CN102517253B - In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood - Google Patents
In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood Download PDFInfo
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Abstract
The invention relates to an in vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood. The method comprises the following steps of: separating a CD4+CD25+T cell group rich in the regulatory T cells from the umbilical cord blood; amplifying the enriched CD4+CD25+T cell group to obtain high-purity regulatory T cells; and storing the amplified regulatory T cells in liquid nitrogen at a low temperature to serve as third-party unrelated donor regulatory T cells for treating clinical diseases. The method has the advantages that: a large quantity of regulatory T cells can be obtained from the umbilical cord blood and stored for a long term in the liquid nitrogen, can meet the requirements of clinic for regulatory T cell products, and are used for treating graft versus host disease, organ transplantation rejection and autoimmune disease.
Description
Technical field
The present invention relates to a kind of cell expansion ex vivo and store method, specifically, is a kind of Cord blood regulatory T cells amplification in vitro and Cryopreservation.
Background technology
Acceptor is the major cause of tissue and Transplanted cells failure to the unmatched graft generation of MHC rejection.In clinical practice at present, the use of tradition immunosuppressive drug, though can effectively prevent transplant rejection, alleviation is by cell-mediated acute and chronic graft versus host disease (GVH disease) (the graft-versus-host disease of donor T, GVHD), but often with the whole lower immune function of receptor, the side effects such as infection chance increase and induced tumor.Therefore, take and induce the cell therapy method that between donor-recipient, immune tolerance is object day by day to come into one's own.CD4+CD25+Foxp3+ regulatory T cells (regulatory T cells, Tregs) as a kind of T cell subsets with powerful immune suppression function, be acknowledged as immunoreactive main negative regulator cell, and cell therapy based on Treg cell is also considered to be hopeful most successful cell therapy.Treg cell can regulate immune response in several ways, comprises secretion SC factor IL-10, TGF-β; A large amount of consumption maintains the cytokine IL-2 of inflammatory cell existence and the contact inhibition of cell surface.Activate the retarding effect that Treg can bring into play wide spectrum, and this retarding effect is not limited by MHC molecule can simultaneously.Bone marrow transplanted mice experimental results show that the mouse Treg cell of fresh separated or amplification in vitro can effectively suppress GVHD, and this explanation people Treg cell also may have such treatment potentiality equally.
Yet the clinical application of people Treg cell exists many problems and challenge.First, seldom, in human body, Treg cell accounts for 5%～10% of CD4+T cell to the interior natural Treg cell number of body.This just need to set up effective Treg cell expansion ex vivo method, could meet a large amount of clinical infusion needs.Secondly, Treg is different from mouse, and people Treg cell can not rely on merely this surface marker of CD25 to carry out separation.The sign that some Treg express, as: FoxP3, CTLA-4 and GITR also express on some activating effect T cell.So, when separated people Treg cell subsets, exist huge difficulty, can not only rely on above-mentioned these signs to carry out separation.And relative, the T cell that derives from bleeding of the umbilicus keeps puerilism substantially, and T subsets distribution is simple, has obvious Treg cell subsets, and its content is also higher than peripheral blood simultaneously.In addition because it contains relatively long telomere, thereby can increase in a large number.Therefore, bleeding of the umbilicus is the source of a high-quality of Treg cell.
Although still have many problems to wait to solve, it is encouraging, the clinical I phase of a plurality of Treg cells tests and carries out, and has obtained impressive progress in the recent period.The U.S. and Italian hemopathy expert have completed the I clinical trial phase that two amplification in vitro bleedings of the umbilicus and peripheral blood Treg cell suppress GVHD, proof obviously alleviates through its GVHD symptom of patient of third party unrelated donor Treg cell therapy, and not there is infection, the situation of recurrence or Deaths.The third party unrelated donor Cord blood Treg cell of this is clear and definite amplification in vitro is suppressing to have significant clinical effectiveness on GVHD, also strong confirmation the clinical application of Cord blood Treg cell be safe.There are some researches show, Cord blood Treg cell its inhibition ability in vivo and in vitro after amplification in vitro is cultivated is stronger than the Treg cell of fresh separated, and Treg cell still can be brought into play its effective inhibit feature after frozen and thawing.In sum, Cord blood Treg cell is carried out amplification in vitro and carries out profound hypothermia preservation, and the freezing preservation of the third party unrelated donor human cord blood regulatory T cells storehouse of setting up amplification in vitro will greatly promote carrying out of Treg cell clinical treatment hematopoietic stem cell transplantation graft versus host disease (GVH disease), allogene organ-graft refection, autoimmune disorder.
