Preparation method of cells and application of cells in cell effectiveness detection

Abstract

The invention relates to the field of bioengineering, in particular to a preparation method of cells and application of the cells in cell effectiveness detection. The invention provides a cell culture method which comprises the following steps: carrying out first stimulation, first culture, second stimulation, blocking, second culture and cleaning on pretreated cells to obtain the cells. The invention provides a method for determining the effectiveness of an immune cell preparation activated in vitro, according to the characteristics of the immune cell preparation activated in vitro, a two-step stimulation activation mode is adopted, the activation ratio of CD69 + CD44 + is used as a systematic internal control index of the method, and meanwhile, the mechanism of the immune cell preparation activated in vitro is combined to determine the effectiveness of the immune cell preparation activated in vitro. The Tc cell subpopulation secreting granzyme B is used as a method detection index, so that the indirect evaluation of the effectiveness of the immune cell preparation activated in vitro in human body is realized, and the method can be used for release detection of the immune cell preparation activated in vitro in human body.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a method for preparing cells and an application thereof in detecting cell effectiveness. Background Art

[0002] The human in vitro activated immune cell preparation is an in vitro activated tumor-specific non-genetically modified autologous immune cell therapy, which is one of the adoptive cell immunotherapy methods. It is a mixed T lymphocyte obtained by in vitro activation and amplification of mononuclear cells in the patient's own peripheral blood by antibodies and cytokines. After reinfusion, it can supplement the patient's body with specific T lymphocytes with natural anti-tumor ability, achieving direct killing of tumors and inhibiting their growth.

[0003] Immune cell preparations are therapeutic biological products, and effectiveness is an important evaluation indicator. At present, the model animal method is the gold standard method for evaluating the effectiveness of immune cell preparations, but its cycle is too long and the success rate is low, so it cannot be used for daily release testing of cell preparations. Therefore, it is necessary to develop a fast and effective method for testing the effectiveness of cell preparations. Summary of the invention

[0004] In view of this, the present invention provides a method for preparing cells and its application in cell effectiveness detection. The present invention provides a method for determining the effectiveness of immune cell preparations activated in vitro. According to the characteristics of immune cell preparations activated in vitro, a two-step stimulation and activation method is adopted, and CD69 + CD44 + The activation ratio is used as a systematic internal control indicator of the method. At the same time, combined with the mechanism of human in vitro activated immune cell preparations, the Tc cell subset that secretes granzyme B is used as the method detection indicator, thereby realizing an indirect evaluation of the effectiveness of human in vitro activated immune cell preparations, which can be used for the release detection of human in vitro activated immune cell preparations.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a cell culture method, which comprises subjecting the pretreated cells to a first stimulation, a first culture, a second stimulation, blocking, a second culture, and washing to obtain the cells;

[0007] The first stimulation used CD3 antibody at a final concentration of 2 μg / mL;

[0008] The second stimulus used myristoyl phorbol ethyl ester at a final concentration of 2.4 µM and ionomycin at a final concentration of 40.2 µM;

[0009] The blocking was performed using Brefeldin A at a final concentration of 5 μg / mL.

[0010] In some embodiments of the present invention, in the above-mentioned culture method, the cell concentration of the first stimulus is 0.5-6.0×10 6 Pieces / mL.

[0011] In some embodiments of the present invention, in the above culture method, the cell concentration of the first stimulus is 0.5×10 6 / mL, 1.0×10 6 / mL, 2.0×10 6 / mL, 4.0×10 6 / mL or 6.0×10 6 Pieces / mL.

[0012] In some embodiments of the present invention, in the above culture method, the cell concentration of the first stimulus is 2.0×10 6 Pieces / mL.

[0013] In some embodiments of the present invention, in the above-mentioned culture method, the time from the first stimulation to the second culture does not exceed 6 hours.

