CN102517253A - 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
- Publication number
- CN102517253A CN102517253A CN201110425246XA CN201110425246A CN102517253A CN 102517253 A CN102517253 A CN 102517253A CN 201110425246X A CN201110425246X A CN 201110425246XA CN 201110425246 A CN201110425246 A CN 201110425246A CN 102517253 A CN102517253 A CN 102517253A
- Authority
- CN
- China
- Prior art keywords
- cell
- cord blood
- regulatory
- cells
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
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 method.
Background technology
The major cause that rejection is tissue and Transplanted cells failure takes place to the unmatched graft of MHC in acceptor.In clinical practice at present; The use of tradition immunosuppressive drug; Though can effectively prevent transplant rejection, alleviate by the cell-mediated acute and chronic graft versus host disease of donor T (graft-versus-host disease, GVHD); But often with the whole lower immune function of receptor, spinoffs such as infection chance increase and induced tumor.Therefore, be that the cell therapy method of purpose comes into one's own day by day to induce the tolerance of transplantation immunity between the donor-recipient.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 adjusting cell, and also be considered to be hopeful most the cell therapy of success based on the cell therapy of Treg cell.The Treg cell can be regulated immunoreation in several ways, comprises secretion SC factor IL-10, TGF-β; Mass consumption is kept the cytokine IL-2 of inflammatory cell existence and the contact inhibition of cell surface.The Treg of activation simultaneously can bring into play the retarding effect of wide spectrum, and this retarding effect is not limited by the MHC molecule can.The mouse Treg cell of mouse bone marrow cells transplantation experiments proof fresh separated or amplification in vitro can effectively suppress GVHD, and this explanation people Treg cell also possibly have such treatment potentiality equally.
Yet the clinical application of people Treg cell exists many problems and challenge.At first, in the body natural Treg cell number seldom, in human body, the Treg cell accounts for 5%～10% of CD4+T cell.This just need set up effective Treg cell expansion ex vivo method, could satisfy lots of clinical infusion needs.Secondly, Treg is different with mouse, and people Treg cell can not rely on this surface marker of CD25 to separate merely.The sign that some Treg express, as: FoxP3, CTLA-4 and GITR, also express on some activating effect T cell.So, when separation of human Treg cell subsets, exist huge difficulty, can not only rely on above-mentioned these signs to separate.And relative, the T cell that derives from bleeding of the umbilicus keeps puerilism basically, and the T subsets distribution is simple, has tangible Treg cell subsets, and its content also is 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 a fine source of Treg cell.
Although still have many problems to wait to solve, it is encouraging that 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 accomplished 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 occurs infecting, recurring or early stage dead situation.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 the GVHD, also strong confirmation the clinical application of Cord blood Treg cell be safe.There are some researches show that it is stronger than the Treg cell of fresh separated that Cord blood Treg cell is cultivated its inhibition ability in vivo and in vitro of back through amplification in vitro, and the Treg cell through frozen with melt after still can bring into play its effective inhibit feature.In sum; Cord blood Treg cell is carried out amplification in vitro and carries out profound hypothermia and preserve, 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 HSCT graft versus host disease, allogene organ-graft refection, autoimmune disorder.
Chinese patent document CN1509327B discloses a kind of CD4CD25 regulatory T cells from human blood; This invention provides inhibition and/or the human CD4+CD25+T cell of modulability; Be used to breed the method for this cell, and the T cell of inhibition and/or human CD4+CD25+T cell of modulability and propagation is as the purposes of modulability medicament.But also do not appear in the newspapers at present about a kind of Cord blood regulatory T cells amplification in vitro and cryopreservation method.
Summary of the invention
The objective of the invention is provides a kind of Cord blood regulatory T cells amplification in vitro and cryopreservation method to deficiency of the prior art.
A further object of the present invention is that the application of the regulatory T cells after a kind of described method increases is provided.
For realizing above-mentioned purpose, the technical scheme that the present invention takes is: a kind of Cord blood regulatory T cells amplification in vitro and cryopreservation method, and described method may further comprise the steps:
A, separate nucleated cell in the Cord blood for slurries with hydroxyethylamyle; Obtain the Cord blood mononuclearcell with the density gradient centrifugation separation; The Cord blood mononuclearcell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained the CD4+ cell; Cord blood CD4+ cell is further isolated the CD25+ cell with the positive separating step of immunomagnetic beads, obtains to be rich in the bleeding of the umbilicus CD4+CD25+T cell of regulatory T cells;
B, in Tissue Culture Plate or cell culture bags with the increase Cord blood CD4+CD25+T cell of enrichment of magnetic bead that connects CD3/CD28 antibody and recombinant human il-2, obtain highly purified regulatory T cells;
C, with the regulatory T cells cryopreservation after the said amplification in liquid nitrogen.