Chinese patent literature CN1509327B discloses a kind of CD4CD25 regulatory T cells from human blood, this invention provides inhibition and/or modulability mankind CD4+CD25+T cell, for breeding the method for this cell, and the T cell of inhibition and/or modulability mankind CD4+CD25+T cell and propagation is as the purposes of modulability medicament.But yet there are no report about a kind of Cord blood regulatory T cells amplification in vitro and Cryopreservation.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of Cord blood regulatory T cells amplification in vitro and Cryopreservation are provided.
A further object of the present invention is that the application of the regulatory T cells after a kind of described method amplification is provided.
For achieving the above object, the technical scheme that the present invention takes is: a kind of Cord blood regulatory T cells amplification in vitro and Cryopreservation, and described method comprises the following steps:
A, use hydroxyethylamyle are for the separated umbilical cord blood nucleated cells of slurries, with density gradient centrifugation separation, obtain human umbilical cord blood mononuclear cell, human umbilical cord blood mononuclear cell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained CD4+ cell, Cord blood CD4+ cell is further isolated CD25+ cell with the positive separating step of immunomagnetic beads, obtains the bleeding of the umbilicus CD4+CD25+T cell that is rich in regulatory T cells;
B, in Tissue Culture Plate or cell culture bags with connecting the increase Cord blood CD4+CD25+T cell of enrichment of the magnetic bead of CD3/CD28 antibody and recombinant human il-2, obtain highly purified regulatory T cells;
C, by the regulatory T cells cryopreservation after described amplification in liquid nitrogen.
In described step a, the separated step that obtains human umbilical cord blood mononuclear cell comprises: Transfusion Transmission transmissible disease detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries, 4 ℃ of refrigerators are placed precipitation red corpuscle, get the upper plasma leafing heart, abandon most of blood plasma, the white corpuscle of precipitation is resuspended rear separated with Ficoll lymphocyte separation medium, with density gradient centrifugation separation, obtains human umbilical cord blood mononuclear cell.
In described step b, proliferation time is 2-3 week, comprising the amplifications of each 6-8 of two-wheeled days and two-wheeled amplification intermediate cell rest 2-4 days.
After regulatory T cells and cryoprotectant pre-treatment in described step c after amplification, be distributed into liquid nitrogen-196 ℃ preservation in freeze pipe or freezer bag.
Described cryoprotectant is by 10% dimethyl sulfoxide (DMSO), 20% human albumin, and 70% hydroxyethylamyle forms for slurries.
After described CD4+CD25+T cell amplification, the cell of 80-99% is CD4+CD25+T cell, and after amplification, in CD4+CD25+T cell, FoxP3+CD127-cell proportion is greater than 80%.
The 1000-3000 that after described CD4+CD25+T cell amplification, quantity is initiator cell doubly.
For realizing above-mentioned second object, the technical scheme that the present invention takes is: the application in preparation treatment graft versus host disease (GVH disease), organ-graft refection, autoimmune disorder medicine as third party unrelated donor cell of described method amplification or the regulatory T cells after cryopreservation.
The invention has the advantages that:
The method of a kind of Cord blood regulatory T cells amplification in vitro of the present invention and cryopreservation, can from Cord blood, obtain a large amount of regulatory T cells, in liquid nitrogen, preserve for a long time, the clinical demand to regulatory T cell product be can meet, graft versus host disease (GVH disease), organ-graft refection, autoimmune disorder are used for the treatment of.
Accompanying drawing explanation
Fig. 1 is the phenotype of 14 days flow cytometer detection bleeding of the umbilicus Treg cells of amplification.Cord blood Treg cell coexpression CD4 and CD25 after amplification, more than 90% cell expressing FoxP3., compare these cell high expression levels CTLA-4 (68%), CD39 (71%) and LAG-3(45%) with the CD4+CD25+T cell of fresh sorting meanwhile.
