume: 50 vol% of culture medium, 40 vol% of human AB plasma and 10 vol% of dimethyl sulfoxide.
In the preparation process, after the culture medium is supplemented for maintaining the cell density, cell bottling or bag transferring is carried out according to actual conditions.
Example 1
Preparing the cord blood Treg cells according to the following steps:
step 1: 20mL of fresh cord blood was collected, diluted with 2-fold volume of PBS, and mononuclear cells from cord blood were isolated by density gradient centrifugation to give 6.2X 107And (4) cells.
Step 2: mononuclear cells were adjusted to 1X 10 with culture medium6cells/mL, which were inoculated into culture flasks; specifically, it is 5 × 107Cord blood mononuclear cells were resuspended in 50mL of X-VIVO 15 medium and inoculated into a T175 flask and added at 1X 108CD3/CD28 immunomagnetic beads (the ratio of the number of the beads to the number of the cells is 2: 1); simultaneously adding human AB plasma and IL-2; the added AB plasma is 5vol% and the final concentration of IL-2 is 500 IU/mL.
Resuscitating 3 rd generation umbilical cord Wharton's jelly mesenchymal stem cells as trophoblast cells, inoculating 1 × 107The cells were co-cultured in the above T175 flask (cord blood mononuclear cells to trophoblast cells number ratio of 5: 1).
And step 3: the next day, 50mL of X-VIVO 15 medium was added to maintain a suspension cell density of 1X 106cells/mL; and human AB plasma and IL-2 were added, the final concentration of human AB plasma was 5 vol% and IL-2 was 500 IU/mL.
On the fourth day, 100mL of X-VIVO 15 medium was added to maintain the suspension cell density at 1X 106cells/mL; and human AB plasma and IL-2 were added, the final concentration of human AB plasma was 5 vol% and IL-2 was 500 IU/mL.
And 4, step 4: on the sixth day, cells were collected in a number of 2.6X 108Taking 2X 108The individual cells were sorted magnetically according to CD4+CD25+CD127-The kit instruction is operated; labeling cells with CD8, CD19, CD127 and CD123, and removing the cells with LD column to obtain CD 4-enriched cells+A cell. Further sorting of CD25 using CD25 magnetic beads+Cells, 1.8X 107An individual CD4+CD25+CD127-Treg cells.
And 5: resuspension of cells (density 1X 10) was performed using 18mL of X-VIVO 15 medium6cells/mL) into a new T175 flask; then 3.6X 10 are added7CD3/CD28 immunomagnetic beads (the number ratio of the magnetic beads to the cells is 2: 1) are added with optimized amplification factors, and the concentration of each component after the optimized amplification factors are added is as follows: human AB plasma at 5 vol%, IL-2 at 500IU/mL, rapamycin at 100nmol/L, RARA agonist at 5nmol/L, and DNA methyltransferase inhibitor at 5 μmol/L.
Step 6: on the eighth day, 70mL of X-VIVO 15 medium was added to maintain the cell density at 1X 106cells/mL; and adding an optimized amplification factor, wherein the concentration of each component after the optimized amplification factor is added is as follows: human AB plasma at 5 vol%, IL-2 at 500IU/mL, rapamycin at 100nmol/L, RARA agonistThe concentration was 5nmol/L and the concentration of DNA methyltransferase inhibitor was 5. mu. mol/L.
On the tenth day, 250mL of X-VIVO 15 medium was added to maintain the cell density at 1X 106cells/mL; and adding an optimized amplification factor, wherein the concentration of each component after the optimized amplification factor is added is as follows: human AB plasma at 5 vol%, IL-2 at 500IU/mL, rapamycin at 100nmol/L, RARA agonist at 5nmol/L, and DNA methyltransferase inhibitor at 5 μmol/L. After the liquid is added, the cells need to be transferred to a culture bag.
On the twelfth day, 1000mL of X-VIVO 15 medium was added to maintain the cell density at 1X 106cells/mL; and adding an optimized amplification factor, wherein the concentration of each component after the optimized amplification factor is added is as follows: human AB plasma at 5 vol%, IL-2 at 500IU/mL, rapamycin at 100nmol/L, RARA agonist at 5nmol/L, and DNA methyltransferase inhibitor at 5 μmol/L.
