CN111235209A - Method for evaluating immune regulation function of stem cells - Google Patents
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Abstract
The invention provides a method for quantitatively evaluating the immune regulation and control function of stem cells, which comprises the following steps: sorting effector cells of a test body receiving the stem cell preparation, performing standardized effector cell amplification by using antibody-coated amplification magnetic beads, evaluating the proliferation of the effector cells mixed and cultured in the stem cell single cell suspension to be detected based on CFSE staining and labeling the effector cells and flow cytometry, and evaluating the immune regulation and control function of the stem cells to be detected according to the inhibition rate of the proliferation of the effector cells. The method is reliable and good in repeatability, can be used as necessary content for comprehensively reflecting the quality of the stem cells, and can be used for evaluating the immune regulation and control capability of the stem cells in the scientific research field and evaluating the immune regulation and control capability of a clinical infusion-grade stem cell preparation in the clinical application field and controlling the quality.
Description
Technical Field
The invention relates to the field of medical treatment, in particular to a method for evaluating the immune regulation and control function of stem cells.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Stem cells are cells with self-renewal/multidirectional differentiation potential and unique immune regulation function, and cell therapy represented by stem cells breaks through two traditional treatment modes of medicines and operations, and become the forefront and most promising disease intervention strategy in the field of biomedicine. At present, the rest ten stem cell products are approved to be on the market by all countries around the world, mainly mesenchymal stem cells, but no stem cell product is on the market at present at home, but in order to ensure the normal, ordered, safe and healthy cell therapy industry prospect, the countries set a series of policy and regulations to promote the clinical development of stem cell therapy, and the stem cell therapy is mainly carried out from two directions of stem cell clinical research and record projects and stem cell new drug clinical research. By the end of 2019, the number of national approved medical institutions for stem cell clinical treatment research has increased to 118 (including 12 military system hospitals), and the number of record projects has increased to 62. The perfection of regulatory policies will standardize the clinical transformation of stem cell therapy and direct the direction of development in this field.
In addition to self-renewal and multipotentiality, stem cells have unique immunological properties and exhibit unique immune regulatory functions. Stem cells have been used to treat various types of immune disorders such as autoimmune responses, disorders resulting from uncontrolled inflammation, such as type 1 diabetes, Systemic Lupus Erythematosus (SLE), acute and chronic graft-versus-host-disease (GVHD), and the like. In addition, researchers are actively advancing research and exploration on clinical curative effects in severe cases including convalescent plasma, stem cells and the like, and the immune regulation and control capability of the stem cells is expected to be hopeful in assisting novel coronavirus blocking fighting, and particularly, the immune regulation and control capability of the stem cells is expected to have the advantage that other treatments cannot be replaced in the aspects of preventing and improving severe inflammatory waterfall reactions and pulmonary fibrosis. Currently, a large number of preclinical studies at home and abroad verify the effectiveness and safety of stem cells in treating autoimmune diseases, and a lot of studies enter a phase II-III clinical test stage. In recent years, the immune regulation function of stem cells is gradually taken as a key effectiveness attribute of the stem cells as a therapeutic product internationally, and a consensus is formed and some constructive opinions are proposed, such as that International Society of Cell Therapy (ISCT) suggests in 2013 that a set of reliable and well-reproducible standardized detection technology is established for each immune regulation function of the stem cells, so as to evaluate the quality of the stem cells as the therapeutic product. However, the quality control and preclinical research guiding principle of stem cell preparations, which are released from the stem cell clinical research management method, also clearly put forward 'the quality evaluation content of the biological effectiveness of stem cells by immune regulation and control function'.
However, the inventors found that the current quality tests for stem cell preparations at home and abroad generally only involve general biological attributes (quantity, activity, proliferation capacity, and the like), microbial safety (detection of bacteria, viruses, fungi, endotoxicity, and the like), and biological safety (detection of antigenicity, tumorigenicity, and the like), and the quality test means for biological effectiveness of cell preparations is relatively lacking, and at present, the quality test means also focuses more on the detection of molecular markers and induced differentiation functions, and the key cell biological performance evaluation of immune regulation and control performance is lacking. Especially for diabetes, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus and the like), acute and chronic inflammatory diseases (such as systemic inflammatory response SIRS caused by infection of novel coronavirus COVID-19, SARS coronavirus, Ebola virus or H7N9 influenza virus and the like), and the like, the immune regulation performance rather than the directional differentiation capacity is the most important treatment mechanism of stem cells. For cell preparations used for the treatment of these diseases, scientific and objective evaluation of their immunoregulatory properties is essential.