Fig. 2 is the cytokine-expressing of the bleeding of the umbilicus Treg cell after amplification.Compare with the CD4+CD25-T cell of activation, the Cord blood Treg cell after amplification is expressed IL-2 gene hardly, expresses a small amount of IFN-γ, and high expression level IL-10 and TGF-β gene.
Fig. 3 is the propagation of the third party unrelated donor bleeding of the umbilicus Treg retarding effect T cell of amplification in vitro.From the separation of peripheral blood magnetic bead, obtain CD4+CD25-cell action effect T cell (T
effect ), after CFSE dyeing, by Treg and T
effect cell different ratios adds the Cord blood Treg cell of the third party unrelated donor after amplification, and CD3/CD28 monoclonal antibody or mature dendritic cell (mDC) thorn activating signal activation for every hole are cultivated after 4 days collecting cell and carry out interpretation of result with flow cytometer.Third party unrelated donor's Cord blood Treg cell is at Treg:T
effect the effective amplification in vitro of retarding effect T cell when cell proportion is 1:1,1:4,1:16.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
A kind of Cord blood regulatory T cells amplification in vitro of the present invention and Cryopreservation comprise the following steps:
Transfusion Transmission transmissible disease detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries, 4 ℃ of refrigerators are placed precipitation red corpuscle, get the upper plasma leafing heart, abandon most of blood plasma, the white corpuscle of precipitation is resuspended rear separated with Ficoll lymphocyte separation medium, with density gradient centrifugation separation, obtains human umbilical cord blood mononuclear cell.
2. human umbilical cord blood mononuclear cell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained CD4+ cell, and Cord blood CD4+ cell is further isolated CD25+ cell with the positive separating step of immunomagnetic beads and obtained the bleeding of the umbilicus CD4+CD25+T cell that is rich in regulatory T cells.
In Tissue Culture Plate or cell culture bags with connecting the increase Cord blood CD4+CD25+T cell of enrichment of the magnetic bead of CD3/CD28 antibody and recombinant human il-2, proliferation time is 2-3 week, comprising the amplifications of each 6-8 of two-wheeled days and two-wheeled amplification intermediate cell rest 2-4 days.
4. after amplification, Cord blood regulatory T cells is removed CD3/CD28 magnetic bead, after cryoprotectant pre-treatment, is distributed into liquid nitrogen-196 ℃ preservation in freeze pipe or freezer bag.Cryoprotectant is by 10% dimethyl sulfoxide (DMSO) (DMSO), 20% human albumin, and 70% hydroxyethylamyle forms for slurries (HES).
After the recovery of frozen Cord blood regulatory T cells as third party unrelated donor infusion of therapeutic graft versus host disease (GVH disease), organ-graft refection, autoimmune disorder.
Above-mentioned all processes all carries out in gnotobasis.
It should be noted that, after described CD4+CD25+T cell amplification, the cell of 80%-99% is CD4+CD25+T cell, and after amplification, in CD4+CD25+T cell, FoxP3+CD127-cell proportion is greater than 80%.The 1000-3000 that after described CD4+CD25+T cell amplification, quantity is initiator cell doubly.
The Cord blood by volume ratio of 4:1 adds 6% hydroxyethylamyle for slurries, 4 ℃ of refrigerators are placed 1h precipitation red corpuscle, get the upper plasma leafing heart (2000rpm/min, 5min), abandon most of blood plasma, the white corpuscle of precipitation is resuspended rear with Ficoll lymphocyte separation medium separated (2000rpm/min, 20min), with PBS washing three times, obtain human umbilical cord blood mononuclear cell.According to the explanation of producer, use standard separating kit, for example use CliniMACS, anti-human CD8, the CD14 of Miltenyi Biotec company, CD19, CD56 monoclonal antibody immunity magnetic bead are removed the non-cd4 cell in human umbilical cord blood mononuclear cell, then the enrichment with magnetic bead CD4+CD25+ cell with anti-human CD25 antibody coupling to Cord blood CD4+T cell.After Cord blood CD4+CD25+T cell purification, with flow cytometer, detect the purity of isolated cell.
