Method for detecting color of cell culture fluid

Abstract

The invention relates to a method for detecting the color of a cell culture solution, and belongs to the technical field of cell detection. The method comprises the following steps: acquiring a color detection value a * actual detection of a to-be-detected cell culture fluid; judging whether the color detection value a * actual detection is smaller than a preset value a * preset of a cell culture fluid or not, and if yes, supplementing or replacing a cell culture medium; otherwise, putting back the culture box to continue culturing for one day, detecting the color detection value of the cell culture solution again on the second day, and supplementing or replacing the cell culture medium until the judgment condition is met. According to the method, the cell growth is not influenced, the color change time of the cell culture fluid can be correctly judged, the cell culture fluid is supplemented or replaced for the cells in time, the detection efficiency is improved, and the labor cost is reduced.

Description

Technical Field

[0001] The present application relates to the technical field of cell detection, and in particular to a method for detecting the color of a cell culture fluid. Background Art

[0002] Cell immunotherapy is a treatment method that improves the patient's immunity by activating and amplifying immune cells taken from the patient in vitro and then returning them to the patient's body. It is also called "adoptive immunotherapy." The core component of cell immunotherapy-related products is cells, which are usually living cell products of human origin, including genetically modified or non-genetically modified cells, such as autologous or allogeneic immune cells, stem cells, tissue cells or cell lines. The key process in cell immunotherapy-related products is the cell culture process, which specifically involves steps such as the activation and amplification of immune cells. In order to activate and amplify immune cells in vitro, fluid replacement or rehydration becomes a necessary key point in the cell culture process.

[0003] At present, in the process of cell culture, there are the following problems in determining the timing of fluid replacement or fluid replenishment:

[0004] (I) Individual differences in human visual observation:

[0005] When phenol red is added to the culture medium, the color of the culture medium will change with the change of pH. When the fluid needs to be replaced or replenished, the operator needs to judge the change of the color of the cell culture medium based on actual experience. Due to the large individual differences in color cognition and vision between individuals, the judgment of the timing of replenishment is not accurate, which usually leads to the early or delayed replacement of the cell culture medium. Therefore, in addition to the operator, a reviewer is usually added, and the cells are generally located in a sterile clean area. When personnel enter the sterile environment to replace the cell culture medium, they need to wear clean clothes to enter, which may cause pollution to the sterile environment, increase production costs, and reduce operating efficiency. The time that cells are exposed outside the incubator is also extended.

[0006] (II) Sampling and measuring pH value may cause the risk of damaging the closed system:

[0007] The method of sampling and measuring pH value is to sample the cell fluid in the culture container such as the culture bottle and the culture bag to measure the pH value, and judge whether the fluid needs to be replaced or replenished according to the pH value result. This method has the risk of destroying the closed system, which may destroy the growth state and environment of the cells in the culture container, affect the subsequent growth of the cells, and there is a risk of contaminating cells. In severe cases, it may lead to a decrease in cell number, a decrease in viability or even death. In addition, this method also has a time difference between sampling and measurement, and there is a problem of deviation between the measured value and the actual value.

[0008] (III) Online pH probe detection capabilities are limited:

[0009] Compared with the sampling pH measurement method, this method has the advantage of good real-time performance; however, the process of realizing online pH measurement requires special consumables combined with pH probes, which are generally disposable; the cost is usually much higher than ordinary consumables; at the same time, the probe combined in the special consumables is an integrated component with low sensitivity and narrow measurement range. The debugging and calibration process is relatively cumbersome, and it is a one-to-one measurement relationship. Summary of the invention

[0010] The present application provides a method for detecting the color of a cell culture fluid, so as to improve the problem of low efficiency of existing methods for observing the color of a cell culture fluid.