[0014] In some embodiments of the present invention, in the above-mentioned culture method, the pretreatment comprises the following steps: activating, culturing and collecting PBMC cells to obtain;

[0015] The activation includes: a first activation and a second activation;

[0016] The first activation used 2 μg / mL CD3 monoclonal antibody for 3 h;

[0017] The second activation uses cytokines for 5 h;

[0018] The cytokines include: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α, and 6000 IU / mL of rhIFN-γ;

[0019] The culture was carried out using serum-free medium for 120 h.

[0020] In some embodiments of the present invention, in the above culture method, the concentration of the PBMC cells is 1.0×10 6 / mL.

[0021] In some embodiments of the present invention, in the above-mentioned culture method, the step of freezing is further included after the collection; the freezing adopts a freezing solution; the freezing solution comprises: 25% (v / v) human serum albumin, 5% (v / v) DMSO, 35% (v / v) hydroxyethyl starch solution, 150 μg / mL recombinant human interleukin-2 and 25 μg / mL human interferon γ.

[0022] The present invention also provides the use of the cells obtained by the above-mentioned culture method in preparing a product for detecting the in vitro effectiveness of an immune cell preparation.

[0023] In some embodiments of the present invention, in the above application, the detection comprises the following steps: staining the cell surface, fixing, permeabilizing, intracellular staining, and detecting, and obtaining the detection result according to the proportion of Tc cell subpopulations secreting granzyme B and internal control indicators;

[0024] The internal control indicators include: CD69 + CD44 + The activation rate is greater than 90%.

[0025] In some embodiments of the present invention, in the above application, the surface antibodies used in the surface staining are CD3 and CD8 fluorescent antibodies;

[0026] In some embodiments of the present invention, in the above application, the membrane permeabilization buffer used for permeabilization is a PBS buffer containing saponin, sodium azide and fetal bovine serum;

[0027] In some embodiments of the present invention, in the above application, the antibodies used for intracellular staining are CD69 fluorescent antibody, CD44 fluorescent antibody and granzyme B fluorescent antibody.

[0028] In some embodiments of the present invention, the above application, the Tc cell subsets include: CD3 + CD8 + Granzyme B + / CD3 + CD8 + .

[0029] In some embodiments of the present invention, in the above application, the proportion of the Tc cell subpopulation secreting granzymes greater than 60% is used as an evaluation indicator of the in vitro effectiveness of the cell preparation.

[0030] The present invention provides a cell culture method, which comprises subjecting the pretreated cells to a first stimulation, a first culture, a second stimulation, blocking, a second culture, and washing to obtain the cells;

[0031] The first stimulation used CD3 antibody at a final concentration of 2 μg / mL;

[0032] The second stimulus used myristoyl phorbol ethyl ester at a final concentration of 2.4 µM and ionomycin at a final concentration of 40.2 µM;

[0033] The blocking was performed using Brefeldin A at a final concentration of 5 μg / mL.

[0034] The present invention provides a method for determining the effectiveness of an in vitro activated immune cell preparation. The detection method adopts a two-step stimulation method based on the characteristics of the in vitro activated immune cell preparation, and uses CD69 + CD44 + The stimulation ratio was used as the method's internal control index, and the Tc cell subsets secreting granzyme B (CD3 + CD8 + Granzyme B + / CD3 + CD8 + ) as a method detection indicator, thereby achieving an indirect evaluation of the effectiveness of human in vitro activated immune cell preparations, which can be used for the release detection of preparations. The present invention changes the problem of insufficient stimulation and lack of systematic internal control standards of the traditional cytokine secretion detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0036] Figure 1 The flow cytometry diagram of the test sample alone without stimulation and blocking is shown;

[0037] Figure 2 Shown is the flow cytometry image with stimulation only and no blocking;

[0038] Figure 3 Shown is the flow cytometry image with only blocking but no stimulation;

[0039] Figure 4 Shows the flow cytometry diagram of the test article after stimulation and blocking treatment;

[0040] Figure 5 Shows the flow cytometry detection diagram of the mixed stimulation group alone;