Separating the step obtain the Cord blood mononuclearcell among the described step a comprises: blood transfusion disseminate infection detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries; 4 ℃ of refrigerators are placed the deposition red corpuscle; Get the upper plasma leafing heart; Abandon most of blood plasma, separate with the Ficoll lymphocyte separation medium the resuspended back of sedimentary white corpuscle, separates obtaining the Cord blood mononuclearcell with density gradient centrifugation.
Proliferation time is 2-3 week among the described step b, comprising intermediate cell rest 2-4 days of increasing of each 6-8 of two-wheeled days amplifications and two-wheeled.
Behind the regulatory T cells and cryoprotectant pre-treatment among the described step c after the amplification, divide liquid nitrogen-196 ℃ preservation in pack into freeze pipe or the freezer bag.
Described cryoprotectant is by 10% DMSO 99.8MIN., 20% human albumin, and 70% hydroxyethylamyle is formed for slurries.
The cell of 80-99% is the CD4+CD25+T cell behind the described CD4+CD25+T cell amplification, and the FoxP3+CD127-cell proportion is greater than 80% in the CD4+CD25+T cell of amplification back.
Behind the described CD4+CD25+T cell amplification quantity be initiator cell 1000-3000 doubly.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: the regulatory T cells behind described method amplification or the cryopreservation is as the application of third party unrelated donor cell in preparation treatment graft versus host disease, organ-graft refection, autoimmune disorder medicine.
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 the medium-term and long-term preservation of liquid nitrogen; Clinical demand be can satisfy, graft versus host disease, organ-graft refection, autoimmune disorder are used to treat the regulatory T cell product.
Description of drawings
Fig. 1 is the phenotype of amplification flow cytometer detection in 14 days bleeding of the umbilicus Treg cell.Cord blood Treg cell coexpression CD4 and CD25 after the amplification, the cell expressing FoxP3 more than 90%.Simultaneously, compare these cell high expression levels CTLA-4 (68%), CD39 (71%) and LAG-3 (45%) with the CD4+CD25+T cell of fresh sorting.
Fig. 2 is the cytokine-expressing of the bleeding of the umbilicus Treg cell after the amplification.Compare with activatory CD4+CD25-T cell, the Cord blood Treg cell after the amplification is expressed the IL-2 gene hardly, expresses a spot 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.Obtain CD4+CD25-cell action effect T cell (T from the separation of peripheral blood magnetic bead
Effect ), after CFSE dyeing, press Treg and T
Effect The cell different ratios adds the third party unrelated donor's after the amplification Cord blood Treg cell, and activating signal activation is stung with CD3/CD28 monoclonal antibody or mature dendritic cell (mDC) in every hole, cultivates after 4 days collecting cell and carries out interpretation of result with flow cytometer.Third party unrelated donor's Cord blood Treg cell is at Treg:T
Effect Effectively retarding effect T cells in vitro amplification 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 method may further comprise the steps:
Blood transfusion disseminate infection detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries; 4 ℃ of refrigerators are placed the deposition red corpuscle; Get the upper plasma leafing heart; Abandon most of blood plasma, separate with the Ficoll lymphocyte separation medium the resuspended back of sedimentary white corpuscle, separates obtaining the Cord blood mononuclearcell with density gradient centrifugation.
2. the Cord blood mononuclearcell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained the CD4+ cell, and Cord blood CD4+ cell is further isolated the bleeding of the umbilicus CD4+CD25+T cell that the CD25+ cell obtains to be rich in regulatory T cells with the positive separating step of immunomagnetic beads.
In Tissue Culture Plate or cell culture bags with the increase Cord blood CD4+CD25+T cell of enrichment of magnetic bead that connects CD3/CD28 antibody and recombinant human il-2; Proliferation time is 2-3 week, comprising intermediate cell rest 2-4 days of increasing of each 6-8 of two-wheeled days amplifications and two-wheeled.
