CN114317665B - Application and method of CTLL-2 cells domesticated by CAR-T culture medium for detecting activity of IL-2 - Google Patents
Application and method of CTLL-2 cells domesticated by CAR-T culture medium for detecting activity of IL-2 Download PDFInfo
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Abstract
The invention relates to the field of IL-2 activity detection, in particular to an application of CTLL-2 cells domesticated by a CAR-T culture medium to the activity detection of IL-2 and a method thereof, and provides a CTLL-2 cell bank domesticated by the CAR-T culture medium, and a method for detecting the activity of the domesticated cells for IL-2, which comprises the following steps: preparing IL-2 standard sample solutions with different concentrations by taking a CAR-T basic culture medium as a diluent; mixing CTLL-2 cells domesticated by the CAR-T culture medium with standard sample solutions and sample solutions to be tested with different concentrations respectively, and incubating simultaneously in an environment suitable for CTLL-2 cell growth; adding CCK8 solution for incubation, and then developing and detecting absorbance; and calculating the relative activity of IL-2 in the sample to be tested. The domesticated CTLL-2 cells are suitable for detecting the IL-2 activity in the CAR-T culture medium, avoid detection interference caused by the existence of the RPMI1640 culture medium in a general IL-2 activity detection method, and have good accuracy and repeatability.
Description
Technical Field
The invention relates to the field of IL-2 activity detection, in particular to an application and a method of CTLL-2 cells domesticated by a CAR-T culture medium for detecting the activity of IL-2.
Background
The current IL-2 activity assay is most reported to be based on CTLL-2 cells (1640 medium+fetal bovine serum culture system) under which it has good reactivity to human IL-2. This method is sometimes referred to as the "universal IL-2 activity assay". The method is also written into 2015 pharmacopoeia 3524 recombinant human interleukin-2 biological Activity assay
Tumor cell immunotherapy is an emerging therapeutic modality, and is currently considered to be the most effective one. Currently, CAR-T cells, which are immunotherapy techniques, show very good clinical effects on a variety of hematological tumors and also have great potential for solid tumor treatment. The CAR-T technology modifies and expands T cells in vitro and then infuses them back to the patient for treatment. In vitro amplification is a culture system of CAR-T culture medium and human serum, and IL-2 is an important exogenous additive factor used for stimulating T cell proliferation in the amplification stage. The addition amount of IL-2 is closely related to the harvest time of CART cells and the quality of the product. Therefore, establishment of an activity detection method for IL-2 which is suitable for a CAR-T medium system and has good reactivity is very important. For example: stability study of IL-2 in CAR-T medium; IL-2 activity change research at different stages of CAR-T process and the like are important research points in process development.
In the general IL-2 activity detection method 3524, an RPMI1640 basic culture system is used for diluting a sample to be detected to IL-200IU/ml in a large proportion as an initial concentration point for detection (the large proportion dilution can eliminate the interference of the original matrix of the sample on detection if any). However, based on CAR-T process parameters, we predict that the concentration of IL-2 in CAR-T medium is 0-500IU/ml. Too low IL-2 is unfavorable for improving the proliferation rate of cells and slow proliferation of cells; too high IL-2 tends to result in an increased proportion of Treg cells, thereby compromising CAR-T killing function. Thus, the IL-2 concentration in the CAR-T process medium cannot be diluted in large proportion (e.g. > 10-fold dilution) to reduce potential interference of the matrix itself, since the pre-dilution concentration is already near the starting point concentration level required in the universal IL-2 activity assay. The CAR-T culture medium (X-vivo culture medium) is an optimized culture medium beneficial to the growth of human T lymphocytes, if the culture medium cannot be diluted in a large proportion, and under the condition of no dilution, referring to other steps of the method of pharmacopoeia 3524, 100ul of cells (RPMI 1640 culture medium) and 100ul of to-be-detected substances (CART culture medium) are respectively added into a test hole, so that the problems that unknown interference is caused by analysis caused by higher proportion of the CAR-T culture medium in a test system, the IL-2 activity cannot be accurately measured, and in addition, when CTLL-2 cells are applied to the detection of the IL-2 activity, the time consumption is long, the recovery of the cell state is slow, and the recovery is difficult are solved. Thus, we consider that there is a need to establish a method of detecting IL-2 activity that is well-behaved and suitable for use in CAR-T medium systems.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for detecting IL-2, which is used for solving the problems of long time consumption during CTLL-2 cell resuscitation and potential interference of CART medium in detection of general IL-2 activity in the prior art.
To achieve the above and other related objects, the present invention provides a use of CTLL-2 cells acclimatized to a CAR-T medium for activity detection of IL-2 in a medium at each stage of CAR-T process.
In one embodiment, CTLL-2 cells acclimatized to the CAR-T medium are used for detection of IL-2 activity in the medium when the CAR-T medium is used for CAR-T culture.
In one embodiment, the concentration of IL-2 in the culture medium is greater than or equal to 100IU/ml and less than 200IU/ml when the CAR-T cells are cultured.
The invention also provides an activity detection method of IL-2, which comprises the following steps:
1) Preparing IL-2 standard sample solutions with different concentrations by taking a CAR-T basic culture medium as a diluent;
2) Preparing sample solutions to be tested with different concentrations by taking a CAR-T basic culture medium as a diluent;
3) Mixing the recovered CTLL-2 cells domesticated by the CAR-T culture medium with standard sample solutions with different concentrations and sample solutions to be detected with different concentrations respectively, and incubating simultaneously in an environment suitable for the growth of the CTLL-2 cells;
4) Developing and detecting absorbance;
5) And calculating the relative activity of the IL-2 of the sample to be tested.
In one embodiment, the cell viability of the CTLL-2 of step 3) is greater than 85%.
In one embodiment, the step 5) specifically includes: and respectively reading the IL-2 activity concentration of half inhibition effect in the standard drug curve and the curve of the sample to be detected, and calculating the relative activity of the sample to be detected.
In one embodiment, the domesticated CTLL-2 cells are prepared by a method comprising the steps of: and increasing the volume ratio of the CAR-T culture medium in the CTLL-2 cell mixed culture medium by generations until the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium is 100%, and domesticating and culturing the CTLL-2 cells for 5-8 generations.
In one embodiment, the volume ratio of the CAR-T medium in the CTLL-2 cell culture medium is increased by 10-20% each time.
In one embodiment, the CTLL-2 cell culture medium contains IL-2 for the growth of CTLL-2 cells.
In one embodiment, the CTLL-2 cells are seeded at a density of 1 to 3 x 10 at acclimation passaging 5 cells/ml。
In one embodiment, the CTLL-2 cells have a density of 0.8 to 1.6X10 6 cells/ml were harvested.
In one embodiment, the culture conditions of the CTLL-2 cells during domestication are at a temperature of 35-37 ℃ and 4-6% CO 2 。
In one embodiment, in step 3), the resuscitated CTLL-2 cells acclimatized with CAR-T medium are washed and then diluted with CAR-T basal medium.
As described above, the method for detecting IL-2 has the following beneficial effects: the domesticated CTLL-2 cells are suitable for detecting the IL-2 activity in the CAR-T culture medium, avoid detection interference caused by mixing two culture mediums in a general IL-2 activity detection method 3524, and have good accuracy and repeatability. The time consumption for recovering CTLL-2 cells which are not domesticated by the CAR-T culture medium is long, the recovery of cell states is slow, the recovery of the domesticated CTLL-2 cells is difficult, the time consumption for recovering the domesticated CTLL-2 cells is short, the state recovery is faster, and the activity detection time of IL-2 is saved.
Drawings
FIG. 1 shows a graph of growth doubling time after CTLL-2 cell resuscitation.
FIG. 2 shows a schematic of the accuracy of different relative active sites for IL-2.
Fig. 3 shows a linear fit of the average of the measurement results.
FIG. 4 shows a summary of curves for different concentrations of IL-2.
Detailed Description
The invention firstly provides an application of CTLL-2 cells domesticated by a CAR-T culture medium to activity detection of IL-2.
The CTLL-2 cells domesticated by the CAR-T culture medium provided by the invention are suitable for detecting the activity of IL-2 in various samples containing IL-2, and are particularly suitable for detecting the activity of IL-2 in the culture medium when the CAR-T cells are cultured.
In CAR-T culture, the concentration of IL-2 in the culture medium is usually 0-500IU/ml, CTLL-2 cells acclimatized by the CAR-T culture medium are suitable for each stage of CAR-T cell culture, and the activity of IL-2 in the culture medium is detected. When the concentration of IL-2 is lower than 200IU/ml, the existing IL-2 activity detection method is not applicable, and compared with the prior art, the method disclosed by the invention is more applicable to detecting CART culture media with the concentration of IL-2 lower than 200IU/ml after CTLL-2 cells are domesticated by the CAR-T culture media. Therefore, it is preferred that CTLL-2 cells acclimatized to CAR-T medium are used for detection of CART medium samples having IL-2 concentration of less than 200IU/ml. In order to obtain a complete S-shaped curve of the sample to be tested, more preferably, the concentration of IL-2 in the sample of the CAR-T medium to be tested is greater than or equal to 100IU/ml and less than 200IU/ml.
In the present invention, the CAR-T medium refers to a medium suitable for culturing CAR-T cells. The media suitable for culturing CAR-T cells, either commercially available or described in the literature, are suitable for use in the present invention. In a specific embodiment, the CAR-T Medium comprises 53ml of Human AB Serum, 10.6ml of Glutamax, 2.1ml of Poloxamer 188 and IL-2 per 1000ml of X-vivo Medium, and the practitioner selects the amount of IL-2 to be added according to the needs and morphology of the cells.
CTLL-2 cells domesticated by the CAR-T culture medium can survive and be passaged in the CAR-T culture medium, the CTLL-2 cells domesticated by the CAR-T culture medium can be applied to the activity detection of IL-2 in the CART culture medium, and the detection interference caused by the existence of RPMI1640 culture medium in the general IL-2 activity detection method 3524 is avoided in a single detection environment.
The invention also provides an activity detection method of IL-2, which comprises the following steps:
1) Preparing IL-2 standard sample solutions with different concentrations by taking a CAR-T basic culture medium as a diluent;
2) And preparing sample solutions to be tested with different concentrations by taking the CAR-T basal medium as a diluent.
3) Mixing the recovered CTLL-2 cells domesticated by the CAR-T culture medium with standard sample solutions with different concentrations and sample solutions to be detected with different concentrations respectively, and incubating simultaneously in an environment suitable for the growth of the CTLL-2 cells;
4) Developing and detecting absorbance;
5) And calculating the relative activity of IL-2 in the sample to be tested.
The CAR-T basal medium is a CAR-T medium which does not contain IL-2.
In step 1), IL-2 standard sample solutions with different concentrations are prepared by taking a CAR-T basal medium as a diluent. In one embodiment, a stock solution of frozen standard IL-2 is diluted to 400IU/mL with CAR-T basal medium and then diluted to various concentrations as required.
In the step 2), the CAR-T basic culture medium is used as a diluent to prepare sample solutions to be tested with different concentrations. The sample solution to be tested was diluted with CAR-T basal medium. When the CAR-T basal medium dilutes the sample solution to be detected, the dilution gradient can be consistent with the step 1) or inconsistent with the step 1). For the convenience of reading, preferably, when the CAR-T basal medium dilutes the sample solution to be detected, the dilution gradient is consistent with that of the IL-2 standard sample diluted by the CAR-T basal medium in the step 1, so as to obtain the sample solution to be detected, the concentration gradient of which is the same as that of the IL-2 standard sample solution with different concentrations.
In step 3), the recovered CTLL-2 cells acclimatized by the CAR-T medium are respectively mixed with standard sample solutions with different concentrations and sample solutions to be tested with different concentrations, and incubated simultaneously in an environment suitable for the growth of the CTLL-2 cells. Resuscitates and cultures the domesticated CTLL-2 cells, dilutes the domesticated CTLL-2 cells by using CAR-T basic culture, calculates the cell density and the activity of the domesticated CTLL-2 cells, mixes the CTLL-2 cells with the cell density and the activity reaching the requirements with standard sample solutions with different concentrations and sample solutions to be detected with different concentrations, and then places the mixture into an environment suitable for the growth of the CTLL-2 cells and incubates the mixture for about 18 to 24 hours.
In step 4), color development and absorbance detection are performed. The color may be developed by conventional methods, for example, in one embodiment, after incubation is completed, CCK8 color developing solution is added to each mixed solution separately and then placed into CO 2 Incubator 37 ℃, CO 2 The incubation is continued for 2.5-4h, after the incubation is completed, the absorption wavelength is set to 450nm, the reference wavelength is set to 650nm, and the OD value of each mixed solution is respectively read.
In step 5), the relative activity of IL-2 in the test sample is calculated. The data can be processed by software to obtain a standard curve of concentration (ng/ml) versus absorbance (OD 450nm-650 nm), and the relative activity of IL-2 in the sample to be detected can be obtained through a relative activity calculation formula.
TBD Sample IU/ml＝ST(con)*C(ST)/C(SA)；
TBD Sample IU/ml: the active concentration (IU/ml) of IL-2 in a sample to be tested;
ST (Con): the activity concentration (IU/mL) of the IL-2 in the diluted standard sample is 400IU/mL;
c (ST): the standard achieves IL-2 activity concentration (IU/ml) of half inhibition effect of the standard curve;
c (SA): the samples reached the IL-2 activity concentration (IU/ml) of half of the inhibitory effect of the sample curve.
In one embodiment, the cell viability of the CTLL-2 of step 3) is greater than 85%. The cell viability can be obtained by the following calculation formula: cell viability = (total number of cells-dead number of cells)/total number of cells × 100%.
In one embodiment, the domesticated CTLL-2 cells are prepared by a method comprising the steps of: and increasing the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium by generations until the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium is 100%, and domesticating and culturing the CTLL-2 cells for 5-8 generations.
In one embodiment, the volume ratio of the CAR-T medium in the CTLL-2 cell cocktail medium is increased by 10-20% each generation. The percentage of improvement in the volume ratio can be selected by the practitioner as desired, and can be, for example, 10-11%, 11-12%, 12-13%, 13-14%, 14-15%, 15-16%, 16-17%, 17-18%, 18-19%, and 19-20%.
In one embodiment, the CTLL-2 cell culture medium contains IL-2 for the growth of CTLL-2 cells. Preferably, the IL-2 concentration in the CTLL-2 cell culture medium of the first generation is 400IU/ml. IL-2 was added to stimulate CTLL-2 cell proliferation, counteracting the effects of medium changes.
In acclimating the CTLL-2 cells, the CTLL-2 cells are prevented from depending on high concentration of IL-2, resulting in reduced examination sensitivity. Preferably, the IL-2 concentration is reduced every second generation or every third generation until the IL-2 concentration is 200IU/ml.
In one embodiment, the CTLL-2 cells are seeded at a density of 1 to 3 x 10 at acclimation passaging 5 cells/ml. For the first acclimation, more cells are required, e.g. a selected seeding density of 3×10 5 cells/ml, with acclimation, cells gradually adapt to new environment, some cells can be inoculated less, and experimenters determine specific inoculation density according to cell state judgment, for example, the inoculation density can be 1-1.5×10 5 cells/ml、1.5～2*10 5 cells/ml、2～2.5*10 5 cells/ml and 2.5-3 x 10 5 cells/ml。
In one embodiment, the CTLL-2 cells have a density of 0.8 to 1.6X10 6 cells/ml were harvested. The density of CTLL-2 cells is 0.8-1.6x10 6 When cells/ml are nearly saturated, if continuous culture is carried out, the cell viability is greatly reduced without supplementing new culture medium and IL2, and experimenters select C according to the cell stateThe TLL-2 cells are harvested when the density reaches a certain value, for example, 0.8-1.0 x 10 6 cells/ml、1.0～1.2*10 6 cells/ml、1.2～1.4*10 6 cell/ml and 1.4-1.6 x 10 6 cells/ml。
In one embodiment, the culture conditions of the CTLL-2 cells during domestication are at a temperature of 35-37 ℃ and 4-6% CO 2 . The culture conditions of CTLL-2 cells can be selected by the operators according to the requirements, and the temperature can be 35-35.5 ℃, 35.5-36 ℃, 36-36.5 ℃ and 36.5-37 ℃, CO 2 The volume ratio may be 4-5% and 5-6%.
In one embodiment, in step 3), the resuscitated CTLL-2 cells acclimatized with CAR-T medium are washed and then diluted with CAR-T basal medium. In one embodiment, the recovered CTLL-2 cells acclimatized with the CAR-T medium are washed with PBS solution to remove IL-2 attached to the CTLL-2 cells, and diluted with the CAR-T basal medium to prevent detection interference caused by different media.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Example 1
1 culture Medium configuration
1.1 RPMI1640 medium
| Name of the name | Goods number | Quantity (ml) |
| RPMI 1640 Medium | Gibco 22400-089 | 450 |
| FBS | Gibco 35-081-CV | 50 |
| Pyruvic acid sodium salt | Gibco 11360-070 | 5 |
| Double antibody | Hyclone SV30010 | 5 |
1.2 CAR-T basal medium
1.3 CAR-T medium
The concentration of IL-2 in CAR-T medium can be selected as desired.
1.4 IL-2 mother liquor configuration
Il-2 manufacturer GE healthcare, cat# 29062789, prepared by dissolving with sterile water based on factory CoA, to a concentration of 100000IU/ml, and a storage expiration date of-20 ℃ is tentatively set for 1 month.
2 CTLL-2 cell line resuscitation
2.1 CTLL-2 cell resuscitation: CTLL-2 was purchased from ATCC under the following designations: TIB-214. The CTLL-2 cells are rapidly taken out from a liquid nitrogen tank and placed in a water bath kettle at 37 ℃, and a cell freezing tube is rapidly rocked, so that the cells are re-melted as soon as possible.
2.2 transfer the thawed cells to a centrifuge tube containing 9ml of RPM 1640 basal medium, centrifuge 400g to remove supernatant, then resuspend the cells with 25ml of RPM 1640 basal medium and transfer to T75 flasks, and finally add 50ul IL-2 stock solution per flask and culture at 37℃under 5% CO 2.
2.3 the state recovery of the cell takes longer (> 15 days).
2.4 after the cell state is recovered, passaging is carried out for 2-3 times.
Domestication culture of 3 CTLL-2 cells
3.1 domestication
The following table shows examples of one-time acclimation culture:
3.2 maintenance of growth
After 5 generations of growth domestication, the cells can be maintained, amplified and frozen for normal growth and passage.
TABLE 2 CTLL-2 cell passaging Density
| Passage time | Seed Density (10) 5 cells/ml) |
| Passaging 1 time every 1 day | 1.0～1.5 |
| Passaging 1 time every 2 days | 0.5～1.0 |
Determination of IL-2 Activity in 4 CART Medium
4.1 preparation of cells:
4.1.1 referring to Table 2, cell growth passaging was performed in a CO2 incubator (37 ℃ C. 5% CO 2).
4.1.2 cell harvesting: cells in T75 flasks were blown apart and then transferred in their entirety to 50ml EP tubes.
4.1.3 Centrifuging at 400g for 5min, and removing supernatant.
4.1.4 pre-warmed 10ml PBS was re-suspended and washed 1-2 times.
4.1.5 Centrifuging at 400g for 5min, and removing supernatant; resuspended with 10ml of CAR-T basal medium and counted. And respectively carrying out more than two groups of parallel experiments, and calculating the average activity rate of the cells.
4.1.6 cell viability >85% and can be used for further experiments.
4.1.7 cells were diluted to 1.5 x 10 with CAR-T basal medium 6 cells/mL。
4.2 preparation of standard curve:
4.2.1 taking the mother liquor of frozen standard IL-2, firstly diluting to 400IU/mL with CAR-T basal medium, and then serially diluting with reference to the following table:
4.2.2 preparation of samples to be tested:
the sample to be tested is directly diluted according to the table 4.2.1.
4.2.3 dosing and plating
Using a row gun, adding 4.2.1&4.2.2 standard substances and samples into a designated position of a 96-well plate, wherein the ratio is 150ul/well;
using a gang gun, 4.1.7 cells were added to the 96-well plate at the indicated location, 50ul/well;
the loading layout is as follows:
4.2.4 incubation:
placing the above 96-well plate into CO 2 Incubator incubation, 37 ℃,5% co2, for 18-24h.
4.2.5 color development
After incubation for about 18-24 hours, the 96-well plate was removed and CCK 8-developed solution, 25 ul/well, was added. Then put into a CO2 incubator at 37 ℃ and incubated with 5% CO2 for 2.5-4 hours.
4.2.6 read plate:
after incubation, the plate was read by setting an absorption wavelength of 450nm and a reference wavelength of 650 nm.
4.2.7 data analysis:
1) Data were processed using Softmax software and standard curves were plotted as concentration (ng/ml) versus absorbance (450 nm-650nm OD), with a 4-parameter fit selected.
2) Calculation of relative Activity:
TBD Sample IU/ml＝ST(con)*C(ST)/C(SA)；
TBD Sample IU/ml: the active concentration (IU/ml) of IL-2 in a sample to be tested;
ST (Con): the activity concentration (IU/mL) of the IL-2 in the diluted standard sample is 400IU/mL;
c (ST): the standard achieves IL-2 activity concentration (IU/ml) of half inhibition effect of the standard curve;
c (SA): the samples reached the IL-2 activity concentration (IU/ml) for half of the inhibitory effect of the curve.
As shown in fig. 4, the ST standard curve is Plot 4; c value: half inhibition concentration was 22.1IU/ml, one TBD curve was Plot 3, C: half inhibition concentration was 30.3IU/ml, tbd=400×22.1/30.3=291.7 IU/ml.
As shown in FIG. 4, when the concentration of a sample to be detected is 100IU/ml, an S-shaped curve can be displayed through testing and fitting, and the method can be used for detecting the activity of IL-2 with the concentration of more than or equal to 100IU/ml in a CAR-T culture medium, and the detection result can be quantified and can be expressed in a numerical form.
Comparison of cell resuscitation before/after CTLL-2 cell acclimation:
as shown in figure 1 of the drawings,
remarks: doubling time refers to the time required for the number of cells to double, reflecting the growth rate of the cells. The shorter the doubling time, the more vigorous the cell growth.
Repeatability of the method:
1. repeatability of
Different active points within the range of 100IU/mL-800IU/mL are selected for a repeatability test:
2. accuracy of different relative active points
As shown in fig. 2, all the detection results are within the control line, indicating that the detection results are valid.
3. Linearity of
As shown in FIG. 3, a linear fit was performed by taking the average of the measurements of a total of 8 points of 100/200/300/400/500/600/700/800IU/mL, y=1.01188 x+2.0583, R 2 ＝0.9987。
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (8)
1. Use of CTLL-2 cells acclimatized to CAR-T medium for activity detection of IL-2 in CAR-T medium; the domesticated CTLL-2 cells are prepared by a method comprising the steps of: and increasing the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium by generations until the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium is 100%, and domesticating and culturing the CTLL-2 cells for 5-8 generations.
2. Use according to claim 1, characterized in that: when CTLL-2 cells acclimatized to the CAR-T medium are used for CAR-T cell culture, the activity of IL-2 in the medium is detected.
3. Use according to claim 2, characterized in that: when the CAR-T cells are cultured, the concentration of IL-2 in the culture medium ranges from greater than or equal to 100IUml to less than 200IUml.
4. A method for detecting the activity of IL-2, comprising the steps of:
1) Preparing IL-2 standard sample solutions with different concentrations by taking a CAR-T basic culture medium as a diluent;
2) Preparing sample solutions to be tested with different concentrations by taking a CAR-T basic culture medium as a diluent;
3) Mixing the recovered CTLL-2 cells domesticated by the CAR-T culture medium with standard sample solutions with different concentrations and sample solutions to be detected with different concentrations respectively, and incubating simultaneously in an environment suitable for the growth of the CTLL-2 cells;
4) Adding CCK8 color development liquid for color development and detecting absorbance;
5) Calculating the relative activity of IL-2 in a sample to be detected;
the domesticated CTLL-2 cells are prepared by a method comprising the steps of: increasing the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium from generation to generation until the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium is 100%, and domesticating and culturing the CTLL-2 cells for 5-8 generations;
the cell viability of CTLL-2 in step 3) is greater than 85%;
step 5) specifically comprises the steps of respectively reading the IL-2 activity concentration of half inhibition effect in a standard drug curve and a curve of a sample to be detected, and calculating the relative activity of the sample to be detected.
5. The method according to claim 4, wherein: in step 3), the resuscitated CTLL-2 cells acclimatized with CAR-T medium are washed and diluted with CAR-T basal medium.
6. The method according to claim 4, wherein: the volume ratio of the CAR-T culture medium in the CTLL-2 cell culture medium is increased by 10-20% each time.
7. The method of claim 4, wherein the CTLL-2 cell culture medium comprises IL-2 for CTLL-2 cell growth.
8. The method of claim 4, comprising one or more of the following features:
the inoculation density of CTLL-2 cells in acclimatization and passage is 1-3 x 10 5 cell/ml；
The harvesting density of the CTLL-2 cells after domestication and passage is 0.8-1.6x10 6 cell/ml；
The culture condition of the CTLL-2 cells in domestication is that the temperature is 35 ℃ to 37 ℃ and the CO content is 4 to 6 percent 2 。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134980A (en) * | 2006-08-31 | 2008-03-05 | 上海新生源医药研究有限公司 | Method for determining interleukin 15 biological activity |
| CN101139627A (en) * | 2006-09-06 | 2008-03-12 | 上海新生源生物医药有限公司 | Method for determining cytokine interleukin 15 biological activity |
| CN110964695A (en) * | 2019-12-10 | 2020-04-07 | 康立泰药业有限公司 | Cell strain and method for detecting rhIL-12 in vitro activity by proliferation thereof |
-
2020
- 2020-09-29 CN CN202011047320.4A patent/CN114317665B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134980A (en) * | 2006-08-31 | 2008-03-05 | 上海新生源医药研究有限公司 | Method for determining interleukin 15 biological activity |
| CN101139627A (en) * | 2006-09-06 | 2008-03-12 | 上海新生源生物医药有限公司 | Method for determining cytokine interleukin 15 biological activity |
| CN110964695A (en) * | 2019-12-10 | 2020-04-07 | 康立泰药业有限公司 | Cell strain and method for detecting rhIL-12 in vitro activity by proliferation thereof |
Non-Patent Citations (2)
| Title |
|---|
| A rapid alternative method to evaluate T-cell hybridoma activation using an improved cytokine (IL-2) secretion assay;Paola Gastelum-Aviña等;《J Immunol Methods》;第438卷;42-50 * |
| 可溶性噻唑盐WST-8用于重组人白细胞介素-2的生物学活性测定;郭桥;肖艳玲;左毅;许国杰;鲁文艳;张岩;崔颖杰;;《微生物学免疫学进展》(第04期);33-36 * |
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Application publication date: 20220412 Assignee: Shanghai Mingju Biotechnology Co.,Ltd. Assignor: Shanghai Yaoming junuo Biotechnology Co.,Ltd. Contract record no.: X2024980013242 Denomination of invention: The use and method of CTLL-2 cells domesticated in CAR-T medium for IL-2 activity detection Granted publication date: 20240109 License type: Common License Record date: 20240828 |
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