[0041] Figure 6 Shows the flow cytometry detection diagram of CD3 monoclonal antibody + mixed stimulation group;

[0042] Figure 7 Schematic diagram of flow cytometry in Example 2;

[0043] Figure 8 Example 3 Batch 1 CD3 + CD8+ Granzyme B + Linear regression plot of ;

[0044] Fig. 9 Example 3 Batch 2 CD3 + CD8 + Granzyme B + Linear regression plot of ;

[0045] Fig.10 Example 3 Batch 3 CD3 + CD8 + Granzyme B + Linear regression plot of . DETAILED DESCRIPTION

[0046] The invention discloses a cell preparation method and application thereof in cell effectiveness detection.

[0047] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0048] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0049] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0050] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0051] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0052] The present invention provides a method for determining the effectiveness of an in vitro activated immune cell preparation. The detection method adopts a two-step stimulation method based on the characteristics of the in vitro activated immune cell preparation, and uses CD69 + CD44 + The stimulation ratio of 100 μg / mL was used as the method's systemic internal control indicator, and the mechanism of human in vitro activated immune cell preparations was combined with the Tc cell subsets (CD3 + CD8 + Granzyme B + / CD3 + CD8 + ) as a method detection indicator, thereby realizing an indirect evaluation of the effectiveness of human in vitro activated immune cell preparations, which can be used for the release detection of human in vitro activated immune cell preparations, and specifically includes the following steps:

[0053] Step 1: Test sample treatment: resuscitate the human in vitro activated immune cell preparation according to conventional methods, wash it, and dilute it with culture medium for later use.

[0054] Step 2: Stimulation and blocking. A two-step stimulation method was used. The primary stimulant was first coated on the carrier, incubated at 2-8°C overnight, the coating solution was discarded, and the treated test sample was added after washing. After incubation in a CO2 incubator for 2 hours, the mixed stimulant and blocking agent were added at the same time, and incubated in a CO2 incubator for 3 hours.

[0055] Step 3: Washing: Collect cells, resuspend in DPBS, centrifuge at 500g for 5 minutes, and wash 1-2 times.

[0056] Step 4: Surface staining: Add 1~5μL of surface antibody, incubate at 2~8℃ for 20~30 minutes in the dark, then use cell staining buffer, centrifuge at 350g for 5 minutes, and wash twice.

[0057] Step 5: Fixation. Incubate the cells in the fixation buffer at room temperature in the dark for 20 minutes. Then wash the cells 1-2 times with cell staining buffer, centrifuge at 350g for 5 minutes.

[0058] Step 6: Permeabilization. Resuspend the fixed cells with permeabilization buffer (1 mL / tube), centrifuge at 350 g for 5 minutes, and discard the supernatant. Repeat this step once.

[0059] Step 7: Intracellular staining. Add 1~5μL intracellular antibody, incubate at room temperature in the dark for 20~30 minutes, centrifuge at 350g for 5 minutes with permeabilization buffer, wash cells twice. Resuspend in cell staining buffer and detect on flow cytometer.

[0060] Step 8: Flow cytometry detection: The proportion of Tc cell subsets secreting granzyme B was obtained by flow cytometry detection.

[0061] The present invention is applicable to the detection of the effectiveness of immune cell preparations activated in vitro, wherein:

[0062] The cell viability of the human in vitro activated immune cell preparation after recovery in step 1 is not less than 80%. The concentration of the culture medium after dilution is 0.5~6.0×10 6 / mL, preferably 2×10 6 Pieces / mL.

[0063] The primary stimulator in step 2 is CD3 antibody, and the concentration is 2 μg / mL. The carrier is a 6-well cell culture plate. The mixed stimulator is an in vitro polyclonal activator, which is a mixture of PMA (myristoylphorbol ethyl ester) and ION (ionomycin), with concentrations of 40.5 μM and 699.3 μM, respectively, and the concentrations used are 2.4 μM and 40.2 μM, respectively. The blocking agent is BFA (Brefeldin A), with a concentration of 5 mg / mL, and the concentration used is 5 μg / mL. The culture conditions are a temperature of 38.5±0.5°C and a CO2 concentration of 6.5%.