4. amplification back Cord blood regulatory T cells is removed the CD3/CD28 magnetic bead, after the cryoprotectant pre-treatment, divides liquid nitrogen-196 ℃ preservation in pack into freeze pipe or the freezer bag.Cryoprotectant is by 10% DMSO 99.8MIN. (DMSO), 20% human albumin, and 70% hydroxyethylamyle is formed for slurries (HES).
5. frozen Cord blood regulatory T cells recovery back is as third party unrelated donor infusion of therapeutic graft versus host disease, organ-graft refection, autoimmune disorder.
Above-mentioned all processes all carries out in gnotobasis.
Need to prove that the cell of 80%-99% is the CD4+CD25+T cell behind the described CD4+CD25+T cell amplification, the FoxP3+CD127-cell proportion is greater than 80% in the CD4+CD25+T cell of amplification back.Behind the described CD4+CD25+T cell amplification quantity be initiator cell 1000-3000 doubly.
The Cord blood ratio of 4:1 by volume adds 6% hydroxyethylamyle for slurries, and 4 ℃ of refrigerators are placed 1h deposition red corpuscle, get the upper plasma leafing heart (2000rpm/min; 5min); Abandon most of blood plasma, the resuspended back of sedimentary white corpuscle with the separation of Ficoll lymphocyte separation medium (2000rpm/min, 20min); With PBS washing three times, obtain the Cord blood mononuclearcell.Explanation according to producer; Use the standard separating kit; For example use CliniMACS; The anti-people CD8 of Miltenyi Biotec company, CD14, CD19, CD56 monoclonal antibody immunity magnetic bead are removed the non-cd4 cell in the Cord blood mononuclearcell, then to the enrichment with magnetic bead CD4+CD25+ cell of Cord blood CD4+T cell with anti-people CD25 antibody coupling.Detect the purity of isolated cell behind the Cord blood CD4+CD25+T cell purification with flow cytometer.
In cell culture bags that is purchased or Tissue Culture Plate, the Cord blood CD4+CD25+T cell of purifying is carried out amplification in vitro with the magnetic bead of anti-people CD3/CD28 antibody coupling and recombinant human il-2 (200U/ml); Use the X-VIVO15 complete culture solution to cultivate and (contain 10% deactivation people AB serum; The two anti-50U/ml of blue or green, Streptomycin sulphate); The Dynal Beads that CD3/CD28 encapsulates in the culture system 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 each 6-8 days two-wheeleds amplifications and two-wheeled amplification intermediate cell rest 2-4 days, promptly cell is removed behind the magnetic bead the X-VIVO15 complete culture solution cultivation that contains IL-2 (20U/ml) 2 days.Amplification in vitro finishes the back cell quantity and increases about 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 Cord blood Treg cellular immunization phenotype analytical.Use CD4 antibody, the CD25 antibody of PE-Cy5 mark, the CD127 antibody of APC mark, the CTLA-4 antibody of PE mark, CD39 antibody, the LAG3 antibody of FITC mark to carry out cell surface marker; After stationary liquid and rupture of membranes liquid are handled, carry out cell inner mark with the Foxp3 of PE mark.As shown in Figure 1, Cord blood Treg cell coexpression CD4 and CD25 after the amplification, the cell expressing FoxP3 more than 90%.Simultaneously, with the CD4 of fresh sorting
+CD25
+The T cell is compared, amplification back Cord blood Treg cell high expression level CTLA-4 (68%), CD39 (71%) and LAG-3 (45%).
Adopt micro-extracting RNA method to obtain activatory peripheral blood CD4+CD25-T cell and amplification back Treg cell total rna, another mistake is transcribed into cDNA, and the cDNA that is obtained is used for follow-up Real-time PCR system as the amplified target gene fragment.Measure as reference with β-actin; Cytokine IL-2, IFN-γ, IL-10, TGF-β compare the difference of activatory peripheral blood CD4+CD25-T cell and amplification back Treg cell IL-2, IFN-γ, IL-10, TGF-beta gene expression as target gene through Real-time PCR.As shown in Figure 2, with activatory CD4
+CD25
-The T cell is compared, and the Cord blood Treg cell after the amplification is expressed the IL-2 gene hardly, expresses a spot of IFN-γ, and high expression level IL-10 and TGF-β gene.