Disclosure of Invention
Therefore, it is an object of the present invention to provide a standardized protocol for assessing the immune regulatory function of stem cells. The method is based on the basic principle of mixed lymphocyte reaction, can evaluate the inhibition capacity of stem cells on the proliferation of total lymphocytes, and is used as the basic performance evaluation of the immune regulation function of the stem cells; the stem cell pair CD4 can also be further evaluated+The regulation and control efficiency of T lymphocyte subgroup proliferation and differentiation, and the regulation and control ability of stem cells to release inflammatory factors from different immune cells. The method is a reliable and good-repeatability standardized detection method, can be used as necessary content for comprehensively reflecting the quality of the stem cells, and can reflect the biological effectiveness of the stem cell preparation.
Specifically, the technical scheme of the invention is as follows:
the invention provides a method for evaluating the immune regulation function of stem cells, which comprises the following steps: the method comprises the steps of sorting the effector cells of a recipient (namely an individual receiving stem cell treatment) receiving a stem cell preparation or an identical standard donor (serving as a reference) based on immunomagnetic bead sorting (MACS), performing standardized effector cell amplification by using antibody-coated amplified magnetic beads, staining and marking the effector cells based on carboxyfluorescein diacetate succinimidyl ester (CFSE), evaluating the proliferation of the effector cells mixed and cultured in a stem cell suspension to be detected by flow cytometry, and evaluating the immunoregulation function of the stem cells to be detected by the inhibition rate of the effector cell proliferation.
Specifically, the method for evaluating the immune regulation function of the stem cells comprises the following steps: separating and preparing effector cells; CFSE labeling effector cells; inoculating effector cells; carrying out mixed culture on effector cells and stem cells to be detected; detecting the proliferation condition of effector cells in the mixed cells by flow cytometry; evaluating the immune regulation function of the stem cells to be detected according to the inhibition rate of the proliferation of the effector cells.
The effector cells of the invention are PBMCs (human peripheral blood mononuclear cells) or Tresp cells (effector T cells, which are not effector T cell-effector T cells but effector cells that can be regulated by stem cells in T cells).
In some embodiments of the invention, the method for isolating PBMCs comprises: taking PBMCs of a separated stem cell preparation receptor (an individual to be treated by stem cell infusion), removing cell clusters in suspension cells, centrifuging, and removing dead cells by discarding a stem supernatant; in some embodiments, the method for removing the cell mass in the suspension cells can be a conventional method in the art, or can be a method of sieving with a 30um nylon mesh.
In some embodiments of the invention, the Tresp cells are prepared using a T cell sorting kit. The T cell sorting kit can be used for sorting regulatory T cells and effector T cells Tresp and can be obtained by purchasing.
In some embodiments of the invention, the Tresp comprises, but is not limited toRestricted to CD4+CD25-T cells, and CD4+CD25-The method for preparing the T cell comprises the following steps: label non-CD 4+Cell, antibody marker CD25+And (4) selecting Tresp cells from the MS column.