[0064] The surface antibodies in step 4 are CD3 and CD8 fluorescent antibodies. The cell concentration before staining was adjusted to 1×10 6 / 100μL / reaction tube, the amount of antibody added is 1~5μL / reaction tube, preferably 2μL / reaction tube. The cell staining buffer is PBS buffer containing fetal bovine serum and sodium azide.

[0065] The fixing buffer in step 5 is a PBS buffer containing 4% paraformaldehyde.

[0066] The membrane permeabilization buffer in step 6 is a PBS buffer containing saponin, sodium azide and fetal bovine serum.

[0067] The intracellular antibodies in step 7 are CD69 fluorescent antibodies, CD44 fluorescent antibodies and granzyme B fluorescent antibodies. The ratio of CD69CD44 double positive cells is not less than 90% as a systematic internal control indicator of the method to evaluate whether the activity of the cells to be tested meets the product release standards.

[0068] In Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0069] The present invention will be further described below in conjunction with embodiments:

[0070] Example 1 Preparation method of human in vitro activated immune cell preparation

[0071] 1.1 Methods

[0072] 1.1.1 Isolation of mononuclear cells: After single blood or peripheral blood is diluted with compound electrolyte injection, it is separated by density gradient centrifugation using lymphocyte separation solution; after the cells are mixed with serum-free culture medium, the viability of the obtained cells is analyzed by automatic image analysis.

[0073] 1.1.2 Mononuclear cells were inoculated in serum-free medium at a certain concentration in T25 flasks pre-coated with CD3 monoclonal antibody for the first activation. The concentration of CD3 monoclonal antibody was 2 μg / mL and the culture time was 3 h.

[0074] 1.1.3 The activated PBMCs were removed and a combination of rhIL-2, rhIL-4, rhG-CSF, rhTNF-α, and rhIFN-γ cytokines were added for the second step of in vitro activation culture. The concentrations of each cytokine were 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α, and 6000 IU / mL of rhIFN-γ. The culture time was 5 h.

[0075] 1.1.4 Collect the cell suspension and centrifuge it. Mix the cells with serum-free medium and add 3.0×10 5 The cells were inoculated into a culture flask or culture bag at a concentration of 10 viable cells / mL and cultured continuously for 120 h.

[0076] 1.1.5 After the cell suspension is collected and centrifuged, the cells are mixed with compound electrolyte injection solution, the cell viability is analyzed by automatic image analysis, and then centrifuged. The human in vitro activated immune cell preparation is cryopreserved with a special cryopreservation solution. The composition and content of the cryopreservation solution are 25% (v / v) human serum albumin, 5% (v / v) DMSO, 35% (v / v) hydroxyethyl starch solution, 150 μg / mL recombinant human interleukin-2 and 25 μg / mL human interferon γ.

[0077] 1.1.6 Take 50μL of the cell suspension prepared in step 1.1.5 and put it into a 96-well cell culture plate. Add 50μL of AO / PI dye solution to the cell suspension and mix it by pipetting with a 100μL pipette. Take 20μL from the middle of the suspension and add it to one side of the disposable cell counting plate of the analyzer. Insert the end of the sample into the sample slot of the Cellometer K2 dual fluorescence cell analyzer. Use the program set by the analyzer software to count live cells and evaluate the viability.

[0078] Example 2 Rapid determination of the effectiveness of human in vitro activated immune cell preparations

[0079] 2.1 Basic steps

[0080] 2.1.1 Plate coating

[0081] Use DPBS to prepare a CD3 antibody coating solution with a concentration of 2 μg / mL, add 1.5 mL / well to a 6-well plate, and place in a refrigerator at 2-8°C overnight before use.

[0082] 2.1.2 Washing the plate

[0083] Take a 6-well coated plate, discard the coating solution, add 1 mL of DPBS water to each well, rinse once and then discard.