[0011] A method for detecting the color of a cell culture fluid comprises the following steps:

[0012] S1, obtain the color detection value a* of the cell culture fluid to be tested 实际检测 ;

[0013] S2, determining the color detection value a* 实际检测 Is it less than the preset value a* of the cell culture medium? 预设 If yes, then the cell culture medium is supplemented or replaced; otherwise, the color detection value a* of the cell culture medium is returned. 实际检测 .

[0014] Optionally, before step S1, the method further includes: before step S1, the method further includes: measuring the color of the cell culture fluid reference solution to obtain a preset value a* of the cell culture fluid. 预设 .

[0015] Optionally, obtain a preset value a* of the cell culture medium 预设 , specifically including: during the process of artificial cell culture, synchronously detecting the color detection value a* of the cell culture fluid 预设检测 , the preset value a* 预设 It is the optimal value that meets the process conditions during manual operation. The optimal value includes one of the following:

[0016] ①a* 预设检测 Maximum, minimum, median, and quartiles;

[0017] ②a* 预设检测 The average value μ;

[0018] ③a* 预设检测 Combination of mean μ and standard deviation σ: μ+σ, μ+2σ or μ+3σ.

[0019] Optionally, before step S1, the method further includes: detecting a correction value, and determining whether the correction value satisfies a preset value.

[0020] Further optionally, the calibration value includes a color detection value of a standard color plate and / or a standard solution;

[0021] Further optionally, the standard color plate is a white plate;

[0022] Optionally, the color preset value of the standard solution is a* 标准品溶液预设 It is 2.5±10%.

[0023] Optionally, between step S1 and step S2, the following step is further performed: the container containing the cell culture fluid to be tested is allowed to stand for more than 5 seconds, and then the color detection value a* of the cell culture fluid to be tested is detected. 实际检测 .

[0024] Optionally, the color detection value a* of the cell culture fluid to be tested is detected 实际检测 Specifically, it includes: testing one or more detection points on the outside of the container containing the cell culture fluid to be tested. When there are more than two detection points, the color detection value a* of the cell culture fluid is 实际检测 is the average value of all detection points.

[0025] Further optionally, the number of the detection points is three.

[0026] Optionally, the container containing the cell culture fluid is a cell culture flask or a cell culture bag.

[0027] Further optionally, when the container of the cell culture fluid is a cell culture flask, the three detection points are located at the edge of the cell flask; when the container is a cell culture bag, the detection points are selected according to actual needs and may be located in the middle of the cell culture bag, may be located at the edge of the cell culture bag, or may be both.

[0028] Optionally, each detection point is detected once, and the detection time interval between different detection points is 5-15 seconds.

[0029] Further optionally, the detection time interval between different detection points is 8-12 seconds.

[0030] Optional, cell culture fluid color detection value a* 实际检测 The detection time t 实际检测 Less than the preset time t 预设 .

[0031] Further optional, t 预设 2-5min.

[0032] The above technical solution provided by this application has the following advantages compared with the prior art:

[0033] The present application provides a method for detecting the color of a cell culture fluid. The method uses a colorimeter to detect the cell culture fluid, obtains a color detection value, and compares it with a preset value of the cell culture fluid. Once the color is lower than the preset value, the cell culture medium is immediately supplemented or replaced, thereby avoiding individual differences and excluding the risk of damaging the closed system. At the same time, one-to-many measurement is achieved, which not only ensures that cell growth is not affected, but also can correctly judge the timing of the color change of the cell culture fluid, replenish or replace the cell culture fluid for the cells in time, improve detection efficiency, and reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0035] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0036] Figure 1 A flowchart of the method provided for this application.

[0037] Figure 2 It is a schematic diagram of the detection method provided in the preferred embodiment of the present application.

[0038] Figure 3 This is a schematic diagram of the detection points of a cell culture flask, 1 is the detection point, and 2 is the bottom surface of the culture flask.

[0039] Figure 4 is (a) a* during the first rehydration process of the embodiment 实际检测1 Frequency distribution histogram of values, (b) quantile plot (Normal QQ plot).

[0040] Figure 5 (a) a* during the second rehydration process in the embodiment 实际检测2 Frequency distribution histogram of values, (b) quantile plot (Normal QQ plot).

[0041] Figure 6 : (a) frequency distribution histogram of a* values ​​and (b) quantile plot (Normal QQplot) during bagging in the embodiment. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0044] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0045] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0046] The present application provides a method for detecting the color of a cell culture fluid, such as Figure 1 As shown, the following steps are included:

[0047] S1, obtain the color detection value a* of the cell culture fluid to be tested 实际检测 ;

[0048] S2, determining the color detection value a* 实际检测 Is it less than the preset value a* of the cell culture medium? 预设 If yes, then perform cell rehydration or cell replacement; otherwise, return the color detection value a* of the cell culture fluid. 实际检测 .

[0049] In some embodiments, before step S1, the following may be included: measuring the color of the cell culture fluid reference solution to obtain a preset value a* of the cell culture fluid. 预设 The whole process diagram is as follows: Figure 2 shown.

[0050] In some implementations, a preset value a* is obtained. 预设 Specifically, the following steps are included: during the process of artificially culturing cells, the color detection value a* of the cell culture fluid reference solution is synchronously detected. 预设检测 , the preset value a* 预设 It is the optimal value that meets the process conditions during manual operation. The optimal value includes one of the following:

[0051] ①a* 预设检测 Maximum, minimum, median, and quartiles;

[0052] ②a* 预设检测 The average value μ;

[0053] ③a* 预设检测 Combination of mean μ and standard deviation σ: μ+σ, μ+2σ or μ+3σ.

[0054] In some embodiments, before step S1, the method further includes: detecting a calibration value and determining whether the calibration value satisfies a preset value. In some embodiments, the calibration value includes a color detection value of a standard color plate and / or a standard solution, such as only using a standard color plate for detection, only using a standard solution for detection, or first performing a standard color plate detection and then using a standard solution for detection, thereby obtaining the calibration value.

[0055] In some embodiments, the standard color palette is a white palette.

[0056] In some embodiments, the color preset value of the standard solution is a* 标准品溶液预设 It is 2.5±10%.

[0057] It is not necessary to re-obtain the preset value a* before each test. 预设 , it only needs to be determined once in each step, for example, the preset value can be determined once in the liquid replenishment process and the bag filling process respectively.

[0058] In some embodiments, in order to ensure that the solution in the container, i.e., the cell culture fluid, is in a relatively static state and effectively avoid detection errors caused by shaking of the cell culture fluid, before step S1, the following step is further included: leaving the container containing the cell culture fluid to be tested to stand for more than 5 seconds, and then detecting the color detection value a* of the cell culture fluid to be tested. 实际检测 .

[0059] In some embodiments, the color detection value a* of the cell culture fluid to be tested is detected. 实际检测 Specifically, it includes: testing one or more detection points on the outside of the container containing the cell culture fluid to be tested, when there are more than two detection points, the cell culture fluid color detection value a* 实际检测 is the average value of all detection points.

[0060] Although the detection data will be more accurate when the number of detection points increases, the inventors have found that too many detection points will increase the workload and increase the time for cells to leave the incubator, thereby affecting the growth state of the cells; too few detection points may cause inaccurate data due to various factors such as the different states of the cell culture fluid at different locations. Therefore, in a more preferred embodiment, the number of detection points is three, which can ensure that the cells will not leave the incubator for too long and can also ensure the accuracy of the data as much as possible.

[0061] In some embodiments, the container containing the cell culture fluid is a cell culture flask or a cell culture bag.

[0062] In some embodiments, when the container of the cell culture fluid is a cell culture bottle, the detection point is located at the edge of the cell bottle, such as Figure 3 When the container is a cell culture bag, the detection point position is selected according to the needs, which can be the middle of the cell culture bag, or the edge, or both.

[0063] In some embodiments, when the number of detection points exceeds two, each detection point only needs to be detected once, and the detection time interval between different detection points is 5-15 seconds.

[0064] In a more preferred embodiment, the detection time interval between different detection points is 8-12 seconds. If the interval is too long or there are too many detection points, the cells will be out of the incubator for too long, affecting the growth state of the cells.

[0065] In some embodiments, the cell culture fluid color detection value a* 实际检测 The detection time t 实际检测Less than the preset time t 预设 , which can reduce the time that cells are out of the incubator.

[0066] In some embodiments, when the number of detection points is 3, t 预设 2-5min.

[0067] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0068] Example

[0069] This embodiment provides a method for detecting the color of cell culture fluid. The detection means adopts a non-invasive method, does not destroy the growth state and environment of cells in the culture container, can effectively ensure that the subsequent growth of cells is not affected, and can also effectively reduce the risk of cell contamination.

[0070] In the present invention, a* is a* in CIE1976 L*a*b* color space (CIE LAB color space), and CIE1976 L*a*b* color space is currently the most widely used color measurement system. The position of a color in the color space is represented by lightness L* and chromaticity coordinates a* and b*. L* represents the lightness of the color, a* positive value represents reddish, and negative value represents greenish; b* positive value represents yellowish, and negative value represents bluish. In the present invention, a* is used alone as the detection value to measure the color change of the cell culture fluid. Compared with L* and b*, the inventors found that a* is significantly correlated with the physical and chemical indicators and pH value of the cell suspension, and the correlation coefficient is the largest. A* positive value represents reddish, and the higher a* is, the redder the color is. When a* changes from large to small, the color of the cell suspension gradually changes from red to yellowish. When a* drops to near 0, the cell suspension has become obviously yellow. Therefore, the present invention selects a* value as a standard for measuring the color change of the cell culture fluid.

[0071] The cell fluid replacement mentioned in the present invention refers to removing the old cell culture medium and replacing it with a new complete cell culture medium to achieve the purpose of removing metabolic waste in the cell growth process and replenishing new nutrients; cell fluid replenishment refers to replenishing the cell culture medium into the culture system without removing the old cell culture medium to achieve the purpose of replenishing new nutrients for cell growth.

[0072] The color detection value of the cell culture fluid described in the present invention is mainly obtained through input devices such as probes and cameras. The preferred acquisition method of the present invention is to use a probe to obtain; a D65 light source is used as a color light source for detection. The D65 light source is an artificial light source that simulates daylight. Its spectral characteristics are closest to natural light and is a color light source commonly used in the field of color detection. In addition, in order to facilitate the detection operation, a stage can be set in front of the probe, and a container containing cell culture fluid, such as a cell culture bottle or a cell culture bag, is placed on the stage.

[0073] Preferably, the color detection value a* of the cell culture fluid to be tested in the detection method of the present invention is 实际检测 , Color detection value a* of cell culture fluid reference solution 预设检测 、Color detection value of standard solution a* 标准品溶液预设 This can be achieved using a colorimeter.

[0074] The cell culture and / or harvesting process described in the present invention is a process known in the art. As an exemplary description, reference may be made to the methods for culturing cells in existing patents such as CN103436492B on a method for amplifying activated lymphocytes by serum-free culture and CN101302491B on a method for efficiently amplifying activated lymphocytes.

[0075] The cell culture medium described in the present invention is a culture medium conventionally used for culturing cells in the art, as an exemplary illustration, such as a mixture of one or more of RPMI-1640, DMEM, IMEM, AIM-V, KBM581, etc., preferably a serum-free cell culture medium; the cell culture medium may include components such as the cell culture medium, cultured cells, and cytokines; the cell culture medium contains phenol red, which is an acid-base indicator with a color change range of pH: 6.8 (yellow)-8.4 (red). When cells are cultured, lactic acid and carbon dioxide are produced due to growth metabolism, which reduces the pH value of the cell culture medium, and then the cell culture medium turns yellow. Operators in the art confirm whether to supplement or replace the cell culture medium according to the timing when the cell culture medium turns yellow.

[0076] Cell culture fluid color detection

[0077] 1) Detect a* 实际检测 value:

[0078] Place the cell culture bottle / culture bag horizontally on the stage, perpendicular to the colorimeter probe, so that the detection point is directly above the probe. After standing for 5-10 seconds, the detection can be performed after the liquid inside is relatively still.

[0079] The a* value is detected at the position of the specified detection point on the cell culture bottle, that is, a* 实际检测When there are multiple detection points, each detection point is detected once, with an interval of 10 seconds each time, and a* of all detection points is calculated. 实际检测 When the detection object is a cell culture bottle, the detection point is selected on the edge of the cell bottle; when a cell culture bag is used, the detection point can be selected according to actual needs, and the detection points are all located in the middle, at the edge, or both. The number of detection points can preferably be three.

[0080] 2) Determine the color detection value a* of the cell culture fluid 实际检测 Whether the preset conditions are met, if yes, then the cell culture medium is supplemented (replenished) or replaced (replaced), if not, then the next round of testing is returned, that is, re-testing after continued culture, such as returning to the incubator to continue culture for one day, and re-testing the color of the cell culture fluid on the second day. 实际检测 , until the preset conditions are met, that is, the color detection value of the cell culture fluid a* 实际检测 Less than the preset value a* 预设 , to supplement or replace cell culture medium.

[0081] The preset value a* in each step of the first rehydration, the second rehydration, the second expansion culture-bag division 预设 Sure

[0082] The manual operation process includes the first rehydration, the second rehydration, and the second expansion culture-bag division. Before each step, the preset value a* of the cell culture medium is required. 预设 Determine. During the manual operation, the color detection value a* of the cell culture fluid reference solution is detected in each step of the first rehydration, the second rehydration, the second expansion culture-bag division. 预设检测 , and then obtain the preset value a* of the cell culture medium in each step 预设 .

[0083] The first fluid infusion preset value a* 预设1 Determination of the scope:

[0084] 1) Test the standard color solution, and the test value of the standard color solution is a* 标准品溶液预设1 , where a* 标准品溶液预设1 Must meet a* 标准品溶液预设1 2.5±10%;

[0085] 2) During the first refilling, the color detection value a* of the cell culture fluid reference solution is synchronously detected during the manual operation. 预设检测1 , default value a* 预设1 It is the optimal value that meets the process conditions during manual operation.

[0086] Second fluid infusion preset value a*预设2 The determination includes the following steps:

[0087] 1) Test the standard color solution, and the test value of the standard color solution is a* 标准品溶液预设2 , where a* 标准品溶液预设2 Must meet 2.5±10%;

[0088] 2) During the second rehydration, the color detection value a* of the cell culture fluid reference solution is synchronously detected during the manual operation. 预设检测2 , default value a* 预设2 It is the optimal value that meets the process conditions during manual operation.

[0089] First expansion culture-bag preset value a* 预设3 The determination includes the following steps:

[0090] 1) Test the standard color solution, and the test value of the standard color solution is a* 标准品溶液预设3 , where a* 标准品溶液预设3 Must meet 2.5±10%;

[0091] 2) During the first amplification culture, the color detection value a* of the cell culture fluid reference solution is synchronously detected during the manual operation. 预设检测3 , default value a* 预设3 It is the optimal value that meets the process conditions during manual operation.

[0092] The preset values ​​determined above are applied to the cell culture process. The specific process is as follows:

[0093] In this example, 37 PBMCs from different donors were used as the starting raw materials for immune cell products for cell culture. During the entire cell culture process, the cell culture fluid was observed using the method for detecting the color of the cell culture fluid provided in the present invention. The specific process is as follows:

[0094] (1) PBMC inoculation and activation culture

[0095] After PBMC isolation, the PBMC cells were resuspended in serum-free cell culture medium and inoculated onto a 225 cm 2 The cells are placed in a cell culture flask. Samples are taken, sterility tested and labeled, and the labeled cells are placed in a cell culture incubator for culture.

[0096] (2) First fluid replacement

[0097] After 3-6 days of inoculation, the color value of the cell culture fluid is detected and a* is obtained. 实际检测1 At this time, the color of the cell culture medium usually becomes lighter and yellower, and the color detection value of the cell culture fluid is a*实际检测1 Is it less than the preset value a* 预设1 , when a* 实际检测1 Less than a* 预设1 When the cells are in good condition, observe the cell morphology under a microscope, count the cells, add 50 ml of serum-free cell culture medium into the cell culture flask, and put it back into the cell culture incubator for culture;

[0098] when a* 实际检测1 Greater than a* 预设1 When the color of the cell culture fluid is detected, return to the test to obtain the color value a* of the cell culture fluid 实际检测1 , the cells can be placed back in the incubator for another day and retested the next day.

[0099] For 37 cases of a* 实际检测1 The data were analyzed for normal distribution, and the results were as follows Figure 4 shown.

[0100] (3) Second fluid replacement

[0101] After the first rehydration, culture for 1-3 days and obtain the color value a* of the cell culture fluid. 实际检测2 At this time, the color of the cell culture medium usually becomes lighter and yellower, and the color detection value of the cell culture fluid is a* 实际检测2 Is it less than the preset value a* 预设2 , when a* 实际检测2 Less than a* 预设2 When the cells are in good growth condition, observe the cell morphology under a microscope, count the cells, add 140 ml of serum-free cell culture medium into the cell culture flask, and return it to the cell culture incubator for culture;

[0102] when a* 实际检测2 Greater than a* 预设2 When the color of the cell culture fluid is detected, return to the test to obtain the color value a* of the cell culture fluid 实际检测2 , the cells can be placed back in the incubator for another day and retested the next day.

[0103] For 37 cases of a* 实际检测2 The data were analyzed for normal distribution, and the results were as follows Figure 5 shown.

[0104] (4) First expansion culture - bagging

[0105] After the second rehydration, culture for 1-3 days and obtain the color value a* of the cell culture fluid. 实际检测3 At this time, the color of the cell culture medium usually becomes lighter and yellower, and the color detection value of the cell culture fluid is a* 实际检测3 Is it less than the preset value a* 预设3 , when a* 实际检测3 Less than a* 预设3When the viable cell density is not less than 1×10 6 When the cell culture medium and 750 ml serum-free cell culture medium are reached, pour the cell culture solution into the cell culture bag and put it back into the cell culture incubator for culture;

[0106] when a* 实际检测3 Greater than a* 预设3 When it is out of range, return to the test to obtain the cell culture fluid color detection value a* 实际检测 , the cells can be placed back in the incubator for another day and retested the next day.

[0107] For 37 cases of a* 实际检测3 The data were analyzed for normal distribution, and the results were as follows Figure 6 shown.

[0108] (5) Second expansion culture - bagging

[0109] After culturing for 3-5 days after bagging, take a new cell culture bag, add 1000ml of serum-free cell culture medium (bag A), take out the cell culture bag containing the cultured cells (bag B) from the incubator, connect the two bags and mix them evenly so that the liquid volume in both bags is 1000ml. Seal the tube mouth, extract samples for sterility inspection; observe the cell morphology under a microscope, and put the two bags of cells back into the incubator for culture;

[0110] (6) Purification, centrifugation and concentration to collect cells and make cell stock solution

[0111] On the 12th to 19th day after the two cell culture bags were placed, the cell culture bags were removed from the incubator, the cell culture fluid was centrifuged and the supernatant was discarded, the cells were resuspended with sodium chloride injection, and samples were taken for cell counting, cell phenotype examination, sterility examination, biological activity examination, endotoxin examination and other examinations. The cell culture fluid was centrifuged and the supernatant was discarded. The supernatant was poured off to obtain the activated and expanded lymphocyte stock solution.

[0112] (7) Preparation of immune cell products

[0113] The activated and expanded lymphocytes were expressed at a concentration of (2-18)×10 9 Resuspend the cell stock solution at 100 cells / ml in normal saline for injection containing human albumin to obtain the product.

[0114] Sampling and testing are performed to obtain parameters such as cell number and viability, total cultured cell volume, expansion multiple, viable cell density, and finished product CD3+CD8+(%).

[0115] The above test results show that the immune cell products made from PBMCs from 37 different donors as the starting raw materials of immune cell products all meet the standards.

[0116] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for detecting the color of a cell culture fluid, characterized in that: The following steps are involved: S1, obtain the color detection value a* of the cell culture fluid to be tested 实际检测 ; S2, determining the color detection value a* 实际检测 Is it less than the preset value a* of the cell culture medium? 预设 If yes, then the cell culture medium is supplemented or replaced; otherwise, the color detection value a* of the cell culture medium is returned. 实际检 Test.

2. The method for detecting the color of a cell culture fluid according to claim 1, characterized in that: Before step S1, the method further includes: measuring the color of the cell culture fluid reference solution to obtain a preset value a* of the cell culture fluid. 预设 .

3. The method for detecting the color of a cell culture fluid according to claim 2, characterized in that: The preset value a* of the cell culture fluid is obtained 预设 , specifically including: during the process of artificial cell culture, synchronously detecting the color detection value a* of the cell culture fluid reference solution 预设检测 , the preset value a* 预设 It is the optimal value that meets the process conditions during manual operation. The optimal value includes one of the following: ①a* 预设检测 Maximum, minimum, median, and quartiles; ②a* 预设检测 The average value μ; ③a* 预设检测 Combination of mean μ and standard deviation σ: μ+σ, μ+2σ or μ+3σ.

4. The method for detecting the color of a cell culture fluid according to claim 1 or 2, characterized in that: Before step S1, the method further includes: detecting a correction value and determining whether the correction value satisfies a preset value; Preferably, the calibration value includes a color detection value of a standard color plate and / or a standard solution; Preferably, the standard color plate is a white plate.

5. The method for detecting the color of a cell culture fluid according to claim 1, characterized in that: Before step S1, the step further includes: the container containing the cell culture fluid to be tested is allowed to stand for more than 5 seconds, and then the color detection value a* of the cell culture fluid to be tested is detected. 实际检测 .

6. The method for detecting the color of a cell culture fluid according to claim 5, characterized in that: The color detection value a* of the cell culture fluid to be tested is detected 实际检测 Specifically, it includes: testing one or more detection points on the outside of the container containing the cell culture fluid to be tested. When there are more than two detection points, the color detection value a* of the cell culture fluid is 实际检测 is the average value of all detection points.

7. The method for detecting the color of a cell culture fluid according to claim 6, characterized in that: The number of the detection points is three.

8. The method for detecting the color of a cell culture fluid according to claim 6 or 7, characterized in that: The container containing the cell culture fluid to be tested is a cell culture bottle or a cell culture bag; Preferably, when the container is a cell culture flask, the detection point is located at the edge of the cell flask.

9. The method for detecting the color of a cell culture fluid according to claim 6, characterized in that: Each detection point is tested once, and the detection time interval between different detection points is 5-15 seconds; Preferably, the detection time interval between different detection points is 8-12 seconds.

10. The method for detecting the color of a cell culture fluid according to claim 1, characterized in that: In step S1, the color detection value a* of the cell culture fluid is obtained. 实际检测 The detection time t 实际检测 Less than the preset time t 预设 ; Preferably, t 预设 2-5min.

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