In some specific embodiments, the operation process comprises:
label non-CD 4+Cell: resuspend with 90. mu.l MACS buffer (10 each)7Cells), 10. mu.l of Biotinlabelled Cocktail antibody was added, mixed well, and incubated at 4 ℃ for 10 min; adding 20. mu.l of anti-Biotin microbeads, and incubating for 15min at 4 ℃; adding 1mL of MACS buffer, centrifuging at 4 ℃ for 10min at 300g, and removing the supernatant by using a gun; adding 500. mu.l Buffer for resuspension; negative selection of non CD4 from LD separation column+Cells (placing an LD column separation column on a Separator (purple) on a magnetic field STAND, adding 2mL of buffer for rinsing, naturally flowing the cell suspension through the column, continuously adding 1mL of buffer for two times, and collecting all cell suspensions (namely CD4+ cell suspensions including washing liquid) flowing through the column;
antibody marker CD25+Cell: the CD4+The cell suspension was centrifuged at 300g for 10min, the supernatant removed, and 90. mu.l buffer added for resuspension (10 aliquots)7Cells); add 10. mu.l of anti-CD25 microbeads (10 each)7Cells), mixing well, incubating for 15min at 4 ℃; adding 1mL buffer, centrifuging at 4 deg.C for 10min at 300g, and removing supernatant with gun; adding 500 mu l of Buffer for resuspension to obtain cell suspension marked by anti-CD25 magnetic beads;
and (3) selecting Tresp cells on the MS column: placing the LD column on a small Separator (Green) in a magnetic field STAND, and adding 500 μ l buffer for rinsing; adding the above cell suspension, flowing through the separation column, continuously adding 500L buffer for 3 times, and collecting the cell suspension (i.e. Tresp cells: CD 4) flowing through the separation column+CD25-A cell suspension). Removing the magnetic field from the MS column, placing in a collected EP tube, adding 1mL buffer, immediately plugging a piston to extrude the cell suspension, and collecting the cell suspension in a sterile EP tube to obtain purified regulatory T cells (Treg cells, CD 4)+CD25+A cell). The purity of the cells obtained by sorting is determined by a flow cytometer
In embodiments of the invention, the detection of effector cell proliferation is preferably by evaluation of the CSFE marker, other assays such as LDH-based assays (e.g., MTT colorimetric assay), 3H-TdR incorporation, or BrdU labeling may be substituted, but in the present study, CSFE markers are more preferred in all respects. The process for labeling effector cells with CSFE comprises the following steps: mixing the CSFE stock solution and PBS in a dark place, and quickly mixing the effect cells with the mixed solution in the dark place after resuspending the effect cells by the PBS; after incubation and centrifugation, the supernatant was removed and resuspended in complete T cell culture medium.
In some embodiments of the invention, the effector cells are resuspended in an equal volume of PBS.
The mixing volume of the PBS resuspension of the effector cells, the CSFE storage solution and the PBS light-proof uniform mixing solution is 1: 1;
the incubation conditions were: incubating at room temperature (25 ℃) for 5min in the dark; or incubating at 4-10 ℃ in a dark place;
after incubation and centrifugation at 300g for 5min, the supernatant was removed and resuspended in 2mL of T cell complete medium containing 10% FBS.
The CSFE stock solution of the present invention can adopt a stock solution conventionally used in the art, and in the present invention, CSFE dissolved in DMSO is preferable, and the concentration of CSFE dissolved in DMSO is 5 mmol/L.
In an embodiment of the invention, the process of seeding the effector cells comprises: CSFE-labeled effector cells were resuspended in complete T cell medium with added expanded magnetic beads and plated in well plates, with 2 × 3 effector cell replates per set.
The amplification magnetic beads are magnetic beads pre-coated with anti-human CD3 antibodies and CD28 antibodies. The anti-human CD3 antibody and CD28 antibody pre-coated magnetic beads are called expanded magnetic beads, because the magnetic beads can replace antigen stimulated and Antigen Presenting Cells (APC) to contact T cells and directly and nonselectively activate TCR first signals and CD28 second signals, and therefore activation of T cell proliferation is achieved without APC presentation. Wherein the magnetic beads are self-prepared or commercially available from MACSiBead available from MiltenyiTMOr Dynabeads from Invitrogen corporationTM。
In some embodiments of the invention, the ratio of the number of expanded magnetic bead particles to the number of CSFE-labeled effector cells is from 4:1 to 1:1, preferably 2:1, depending on the effector cell mixed culture time and the number of expected expansion passages;
in the present embodiment, the number of cells after resuspension is 5X 105To 1X 106Individual cell/mL, preferably 5X 105Individual cells/mL;
in an embodiment of the invention, the number of cells per well is 1X 104To 1X 105Individual cells, preferably 5X 10, according to the desired seeding density and expansion of the effector cells4And (4) cells.
In some embodiments of the present invention, the method for preparing the complete T cell culture medium containing the expanded magnetic beads comprises: the resuspended beads were mixed well in the visual, and the mixed beads (4X 10) were immediately aspirated7beads/mL) to a sterile EP tube, adding an equal volume or at least 500. mu.l of minimal medium, centrifuging to remove the supernatant or placing on MACS STAND, sucking off the liquid after the magnetic beads are fully adsorbed to the tube wall, and suspending with a complete T cell culture medium. Before the complete amplification medium is prepared, magnetic bead washing is required.