Embodiment 5
The CD4+CD25-cell action effect T cell that obtains from the separation and purification of peripheral blood immunomagnetic beads is after CFSE (5 μ M) dyeing, with 5 * 10
4/ hole is inoculated in 96 well culture plates; Press the Cord blood Treg cell that Treg and effector T cell different ratios (1:1,1:4,1:16,1:64) add the third party unrelated donor after increasing again; Every hole is with CD3/CD28 monoclonal antibody (300ng/ml) or the thorn of the allogene mature dendritic cell (mDC) after MTC is handled activating signal activation, in 37 ℃, 5% CO
2And cultivated 4 days collecting cell and carry out interpretation of result after 4 days under the saturated humidity condition with flow cytometer.As shown in Figure 3, third party unrelated donor's Cord blood Treg cell is at Treg:T
Effect Effectively retarding effect T cells in vitro amplification when cell proportion is 1:1,1:4,1:16.
Embodiment 6
Amplification back Cord blood regulatory T cells is removed the CD3/CD28 magnetic bead, after the cryoprotectant pre-treatment, divides liquid nitrogen-196 ℃ preservation in pack into freeze pipe or the freezer bag.Cryoprotectant is by 10% DMSO 99.8MIN. (DMSO), 20% human albumin, and 70% hydroxyethylamyle is formed for slurries (HES).Carry out enrichment and amplification in vitro Treg cell after the human cord blood strict trace routine of process (syphilis antibody detection, HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test etc.) detection is qualified; The Cord blood Treg cell that obtains is frozen in liquid nitrogen after purity and the detection of inside and outside inhibit feature are qualified, is used to treat graft versus host disease, organ-graft refection, autoimmune disorder.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.
Claims (8)
1. Cord blood regulatory T cells amplification in vitro and cryopreservation method is characterized in that described method may further comprise the steps:
A, separate nucleated cell in the Cord blood for slurries with hydroxyethylamyle; Obtain the Cord blood mononuclearcell with the density gradient centrifugation separation; The Cord blood mononuclearcell is removed non-cd4 cell with the negative separating step of immunomagnetic beads and is obtained the CD4+ cell; Cord blood CD4+ cell is further isolated the CD25+ cell with the positive separating step of immunomagnetic beads, obtains to be rich in the bleeding of the umbilicus CD4+CD25+T cell of regulatory T cells;
B, in Tissue Culture Plate or cell culture bags with the increase Cord blood CD4+CD25+T cell of enrichment of magnetic bead that connects CD3/CD28 antibody and recombinant human il-2, obtain highly purified regulatory T cells;
C, with the regulatory T cells cryopreservation after the said amplification in liquid nitrogen.
2. method according to claim 1; It is characterized in that, separate the step obtain the Cord blood mononuclearcell among the described step a and comprise: blood transfusion disseminate infection detect qualified Cord blood by volume the ratio of 4:1 add 6% hydroxyethylamyle for slurries, 4 ℃ of refrigerators are placed the deposition red corpuscle; Get the upper plasma leafing heart; Abandon most of blood plasma, separate with the Ficoll lymphocyte separation medium the resuspended back of sedimentary white corpuscle, separates obtaining the Cord blood mononuclearcell with density gradient centrifugation.
3. method according to claim 1 is characterized in that, proliferation time is 2-3 week among the described step b, comprising intermediate cell rest 2-4 days of increasing of each 6-8 of two-wheeled days amplifications and two-wheeled.
4. method according to claim 1 is characterized in that, behind the regulatory T cells and cryoprotectant pre-treatment among the described step c after the amplification, divides liquid nitrogen-196 ℃ preservation in pack into freeze pipe or the freezer bag.
5. method according to claim 4 is characterized in that, described cryoprotectant is by 10% DMSO 99.8MIN., 20% human albumin, and 70% hydroxyethylamyle is formed for slurries.
6. method according to claim 3 is characterized in that, the cell of 80-99% is the CD4+CD25+T cell behind the described CD4+CD25+T cell amplification, and the FoxP3+CD127-cell proportion is greater than 80% in the CD4+CD25+T cell of amplification back.
7. method according to claim 3 is characterized in that, behind the described CD4+CD25+T cell amplification quantity be initiator cell 1000-3000 doubly.
