CN115266664B - Method for evaluating in-vitro killing efficacy of immune cell therapeutic preparation and application thereof - Google Patents
Method for evaluating in-vitro killing efficacy of immune cell therapeutic preparation and application thereof Download PDFInfo
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Abstract
The invention provides an immune cell therapeutic preparation in-vitro killing efficacy evaluation method and application thereof. The indole heptamethine cyanine dye is a macromolecular dye and cannot freely pass through a cell membrane, the indole heptamethine cyanine dye is led into a target cell to mark the target cell through an electroporation technology, the heptamethine cyanine dye cannot freely pass through the cell membrane, a phenomenon of spontaneously releasing a marker cannot occur after the label is led in, the problem that a detection result is large because a spontaneously released marking reagent and a marking substance released into a supernatant after cell damage cannot be distinguished is avoided, and the method has better accuracy when being used as an in-vitro killing efficacy evaluation method of an immune cell therapeutic agent.
Description
Technical Field
The invention relates to the field of immune cell efficacy evaluation, in particular to an immune cell therapeutic preparation in-vitro killing efficacy evaluation method and application thereof.
Background
Immune cell therapy is one of the innovative therapies considered to be the most promising for combating tumors. Immunotherapy, also known as Adoptive Cell (ACT) therapy, is based on immunological principles and methods by collecting human immune cells, culturing and amplifying them in vitro to enhance the targeted killing function, and then infusing them back into the human body, killing pathogens, cancer cells and mutant cells in blood and tissues by mobilizing the immune system of the body, inhibiting tumor growth, and enhancing the immunity of the body. The cost of treatment corresponding to such personalized treatment regimens is relatively expensive, but the many advantages offered by immune cell therapy still make global oncology patients feel more attractive. Immune cell therapy is a complex process, which comprises six links of cell extraction, cell separation, cell culture, quality control, cell feedback and efficacy evaluation, and each link has high requirements on technology and experimental environment. Among them, quality control of killing efficacy is one of the difficulties.
As a cell killing test method, there are a cadmium 51 release test, a Lactate Dehydrogenase (LDH) release method, a BATDA method, a CAM method, a Cytotox-Glo method, a PKH method and the like. The classical method is a cadmium 51 release experiment, and the method has good repeatability, but because the method uses isotope to label target cells, various limiting factors such as short half-life, high isotope waste treatment and experiment protection requirements exist, and the use of radioactive isotopes has great threat to health and environment, and other radioisotope to label target cells such as H3 also has such defects, the application of the method is limited, so many researchers can adopt other alternative methods to carry out biological efficacy detection. The traditional LDH method has unstable sensitivity and repeatability, longer time, larger variation among batches and incapability of adapting to the characteristic of short immune cell effect period.
Cadmium 51 release experiments, LDH method, BATDA method and Cytotox-Glo method all 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 damaged by the attack of the immune cells, cell membrane permeability is changed, substances are released into the supernatant, and the activity of the immune cells can be measured by measuring the content of the released substances. However, the labeling reagent in the above method causes a phenomenon that the labeling reagent is spontaneously released from the target cells to the supernatant after the labeling reagent is labeled on the target cells, and the spontaneously released labeling reagent and the labeling substance released into the supernatant after the cell damage cannot be distinguished, resulting in a large detection result.
The CAM method and PKH method reflect killing efficiency by detecting dual signals of living cell markers and death markers by a flow cytometer, and compared with the methods described above, the signals reflected by the two methods also include cells in an apoptotic state. Among them, flow Cytometry (FCM) is a technique for rapidly determining biological properties of individual cells or organelles in a fluid Flow system and sorting and collecting specific cells or organelles from a population. In view of the advantage of high-efficiency and accurate quantitative analysis at the single cell level, various methods for measuring NK cell killing activity based on flow cytometry are currently developed, and four dyes, namely CSFE, MTG, dio and CAM, are commonly used for evaluating the cytotoxic activity of NK cells in a method for marking cells by using fluorescent dyes based on flow cytometry, but after marking cells by using the fluorescent dyes, marked living cells can have spontaneous release phenomena of the dyes, which are unfavorable for distinguishing effective cells from target cells, and can cause large detection deviation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an in-vitro killing efficacy evaluation method of an immune cell therapeutic preparation and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a method for evaluating the in vitro killing efficacy of an immune cell therapeutic formulation, the method comprising the steps of:
(1) Introducing indole heptamethine cyanine dye into target cells by adopting an electroporation technology, and marking the target cells;
(2) Co-culturing target cells of the labeled indoheptamethine cyanine dye with immune cells to obtain a system A; separately culturing target cells of the labeled indoheptamethine cyanine dye to obtain a system B; the system B is identical to the system A except that the system B does not contain immune cells;
(3) Collecting supernatant A of the system A after culture and detecting fluorescent signal intensity Ia;
(4) After destroying target cells in the system B, collecting supernatant B to detect fluorescence signal intensity Ib;
(5) The in vitro killing efficacy of the immunocytotherapeutic formulation was evaluated as target cell mortality w, i.e. w=ia/ib×100%.