In an embodiment of the present invention, the process of culturing the effector cells and the stem cells to be detected in a mixed manner comprises: adding the stem cell single cell suspension to be detected into 3 multiple holes of the hole plate inoculated with effector cells to serve as detection holes; the single cell suspension of effector cells not labeled with CSFE was added to the other 3 multiple wells of the effector cell-seeded well plate as control wells, mixed well and cultured.
In the embodiments of the present invention, the culture conditions may employ conditions suitable for culturing stem cells and effector cells in the art. In some embodiments of the invention, preferred culture conditions are: 37 ℃ and 5% CO2Culturing under dark and saturated humidity conditions for 48-96h, preferably 48 h.
In an embodiment of the present invention, detecting the proliferation of effector cells in the mixed cells by flow cytometry comprises: collecting mixed cultured cells, fully blowing dispersed cells and magnetic beads, collecting cell suspension in a strong magnetic field, centrifuging to remove a culture medium, carrying out flow-type detection after resuspension by PBS, and selecting and analyzing the proportion of CFSE positive cells, namely proliferating cells of effector cells.
In an embodiment of the present invention, the evaluation of the inhibition rate of the proliferation of the effector cell for the immune regulatory function of the stem cell to be tested calculates the inhibition rate of the proliferation of the effector cell according to the following formula:
the inhibition rate of effector cell proliferation (average value of proliferating cell ratio in control well-average value of proliferating cell ratio in detection well)/average value of proliferating cell ratio in control well × 100%.
The method can evaluate the inhibition capacity of the stem cells on effector cells (such as total mononuclear cells, certain effector T cell subsets and the like) and is used for evaluating the basic performance of the immune regulation function of the stem cells; the stem cells can also be further evaluated for effector cells (e.g., CD 4)+T lymphocyte subpopulation) and the regulatory capacity of stem cells to release inflammatory factors from different immune cells. In specific application, the method can be used for evaluating the regulation and control capability of the stem cells on the aspect of releasing inflammatory factors from different immune cells in the treatment of diabetes, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus and the like) and acute and chronic inflammatory diseases (such as systemic inflammatory response SIRS caused by infection of novel coronavirus COVID-19, SARS coronavirus, Ebola virus or H7N9 influenza virus and the like), and has good repeatability and comparability.
The method of the invention can realize the effect of stem cells, especially different batches of stem cells on receptor effector cells (such as peripheral blood mononuclear cells or CD 4)+CD25-Effector cells) are involved. One skilled in the art can adjust the selection of other subpopulations of cells that can be activated by antigen presenting cells as effector cells (including but not limited to CD 3) as needed for clinical therapeutic purposes based on the disclosure of the present invention+T cell subset, CD3+CD8+T cell subset, CD3+CD4+T cell subsets and lymphocyte subsets specific for a particular antigen, etc.). In addition, the method of the invention can also be used for detecting and controlling other cell preparations for cell therapy, such as other immune negative regulatory cells (including but not limited to regulatory T cells).
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a diagram of the data of the flow cytometry machine test in the test example.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Kit contents and instructions：
T cell culture medium supplement (Supplements, 100X) 10mLRPMI1640 minimal medium containing glutamine 0.2923g HEPES 5.9575g β -mercaptoethanol 3.52. mu.l (filter sterilization), 1mL of the above Supplements, RPMI164088mL + inactivated FBS 10mL +5000U/mL IL-21 mL + double antibody (1000X) 100. mu.l to prepare complete T cell culture medium (complete medium with IL-2 added stored at 4 ℃ for 1 week of validity period);
standardized amplified magnetic beads (4X 10)7Buffer phosphate buffer 0.1% human serum albumin): the magnetic beads pre-coated with anti-human CD3 antibody and CD28 antibody are called expanded magnetic beads, because the magnetic beads can replace antigen-stimulated and antigen-presenting cells (APC) to contact T cells and directly activate TCR first signal without selectionCD28 second signal, to effect activated T cell proliferation without APC presentation. The magnetic beads can be prepared by themselves or selected from MACSiBead of MiltenyiTMOr Dynabeads from Invitrogen corporationTM。
CFSE (5mmol/L in DMSO) -25 ℃ is frozen and stored in dark place;
human recombinant interleukin-2 (IL-2) (5000U/mL, 100X);
rapamycin (rapamycin).