8. treat application in graft versus host disease, organ-graft refection, autoimmune disorder medicine as third party unrelated donor cell in preparation according to the regulatory T cells behind arbitrary described method amplification of claim 1-7 or the cryopreservation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110425246.XA CN102517253B (en) | 2011-12-19 | 2011-12-19 | In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110425246.XA CN102517253B (en) | 2011-12-19 | 2011-12-19 | In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102517253A true CN102517253A (en) | 2012-06-27 |
| CN102517253B CN102517253B (en) | 2014-04-02 |
Family
ID=46288322
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110425246.XA Active CN102517253B (en) | 2011-12-19 | 2011-12-19 | In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102517253B (en) |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN104357389A (en) * | 2014-10-15 | 2015-02-18 | 湖南赛诺生物科技有限责任公司 | Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium |
| CN107349219A (en) * | 2017-07-25 | 2017-11-17 | 中南大学湘雅二医院 | Application and its spread cultivation liquid and propagation method of the regulatory T cells in the immune diabetes medicament for the treatment of is prepared |
| CN107771780A (en) * | 2016-08-29 | 2018-03-09 | 灏灵赛奥（天津）生物科技有限公司 | A kind of CAR T cells freezing media and cryopreservation methods |
| 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 |
| 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 |
| 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 |
| CN110195041A (en) * | 2019-06-20 | 2019-09-03 | 威海正生生物科技有限公司 | The method for obtaining high activity Tregs cell using ASC dimensional culture system |
| CN110753752A (en) * | 2017-03-29 | 2020-02-04 | 桑昆血液供给基金会 | Isolation of stable regulatory T cells and uses thereof |
| CN112135901A (en) * | 2018-03-01 | 2020-12-25 | 格雷戈里奥马拉尼翁医院生物医学研究基金会 | Method for obtaining regulatory T cells derived from thymus tissue and use of said cells as a cellular immunotherapy for the dysregulation of the immune system |
| CN112458053A (en) * | 2020-11-26 | 2021-03-09 | 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 |
| CN112662625A (en) * | 2021-01-18 | 2021-04-16 | 杭州原生生物科技有限公司 | T cell culture medium and method for expanding and culturing T cells by using same |
| CN113564117A (en) * | 2021-08-23 | 2021-10-29 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation umbilical cord blood source regulatory T cell in-vitro amplification optimization method |
| 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 |
| US11384336B2 (en) | 2016-12-07 | 2022-07-12 | East Carolina University | Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells |
| WO2024015813A1 (en) * | 2022-07-11 | 2024-01-18 | Ossium Health, Inc. | Methods and compositions for cryopreserving subpopulations of lymphocytes |
| CN110753752B (en) * | 2017-03-29 | 2024-04-26 | 桑昆血液供给基金会 | Isolation of stable regulatory T cells and uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1981031A (en) * | 2004-03-05 | 2007-06-13 | 宾久法尼亚大学理事会 | Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses |
| CN101864396A (en) * | 2010-05-17 | 2010-10-20 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing megakaryoblast and megakaryocyte in vitro |
-
2011
- 2011-12-19 CN CN201110425246.XA patent/CN102517253B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1981031A (en) * | 2004-03-05 | 2007-06-13 | 宾久法尼亚大学理事会 | Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses |
| CN101864396A (en) * | 2010-05-17 | 2010-10-20 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing megakaryoblast and megakaryocyte in vitro |
Non-Patent Citations (1)
| Title |
|---|
| 刘清泉等: "脐带血、成人外周血中CD4+ CD25high调节性T细胞分布的比较", 《细胞与分子免疫学杂志》, vol. 22, no. 3, 31 December 2006 (2006-12-31), pages 411 - 412 * |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN104357389A (en) * | 2014-10-15 | 2015-02-18 | 湖南赛诺生物科技有限责任公司 | Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium |
| CN107771780A (en) * | 2016-08-29 | 2018-03-09 | 灏灵赛奥（天津）生物科技有限公司 | A kind of CAR T cells freezing media and cryopreservation methods |
| US11384336B2 (en) | 2016-12-07 | 2022-07-12 | East Carolina University | Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells |
| CN110753752A (en) * | 2017-03-29 | 2020-02-04 | 桑昆血液供给基金会 | Isolation of stable regulatory T cells and uses thereof |
| CN110753752B (en) * | 2017-03-29 | 2024-04-26 | 桑昆血液供给基金会 | Isolation of stable regulatory T cells and uses thereof |
| CN107349219A (en) * | 2017-07-25 | 2017-11-17 | 中南大学湘雅二医院 | Application and its spread cultivation liquid and propagation method of the regulatory T cells in the immune diabetes medicament for the treatment of is prepared |
