CN115266664A - Method for evaluating killing efficacy of immune cell therapeutic preparation in vitro and application thereof - Google Patents
Method for evaluating killing efficacy of immune cell therapeutic preparation in vitro and application thereof Download PDFInfo
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Abstract
The invention provides an evaluation method of killing efficiency of an immune cell therapy preparation in vitro and application thereof. The indole heptamethine cyanine dye is a macromolecular dye and cannot freely penetrate through a cell membrane, the indole heptamethine cyanine dye is introduced into a target cell to mark the target cell through an electroporation technology, the heptamethine cyanine dye cannot freely penetrate through the cell membrane, and the phenomenon of spontaneous release of a marker cannot occur after marking introduction, so that the problem that a detection result is larger due to the fact that a spontaneously released marking reagent and a marking substance released into supernatant after cell damage cannot be distinguished is solved, and the indole heptamethine cyanine dye has better accuracy when being used as an in-vitro killing efficacy evaluation method of an immune cell therapy preparation.
Description
Technical Field
The invention relates to the field of immune cell efficacy evaluation, in particular to an in-vitro killing efficacy evaluation method of an immune cell therapeutic preparation and application thereof.
Background
Immune cell therapy is one of the innovative therapies that are considered to be the most promising for combating tumors. The immunotherapy, also called Adoptive Cell (ACT) therapy, is based on the immunological principle and method, and is characterized by that it collects human body immune cells, makes them implement in-vitro culture and amplification so as to raise target killing function, and then utilizes the regulation of immune system of human body to kill pathogen, cancer cell and mutant cell in blood and tissue, inhibit tumor growth and raise immunological competence of human body. The treatment cost corresponding to the personalized treatment scheme is expensive, but the advantages of immune cell treatment still lead the global tumor patients to be focused. Immune cell therapy is a complex process, comprising six links of cell extraction, cell separation, cell culture, quality control, cell feedback and curative effect evaluation, wherein each link has higher requirements on technology and experimental environment. Among them, the quality control of killing efficacy is one of the difficulties.
The commonly used cell killing detection methods include cadmium 51 release test, lactate Dehydrogenase (LDH) release method, BATDA method, CAM method, cytoTox-Glo method, PKH method and the like. The classical method is a cadmium 51 release experiment, which has good repeatability, but has various limiting factors such as short half-life, high isotope waste treatment and experiment protection requirements and the like due to the fact that isotope is used for labeling target cells, especially the use of radioactive isotopes has great threat to health and environment, and other radioactive isotope labeled target cells such as H3 also have the defects, so that the application of the method is limited, and therefore, a plurality of researchers can adopt other alternative methods for biological efficacy detection. The traditional LDH method has the characteristics of unstable sensitivity and repeatability, long required time, large batch-to-batch variation and short immune cell expiration date.
The cadmium 51 release experiment, the LDH method, the BATDA method and the CytoTox-Glo method belong to indirect methods, namely, a certain reagent is firstly used for marking target cells, then the target cells are incubated with immune cells with different concentrations, when the target cells are attacked and damaged by the immune cells, the permeability of cell membranes is changed, the substances can be released into supernatant, and the activity of the immune cells can be determined by determining the content of the released substances. However, in the above method, when the labeling reagent labels the target cells, the target cells spontaneously release the labeling reagent, the labeling reagent is spontaneously released from the target cells into the supernatant, and the spontaneously released labeling reagent and the labeling substance released into the supernatant after the cells are damaged cannot be distinguished from each other, resulting in a large detection result.
The CAM method and the PKH method reflect the killing efficiency by detecting a double signal of a live cell marker and a death marker by a flow cytometer, and the signals reflected by the two methods also include cells in an apoptotic state, compared with the aforementioned methods. Among them, flow Cytometry (FCM) is a technique for rapidly measuring the biological properties of a single cell or organelle in a Flow system and classifying and collecting specific cells or organelles from a population. In view of the advantages of efficient and accurate quantitative analysis on the single cell level, a plurality of flow cytometry-based NK cell killing activity determination methods are developed at present, and in the flow cytometry-based fluorescent dye cell labeling method, four dyes including CSFE, MTG, dio and CAM are commonly used for evaluating the cytotoxic activity of NK cells, but after the fluorescent dye labels the cells, the labeled living cells can generate spontaneous release phenomena of the dyes, so that the method is not favorable for distinguishing effective cells from target cells, and can cause great detection deviation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an in vitro killing efficacy evaluation method of an immune cell therapeutic preparation and application thereof.