Other key reagents and devices：
Peripheral Blood Mononuclear Cell (PBMC) isolates;
t cell sorting kits (useful for regulatory T cell, effector T cell Tresp sorting);
magnetic field cell sorting rack MACS multi Sand, purchased from Miltenyi Biotec;
the method for evaluating the immune regulation function of the stem cells comprises the following steps:
A. taking PBMCs of an isolated stem cell preparation receptor (an individual to be treated by stem cell infusion) to pass through a 30um nylon mesh to remove cell masses in suspension cells, centrifuging for 10 minutes at 300g, and removing dead cells by discarding a stem supernatant;
B. optionally, effector cell sorting (with CD 4)+CD25-T cells are examples): and (3) sorting and preparing Tresp cells by adopting a T cell sorting kit:
a. labeling non-CD 4+ cells: resuspend with 90. mu.l MACS buffer (10 each)7Cells), 10. mu.l of Biotinlabelled Cocktail antibody was added, mixed well, and incubated at 4 ℃ for 10 min; adding 20. mu.l of anti-Biotin microbeads, and incubating for 15min at 4 ℃; adding 1mL of MACS buffer, centrifuging at 4 ℃ for 10min at 300g, and removing the supernatant by using a gun; adding 500. mu.l Buffer for resuspension; removing non CD4+ cells by negative selection of an LD separation column (placing the LD column on a Separator (purple) on a magnetic field STAND, adding 2mL of buffer for rinsing, adding the cell suspension, naturally flowing through the separation column, continuously adding 1mL of buffer for two times, and collecting all cell suspensions (namely CD4+ cell suspensions including washing liquid) flowing through the separation column;
b. antibody-labeled CD25+ cells: the CD4+ cell suspension was centrifuged at 300g for 10min, the supernatant removed, and resuspended in 90. mu.lbuffer (every 10. mu.l of suspension)7Cells); add 10. mu.l of anti-CD25 microbeads (10 each)7Cells), mixing well, incubating for 15min at 4 ℃; adding 1mL buffer, centrifuging at 4 deg.C for 10min at 300g, and removing supernatant with gun; adding 500 mu l of Buffer for resuspension to obtain cell suspension marked by anti-CD25 magnetic beads;
MS column negative selection of Tresp cells: placing the LD column on a small Separator (Green) in a magnetic field STAND, and adding 500 μ l buffer for rinsing; adding the above cell suspension, flowing through the separation column, continuously adding 500L buffer for 3 times, and collecting the cell suspension (i.e. Tresp cells: CD 4) flowing through the separation column+CD25-T cell suspension); removing the magnetic field from the MS column, placing the MS column in a collected EP tube, adding 1mL buffer, immediately plugging a piston to extrude the cell suspension and collect the cell suspension into a sterile EP tube, namely the purified Treg cell (CD 4)+CD25-A cell). The purity of the cells obtained by sorting is identified by a flow cytometer;
C. preparing amplified magnetic beads: before preparing the complete amplification culture medium, magnetic bead cleaning is required: fully mixing the resuspended magnetic beads in the visual, immediately sucking a proper volume of the uniformly mixed magnetic beads (4 multiplied by 107beads/mL) to a sterilized EP tube, adding an equal volume or at least 500 mu l of a basic culture medium, centrifuging for 5min at 300g to remove supernatant (or putting on MACS STAND for 1min, sucking liquid after the magnetic beads are fully adsorbed to the tube wall), and then suspending by a proper volume of complete T cell culture medium;
csfe-labeled effector cells: CSFE stock solution (1000X) 2. mu.l add 1mL PBS (or proportional volume) in EP tube and mix well in dark, effector cells (above separation of PBMC or Tresp) with equal volume of PBS heavy suspension; adding the two into the mixture at a ratio of 1:1, quickly and uniformly mixing, and incubating for 5min at room temperature in a dark place; centrifuging at 300g for 5min to remove supernatant, and adding 2mL of T cell complete culture medium containing 10% FBS for resuspension; centrifuge 300g for 5min and wash once more.