And 7: on the fourteenth day, cord blood Treg cells were collected and the residual CD3/CD28 immunomagnetic beads were removed to a total cell count of 3.2X 109. Samples were taken for phenotypic characterization of cells and for in vitro functional assays. Storing the tregs of the cord blood qualified by detection in liquid nitrogen, wherein the frozen stock solution comprises the following components in percentage by volume: 50 vol% of culture medium, 40 vol% of human AB plasma and 10 vol% of dimethyl sulfoxide.
The method for identifying the cell phenotype comprises the following steps: take 1X 106Incubating the cells for 30min by using CD4-APC and CD25-PE antibodies, then fixing and breaking membranes, and incubating for 60min by using FoxP3-FITC antibodies; and finally, loading and detecting. The detection of the other surface molecules CD3, CD8, CD19, CD45RA, CD56 and HLA-DR was carried out according to a conventional method.
The in vitro function test detection method comprises the following steps: freshly isolated peripheral blood mononuclear cells were used as Effector cells (Effector T cells, Teff), labeled with 5. mu.M CSFE dye and labeled at 2X 105One was inoculated into a 96-well plate (U-bottom); cord blood Treg cells were then added in varying numbers (Teff: Treg ═ 1: 1, 2: 1, 4: 1, 8: 1, 16: 1). Sampling after 3-4 days of co-culture, carrying out flow detection, and analyzing CD8+And CD4+Proliferation of cell subsets.
The results show that the number ratio of trophoblast cells to mononuclear cells in this example is 1: 5, the compound can obviously induce the proliferation of Treg cells in mononuclear cells, wherein the CD25+FoxP3+The proportion of positive cells increased from 2.5% at day 0 to 14.6% at day 6 (as shown in figure 2). When CD4 is stimulated by optimized amplification factor+CD25+CD127-After Treg cells, the cells expanded very rapidly and formed clumps of cells by CD3/CD28 immunomagnetic beads, and the clumps became larger and larger with increasing culture time (as shown in fig. 3). The expansion of Treg cells at day 14 was 3780.
The results of the cell phenotype assay are shown in FIG. 4, in this example, CD4 was observed on day 10 of cord blood Treg cells+CD25+The positive rate is 94.03%, and the positive rate of FoxP3 is 95.80%; CD4 at day 14+CD25+The positive rate is 92.95%, and the positive rate of FoxP3 is 89.98%. The cord blood Treg cells in this example are of higher purity and contain almost no other miscellaneous cells. Wherein CD8+Example of T cells 2.26%, CD19+The proportion of B cells was 0.06%, CD3-CD56+The NK cell proportion was 0.07%, CD3+CD56+The NKT cell ratio was 2.73% (as shown in figure 5). The inhibitory capacity of cord blood Treg cells on lymphocyte proliferation was analyzed by in vitro co-culture and the results are shown in fig. 6 when Treg: teff is 1: 1, cord blood Treg cells to CD8+The inhibition rate of T cell proliferation was 86.1% for CD4+The proliferation inhibition rate of the T cells is 70.9%; and the inhibition efficiency of the cord blood Treg cells on lymphocyte proliferation is closely related to the effective target ratio.
Example 2
Cord blood Treg cells were prepared following the same procedure as in example 1, except that the final concentrations of the components after addition of the optimized expansion factors in steps 5 and 6 were: human AB plasma at 10 vol%, IL-2 concentration at 1000IU/mL, rapamycin concentration at 100nmol/L, RARA agonist concentration at 10nmol/L, and DNA methyltransferase inhibitor concentration at 10. mu. mol/L. The results show that the phenotype of the Treg cells in this example is similar to that of example 1, where CD4 is present+CD25+Positive rate is greater than 90%, FoxP3 positive rate is more than 80%; and the expansion fold of Treg cells at day 14 was 3059.