In order to realize the purpose, the invention adopts the technical scheme that: a method for evaluating killing efficacy of an immune cell therapeutic agent in vitro, the method comprising the steps of:
(1) Adopting an electroporation technology to lead the indoheptamethine cyanine dye into target cells to mark the target cells;
(2) Co-culturing target cells marked with indole heptamethine cyanine dyes and immune cells to serve as a system A; independently culturing target cells marked with the indole heptamethine cyanine dye to serve as a system B; compared with the system A, the system B is consistent except that immune cells are not contained;
(3) Collecting supernatant A of the system A after culture and detecting the fluorescence signal intensity Ia;
(4) Destroying target cells in the system B, collecting supernatant B, and detecting fluorescence signal intensity Ib;
(5) The killing efficacy of the immunocytotherapeutic preparation in vitro was evaluated as target cell mortality w, i.e. w = Ia/Ib × 100%.
According to the in-vitro killing efficacy evaluation method of the immune cell therapeutic preparation, the target cell is marked by the indole heptamethine cyanine dye, the indole heptamethine cyanine dye is a macromolecular dye and cannot freely penetrate through a cell membrane, the indole heptamethine cyanine dye is guided into the target cell through an electroporation technology to mark the target cell, the heptamethine cyanine dye cannot freely penetrate through the cell membrane and cannot spontaneously release a marker after being marked and guided, the problem that the detection result is larger due to the fact that the spontaneously released marker reagent and a marker substance released into supernatant after the cells are damaged cannot be distinguished is solved, and the in-vitro killing efficacy evaluation method of the immune cell therapeutic preparation has better accuracy.
Preferably, the indole heptamethine cyanine dye is indole heptamethine cyanine dye IR-780, indole heptamethine cyanine dye IR-783, or indole heptamethine cyanine dye IR-808.
Indole heptamethine cyanine dye IR-780, indole heptamethine cyanine dye IR-783 and indole heptamethine cyanine dye IR-808 are used as target cell markers, so that the phenomenon of spontaneous marker release is avoided, and the cytotoxicity is extremely low.
Preferably, the introduction of the indoheptamethine cyanine dye into the target cells using the electroporation technique comprises the steps of:
mixing a low-conductivity electroporation buffer solution with a culture medium in which target cells are suspended to obtain a mixed solution, wherein the low-conductivity electroporation buffer solution comprises magnesium chloride, phosphate and indoheptamethine cyanine dye;
(II) adding propidium iodide to the mixture, and applying a pulse voltage.
The low-conductivity electroporation buffer solution and the propidium iodide have higher dyeing efficiency, and are favorable for improving the sensitivity of the evaluation method.
Preferably, the electric field strength of the pulse voltage is 0.2 to 2.5kV/cm.
Preferably, 6 to 10 pulses are set when a pulse voltage is applied, the pulse duration of the pulse voltage is 80 to 120 mus, and the pulse frequency of the pulse voltage is 0.8 to 1.2Hz.
The pulse voltage with the parameters has higher dyeing efficiency and is beneficial to improving the sensitivity of the evaluation method.
Preferably, the pH of the low conductivity electroporation buffer is 7.3 to 7.5, the concentration of the indoheptamethylcyanine dye in the mixture is 200 to 300mM, the concentration of the cells in the mixture is (0.5 to 1.5). Times.107 cells/mL, and the concentration of the magnesium chloride in the mixture is 0.8 to 1.2mM.
Preferably, in the step (II), propidium iodide is added to the mixed solution to a concentration of 120 to 180. Mu.M.
Preferably, in the step (4), the target cells are destroyed by water absorption and burst of distilled water cells.
The invention also provides the use of any of the above described evaluation methods for non-disease diagnostic and therapeutic purposes, for detecting cytocidal activity, or for preparing an immune cell preparation.