According to the method for evaluating the in-vitro killing efficacy of the immune cell therapeutic preparation, the target cells are marked by the indole heptamethine cyanine dye, the indole heptamethine cyanine dye is a macromolecular dye and cannot freely penetrate through cell membranes, the indole heptamethine cyanine dye is led into the target cells to mark the target cells by electroporation technology, the heptamethine cyanine dye cannot freely penetrate through the cell membranes, the phenomenon of spontaneous release of a marker after the label is led in is avoided, the problem that the detection result is large due to the fact that a spontaneously released marking reagent and a marking substance released into supernatant after cell injury cannot be distinguished is avoided, and the method for evaluating the in-vitro killing efficacy 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.
The indole heptamethine cyanine dye IR-780, the indole heptamethine cyanine dye IR-783 and the indole heptamethine cyanine dye IR-808 are used as target cell markers, so that the phenomenon of spontaneous release of the markers can not occur, and the cytotoxicity is extremely low.
Preferably, the introduction of the indoheptamethine cyanine dye into the target cell using electroporation technique comprises the steps of:
mixing a low-conductivity electroporation buffer with a culture medium in which target cells are suspended to obtain a mixed solution, wherein the low-conductivity electroporation buffer comprises magnesium chloride, phosphate and indoheptamethine cyanine dye;
and (II) adding propidium iodide into the mixed solution, and applying pulse voltage.
The low-conductivity electroporation buffer solution and the propidium iodide have higher dyeing efficiency, and are beneficial to improving the sensitivity of an evaluation method.
Preferably, the electric field strength of the pulsed 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 mu s, 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 solution is 7.3-7.5, the concentration of the indoheptamethine cyanine dye in the mixed solution is 200-300 mM, the concentration of cells in the mixed solution is (0.5-1.5) multiplied by 107/mL, and the concentration of magnesium chloride in the mixed solution is 0.8-1.2 mM.
Preferably, in the step (II), propidium iodide is added to the mixed solution to a concentration of 120-180 mu M.
Preferably, in the step (4), the target cell is destroyed by swelling the distilled water cells with water.
The invention also provides application of any of the evaluation methods in diagnosis and treatment of non-diseases, detection of cell killing power or preparation of immune cell preparations.
The invention has the beneficial effects that: the invention provides an immune cell therapeutic preparation in-vitro killing effect evaluation method and application thereof, wherein the immune cell therapeutic preparation in-vitro killing effect evaluation method is characterized in that indole heptamethine cyanine dye is used for marking target cells, the indole heptamethine cyanine dye is a macromolecular dye and cannot freely pass through cell membranes, the indole heptamethine cyanine dye is introduced into the target cells to mark the target cells through electroporation technology, the heptamethine cyanine dye cannot freely pass through the cell membranes, the phenomenon of self-release of a marker cannot occur after the introduction of the marker, the problem that the detection result is large because the self-release marking agent and a marker substance released into supernatant after cell injury cannot be distinguished is avoided, and the immune cell therapeutic preparation in-vitro killing effect evaluation method has better accuracy.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1
As an in vitro killing efficacy evaluation method of an immune cell therapeutic agent according to an embodiment of the present invention, an in vitro killing efficacy evaluation method of an immune cell therapeutic agent, the method comprising the steps of:
(1) Introducing indole heptamethine cyanine dye into target cells by adopting an electroporation technology, and marking the target cells;
(2) Co-culturing target cells of the labeled indoheptamethine cyanine dye with immune cells to obtain a system A; separately culturing target cells of the labeled indoheptamethine cyanine dye to obtain a system B; the system B is identical to the system A except that the system B does not contain immune cells;
(3) Collecting supernatant A of the system A after culture and detecting fluorescent signal intensity Ia;
(4) After destroying target cells in the system B, collecting supernatant B to detect fluorescence signal intensity Ib; the method for destroying the target cells comprises centrifuging the system B at a low speed of 500g, suspending cells with distilled water, swelling the cells with water, and collecting supernatant B;
(5) The in vitro killing efficacy of the immunocyte therapeutic formulation was evaluated as target cell mortality 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 an electroporation technology comprises the following steps of:
(I) 20mM K 2 HPO 4 、20mM KH 2 PO 4 The 2mM magnesium chloride and 500mM indoheptamethine cyanine dye IR-780 were formulated as a low conductivity electroporation buffer having a pH of 7.4 and suspended with 2X 10 7 Mixing the culture medium of each/mL target cell according to the volume ratio of 1:1 to obtain a mixed solution;
and (II) adding propidium iodide into the mixed solution to a concentration of 150 mu M, transferring the mixed solution into a disposable electroporation cuvette, applying pulse voltage with a distance of 2mm, wherein the electric field strength 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 in vitro killing efficacy of an immunocytotherapeutic agent of the present embodiment, the only difference between this embodiment and embodiment 1 is: indole heptamethine cyanine dye IR-783.
Example 3
As a method for evaluating in vitro killing efficacy of an immunocytotherapeutic agent of the present embodiment, the only difference between this embodiment and embodiment 1 is: indole heptamethine cyanine dye IR-808.
Test examples
1. Sample material:
1. target cell culture: human melanoma M21 cells were cultured in RPMI1640 medium containing 10% FBS, target cells in logarithmic growth phase were collected, washed once with buffer solution (PBS), and the medium was resuspended to 2X 10 7 Cells/ml.
2. Immune cell culture: NK-92 cells in GH T551H 3 medium containing 500IU/ml IL-2Culturing, collecting target cells in logarithmic growth phase, washing with buffer solution (PBS) once, and re-suspending the culture medium to 1×10 6 Cells/ml.
2. Test method
1. Target cell markers: 20mM K 2 HPO 4 、20mM KH 2 PO 4 2mM magnesium chloride and 500mM Indol heptamethine cyanine dye IR-780 were formulated as a low conductivity electroporation buffer having a pH of 7.4 and suspended with 2X 10 7 Mixing the culture medium of each/mL target cell according to the volume ratio of 1:1 (100 mu L:100 mu L) to obtain a mixed solution; propidium iodide was added to the mixture to a concentration of 150. Mu.M and transferred to a disposable electroporation cuvette, a pulse voltage was applied at a distance of 2mm from 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 500g low-speed centrifugation after the end of the pulse, the cells were resuspended to a cell concentration of 1X 10 using RPMI1640 medium containing 10% FBS 5 And each mL. Is used for cell culture.
The same thing was labeled with indole heptamethine cyanine dye IR-783 and indole heptamethine cyanine dye IR-808.
2. Immune cell killing experiments: sucking 100 mu L of marked target cells into a 96-well plate, and mixing NK-92 cells with the marked target cells, wherein the ratio of the NK-92 cells to the marked target cells is 10:1, a step of; at 37 ℃,5% CO 2 For 2 hours under the condition of 500g of 96-well plate for 5min, and collecting supernatant to detect fluorescent signal intensity Ia. 6 parallels were set.
100. Mu.L of labeled target cells were pipetted into a 96-well plate, mixed with labeled cells in a phosphate buffer of the same volume as NK-92 cells, and incubated at 37℃with 5% CO 2 Under the condition of (2) hours, 500g of a 96-well plate is placed for 5 minutes, and the supernatant is collected to detect the fluorescence signal intensity I 1 . The centrifuged cells were resuspended in distilled water, the cells were broken by swelling with water, the supernatant B was collected, and the fluorescence signal intensity Ib was measured. 6 parallels were set.
3. Blank test: 20mM K 2 HPO 4 、20mM KH 2 PO 4 The 2mM magnesium chloride and 500mM indoheptamethine cyanine dye IR-780 were formulated as a low conductivity electroporation buffer having a pH of 7.4 and suspended with 2X 10 7 Mixing the culture medium of each/mL target cell according to the volume ratio of 1:1 (100 mu L:100 mu L) to obtain a mixed solution; the mixture was stirred at 37℃with 5% CO 2 Is incubated for 30min. After 500g centrifugation, washing the cells with phosphate buffer solution, centrifuging again after washing, re-suspending the centrifuged cells in distilled water, swelling the cells by absorbing water, collecting supernatant, and detecting fluorescent signal intensity I 2 。
4. Supernatant detection fluorescence signal intensity detection: the Thermo enzyme label instrument detects the fluorescence signal intensity I of the corresponding label test at the wavelength positions of 780nm, 783nm and 808nm in the wavelength range of 340-850 nm.
Experimental results:
1. in the supernatant of the labeled human melanoma M21 cells after culture, the fluorescent signal intensity I1 can not be detected, which indicates that the target cells marked by the indoheptamethine cyanine dye IR-780, the indoheptamethine cyanine dye IR-783 and the indoheptamethine cyanine dye IR-808 do not have the phenomenon of spontaneously releasing the labeling substance indoheptamethine cyanine dye in the culture process.
2. In the supernatant of the blank test, the fluorescence signal intensity I2 cannot detect the signal intensity, which indicates 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 cannot spontaneously enter the target cell.
3. The in vitro killing efficacy of the immunocyte therapeutic formulations was evaluated as target cell mortality w, i.e. w=ia/ib×100%, and the results are shown in table 1.
TABLE 1 target cell mortality (%)
The experimental results show that the RSD of the in-vitro killing efficacy evaluation method of the immune cell therapeutic preparation of the embodiment 1-embodiment 3 is between 2.69% and 11.9%, the precision is better and lower than 15%, and the requirement of the precision of the analysis method is met. According to the blank experiment, the indomethacin cyanine dye is a macromolecular dye and cannot freely penetrate through a cell membrane, the indomethacin cyanine dye is led into a target cell to mark the target cell through an electroporation technology, the indomethacin cyanine dye cannot freely penetrate through the cell membrane, the phenomenon of spontaneous release of a marker cannot occur after the introduction of the marker, and the problem that a detection result is large because a spontaneously released marking reagent and a marking substance released into a supernatant after cell damage cannot be distinguished is avoided.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. A method for evaluating the in vitro killing efficacy of an immune cell therapeutic formulation, comprising the steps of:
(1) Introducing indole heptamethine cyanine dye into target cells by adopting an electroporation technology to mark the target cells; the method for introducing the indole heptamethine cyanine dye into the target cells by adopting an electroporation technology comprises the following steps of:
mixing a low-conductivity electroporation buffer with a culture medium in which target cells are suspended to obtain a mixed solution, wherein the low-conductivity electroporation buffer comprises magnesium chloride, phosphate and indoheptamethine cyanine dye; the pH of the low-conductivity electroporation buffer solution is 7.3-7.5, the concentration of the indoheptamethine cyanine dye in the mixed solution is 200-300 mM, and the concentration of cells in the mixed solution is (0.5-1.5) multiplied by 10 7 The concentration of magnesium chloride in the mixed solution is 0.8-1 per mL.2mM；
(II) adding propidium iodide into the mixed solution until the concentration is 120-180 mu M, and applying pulse voltage; the electric field intensity of the pulse voltage is 0.2-2.5 kV/cm; setting 6-10 pulses when pulse voltage is applied, wherein the pulse duration of the pulse voltage is 80-120 mu s, and the pulse frequency of the pulse voltage is 0.8-1.2 Hz;
(2) Co-culturing target cells of the labeled indoheptamethine cyanine dye with immune cells to obtain a system A; separately culturing target cells of the labeled indoheptamethine cyanine dye to obtain a system B; the system B is identical to the system A except that the system B does not contain immune cells;
(3) After culturing, collecting supernatant A of system A and detecting fluorescence signal intensity I a ；
(4) After target cells in the system B are destroyed, collecting supernatant B to detect fluorescence signal intensity I b ；
(5) The in vitro killing efficacy of the immunocytotherapeutic formulation was evaluated as target cell mortality w, i.e. w=ia/ib×100%.
2. The method for evaluating the in vitro killing efficacy of an immune cell therapeutic preparation according to claim 1, wherein the indoheptamethine cyanine dye is indoheptamethine cyanine dye IR-780, indoheptamethine cyanine dye IR-783, or indoheptamethine cyanine dye IR-808.
3. The method for evaluating the in vitro killing efficacy of an immunocytotherapeutic agent according to claim 1, wherein in the step (4), the target cell destruction method is a swelling of distilled water cells.
4. Use of the evaluation method according to any one of claims 1 to 3 for the diagnosis and treatment of non-diseases, for the detection of cell killing or for the preparation of immune cell preparations.
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