Example 3
Cord blood Treg cells were prepared following the same procedure as in example 1, except that the final concentrations of the components after addition of the optimized expansion factors in steps 5 and 6 were: human AB plasma at 5 vol%, IL-2 at 300IU/mL, rapamycin at 100nmol/L, RARA agonist at 1nmol/L, and DNA methyltransferase inhibitor at 1 μmol/L. The results show that the phenotype of the Treg cells in this example is similar to that of example 1, where CD4 is present+CD25+The positive rate is more than 90%, and the positive rate of FoxP3 is more than 80%; and the expansion of Treg cells on day 14 was 1446.
Comparative example 1
Other procedures for preparing cord blood Treg cells were as in example 1, except that no trophoblast cells were added in step 2. From fig. 7, it can be seen that the average expansion fold of the cord blood Treg cells obtained by the trophoblast-based culture method is significantly higher than that of the non-trophoblast culture method. From FIG. 8, it can be seen that there are two ways of trophoblasts and non-trophoblasts to obtain CD4 in Treg cells from cord blood+CD25+The positive rate and the positive rate of FoxP3 are not obviously different. Therefore, the culture method based on the trophoblast cells is more beneficial to the expansion of the Treg cells of the cord blood. This is because mesenchymal stem cells are pluripotent stem cells having an immunoregulatory function, and have an important regulatory role in both innate immunity and adaptive immune response. Research has proved that mesenchymal stem cells can promote the proliferation and differentiation of Treg cells by secreting various cytokines (such as TGF-beta, PGE 2).
Comparative example 2
Other steps for preparing cord blood Treg cells were as in example 1, except that the amplification system used in steps 5 and 6 was not supplemented with optimized amplification factors, but with human AB plasma and IL-2 alone. As can be seen from fig. 9, there was no significant difference in the fold expansion of the cord blood Treg cells obtained in the control group and the optimized group. As can be seen from fig. 10, the FoxP3 positive rate in cord blood Treg cells in the optimized group was 90.28%, and the FoxP3 positive rate in cord blood Treg cells in comparative example 2 was 59.44%; therefore, the optimized amplification factor is more beneficial to maintaining the stable expression of the cord blood Treg cells FoxP 3.
Comparative example 3
Other procedures for preparing cord blood Treg cells were as in example 1, except that the cord blood in step 1 was replaced with peripheral blood. As can be seen from fig. 11, the cord blood Treg cells highly express CD45RA, but hardly express HLA-DR, compared to the peripheral blood Treg cells, and thus have lower immunogenicity, and can be used as universal cells for clinical treatment.
According to the invention, firstly, umbilical cord Wharton's jelly mesenchymal stem cells are used as trophoblast cells to induce the primary proliferation of Treg cells in cord blood mononuclear cells, so that the proportion of Treg cells in cord blood mononuclear cells can be increased, and the subsequent magnetic bead sorting and rapid cell expansion are facilitated. Then, purer Treg cells are obtained through magnetic bead sorting, and the optimized amplification factors are used for stimulating the rapid expansion of the Treg cells. The cord blood Treg cell product with high quantity, purity and activity can be obtained in a short time. Can maintain the stable expression of FoxP3 and enhance the activity of cord blood Treg cells. The fresh cord blood or cord blood hematopoietic stem cells stored by liquid nitrogen are used as raw materials for expanding the Treg cells, so that the quality fluctuation of the Treg cells caused by individual difference of samples can be reduced, and the mass preparation can be realized. In addition, the Treg cells of the cord blood have low immunogenicity, and can be used as universal cells for clinical research. The cord blood Treg cells obtained by the preparation method have high expansion multiple and strong inhibition activity. The Treg cells of the cord blood can be produced in batches and stored in liquid nitrogen, and can be used as a general cell product for clinical disease treatment, such as autoimmune diseases, graft-versus-host diseases, organ transplantation rejection and the like.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all additions and modifications made on the basis of the technical method of the present invention are included in the scope of the present invention.