The invention has the beneficial effects that: the invention provides an in vitro killing efficacy evaluation method of an immune cell therapy preparation and application thereof, the in vitro killing efficacy evaluation method of the immune cell therapy preparation marks target cells by indole heptamethine cyanine dye, the indole heptamethine cyanine dye is macromolecular dye and cannot freely penetrate cell membranes, the indole heptamethine cyanine dye is guided into the target cells by an electroporation technology to mark the target cells, the heptamethine cyanine dye cannot freely penetrate the cell membranes, the phenomenon of self-releasing markers cannot occur after the marker is guided, the problem that the detection result is large due to the fact that a marking reagent which is spontaneously released and a marking substance which is released into a supernatant after the cells are damaged cannot be distinguished is avoided, and the evaluation method has better accuracy as the in vitro killing efficacy evaluation method of the immune cell therapy preparation.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
An in vitro killing efficacy evaluation method of an immune cell therapy preparation, which is an embodiment of the present invention, includes the following steps:
(1) Adopting an electroporation technology to lead the indole heptamethine cyanine dye into target cells to mark the target cells;
(2) Co-culturing target cells marked with indole heptamethine cyanine dyes and immune cells to serve as a system A; independently culturing target cells marked with the indole heptamethine cyanine dye to serve as a system B; compared with the system A, the system B is consistent except that immune cells are not contained;
(3) Collecting supernatant A of the system A after culture and detecting the fluorescence signal intensity Ia;
(4) Destroying target cells in the system B, collecting supernatant B, and detecting fluorescence signal intensity Ib; the method for destroying the target cells comprises the steps of centrifuging the system B at a low speed of 500g, suspending the cells by using distilled water, absorbing water for swelling and breaking the cells, and collecting supernatant B;
(5) The killing efficacy of the immune cell therapy preparation in vitro was evaluated as target cell mortality rate w, i.e. w = Ia/Ib × 100%;
wherein the indole heptamethine cyanine dye is indole heptamethine cyanine dye IR-780;
the method for introducing the indole heptamethine cyanine dye into the target cells by adopting the electroporation technology comprises the following steps:
(I) 20mM K2HPO4、20mM KH2PO42mM magnesium chloride and 500mM indole heptamethine cyanine dye IR-780 as a low conductivity electroporation buffer, the pH of the low conductivity electroporation buffer is 7.4, and 2X 10 suspended with7Mixing the culture media of target cells per mL according to a volume ratio of 1;
(II) adding propidium iodide into the mixed solution to the concentration of 150 mu M, transferring the mixed solution to a disposable electroporation cuvette, applying pulse voltage, wherein the distance between electrodes is 2mm, the electric field intensity of the pulse voltage is 1.0kV/cm, setting 8 pulses, the pulse duration of the pulse voltage is 100 mu s, and the pulse frequency of the pulse voltage is 1Hz.
Example 2
As a method for evaluating the killing efficacy of an immune cell therapy preparation in vitro according to an embodiment of the present invention, the only differences between the embodiment and the embodiment 1 are: indole heptamethine cyanine dye IR-783.
Example 3
As a method for evaluating the killing efficacy of an immune cell therapy preparation in vitro according to an embodiment of the present invention, the only differences between the embodiment and the embodiment 1 are: indole heptamethine cyanine dye IR-808.
Test examples
1. Sample material:
1. and (3) target cell culture: human melanoma M21 cells were cultured in 10-vol FBS-containing RPMI1640 medium, the target cells in the logarithmic growth phase were collected, washed once with a buffer solution (PBS), and the medium was resuspended to 2X 107Cells/ml.
2. And (3) immune cell culture: NK-92 cells were cultured in GH T551H 3 medium containing 500IU/ml IL-2, and the target cells in the logarithmic growth phase were collected, washed once with a buffer solution (PBS), and then the medium was resuspended at 1X 106Cells/ml.