[0084] 2.1.3 Activation of human in vitro activated immune cell preparations

[0085] The frozen human in vitro activated immune cell preparation prepared in Example 1 was taken out, clamped and placed in a 38.5°C constant temperature water bath for recovery, added to a centrifuge tube containing complete culture medium, and centrifuged at 500g for 8 minutes. After the centrifugation, the supernatant was discarded, resuspended with complete culture medium, sampled, counted, centrifuged again at 500g for 8 minutes, and washed twice with DPBS at 1500rpm for 5 minutes. The supernatant was discarded, and the cell staining buffer was added and resuspended to adjust the cell suspension concentration of the human in vitro activated immune cell preparation to 2×10 6 / mL.

[0086] 2.1.4 First stimulus

[0087] Take the cell suspension obtained in 2.1.3, add it to the coated plate, 3 mL / well, and place it in an incubator at 38.5°C and 6.5% CO2 for 2 hours.

[0088] 2.1.5 Second stimulus

[0089] Add 6 μL of mixed stimulation buffer and 3 μL of blocking solution to the cell suspension obtained in 2.1.4, mix well and add to a 6-well plate, 3 mL / well, and place in an incubator at 38.5°C and 6.5% CO2 for 3 hours. Collect the cells from the culture plate into a centrifuge tube, add 5 mL / tube of DPBS, centrifuge at 500g for 5 minutes, wash twice, and resuspend the cells in DPBS to 1×10 7 / mL. The mixed stimulation buffer is an in vitro polyclonal activator, which is a mixture of PMA (phorbol myristate) and ION (ionomycin), with concentrations of 40.5µM and 669.3µM, respectively, and the concentration used is 2μL / mL. The blocking agent is BFA (Brefeldin A), with a concentration of 5mg / mL, and the concentration used is 1μL / mL.

[0090] 2.1.6 Surface dyeing

[0091] 100 μL / tube, divided into 3 tubes (blank, isotype, sample tube), no antibody added to the blank tube, surface antibody added to the sample tube. The surface antibodies are CD3 and CD8 fluorescent antibodies. The cell concentration before staining is adjusted to 1×10 6 / 100μL / reaction tube, antibody addition amount is 1~5μL / reaction tube, add 1mL cell staining buffer to each tube. Cell staining buffer is PBS buffer containing fetal bovine serum and sodium azide. Incubate at 2~8℃ in dark for 30 minutes, add 2μL of antibody each, centrifuge at 350g for 5 minutes and wash once.

[0092] 2.1.7 Fixation

[0093] The fixation buffer is PBS buffer containing 4% paraformaldehyde. 0.5 mL of fixation buffer is used in each tube, and the cells are incubated at room temperature in the dark for 20 minutes. Centrifuge at 350g for 5 minutes and discard the supernatant. Wash the cells once with cell staining buffer, centrifuge at 350g for 5 minutes and discard the supernatant.

[0094] 2.1.8 Membrane rupture

[0095] Resuspend the fixed cells with 1× Permeabilization Buffer, 1 mL / tube, centrifuge at 350g for 5 minutes, and discard the supernatant. Repeat this step once.

[0096] 2.1.9 Intracellular staining

[0097] Resuspend the permeabilized cells with 1× permeabilization buffer (100 μL / tube), add intracellular fluorescent labeled antibodies and intracellular isotype controls to each tube, add granzyme B, CD69 and CD44 to the sample tube, add 2 μL of each antibody, and incubate at room temperature in the dark for 20 minutes. Wash the cells twice with 1 mL permeabilization buffer, centrifuge at 350 g for 5 minutes, and discard the supernatant. Repeat the wash once. Resuspend the fixed stained cells with 200 μL Cell Staining Buffer, and analyze on the machine.

[0098] 2.1.10 Data processing and analysis

[0099] Use flow cytometry to perform the test, adjust the voltage so that the percentage of cells in the negative zone is greater than 98%, circle the target cells and set a gate, read the proportion of granzyme B, and obtain the test results.

[0100] According to the above method, the intracellular cytokines of human in vitro activated immune cell preparations were detected, and the flow cytometry data were obtained as follows: Figure 7 .according to Figure 7 The data show that CD69 in human in vitro activated immune cell preparation samples + CD44 + / The content of immune cells is 93.04%, and the cell stimulation is relatively successful, CD3 + CD8 + Granzyme B + / CD3 + CD8 + The content accounts for 93.84%.

[0101] 2.2 Basic Steps Optimization

[0102] 2.2.1 Optimization of stimulation density and maximum stimulation time of human in vitro activated immune cell preparations

[0103] After the test sample human in vitro activated immune cell preparation was revived according to the conventional method, the cell density was adjusted to 0.5×10 6 / mL, 1.0×10 6 / mL, 2.0×10 6 / mL, 4.0×10 6 / mL, 6.0×10 6 The cells were inoculated into 6-well culture plates and placed in a CO2 incubator (temperature 38.5±0.5℃, CO2 concentration 6.5%) for 6 and 18 hours for viability testing.

[0104] The results in Table 1 show that different densities of human in vitro activated immune cell preparations showed great differences at different culture times. 6The activity rate is the highest, preferably 2.0×10 6 The cell viability has dropped significantly after 6 hours of maintenance and has dropped significantly after 18 hours of maintenance. The longest stimulation time of immune cell preparations activated in vitro is no more than 6 hours.

[0105] Table 1 Cell viability of human in vitro activated immune cell preparations at different stimulation densities and times

[0106]

[0107] 2.2.2 Confirmation of feasibility of quality control of CD44 and CD69 double positive ratio

[0108] After activation of the immune cell preparation, the cells were grouped according to whether stimulation blocking was performed, namely, non-stimulation group, stimulation group, blocking group, and stimulation blocking group. The double positive ratio of CD44 and CD69 was detected to determine whether there was a significant difference in the double positive ratio of CD44 and CD69.

[0109] Figure 1~Figure 4 The results showed that in the unstimulated group, without stimulation and blocking, the CD44 and CD69 double positive ratio was 35.28%; in the group with stimulation but no blocking, the CD44 and CD69 double positive ratio was 53.44%; in the group with blocking but no stimulation, the CD44 and CD69 double positive ratio was 62.33%; after activation and blocking treatment, the CD44 and CD69 double positive ratio was 93.92%. After stimulation and blocking treatment, the CD44 and CD69 double positive ratio of immune cell preparations increased significantly, which was greater than that of the single test group, the test sample + activation group, the test sample + blocking group, and the test sample + activation + blocking group, and there was a significant difference. The ratio of CD44 and CD69 cells can be used as a sign of successful cell activation.

[0110] 2.2.3 Confirmation of the feasibility of two-step stimulus

[0111] After the immune cell preparations were activated, they were divided into mixed stimulation group and CD3 monoclonal antibody + mixed stimulation group according to whether CD3 monoclonal antibody was used for the first stimulation. The double positive ratio of CD44 and CD69 and CD3 + CD8 + Granzyme B + / CD3 + CD8 + ratio.

[0112] Figure 5 and Figure 6 The results showed that after stimulation and blocking, the ratio of CD44 and CD69 double positive in the mixed stimulation group was 74.51%, and CD3 + CD8 + Granzyme B+ / CD3 + CD8 + The ratio was 78.50%; the ratio of CD44 and CD69 double positive in CD3 monoclonal antibody + mixed stimulation group was 93.69%, and CD3 + CD8 + Granzyme B + / CD3 + CD8 + The ratio is 92.84%. Two-step stimulation can effectively increase the ratio of CD44, CD69 and CD3 + CD8 + Granzyme B + / CD3 + CD8 + ratio.

[0113] 2.2.4 Optimization of surface staining fluorescent antibody reaction volume

[0114] The surface staining fluorescent antibody in 2.1.6 of Example 2.1 was set to 1 μL / reaction tube, 2 μL / reaction tube, and 5 μL / reaction tube, respectively, and three analysis batches were set to analyze the effects of different amounts of fluorescent antibody on CD3 + CD8 + Granzyme B + / CD3 + CD8 + The impact of the test results.

[0115] The results in Table 2 show that in the three analysis batches, the same analysis batch of fluorescent antibodies with different addition amounts of CD3 + CD8 + Granzyme B + / CD3 + CD8 + The CV values ​​were all less than 15%, and there was no significant difference among the three fluorescent antibody concentrations. Considering the cost, 2 μL / reaction tube was preferred.

[0116] Table 2 Effect of different antibody dosages on CD3 + CD8 + Granzyme B + / CD3 + CD8 + The test results of the proportion

[0117]

[0118] Example 3 Method Applicability Verification

[0119] According to the assay method optimized in Example 2, by detecting CD3 + CD8+ Granzyme B + / CD3 + CD8 + The method was validated from four aspects: quality control, specificity, precision and linearity.

[0120] 3.1 Quality Control

[0121] In order to ensure the stability and reliability of the instrument, and then ensure the accuracy and repeatability of the experimental data, thus laying the foundation for the smooth progress of subsequent experiments. Quality control is performed every time the flow cytometer is turned on, for a total of 6 times, and the quality control results should meet the following requirements:

[0122] Delay: -5.00us≤Difference from default delay≤5.00us;

[0123] Gain: -20.00%≤% Difference from target gain ≤20.00%;

[0124] Median: -5.00%≤% Difference from the median target value ≤5.00%;

[0125] rCV: rCV (%) ≤ target value rCV (%).

[0126] The results showed that all 6 quality control results met the requirements.

[0127] 3.2 Exclusivity

[0128] 3.2.1 Specificity

[0129] The known negative HCC827 cells were added to the test sample at a certain ratio to prepare the quality control samples, 1:5 (QC1), 1:10 (QC2), 1:20 (QC3), and the test sample control was set to detect the target cell population CD3 + CD8 + Granzyme B + / CD3 + CD8 + Proportion analysis. Statistical analysis of the accuracy of each target cell population within each quality control sample batch, that is, the recovery rate (RE%) of the quality control sample compared with the test sample. Verify that negative cells do not interfere with the stimulation of the test sample, and whether the staining antibody specifically binds to the target cells without interference from foreign cells. The accuracy of each cell subpopulation in the quality control sample is within ±15%.

[0130] 3.2.2 Cross-reactivity

[0131] Three batches of test products were used, all of which were stained single and multiple times, and the CD3 positive target cells in the multicolor tube were analyzed. + CD8 +Granzyme B + / CD3 + CD8 + The recovery rate is calculated based on the percentage of the sample, verifying whether there is cross-reaction between the multiple fluorescent antibody stainings in the sample tube. The recovery rate of each cell subset is within ±15%.

[0132] The results showed that the specificity of each quality control sample CD3 + CD8 + Granzyme B + / CD3 + CD8 + The recoveries were 103.63%, 99.14%, 98.36%, 103.02%, 101.67%, 98.74%, 104.16%, 104.32%, and 98.99%, respectively; the cross-reaction CD3 + The recovery rates were 101.13%, 101.40%, and 99.38%, Granzyme B + The recovery rates were 98.37%, 96.64%, 104.12%, CD8 + The recovery rates were 101.43%, 105.35%, 103.18%, CD69 + The recovery rates were 101.19%, 100.85%, 99.10%, CD44 + The recoveries were 101.03%, 101.17%, and 97.94%, all meeting the validation requirements, and the recoveries were all within the ±15% specificity range.

[0133] 3.3 Precision

[0134] 3.3.1 Repeatability

[0135] Three batches of test samples were used, and six replicate samples were prepared from each batch of samples. The CV% of each target cell population within the sample batch was calculated. The CV% of the test results of each target cell population in the same batch was ≤15%.

[0136] 3.3.2 Intermediate precision

[0137] 3 batches of test products, each batch of test products was tested by 2 analysts, and the CV% of each target cell population tested by 2 analysts from the same batch of samples was analyzed. For the same batch, 2 analysts tested the data, and the CV% of each target cell population was ≤15%.

[0138] The results showed that the repeatability CV% of the three batches of test products were 2.25%, 1.53%, and 1.08%, respectively, and the intermediate precision CV% were 1.70%, 1.31%, and 1.49%, respectively. All of them met the validation requirements, and the CV% of each target cell population was ≤15%, and the precision was qualified.

[0139] 3.4 Linearity

[0140] Standard curve samples S1-S5 were prepared according to dilution factors of 1, 0.5, 0.25, 0.125, and 0.0625. The dilution factor of each cell population was used as the horizontal coordinate, and the proportion of the target population was used as the vertical coordinate. Linear regression was used to fit the standard curve to obtain the regression equation and R 2 Value, at least 3 batches were examined. Standard curve R 2 Should not be less than 0.9800.

[0141] Figure 8~Figure 10 The results show that the linearity of the three batches is 0.9991, 0.9997, and 0.9974, respectively. 2 All values ​​were ≥0.9800, and the linearity was qualified.

[0142] 3.5 Verification conclusion

[0143] According to the determined test parameters, by detecting CD3 + CD8 + Granzyme B + / CD3 + CD8 + The method was verified from four aspects: quality control, specificity, precision and linearity. The verification results showed that the method of the present invention can be used for the rapid determination of the effectiveness of human in vitro activated immune cell preparations.

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for culturing cells, characterized in that: The pretreated cells are subjected to a first stimulation, a first culture, a second stimulation, a blocking, a second culture, and a washing to obtain the cells; The first stimulation used CD3 antibody at a final concentration of 2 μg / mL; The second stimulus used myristoyl phorbol ethyl ester at a final concentration of 2.4 µM and ionomycin at a final concentration of 40.2 µM; The blocking was performed using Brefeldin A at a final concentration of 5 μg / mL.

2. The culture method according to claim 1, characterized in that The cell concentration of the first stimulation is 0.5~6.0×10 6 Pieces / mL.

3. The culture method according to claim 1, characterized in that The time from the first stimulation to the second culture does not exceed 6 hours.

4. The culture method according to claim 1, characterized in that The pretreatment comprises the following steps: activating, culturing and collecting PBMC cells; The activation includes: a first activation and a second activation; The first activation used 2 μg / mL CD3 monoclonal antibody for 3 h; The second activation uses cytokines for 5 h; The cytokines include: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α, and 6000 IU / mL of rhIFN-γ; The culture was carried out using serum-free medium for 120 h.

5. Use of the cells obtained by the culture method according to any one of claims 1 to 4 in the preparation of a product for detecting the in vitro effectiveness of an immune cell preparation.

6. The use according to claim 5, characterized in that The detection comprises the following steps: staining the cell surface, fixing, permeating, intracellular staining, and detecting, and obtaining the detection result according to the proportion of Tc cell subpopulations secreting granzyme B and internal control indicators; The internal control indicators include: CD69 + CD44 + The activation rate is greater than 90%.

7. The use according to claim 6, characterized in that The surface antibodies used in the surface staining are CD3 and CD8 fluorescent antibodies.

8. The use according to claim 6, characterized in that The membrane permeation buffer used in the membrane permeation is a PBS buffer containing saponin, sodium azide and fetal bovine serum.

9. The use according to claim 6, characterized in that The antibodies used for intracellular staining are CD69 fluorescent antibody, CD44 fluorescent antibody and granzyme B fluorescent antibody.

10. The use according to claim 6, characterized in that The Tc cell subsets include: CD3 + CD8 + Granzyme B + / CD3 + CD8 + .

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