E. Effector cell inoculation: the CSFE-labeled PBMC or Tresp were resuspended (5X 10) in a complete T-cell medium containing expanded magnetic beads (number of magnetic bead particles: 2:1)5Individual cells/mL), 100uL were seeded in round bottom 96-well plates at 5 × 10 per well4Individual cells, each group of stem cells to be tested being arranged2 × 3 effector cell repore wells;
F. detecting cell mixed culture: 100uL of stem cell single cell suspension to be detected (5X 10)5Individual cells/mL) was added to 3 duplicate wells of the above effector cell-containing 96-well plate as detection wells, and 100uL of the unlabeled effector cell single cell suspension (5 × 10) obtained in step C was added5Each cell/mL) was added to 3 multiple wells of the 96-well plate containing effector cells as control wells, shaken horizontally and mixed well, and cultured for 48h at 37 ℃ under conditions of 5% CO2 in dark and saturated humidity;
G. detecting on a flow type computer: collecting cells in an EP tube, fully blowing dispersed cells and magnetic beads, placing in a strong magnetic field for 1min, collecting cell suspension, and centrifuging at 300g for 5min to remove culture medium; resuspending in 200. mu.l PBS, detecting by flow machine, selecting CFSE positive cells by drawing a gate, and analyzing the proportion of the proliferating cells of the CFSE positive cells (namely effector cells) by a histogram;
H. evaluating the immune regulation function of the stem cells to be detected according to the inhibition rate of the proliferation of effector cells:
the inhibition rate of effector cell proliferation (average value of proliferating cell in control well-average value of proliferating cell in detection well)/average value of proliferating cell in control well x 100%
Detection examples
The purpose is as follows: and (3) detecting and comparing the immunoregulation performance of the P3 generation umbilical cord mesenchymal stem cells of the conventional culture group and the inflammatory factor pretreatment culture group on the T lymphocytes of the type 1 diabetes subjects.
The method comprises the following steps:
1. preparing the mesenchymal stem cell single cell suspension to be detected:
mesenchymal stem cells (without serum components) derived from umbilical cord of the same healthy donor are purchased or prepared in a conventional manner for conventional culture, and after the cells are expanded to the third generation, the cells are equally divided into two parts, one part is a conventional treatment group and the other part is an inflammatory factor pretreatment group and is marked as B, and the difference between the group B and the group A is that recombinant human cytokine (IFN-gamma) is added to the culture medium at a concentration of 50 ng/ml. After the P3 subculture was completed, A, B two groups of serum-free suspension of stem cells were collected and prepared in the same manner and used for intravenous infusion.
2. Collecting peripheral blood of a type 1 diabetes patient to be treated by stem cells, separating PBMCs, removing cell masses in suspension cells through a 30um nylon mesh, centrifuging for 10 minutes at 300g, and removing dead cells by discarding a dry supernatant;
3. sorting patient's CD4+CD25-And (3) taking the T cells as the Tresp cells, and sorting and preparing the Tresp cells by adopting a T cell sorting kit:
a. labeling non-CD 4+ cells: resuspend with 90. mu.l MACS buffer (10 each)7Cells), 10. mu.l of Biotinlabelled Cocktail antibody was added, mixed well, and incubated at 4 ℃ for 10 min; adding 20. mu.l of anti-Biotin microbeads, and incubating for 15min at 4 ℃; adding 1mL of MACS buffer, centrifuging at 4 ℃ for 10min at 300g, and removing the supernatant by using a gun; adding 500. mu.l Buffer for resuspension; removing non CD4+ cells by negative selection of an LD separation column (placing the LD column on a Separator (purple) on a magnetic field STAND, adding 2mL of buffer for rinsing, adding the cell suspension, naturally flowing through the separation column, continuously adding 1mL of buffer for two times, and collecting all cell suspensions (namely CD4+ cell suspensions including washing liquid) flowing through the separation column;
b. antibody labeling of CD25+ cells: the CD4+ cell suspension was centrifuged at 300g for 10min, the supernatant removed, and resuspended in 90. mu.lbuffer (every 10. mu.l of suspension)7Cells); add 10. mu.l of anti-CD25 microbeads (10 each)7Cells), mixing well, incubating for 15min at 4 ℃; adding 1mL buffer, centrifuging at 4 deg.C for 10min at 300g, and removing supernatant with gun; adding 500 mu l of Buffer for resuspension to obtain cell suspension marked by anti-CD25 magnetic beads;
MS column negative selection of Tresp cells: placing the LD column on a small Separator (Green) in a magnetic field STAND, and adding 500 μ l buffer for rinsing; adding the above cell suspension, flowing through the separation column, continuously adding 500L buffer for 3 times, and collecting the cell suspension (i.e. Tresp cells: CD 4) flowing through the separation column+CD25-T cell suspension); removing the magnetic field from the MS column, placing the MS column in a collected EP tube, adding 1mL buffer, immediately plugging a piston to extrude the cell suspension and collect the cell suspension into a sterile EP tube, namely the purified Treg cell (CD 4)+CD25-A cell). The purity of the cells obtained by sorting is identified by a flow cytometer;
4. expanding devicePreparing an augmented magnetic bead: the resuspended beads were mixed well in the visual, and the mixed beads (4X 10) were immediately aspirated7beads/mL) to a proper volume, adding an equal volume or at least 500 μ l of a minimal medium into the sterilized EP tube, centrifuging for 5min at 300g to remove supernatant (or placing on MACS STAND for 1min, and absorbing liquid after magnetic beads are fully adsorbed to the tube wall), and suspending by a proper volume of complete T cell culture medium;
CSFE-labeled effector cells: CSFE stock solution (1000X) 2. mu.l add 1mL PBS (or proportional volume) in EP tube and mix well in dark, effector cells (above separation of PBMC or Tresp) with equal volume of PBS heavy suspension; adding the two into the mixture at a ratio of 1:1, quickly and uniformly mixing, and incubating for 5min at room temperature in a dark place; centrifuging at 300g for 5min to remove supernatant, and adding 2mL of T cell complete culture medium containing 10% FBS for resuspension; centrifuge 300g for 5min and wash once more.
Seeding of Tresp cells: the CSFE-labeled Tresp was resuspended (5X 10) in a T cell complete medium containing expanded magnetic beads (number of magnetic bead particles: 2:1)5Individual cells/mL), 100uL were seeded in round bottom 96-well plates at 5 × 10 per well4Cells, 3 × 3 effector cells plated in duplicate;
7. detecting cell mixed culture: the A, B two groups of stem cell single cell suspensions (5X 10) to be detected5Each cell/mL) was added to each well at 100uL, 3 duplicate wells of the CSFE-labeled Tresp-seeded 96-well plate were used as assay wells (i.e., 3 assay wells each, total 6 wells), and 100uL of the CSFE-unlabeled patient Tresp single cell suspension obtained in step 4 (5 × 10)5Each cell/mL) is added into 3 multiple wells of the 96-well plate inoculated with the CSFE labeled Tresp as control wells, the control wells are horizontally shaken and fully mixed, and the mixture is cultured for 48 hours at 37 ℃ under the conditions of 5 percent CO2 dark and saturated humidity;
8. detecting on a flow type computer: collecting cells in an EP tube, fully blowing dispersed cells and magnetic beads, placing in a strong magnetic field for 1min, collecting cell suspension, and centrifuging at 300g for 5min to remove culture medium; resuspending in 200. mu.l PBS, detecting by flow machine, selecting CFSE positive cells by drawing a gate, and analyzing the proportion of the proliferating cells of the CFSE positive cells (namely effector cells) by a histogram;
9. evaluating the immune regulation function of the stem cells to be detected according to the inhibition rate of the proliferation of effector cells:
the inhibition rate of effector cell proliferation (average value of proliferating cell in control well-average value of proliferating cell in detection well)/average value of proliferating cell in control well x 100%
The results are shown in the flow cytometry machine test data chart (FIG. 1) and Table 1.
TABLE 1
Analysis of the results, figure 1 and table 1 results show that:
the proliferation inhibition rate of the conventional stem cells of the stem group (group A) on the effector cells is 47.31%, while the proliferation inhibition rate of the stem cells of the inflammatory factor pretreatment strengthening group (group B) on the effector cells is increased to 65.28%. The result shows that the method of the embodiment can well evaluate the regulation and control capability of the stem cells on the aspect of releasing the inflammatory factors by the immune cells in the treatment of the type 1 diabetes. In addition, the coefficient of variation among each group of detection compound holes is within 5%, and the detection repeatability and comparability are good.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of assessing stem cell immune modulatory function comprising: sorting the effector cells of a receptor receiving the stem cell preparation or the same standard donor, performing standardized effector cell amplification by using antibody-coated amplification magnetic beads, marking the effector cells based on CFSE staining, evaluating the proliferation of the effector cells mixed and cultured in the stem cell single-cell suspension to be detected by flow cytometry, and evaluating the immune regulation and control function of the stem cells to be detected according to the inhibition rate of the proliferation of the effector cells.
2. The method according to claim 1, characterized in that it comprises the steps of: separating and preparing effector cells; CFSE labeling effector cells; inoculating effector cells; carrying out mixed culture on effector cells and stem cells to be detected; detecting the proliferation condition of effector cells in the mixed cells by flow cytometry; evaluating the immune regulation function of the stem cells to be detected according to the inhibition rate of the proliferation of the effector cells.
3. The method according to claim 1 or 2, wherein said effector cells are PBMCs or Tresp cells, wherein said Tresp cells include but are not limited to CD4+ CD25-T cells.
4. The method of claim 3, wherein the method of separating the PBMCs comprises: taking PBMCs of the separated stem cell preparation receptor, removing cell clusters in the suspension cells, centrifuging, and removing dead cells by discarding the supernatant;
preferably, the Tresp cells are prepared using a T cell sorting kit.
5. The method of claim 2, wherein the CSFE-labelling effector cells comprises: mixing the CSFE stock solution and PBS in a dark place, and quickly mixing the effect cells with the mixed solution in the dark place after resuspending the effect cells by the PBS; after incubation and centrifugation, removing supernatant and adding the supernatant into a complete T cell culture medium for resuspension;
preferably, the effector cells are resuspended in an equal volume of PBS;
preferably, the mixing volume of the PBS resuspension of the effector cells, the CSFE storage solution and the PBS light-proof mixing solution is 1: 1;
preferably, the incubation conditions are: incubating at room temperature in dark for 5min or at 4-10 deg.C in dark;
preferably, after incubation, centrifugation at 300g for 5min, the supernatant is removed and resuspended in 2mL of T cell complete medium containing 10% FBS.
6. The method of claim 2, wherein the seeding of effector cells comprises: resuspending CSFE-labeled effector cells in a T cell complete culture medium added with expanded magnetic beads, inoculating the effector cells into a pore plate, and arranging 2 x 3 effector cell repolls in each group;
preferably, the amplified magnetic beads are magnetic beads pre-coated with anti-human CD3 antibody and CD28 antibody;
preferably, the ratio of the number of the amplified magnetic bead particles to the number of the CSFE-labeled effector cells is 4:1 to 1:1, preferably 2: 1;
preferably, the number of cells after resuspension is 5X 105-1×106Individual cell/mL, preferably 5X 105Individual cells/mL;
preferably, the number of cells per well is 1X 104-1×105Individual cell, preferably 5X 104And (4) cells.
7. The method of claim 6, wherein the expanded magnetic beads-containing T cell complete medium is prepared by a method comprising: fully mixing the resuspended magnetic beads in the visual, immediately sucking the uniformly mixed magnetic beads to a sterilized EP tube, adding an equal volume or at least 500 mu l of a minimal medium, centrifuging to remove a supernatant or placing on MACS STAND, sucking off a liquid after the magnetic beads are fully adsorbed to the tube wall, and then suspending by using a complete T cell culture basis.
8. The method according to claim 2, wherein the process of culturing the effector cells and the stem cells to be detected in a mixed manner comprises: adding the stem cell single cell suspension to be detected into 3 multiple holes of the hole plate inoculated with effector cells to serve as detection holes; adding the single cell suspension of the effector cells which are not marked by CSFE into the other 3 multiple wells of the pore plate inoculated with the effector cells as control wells, uniformly mixing and culturing;
preferably, the culture conditions are: 37 ℃ and 5% CO2Culturing for 48-96h under dark and saturated humidity conditions.
9. The method of claim 2, wherein detecting effector cell proliferation in the mixed cells using flow cytometry comprises: collecting mixed cultured cells, fully blowing dispersed cells and magnetic beads, collecting cell suspension in a strong magnetic field, centrifuging to remove a culture medium, carrying out flow-type detection after resuspension by PBS, and selecting and analyzing the proportion of CFSE positive cells, namely proliferating cells of effector cells.
10. The method according to claim 8, wherein the stem cell immunoregulatory function to be tested is evaluated as the rate of inhibition of effector cell proliferation, and the rate of inhibition of effector cell proliferation is calculated according to the following formula:
the inhibition rate of effector cell proliferation (average value of proliferating cell ratio in control well-average value of proliferating cell ratio in detection well)/average value of proliferating cell ratio in control well × 100%.
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