2. Test method
1. Labeling of target cells: 20mM K2HPO4、20mM KH2PO42mM magnesium chloride and 500mM indoheptamethine cyanine dye IR-780 as a low conductivity electroporation buffer, the pH of the low conductivity electroporation buffer is 7.4, and 2X 107Mixing the culture mediums of target cells per mL according to a volume ratio of 1 (100. Mu.L: 100. Mu.L) to obtain a mixed solution; propidium iodide was added to the mixed solution to a concentration of 150. Mu.M and transferred to a disposable electroporation cuvette, a pulse voltage was applied with a distance of 2mm to the electrodes, an electric field strength of the pulse voltage was 1.0kV/cm, 8 pulses were set, a pulse duration of the pulse voltage was 100. Mu.s, and a pulse frequency of the pulse voltage was 1Hz.
After completion of the pulse, 500g of the suspension was centrifuged at a low speed, and then resuspended in 10% FBS-containing RPMI1640 medium to a cell concentration of 1X 105one/mL. By usingCulturing the cells.
The labeling is carried out by using indole heptamethine cyanine dye IR-783 and indole heptamethine cyanine dye IR-808 in the same way.
2. Immune cell killing experiment: sucking 100 mu L of labeled target cells into a 96-well plate, sucking NK-92 cells and labeled cells to mix, wherein the ratio of the NK-92 cells to the labeled target cells is 10:1; at 37 ℃,5% CO2And (3) incubating for 2 hours, placing 500g of the 96-well plate for 5min, collecting supernatant, and detecting the fluorescence signal intensity Ia. 6 parallels were set.
Pipetting 100. Mu.L of labeled target cells into a 96-well plate, pipetting phosphate buffer in the same volume as that of NK-92 cells, mixing with labeled cells, and making the ratio of CO 5% at 37%2Incubating for 2 hours under the conditions of (1), standing a 96-well plate for 5min at 500g, collecting supernatant, and detecting fluorescence signal intensity I1. And (3) resuspending the centrifuged cells in distilled water, absorbing water to burst the cells, collecting supernatant B, and detecting the fluorescence signal intensity Ib. 6 parallels were set.
3. Blank test: 20mM K2HPO4、20mM KH2PO42mM magnesium chloride and 500mM indoheptamethine cyanine dye IR-780 as a low conductivity electroporation buffer having a pH of 7.4, with 2X 10 suspended therein7Mixing the culture mediums of target cells per mL according to a volume ratio of 1 (100. Mu.L: 100. Mu.L) to obtain a mixed solution; the mixed solution was heated at 37 ℃ and 5% CO2Was incubated for 30min. Washing cells with phosphate buffer solution after 500g centrifugation, centrifuging again after washing, resuspending the centrifuged cells in distilled water, absorbing water to burst the cells, collecting supernatant, and detecting fluorescence signal intensity I2。
4. Detecting the intensity of a fluorescence signal of the supernatant: and a Thermo enzyme-labeling instrument with the wavelength range of 340-850 nm detects the fluorescence signal intensity I of the corresponding marker labeling test at the wavelength positions of 780nm, 783nm and 808nm respectively.
The experimental results are as follows:
1. the signal intensity can not be detected by the fluorescence signal intensity I1 in the supernatant after the culture of the marked human melanoma M21 cells, which indicates that the phenomenon that the marking substance indole heptamethine cyanine dye is spontaneously released does not exist in the culture process of the target cells marked by the indole heptamethine cyanine dye IR-780, the indole heptamethine cyanine dye IR-783 and the indole heptamethine cyanine dye IR-808.
2. In the supernatant of the blank test, the signal intensity was not detected by the fluorescence signal intensity I2, indicating that the indole heptamethine cyanine dye IR-780, the indole heptamethine cyanine dye IR-783, and the indole heptamethine cyanine dye IR-808 cannot pass through the cell membrane and spontaneously enter the target cell.
3. The killing efficacy of the immunocytotherapeutic preparation in vitro was evaluated as the target cell death rate w, i.e., w = Ia/Ib × 100%, and the results are shown in table 1.
TABLE 1 target cell death rate (%)
From the experimental results, the RSD of the in vitro killing efficacy evaluation method of the immune cell therapeutic preparation of examples 1 to 3 is between 2.69% and 11.9%, and the precision is better, less than 15%, which meets the requirement of the precision of the analysis method. And a blank experiment proves that the indole heptamethine cyanine dye is a macromolecular dye and cannot freely pass through a cell membrane, the indole heptamethine cyanine dye is guided into a target cell to label the target cell by an electroporation technology, the heptamethine cyanine dye cannot freely pass through the cell membrane, and a phenomenon of spontaneous release of a label cannot occur after the label is guided in, so that the problems that a detection result is larger due to the fact that a spontaneously released label reagent and a label substance released into a supernatant after the cell is damaged cannot be distinguished are solved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A method for evaluating killing efficacy of an immune cell therapeutic preparation in vitro, which comprises the following steps:
(1) Adopting an electroporation technology to lead the indole heptamethine cyanine dye into target cells to label the target cells;
(2) Co-culturing target cells marked with indole heptamethine cyanine dyes and immune cells to serve as a system A; independently culturing target cells marked with indole heptamethine cyanine dyes to serve as a system B; compared with the system A, the system B is consistent except that immune cells are not contained;
(3) Collecting supernatant A of the system A after culture and detecting the intensity of a fluorescent signal Ia；
(4) Collecting supernatant B after destroying target cells in the system B and detecting fluorescence signal intensity Ib；
(5) The killing efficacy of immunocytotherapeutic formulations in vitro was evaluated as target cell mortality w, i.e. w = Ia/Ib x 100%.
2. The method for evaluating the in vitro killing efficacy of an immunocytotherapeutic preparation according to claim 1, wherein the indole heptamethine cyanine dye is indole heptamethine cyanine dye IR-780, indole heptamethine cyanine dye IR-783, or indole heptamethine cyanine dye IR-808.
3. The method for evaluating the killing efficacy of an immunocytotherapeutic preparation in vitro according to claim 1, wherein the step of introducing the indoheptamethine cyanine dye into the target cells using an electroporation technique comprises the steps of:
mixing a low-conductivity electroporation buffer solution with a culture medium in which target cells are suspended to obtain a mixed solution, wherein the low-conductivity electroporation buffer solution comprises magnesium chloride, phosphate and indoheptamethine cyanine dye;
(II) adding propidium iodide to the mixture, and applying a pulse voltage.
4. The method for evaluating the killing efficacy of an immunocyte therapeutic agent in vitro according to claim 3, wherein the electric field intensity of the pulse voltage is 0.2 to 2.5kV/cm.
5. The method for evaluating the killing efficacy of an immunocyte therapeutic agent in vitro according to claim 3, wherein 6 to 10 pulses are set when a pulse voltage is applied, the pulse duration of the pulse voltage is 80 to 120 μ s, and the pulse frequency of the pulse voltage is 0.8 to 1.2Hz.
6. The method according to claim 3, wherein the pH of the low conductivity electroporation buffer is 7.3 to 7.5, the concentration of the indoheptamethine cyanine dye in the mixture is 200 to 300mM, the concentration of the cells in the mixture is (0.5 to 1.5). Times.107 cells/mL, and the concentration of the magnesium chloride in the mixture is 0.8 to 1.2mM.
7. The method for evaluating the killing efficacy of an immunocyte therapy preparation according to claim 3, wherein, in the step (II), propidium iodide is added to the mixture to a concentration of 120 to 180. Mu.M.
8. The method for evaluating the killing efficacy of an immunocytotherapeutic preparation in vitro according to claim 1, wherein in the step (4), the target cell is destroyed by the water-absorbing bursting of distilled water cells.
9. Use of the method of any one of claims 1 to 8 for non-disease diagnostic and therapeutic purposes, for detecting cytocidal activity, or for preparing an immune cell preparation.
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| 王昕: "新型近红外小分子NIRCP-61用于干细胞示踪成像和促进组织修复的实验研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117169182A (en) * | 2023-08-30 | 2023-12-05 | 广州沙艾生物科技有限公司 | In-vitro efficacy detection method for stem cell therapeutic preparation and application thereof |
| CN117169182B (en) * | 2023-08-30 | 2024-03-15 | 广州沙艾生物科技有限公司 | In-vitro efficacy detection method for stem cell therapeutic preparation and application thereof |
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| CN115266664B (en) | 2023-05-09 |
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