Claims (10)

1. A method for expanding cord blood Treg cells in vitro based on trophoblast cells is characterized by comprising the following steps:
step 1: isolating mononuclear cells from cord blood;
step 2: adjusting the density of single nuclear cells to 0.5 × 10 using culture medium6～2×106cells/mL, inoculating into a culture bottle, and adding CD3/CD28 immunomagnetic beads, human AB plasma and IL-2;
inoculating umbilical cord Wharton's jelly mesenchymal stem cells, and counting single cell nuclei of umbilical cord blood: the number ratio of the umbilical cord Wharton's jelly mesenchymal stem cells is 2: 1-10: 1;
and step 3: on the second and fourth days, the culture medium is respectively supplemented, and human AB plasma and IL-2 are added;
and 4, step 4: on the sixth day, cells were collected and CD4 was isolated by magnetic bead sorting+CD25+CD127-Treg cells;
and 5: adjusting the Treg cells isolated in step 4 to 0.5X 10 using culture medium6～2×106cells/mL, inoculating into a culture bottle, and adding an optimized amplification factor and CD3/CD28 immunomagnetic beads;
step 6: on the eighth day, the tenth day and the twelfth day, the culture medium is respectively supplemented, and the optimized amplification factors are added;
and 7: collecting the cord blood Treg cells on the fourteenth day, and freezing and storing the cord blood Treg cells after performing quality detection;
the optimized amplification factors include: human AB plasma, IL-2, rapamycin, RARA agonists, DNA methyltransferase inhibitors.
2. The method for the in vitro expansion of the Treg cells in the umbilical blood based on the trophoblast cells as claimed in claim 1, wherein the addition amount of the human AB plasma and the IL-2 in the steps 2 and 3 is as follows: the final concentration of the added solution is 5-10 vol% of human AB plasma, and the concentration of IL-2 is 300-1000 IU/ml.
3. The method for in vitro expansion of Treg cells in umbilical blood based on trophoblast cells according to claim 1, wherein the final concentrations of the components after adding the optimized expansion factors in the steps 5 and 6 are as follows: 5-10 vol% of human AB plasma, 300-1000 IU/mL of IL-2, 10-100 nmol/L of rapamycin, 1-10 nmol/L of RARA agonist and 1-10 mu mol/L of DNA methyltransferase inhibitor.
4. The method for expanding the umbilical blood Treg cells based on the trophoblast cells in vitro as claimed in claim 1, wherein the ratio of the number of CD3/CD28 immunomagnetic beads to the number of the cells in the step 2 and the step 5 is 1: 1-4: 1.
5. the method for the in vitro expansion of the Treg cells in the cord blood based on the trophoblast cells according to claim 1, wherein the cryopreservation solution adopted in the step 7 comprises the following components in volume ratio: 50 vol% of culture medium, 40 vol% of human AB plasma and 10 vol% of dimethyl sulfoxide.
6. The method for expanding Treg cells in cord blood based on trophoblast cells in vitro as claimed in claim 1, wherein the method for isolating mononuclear cells in cord blood in step 1 comprises: cord blood was mixed with PBS buffer at 1: 2, then adding the mixture into separation liquid with the same volume, and separating the mononuclear cells by adopting a density gradient centrifugation method.
7. The method for expanding the umbilical blood Treg cells based on the trophoblast cells in vitro as claimed in claim 1, wherein the cell separation method in the step 4 is as follows: firstly, removing CD4 by magnetic bead negative sorting method-Cells, then enriched for CD25 by magnetic positive sorting+A cell.
8. The method for in vitro expansion of the Treg cells in umbilical blood based on trophoblast cells according to claim 1, wherein the expansion multiple of the Treg cells in umbilical blood expanded in the step 7 is 1400-3800.
9. Cord blood Treg cells obtainable by a method according to any one of claims 1 to 8, wherein said cord blood Treg cells are CD4+CD25+The positive rate is more than 90%, the positive rate of FoxP3 is more than 80%, the positive rate of HLA-DR is less than 5%, and the effective target ratio is 1: cord blood Treg cells to CD8 at 1+The inhibition rate of T cell proliferation is more than 80%.
10. The application of the cord blood Treg cells obtained by the method according to any one of claims 1 to 8 in preparing a medicine for treating immune diseases, wherein the immune diseases are autoimmune diseases and graft-versus-host disease, and the autoimmune diseases are one of multiple sclerosis, systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, inflammatory bowel disease and psoriasis.
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