| CN107349219B (en) * | 2017-07-25 | 2020-07-14 | 中南大学湘雅二医院 | Application of regulatory T cells in preparation of medicine for treating immune diabetes, and expanding culture solution and expanding culture method thereof |
| 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 |
| CN112135901A (en) * | 2018-03-01 | 2020-12-25 | 格雷戈里奥马拉尼翁医院生物医学研究基金会 | Method for obtaining regulatory T cells derived from thymus tissue and use of said cells as a cellular immunotherapy for the dysregulation of the immune system |
| 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 |
| CN110195041A (en) * | 2019-06-20 | 2019-09-03 | 威海正生生物科技有限公司 | The method for obtaining high activity Tregs cell using ASC 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 |
| CN112458053A (en) * | 2020-11-26 | 2021-03-09 | 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 |
| CN112662625A (en) * | 2021-01-18 | 2021-04-16 | 杭州原生生物科技有限公司 | T cell culture medium and method for expanding and culturing T cells by using same |
| 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 |
| CN113564117A (en) * | 2021-08-23 | 2021-10-29 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation umbilical cord blood source regulatory T cell in-vitro amplification optimization method |
| CN113564117B (en) * | 2021-08-23 | 2023-12-26 | 山东省齐鲁干细胞工程有限公司 | In-vitro expansion optimization method for cryopreserved umbilical cord blood-derived regulatory T cells |
| WO2024015813A1 (en) * | 2022-07-11 | 2024-01-18 | Ossium Health, Inc. | Methods and compositions for cryopreserving subpopulations of lymphocytes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102517253B (en) | 2014-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102517253B (en) | In vitro amplification and low-temperature storage method for regulatory T cells of umbilical cord blood | |
| Romano et al. | Treg therapy in transplantation: a general overview | |
| US11160830B2 (en) | Activation of marrow infiltrating lymphocytes in hypoxic alternating with normoxic conditions | |
| CN108220239B (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| Davis et al. | Interleukin-7 permits Th1/Tc1 maturation and promotes ex vivo expansion of cord blood T cells: a critical step toward adoptive immunotherapy after cord blood transplantation | |
| JP6073417B2 (en) | Spontaneous killing cell proliferation method and composition for spontaneous killing cell proliferation | |
| CN105543169A (en) | Preparation method and application of human TSCMs (T memory stem cells) | |
| Berglund et al. | Isolation, expansion and functional assessment of CD4+ CD25+ FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation | |
| TW202128988A (en) | Method of expanding natural killercells in culture, method of increasingcytotoxicity of natural killer cells , method of treating a subject, compositions , and cell composition thereof | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| EP2859092B1 (en) | Therapeutic vaccine for treatment of diabetes type 1 in children, application of the cell sorter and the method of multiplying treg cells to produce therapeutic vaccine for treatment of diabetes type 1 | |
| US10385315B2 (en) | Cells expressing Th1 characteristics and cytolytic properties | |
| RU2391401C2 (en) | METHOD OF OBTAINING POPULATION OF HUMAN CD4+CD25+Foxp3+ T-LYMPHOCYTES ex vivo, DISEASE TREATMENT METHOD | |
| CN102321580A (en) | Regulatory T cells for treating autoimmune diseases, and preparation method thereof | |
| Otto et al. | Human γδ T cells from G-CSF-mobilized donors retain strong tumoricidal activity and produce immunomodulatory cytokines after clinical-scale isolation | |
| CN103074299B (en) | Method for external induction of tolerogenic dendritic cells (DC) by immunosuppressor | |
| Biburger et al. | Activation-induced NKT cell hyporesponsiveness protects from α-galactosylceramide hepatitis and is independent of active transregulatory factors | |
| KR20210149201A (en) | Bead-Free Ex vivo Expansion of Human Regulatory T Cells | |
| RU2437933C1 (en) | METHOD OF HUMAN REGULATORY CD4+CD25+FOXP3+T-CELL ENRICHMENT ex vivo | |
| Zhao et al. | The role of natural killer T cells in liver transplantation | |
| Atreya et al. | Manuel Wiesinger, Diane Stoica1, Susanne Roessner1, Carmen Lorenz1, Anika Fischer 2 | |
| CN116179485A (en) | Novel coronavirus specific T cell and preparation method and application thereof | |
| CN105385656A (en) | ECCE-CIK cell culture method and ECCE-CIK cell preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |