CN115433709A - In-vitro experimental model for predicting myocardial cell transplantation immune rejection - Google Patents
In-vitro experimental model for predicting myocardial cell transplantation immune rejection Download PDFInfo
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Abstract
The invention belongs to the technical field of cells, and relates to an in vitro experimental model for predicting myocardial cell transplantation immune rejection, which is used for obtaining PBMC (peripheral blood mononuclear cell) cells of a target object and iCM cells to be transplanted; PBMC and iCM are subjected to indirect co-culture in vitro, iCM is inoculated in an upper chamber of a Transwell chamber, PBMC is inoculated in a lower chamber of the Transwell chamber, and a culture solution in the upper chamber and a culture solution in the lower chamber can mutually permeate in the culture process; detection of T cells, B cells and NK cells in PBMCActivation and proliferation of (a); inoculation density of said PBMC 2.5 x 10 5 The iCM cells were differentiated via ipscs or ESCs. The indirect co-culture method can effectively improve the accuracy of the immune rejection reaction detection result prediction before clinical application of iCM, and meanwhile, the method has the characteristics of high reproducibility, small experimental data fluctuation and the like.
Description
Technical Field
The invention belongs to the technical field of cells, and relates to an in vitro experimental model for predicting myocardial cell transplantation immune rejection.
Background
One of the early challenges in the treatment of heart disease is that after a heart attack, a part of the heart muscle dies, the heart fails to regenerate muscle tissue, and the dead muscle tissue also causes damage to surrounding muscles, which in turn causes a fatal cardiac dilatation. The replacement of damaged myocardium by injection of cardiomyocytes is a new direction for cardiologists to explore in the treatment of heart disease.
Before the myocardial cells are applied to clinic, possible immune rejection is a necessary key link; if the produced cardiomyocytes were used directly in animal experiments or human experiments, their possible immune rejection would not be expected.
Therefore, the reaction design of immunological rejection reaction in vitro can reduce the risk of in vivo experiment, and is convenient for observing the treatment effect of the cardiac muscle cells on heart system diseases; secondly, a detection index of myocardial cell injection in the clinical application process can be effectively established, so that more comprehensive preclinical research evidence is provided for clinical application of the cardiomyocytes derived from the naive IPSC (iCM).
Disclosure of Invention
The application provides an in vitro experimental model for predicting myocardial cell transplantation immune rejection, and the in vitro verification model is mainly realized based on the design of an indirect co-culture system of PBMC and iCM. The indirect co-culture method can effectively improve the accuracy of the immune rejection reaction detection result prediction before clinical application of iCM, and meanwhile, the method has the characteristics of high reproducibility, small experimental data fluctuation and the like.
To achieve the above technical objects, the present application adopts the technologyThe scheme is that an in vitro experiment model for predicting myocardial cell transplantation immune rejection reaction obtains PBMC cells of a target object and iCM cells to be transplanted; PBMC and iCM are subjected to indirect co-culture in vitro, iCM is inoculated in an upper chamber of a Transwell chamber, PBMC is inoculated in a lower chamber of the Transwell chamber, and a culture solution in the upper chamber and a culture solution in the lower chamber can mutually permeate in the culture process; detecting the activation and proliferation of T cells, B cells and NK cells in PBMCs; inoculation density of said PBMC 2.5 x 10 5 /ml, the iCM cells were differentiated via ipscs or ESCs.
As an improved technical scheme of the application, PBMC and iCM are subjected to indirect co-culture based on a Transwell system, and the culture time is between 48 and 96 hours.
As an improved technical scheme of the application, when PBMC and iCM are subjected to indirect coculture in vitro, the quantity ratio of PBMC to iCM is designed according to a gradient, and at least 2 quantity ratios are selected for detection in the experimental process.
As an improved technical scheme of the application, the establishment mode of the in vitro experimental model comprises the steps of initially establishing the model, verifying the model and judging the result; wherein the content of the first and second substances,
preliminarily establishing a model, co-culturing commercial PBMC and an antibody, screening the dosage of PBMC when the PBMC is activated to the maximum extent, and determining the dosage of PBMC when the PBMC is an in vitro experiment model;
model verification, namely performing indirect co-culture on PBMC (peripheral blood mononuclear cell) of a patient and iCM on the basis of primary model establishment, and comparing the activation state and proliferation condition of T cells, B cells and NK cells in the PBMC in a state of independent culture relative to the PBMC after the indirect co-culture;
judging the result, and evaluating the activation condition of a positive control, the activation condition of a negative control and the activation condition of a test sample; the positive control group is designed to culture PBMC alone, add antibody into culture medium to activate PBMC, and detect the activation condition of the positive control; the negative control group is designed to culture PBMC alone and detect the activation condition of the negative control.
As an improved technical scheme of the application, the judgment of the result further comprises the step of determining that at least T cells in the positive control are activated, the negative control is not activated, and the condition that at least one cell type in the test sample is activated is judged to predict the abnormal immune response.
As an improved technical scheme of the application, the antibody is T Cell TransAct-human, and the addition amount is 0.5ul.
As an improved technical scheme of the application, when PBMC and iCM are subjected to indirect co-culture based on a Transwell system, the PBMC culture medium adopts 1640 culture medium added with FBS, and the volume fraction of the FBS is 10%; iCM medium 1640 medium supplemented with FBS and B27 was used, with FBS volume fraction of 5% and B27 volume fraction of 2%.
As an improved technical scheme of the application, the quantity ratio of PBMC to iCM is designed to be 1 (0.25-1).
As an improved technical scheme of the application, the indirect co-culture requires that the activity rate of iCM is not less than 70% and the purity is not less than 95%.
Advantageous effects
The application selects PBMC and iCM to carry out in-vitro indirect coculture for carrying out in-vitro verification of the myocardial cell transplantation immune rejection. The technical basis of system design is as follows: peripheral Blood Mononuclear Cells (PBMCs) are involved in the immunomodulation of humans. Peripheral blood mononuclear cells are mainly composed of two major cell types, lymphocytes and monocytes, wherein lymphocytes include T lymphocytes, B lymphocytes and NK cells. The T lymphocyte is about 45-70% of the important components of the immune system of the organism, and can be differentiated into various cell subgroups after being activated by conditions such as antigen stimulation and the like, thereby causing certain immune response. The PBMC and iCM indirect coculture can simulate the in-vivo microenvironment after the myocardial cell transplantation, so the indirect coculture in vitro experiment between the myocardial cell and the PBMC can be researched, and more comprehensive preclinical research evidence can be provided for the clinical application of the cardiomyocyte (iCM) derived from the iPSC.
The application adopts the quantity ratio of the PBMC and the iCM which are subjected to gradient design and are subjected to indirect co-culture, can summarize the possibility that the PBMC can be activated at the maximum probability, provides the prediction of the maximum possibility for predicting the immune rejection generated by the myocardial cell transplantation, and effectively improves the verification efficiency.
In the application, the PBMC grows in a suspension manner, the iCM grows in an adherent manner, so that the PBMC and the iCM cells are prevented from being in direct contact during indirect co-culture so as to prevent the surface protein interaction between the PBMC and the iCM cells, and the culture solution can be ensured to be fully interacted between the PBMC and the iCM cells. The application adopts a Transwell culture system, iCM is inoculated in an upper chamber, PBMC is inoculated in a lower chamber, and indirect co-culture of the two is realized.
In order to avoid the mutual influence of the culture mediums of the PBMC and the iCM in the indirect co-culture process on the growth of cells, the culture medium of the PBMC and the iCM culture medium are restrained, and interference items in the experimental process are avoided.
Drawings
FIG. 1 is a graph showing the results of flow cytometry tests using 0.5ul antibody;
FIG. 2 is a graph showing the results of flow cytometry tests using 0.1ul antibody;
FIG. 3 is a graph showing the results of flow cytometry at a PBMC dose of 10W;
FIG. 4 is a graph showing the results of flow cytometry at a PBMC dose of 15W;
FIG. 5 is a graph showing the results of flow cytometry at a PBMC dose of 20W;
FIG. 6 is a graph showing the results of flow cytometry at 25W PBMC;
FIG. 7 is a graph showing the results of flow cytometry at a PBMC dose of 50W;
FIG. 8 is a graph showing the results of a set of flow cytometry tests on patient one;
FIG. 9 is a graph showing the results of a set of two-flow cytometry tests on patient one;
FIG. 10 is a graph depicting the results of a patient one experimental group of three-flow cytometry tests;
FIG. 11 is a graph showing the results of a negative set of flow cytometry tests on patient one;
FIG. 12 is a graph depicting the results of a positive set of flow cytometry tests for patient one;
FIG. 13 is a graph showing the results of a flow cytometry test on the experimental group of patient two;
FIG. 14 is a graph showing the results of two-phase cytometry tests in patient two;
FIG. 15 is a graph depicting the results of a negative set of flow cytometry tests for patient two;
figure 16 is a graph depicting the results of a positive set of flow cytometry tests for patient two.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The invention selects PBMC and iCM to carry out in-vitro indirect co-culture to carry out in-vitro prediction of myocardial cell transplantation immune rejection, and designs a specific verification process, thereby effectively providing more comprehensive preclinical research evidence for clinical application of the myocardial cells (iCM).
The principle of the technical scheme is as follows: the microenvironment in which PBMCs and iCM are positioned in vivo is simulated based on an indirect co-culture technology, and the effect of secretion factors on each other between the PBMCs and iCM is utilized to judge whether iCM secretion factors can stimulate the PBMCs. Since peripheral blood mononuclear cells are mainly composed of two major cell types, lymphocytes and monocytes, which in turn include T lymphocytes, B lymphocytes and NK cells. After being stimulated by antigen, T lymphocyte, B lymphocyte and NK cell can respectively generate corresponding antibody, so that whether iCM stimulates PBMC in the indirect co-culture process is judged, and markers of T lymphocyte, B lymphocyte and NK cell can be respectively detected, such as: the marker of the T cell is CD3-Pc5.5; the B cell marker is CD19-APC; the marker for NK cells is CD56-PE.
In the technical scheme of the application, the PBMC source is obtained by separating and culturing peripheral blood of a patient; the separation and culture of peripheral blood adopt the prior art, which is not the key technology of the application, so the detailed description is omitted, but the understanding of the technical scheme of the application by the technical personnel in the field is not influenced.
The cardiomyocytes (iCM) in the present embodiment were differentiated from ipscs or ESCs. The iPSC differentiated cardiomyocyte and the ESC differentiated cardiomyocyte are both the prior art, and the iPSC differentiated cardiomyocyte and ESC differentiated cardiomyocyte are only applied in the application, so the detailed description is omitted, and the technical scheme of the technical scheme is not influenced by the technical scheme of the technical scheme. In order to ensure iCM (cardiomyocytes) to have optimal activity, it is preferred that iCM is a cell after day 15 of differentiation.
The indirect co-culture of the present application was carried out using Transwell chamber manufacturers: LABSELECT. In the Transwell chamber, iCM was seeded in the upper chamber of the Transwell chamber; PBMC were seeded in the lower chamber of a Transwell chamber. Wherein the pore size of the upper chamber is selected to be 0.4 μm to facilitate interpenetration of the culture fluid between the upper chamber and the lower chamber.
During the indirect co-cultivation, the variables were controlled as follows:
culture conditions for the cells are selected from the conditions of conventional cell culture such as 37 ℃ and 5% CO 2 The incubator of (2) is not changed.
(1) The number ratio of PBMC to iCM cells was designed:
based on the characteristics of cell culture, the present application verifies that the ratio of PBMC to iCM numbers, i.e. PBMC to iCM numbers, increase by geometric multiple, by way of example, experiments design multiple sets, where the ratio of PBMC to iCM in each set of experiments can be designed as 1; verification may also be done incrementally, examples: the PBMC to iCM ratio in each set of experiments can be designed as 1.25, 1:1.25 … … PBMCs from different sources in the actual operation process have different immunological rejection reactions to iCM, and the verification of the maximum range is that the immunological rejection reactions can effectively improve the experimental efficiency.
Based on the principles of text simplification, after a large number of experiments, a group of data with features therein was selected for presentation. Different numbers are different from corresponding secretion factor bases, the PBMC has too large number and has more lymphocytes, and if the iCM cells secrete factors to cause immune rejection reaction, the PBMC has less cells to generate the immune rejection reaction and is difficult to observe. If the number of PBMC cells is small, iCM secretes trace factors to directly trigger PBMC to generate a large amount of antibodies, but the situation cannot truly reflect the in-vivo microenvironment after iCM injection, and cannot be effectively used as a reference basis for immune rejection reaction verification. Therefore, the application designs the quantity ratio of the PBMC to the iCM to be 1 (0.0001-1.25), preferably 1 (0.25-1) by analyzing the lymphocyte content in peripheral blood and combining a large number of medical experiments.
More preferably, when PBMC and iCM are co-cultured indirectly in vitro, the quantity ratio of PBMC to iCM is designed according to gradient, and at least 2 quantity ratios are selected for detection during the experiment.
(2) Requirement of indirect cocultivation time:
in order to achieve the maximum effect of iCM on PBMC, PBMC and iCM were subjected to indirect co-culture based on the Transwell system for not less than 48h, preferably 48h-96h.
(3) Requirement of indirect cocultivation Medium:
to avoid introducing more variables, the PBMC media need to be controlled so as not to affect iCM, while iCM media does not affect PBMC, and any media that can meet this requirement can be used. When PBMC and iCM are subjected to indirect co-culture based on a Transwell system, the PBMC culture medium adopts 1640 culture medium added with FBS, and the volume fraction of the FBS is 10%; iCM medium 1640 medium supplemented with FBS and B27 was used, with FBS volume fraction of 5% and B27 volume fraction of 2%. Effectively ensures that PBMC and iCM are not affected by each other's culture medium.
(4) The inoculation density requirement is as follows:
to ensure that both PBMC and iCM had good status during culture, the PBMC were seeded at a density of 2.5 x 10 5 The seeding density of iCM was determined by the optimal dose ratio of PBMC to iCM.
(5) Cell viability requirements:
the indirect co-culture of PBMC and iCM based on the Transwell system comprises the pretreatment of iCM(ii) a Pretreatment iCM comprises: uniformly planting the myocardial cells in the chamber according to the required myocardial cell density in indirect co-culture, and using 0.5mL of iCM recovery solution (any culture solution capable of realizing myocardial cell recovery can be adopted, such as: the recovery solution comprises 0.5-10% of DMSO, 10-25% of human serum albumin, 0.01-0.05g/mL of hydroxyethyl starch and 0.585-0.805% of sodium chloride solution, or a Cardioeasy myocardial recovery culture medium) for each hole; the next day, the recovery solution of cardiomyocytes (iCM) is changed to cardiomyocyte maintenance solution (a culture medium capable of maintaining the activity of cardiomyocytes, such as the complete culture medium of human cardiomyocytes of model CM-H076 manufactured by Procell), and the temperature is 37 deg.C and 5% CO 2 The cells were attached to the wall, expanded and recovered from beating after 3 days of culture in the incubator, and the medium was changed once every 1 day.
Finally, the activity rate of iCM is required to be not less than 70% and the purity is not less than 95% when indirect co-culture is carried out. In order to ensure the activity of iCM cells, the cardiomyocytes used in the in vitro experimental model were iCM cells after day 15 of differentiation.
Design of verification mode
An in vitro experimental model for predicting myocardial cell transplantation immune rejection reaction is used for obtaining PBMC cells of a target object and iCM cells to be transplanted; indirectly co-culturing PBMC and iCM in vitro; and detecting the activation and proliferation of T cells, B cells and NK cells in the PBMCs.
Specifically, the in vitro experiment model establishment mode comprises the steps of initially establishing a model, verifying the model and judging a result; wherein, the preliminary modeling comprises: co-culturing the commercialized PBMC with the antibody, screening the dosage of the PBMC when the PBMC is activated to the maximum extent, and confirming the dosage of the PBMC as an in vitro experiment model; the model verification comprises the steps of carrying out indirect co-culture on PBMCs of a patient and iCM on the basis of primary model establishment, and comparing the activation state and proliferation state of T cells, B cells and NK cells in the PBMCs in a state of independent culture relative to the PBMCs after the indirect co-culture; the antibody was a T Cell TransAct human antibody (trade name, anti-CD3& Anti-CD28 antibody beads, or Anti-CD3/CD28 magnetic beads (GMP-B038)), and the amount added was 0.5ul. The determination of the result includes assessing positive control activation, negative control activation, test sample activation. Determining the result further comprises determining that at least one of the positive control has activated T cells, the negative control has not activated, and the test sample has activated at least one cell type as predictive of an aberrant immune response.
In particular for the iCM transplantation immune response in vitro experiment: comprises a negative control group and a positive control group; observing iCM transplantation immune response by comparing the activation and proliferation of T cells, B cells and NK cells in PBMCs of negative and positive control groups indirectly co-cultured in vitro with PBMC iCM; wherein, the negative control group is designed to culture PBMCs alone; the positive control group is designed by culturing PBMCs separately and adding antibody microbeads to the culture medium to activate the PBMCs; the antibody (antibody bead) is a T Cell TransAct ™ human, and the addition amount is 0.5ul.
And (3) experimental operation:
1. iCM inoculation
A12-well cell chamber plate was placed in an Upper chamber with 200ul matrigel gel, spread evenly, and placed at 37 ℃ in 5% CO 2 The incubator is pretreated for 1h.
Taking out 12-well cell chamber plates, sucking up matrigel gel of Upper computer, uniformly seeding the cardiomyocytes in the Upper computer according to the density of the cardiomyocytes of the experimental group, and using 0.5ml of iCM recovery solution for each well;
the next day, the myocardial cell resuscitating solution is changed into the myocardial cell maintaining solution at 37 ℃ and 5% CO 2 The cell is attached to the wall, unfolded and recovered from pulsation after 3 days of culture in an incubator, the liquid is changed once every 1 day, and the PC membrane is prevented from being damaged in all operations.
2. Inoculation of PBMC
PBMC recovered one day earlier were collected and centrifuged 400g,7min, cells resuspended in pre-activated PBMC medium, gently pipetted evenly and counted in 250000 cells per well, evenly seeded in 12 well plates Lower cell.
3. iCM and PBMC indirectly co-cultured
1) Indirect co-culture of iCM cells seeded in 12-well plates with PBMC in37℃、5% CO 2 Indirect co-culture for 96 hours, and PBMC-activating antibody microbeads (anti-CD 3) are added to the PBMC culture medium in the positive control group&anti-CD28 antibody beads) 0.5ul, negative control no treatment;
2) After 96 hours, remove Transwell insert and collect PBMC, add 500ul DPBS gently blow the remaining cells, collect all cells and centrifuge at 400g for 7min at room temperature;
3) Preparing flow-type cell antibodies CD3, CD19 and CD56, diluting according to 1;
4) 100ul of DPBS resuspended cells and transferred to a flow tube for flow cytometry.
4. Cell counting after indirect co-culture
Shows the cell number change of T, B, NK cells in PBMC before and after indirect co-culture.
Examples
An experimental group (a PBMC and iCM indirect co-culture group) and a negative control group (a PBMC culture group) and a positive control group (an antibody adding group in the PBMC culture process) are designed.
The inoculation mode of the cells is as follows: the experiment was carried out using a 12-well Transwell system with iCM seeded in the upper chamber and PBMC in the lower chamber.
EXAMPLE 1 screening antibody dosage
In commercial PBMC, T cell content is highest, so this example mainly selects antibodies for activating T cells for relevant validation; the data of NK cells and B cells are only used as reference and have no other significance. The antibody is anti-CD3& anti-CD28 antibody micro-beads.
TABLE 1 State of activation of PBMCs by different antibody doses observed at 25W PBMC doses
| Serial number | Amount of antibody used | B cell content (CD 19 expression level) | NK cell content (CD 56 expression level) | T cell content (CD 3 expression level) |
| 1 | 0.1ul | 2.39% | 3.08% | 72.74% |
| 2 | 0.5ul | 2.77% | 1.38% | 95.95% |
The results of the flow cytometry assays of FIGS. 1 and 2, combined with the data of Table 1, indicate that the requirement for activation of immune cells in PBMCs is already met at an antibody dose of 0.5ul.
Example 2 the optimal PBMC dose was determined by the addition of antibody during PBMC culture.
In commercial PBMC, T cell content is highest, so this example selects antibodies for activating T cells primarily for relevant validation.
TABLE 2 PBMC activated status at different PBMC doses at 0.5ul antibody doses
| Serial number | PBMC dosage | B cell content (CD 19 expression level) | NK cell content (CD 56 expression level) | T cell content (CD 3 expression level) |
| 1 | 10W | 5.95% | 5.07% | 73.40% |
| 2 | 15W | 3.41% | 2.88% | 66.72% |
| 3 | 20W | 3.63% | 2.37% | 75.55% |
| 4 | 25W | 3.37% | 1.97% | 94.96% |
| 5 | 50W | 2.82% | 2.88% | 93.75% |
Combining the results of the assays of Table 2 with the flow cytometry tests of FIGS. 3-7, PBMC was assayed at 20W to 50W, preferably 25W. Meanwhile, the dosage is also the optimal dosage of PBMC in the indirect co-culture process of the PBMC and iCM.
Example 3 patient one PBMC experiments
The PBMC contains T cells, NK cells and B cells, and in the actual transplantation process, if immune rejection reaction is generated, the change of the three cells needs to be verified, so that the T cells, the B cells and the NK cells are verified by a negative control group respectively during experimental design. In addition, the application designs a positive control group, and carries out comprehensive verification on the negative control group and the positive control group for the T cells respectively so as to demonstrate the rationality of indirect co-culture for verifying the immunological rejection reaction.
An experimental group (a PBMC and iCM indirect coculture group) and a negative control group (a PBMC culture group) and a positive control group (an antibody group is added in the PBMC culture process, and the positive control group is only used for comparing T cell change with the experimental group) are designed.
The inoculation mode of the cells is as follows: the experiment was carried out using a 12-well Transwell system with iCM seeded in the upper chamber and PBMC in the lower chamber.
Negative control group: PBMCs were cultured in the lower chamber at a cell inoculum size of 250,000, without iCM;
positive control group: PBMCs were cultured in the lower chamber at a cell inoculum size of 250,000, without iCM; anti-CD3& anti-CD28 antibody beads were added to the medium at 0.5ul.
TABLE 3 patient-experiment based on in vitro experimental model
| PBMC cell count | iCM cell count | B cell content (CD 19 expression level) | NK cell content (CD 56 expression level) | T cell content (CD 3 expression level) | |
| |
|
25W | 2.89% | 25.16% | 11.42% |
| Experimental group 2 | 25W | 12.5W | 2.57% | 24.72% | 9.91% |
| Experimental group 3 | 25W | 6.25W | 3.02% | 24.43% | 10.54% |
| |
25W | Is free of | 1.4% | 1.52% | 3.75% |
| |
25W | Is free of | 1.96% | 0.63% | 94.32% |
Combining the test results of table 3 with the flow cytometry test results of fig. 8-12, the ratio of the number of B cells and NK cells in the positive control group to the number of B cells and NK cells in the negative control group was not much different. The antibody is an activated antibody of the T cell, so that the T cell can be activated and proliferated in the culture process; the B cells and NK cells die in different amounts, so the data of the B cells and NK cells in the positive control group are not compared with the data of the B cells and NK cells in the negative control group and the experimental group. The content of key data T cells in the positive control group is 94.32 percent which is far higher than that in the experimental group and the negative control group, and the T cells are activated. The content of the T cells in the experimental group is respectively compared with the content of the T cells in the negative control group and the content of the T cells in the positive control group, and the conclusion is that: the content of T cells in the experimental group is slightly higher than that in the negative control group and is far lower than that in the positive control group.
The negative control group did not activate any cells with antibodies, so the number of B cells and NK cells in the negative control group was comparable to the number of B cells and NK cells in the experimental group. The comparison conclusion is that: the numbers of B cells and NK cells in the experimental group were increased compared to those in the negative control group.
In conclusion, when PBMC and iCM were indirectly co-cultured in vitro in this patient, the content of T cells, B cells and NK cells was slightly increased compared to the negative control group; and the content of the T cells in the experimental group is obviously lower than that of the T cells in the positive control group, so that the patient is estimated to generate mild immunological rejection on the myocardial cell transplantation according to the verification result of the experimental model.
Example 4 PBMC from patient two was tested
The PBMC contains T cells, NK cells and B cells, and in the actual transplantation process, if immune rejection reaction is generated, the change of the three cells needs to be verified, so that the T cells, the B cells and the NK cells are verified through negative control groups respectively during experimental design. In addition, the application designs a positive control group, and carries out comprehensive verification on the negative control group and the positive control group for the T cells respectively so as to demonstrate the rationality of indirect co-culture for verifying the immunological rejection reaction.
An experimental group (a PBMC and iCM indirect co-culture group) and a negative control group (a PBMC culture group) and a positive control group (an antibody adding group in the PBMC culture process) are designed.
The inoculation mode of the cells is as follows: the experiment was carried out using a 12-well Transwell system with iCM seeded in the upper chamber and PBMC in the lower chamber.
Negative control group: PBMCs were cultured in the lower chamber at a cell inoculum size of 250,000, without iCM;
positive control group: PBMCs were cultured in the lower chamber at a cell inoculum size of 250,000, without iCM; the anti-CD3& anti-CD28 antibody beads were added to the medium at 0.5ul.
TABLE 4 patient two experiments based on in vitro experimental model
| PBMC cell count | iCM cell count | B cell content (CD 19 expression level) | NK cell content (CD 56 expression level) | T cell content (CD 3 expression level) | |
| |
|
25W | 6.68% | 45.15% | 12.48% |
| Experimental group 2 | 25W | 2.5W | 5.94% | 40.67% | 13.16% |
| |
25W | Is free of | 5.79% | 38.16% | 14.54% |
| |
25W | Is free of | 2.71% | 41.67% | 55.29% |
Combining the test results of table 4 with the flow cytometry test results of fig. 13-16, the numbers of B cells and NK cells in the positive control group had a slight change relative to the numbers of B cells and NK cells in the negative control group. The antibody is an activated antibody of the T cell, so that the T cell is activated and proliferated in the culture process, and the B cell and the NK cell die in different amounts, so that the data of the B cell and the NK cell in a positive control group are not compared with the data of the B cell and the NK cell in a negative control group and an experimental group. Of course, positive control groups for B cells or NK cells can be additionally designed.
The content of key data T cells in the positive control group is 55.29 percent and is higher than that of the T cells in the experimental group and the negative control group, which indicates that the T cells in the positive control group are activated and can be used for comparing with the experimental group and the negative control group; the comparison conclusion is that: the content of T cells in the experimental group is almost the same as that of T cells in the negative control group.
The negative control group did not activate any cells with antibodies, so the number of B cells and NK cells in the negative control group was comparable to the number of B cells and NK cells in the experimental group. The comparison conclusion is that: the numbers of B cells and NK cells in the experimental group were only slightly changed compared to the numbers of B cells and NK cells in the negative control group.
In conclusion, in the in vitro experimental model, T cells, B cells and NK cells did not significantly increase compared to the negative control group, and it was estimated that the patient did not produce immune rejection in cardiomyocyte transplantation (iCM transplantation) according to the conclusion of the in vitro experiment.
Claims (9)
1. An in vitro experimental model for predicting myocardial cell transplantation immune rejection, characterized by: obtaining PBMC cells of a transplantation target object and iCM cells to be transplanted; PBMC and iCM were subjected to indirect co-culture in vitro based on the Transwell system, iCM was seeded in the upper chamber of the Transwell chamber, PBMC was seeded in the lower chamber of the Transwell chamber; in the culture process, the culture solution in the upper chamber and the culture solution in the lower chamber can mutually permeate; detecting the activation and proliferation conditions of T cells, B cells and NK cells in the PBMCs after indirect co-culture; inoculation density of said PBMC 2.5 x 10 5 /ml，The iCM cells were differentiated via ipscs or ESCs.
2. An in vitro experimental model for predicting myocardial cell transplant immune rejection according to claim 1, wherein: the culture time is between 48 and 96 hours.
3. The in vitro experimental model for predicting the immune rejection response of myocardial cell transplantation according to claim 1, wherein the PBMC and iCM are cultured indirectly in vitro, the number ratio of PBMC to iCM is designed according to gradient, and at least 2 number ratios are selected for detection during the experiment.
4. The in vitro experimental model for predicting myocardial cell transplantation immune rejection response of claim 1, wherein the in vitro experimental model is established in a manner including preliminary model establishment, model verification and result judgment; wherein the content of the first and second substances,
preliminarily establishing a model, co-culturing commercial PBMC and an antibody, screening the dosage of PBMC when the PBMC is activated to the maximum extent, and determining the dosage of PBMC when the PBMC is an in vitro experiment model;
model verification, namely performing indirect co-culture on PBMC (peripheral blood mononuclear cell) of a patient and iCM on the basis of primary model establishment, and comparing the activation state and proliferation condition of T cells, B cells and NK cells in the PBMC in a state of independent culture relative to the PBMC after the indirect co-culture;
judging the result, and evaluating the activation condition of a positive control, the activation condition of a negative control and the activation condition of a test sample; the positive control group is designed to culture PBMC alone, add antibody into culture medium to activate PBMC, and detect the activation condition of the positive control; the negative control group is designed to culture PBMC alone and detect the activation condition of the negative control.
5. The in vitro experimental model for predicting myocardial cell transplant immune rejection according to claim 4, wherein the determination of the result further comprises specifying that at least one of T cells in the positive control are activated, negative control are not activated, and at least one of T cells, B cells or NK cells in the test sample are activated to predict the occurrence of the abnormal immune response.
6. The in vitro experimental model for the prediction of immune rejection in cardiomyocyte transplantation according to claim 4, wherein said antibody is T Cell TransAct. Human, and is added in an amount of 0.5ul.
7. An in vitro experimental model for predicting myocardial cell transplant immune rejection according to claim 1, wherein: when the PBMC and iCM are subjected to indirect co-culture based on a Transwell system, the PBMC culture medium adopts 1640 culture medium added with FBS, and the volume fraction of the FBS is 10%; iCM medium 1640 medium supplemented with FBS and B27 was used, with FBS volume fraction of 5% and B27 volume fraction of 2%.
8. An in vitro experimental model for predicting myocardial cell transplant immune rejection according to claim 1, wherein: the quantitative ratio of PBMC to iCM was designed to be 1 (0.25-1).
9. The model of claim 1, wherein indirect co-culture requires the survival rate of iCM to be not less than 70% and the purity to be not less than 95%.
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