embodiment 2
Magnetic bead and recombinant human il-2 (200U/ml) with anti-human CD3/CD28 antibody coupling in the cell culture bags being purchased or Tissue Culture Plate carry out amplification in vitro to the Cord blood CD4+CD25+T cell of purifying, use X-VIVO15 complete culture solution to cultivate and (contain 10% deactivation people AB serum, the dual anti-50U/ml of penicillin and streptomycin), in culture system, the coated Dynal Beads of CD3/CD28 and the ratio of cell are 1:1, in 37 ℃, 5%CO
2and cultivate under the condition of saturated humidity, half amount is changed nutrient solution and IL-2 every other day.Whole proliferation time is 2-3 week, and comprising the two-wheeleds amplifications of each 6-8 days and two-wheeled amplification intermediate cell rest 2-4 days, cell is removed after magnetic bead containing IL-2(20U/ml) X-VIVO15 complete culture solution cultivation 2 days.Amplification in vitro finishes rear cell quantity and increases approximately 1500 times, and Cord blood Treg cell is removed the ability that magnetic bead detects surface marker and vitro inhibition effector T cell propagation, and carries out cryopreservation.
embodiment 3
Adopt flow cytometry to carry out the analysis of Cord blood Treg Immunophenotyping.Use CD4 antibody, the CD25 antibody of PE-Cy5 mark, the CTLA-4 antibody of the CD127 antibody of APC mark, PE mark, CD39 antibody, the LAG3 antibody of FITC mark to carry out cell surface marker, after stationary liquid and the processing of rupture of membranes liquid, with the Foxp3 of PE mark, carry out cell inner mark.As shown in Figure 1, Cord blood Treg cell coexpression CD4 and CD25 after amplification, more than 90% cell expressing FoxP3.Meanwhile, with the CD4 of fresh sorting
+cD25
+t cell is compared, Cord blood Treg cell high expression level CTLA-4(68% after amplification), CD39(71%) and LAG-3(45%).
Adopt micro-extracting RNA method to obtain peripheral blood CD4+CD25-T cell and the rear Treg cell total rna of amplification of activation, another mistake is transcribed into cDNA, and the cDNA obtaining is as amplified target gene fragment, for follow-up Real-time PCR system.Using β-actin as reference amount, cytokine IL-2, IFN-γ, IL-10, TGF-β are as target gene, and the peripheral blood CD4+CD25-T cell relatively activating by Real-time PCR and amplification be the difference of Treg cell IL-2, IFN-γ, IL-10, TGF-beta gene expression afterwards.As shown in Figure 2, with the CD4 activating
+cD25
-t cell is compared, and the Cord blood Treg cell after amplification is expressed IL-2 gene hardly, expresses a small amount of IFN-γ, and high expression level IL-10 and TGF-β gene.
embodiment 5
The CD4+CD25-cell action effect T cell obtaining from the separation and purification of peripheral blood immunomagnetic beads, through CFSE(5 μ M) dyeing after, with 5 * 10
4/ hole is inoculated in 96 well culture plates, by Treg and effector T cell different ratios (1:1,1:4,1:16,1:64), add again the Cord blood Treg cell of the third party unrelated donor after amplification, CD3/CD28 monoclonal antibody (300ng/ml) or the thorn of the allogene mature dendritic cell (mDC) after mitomycin is processed activating signal activation for every hole, in 37 ℃, 5% CO
2and under saturated humidity condition, cultivate collecting cell carry out interpretation of result with flow cytometer after 4 days 4 days.As shown in Figure 3, third party unrelated donor's Cord blood Treg cell is at Treg:T
effect the effective amplification in vitro of retarding effect T cell when cell proportion is 1:1,1:4,1:16.
embodiment 6
After amplification, Cord blood regulatory T cells is removed CD3/CD28 magnetic bead, after cryoprotectant pre-treatment, is distributed into liquid nitrogen-196 ℃ preservation in freeze pipe or freezer bag.Cryoprotectant is by 10% dimethyl sulfoxide (DMSO) (DMSO), 20% human albumin, and 70% hydroxyethylamyle forms for slurries (HES).Through strict trace routine, (syphilis antibody detection, HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test etc.) detect after qualified and carry out enrichment and amplification in vitro Treg cell human cord blood, the Cord blood Treg cell obtaining detects after qualified frozen in liquid nitrogen through purity and inside and outside inhibit feature, be used for the treatment of graft versus host disease (GVH disease), organ-graft refection, autoimmune disorder.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
Claims (4)
1. Cord blood regulatory T cells amplification in vitro and a Cryopreservation, is characterized in that, described method comprises the following steps:
A, use hydroxyethylamyle are for the separated umbilical cord blood nucleated cells of slurries, with density gradient centrifugation separation, obtain human umbilical cord blood mononuclear cell, human umbilical cord blood mononuclear cell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained CD4+ cell, Cord blood CD4+ cell is further isolated CD25+ cell with the positive separating step of immunomagnetic beads, obtains the bleeding of the umbilicus CD4+CD25+T cell that is rich in regulatory T cells;
B, in Tissue Culture Plate or cell culture bags with connecting the increase Cord blood CD4+CD25+T cell of enrichment of the magnetic bead of CD3/CD28 antibody and recombinant human il-2, obtain highly purified regulatory T cells, described proliferation time is 2-3 week, comprising the amplifications of each 6-8 of two-wheeled days and two-wheeled amplification intermediate cell rest 2-4 days;
C, by after the regulatory T cells after described amplification and cryoprotectant pre-treatment, be distributed into liquid nitrogen-196 ℃ preservation in freeze pipe or freezer bag, described cryoprotectant is by 10% dimethyl sulfoxide (DMSO), 20% human albumin, 70% hydroxyethylamyle forms for slurries.
2. method according to claim 1, it is characterized in that, in described step a, the separated step that obtains human umbilical cord blood mononuclear cell comprises: Transfusion Transmission transmissible disease detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries, 4 ℃ of refrigerators are placed precipitation red corpuscle, get the upper plasma leafing heart, abandon most of blood plasma, the white corpuscle of precipitation is resuspended rear separated with Ficoll lymphocyte separation medium, with density gradient centrifugation separation, obtains human umbilical cord blood mononuclear cell.
3. method according to claim 1, is characterized in that, after described CD4+CD25+T cell amplification, the cell of 80-99% is CD4+CD25+T cell, and after amplification, in CD4+CD25+T cell, FoxP3+CD127-cell proportion is greater than 80%.
4. method according to claim 1, is characterized in that, the 1000-3000 that after described CD4+CD25+T cell amplification, quantity is initiator cell doubly.
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| CN104357389B (en) * | 2014-10-15 | 2017-04-26 | 湖南赛诺生物科技股份有限公司 | Expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium |
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| WO2018106885A1 (en) | 2016-12-07 | 2018-06-14 | East Carolina University | Compositions and methods for in vitro cultivation and/or expansion of regulatory t cells |
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| CN108060129A (en) * | 2017-12-11 | 2018-05-22 | 上海药明生物技术有限公司 | Regulatory T cells amplification in vitro method |
| CN108004209A (en) * | 2017-12-11 | 2018-05-08 | 山东省齐鲁干细胞工程有限公司 | A kind of bleeding of the umbilicus regulatory T cells amplification in vitro method |
| AU2019226392A1 (en) * | 2018-03-01 | 2020-09-24 | Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañón | Method for obtaining regulatory T cells derived from thymic tissue and use of said cells as cell immunotherapy in immune system disorders |
| CN108617640A (en) * | 2018-06-26 | 2018-10-09 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of cord blood mononuclear cells frozen stock solution and its application |
| CN110195041B (en) * | 2019-06-20 | 2023-04-25 | 威海正生生物科技有限公司 | Method for obtaining high-activity Tregs cells by using ASC three-dimensional culture system |
| CN114112868A (en) * | 2020-08-26 | 2022-03-01 | 上海睿昂基因科技股份有限公司 | Flow cytometry kit for monitoring tumor-related immune microenvironment and monitoring method |
| CN112458053B (en) * | 2020-11-26 | 2021-11-02 | 成都云测医学生物技术有限公司 | Umbilical blood Treg cell in-vitro amplification method based on trophoblast cells and application |
| CN112662625B (en) * | 2021-01-18 | 2023-03-17 | 曹彤 | T cell culture medium and method for expanding and culturing T cells by using same |
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