ROBO1 CAR-NK92 Cell Resuscitation Medium, Kit and Resuscitation Culture Method

Abstract

The present invention relates to the technical field of cell biology, and provides a ROBO1CAR-NK92 cell resuscitation medium, a kit and a resuscitation culture method. The ROBO1CAR-NK92 cell resuscitation medium comprises a basal medium and an additive; the additive is composed of a cell growth factor and vitamin B. Adding IL-2 and nicotinamide to the basal medium HIPP<supgt;TM< / supgt>-T009 can significantly improve the resuscitation survival rate of ROBO1CAR-NK92 cells and shorten the passage time for the cell survival rate to recover to 80%, which is easy for large-scale culture.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a ROBO1 CAR-NK92 cell resuscitation medium, a kit and a resuscitation culture method. Background Art

[0002] Natural killer cells (NK) are an important type of immune cells in the body, mainly distributed in peripheral blood. As the first line of defense in the body's defense system, NK cells can not only exert their ability to kill senescent cells, diseased cells and malignantly transformed tumor cells in the innate immune system, but also secrete different cytokines and chemokines in the early stage of the immune response to regulate the body's acquired immune response. They are important effector cells indispensable for the body to exert immune effects. NK cells lack a T cell receptor (TCR) and are not easily induced to cause graft-versus-host reactions. They are an important type of immune cells that can be used for allogeneic patient reinfusion therapy. Gene-modified cell therapy products based on NK cells have good clinical application prospects.

[0003] Currently, the main method for preparing NK cells is to activate and expand NK cells by using cytokines and feeder cells (aAPC). However, there is still a large gap between the NK cells amplified by this method and the clinical demand, and it cannot meet the usage requirements of more allogeneic clinical patients. Therefore, researchers have begun to focus on using NK cell lines (such as NK-92, YTS, KHYG-1, etc.) instead of primary NK cells derived from peripheral blood for related research. Moreover, different from primary NK cells, NK cells derived from cell lines are easy to expand in vitro, easy to genetically modify and transform, the culture preparation process route is relatively simple, and they have the same anti-tumor effect as primary NK cells, and have a strong killing effect on tumor cells including hematological tumors and solid tumors.

[0004] The applicant's authorized patent CN109810995B discloses a ROBO1 CAR-NK92 cell, which has been verified through a large number of in vitro and in vivo experiments to have the ability to specifically kill ROBO1-highly expressed solid tumor cells, with relatively small toxic side effects and high safety. On this basis, a ROBO1 CAR-NK92 cell bank was prepared, and a cell resuscitation and primary culture method was preliminarily established. However, the original ROBO1 CAR-NK92 cell resuscitation and culture method still has the following disadvantages: the viability of the resuscitated ROBO1 CAR-NK92 cells is relatively low, only about 10%, and it takes about 15 - 20 days to culture the cells to increase the cell viability to about 80%, resulting in too long a culture time and being not conducive to large-scale culture and preparation of cells in the later stage.

[0005] Therefore, how to improve the resuscitation survival rate of ROBO1 CAR-NK92 cells and shorten the time for the cell survival rate to recover to 80% is an urgent problem to be solved. Summary of the Invention

[0006] The object of the present invention is to overcome the above problems existing in the prior art, and to provide a resuscitation medium for ROBO1 CAR-NK92 cells, a kit containing the resuscitation medium, and a method for resuscitation culture of ROBO1 CAR-NK92 cells using the resuscitation medium. By using the resuscitation medium provided by the present invention and combining with the method for resuscitation culture of ROBO1 CAR-NK92 cells provided by the present invention, the resuscitation survival rate of ROBO1 CAR-NK92 cells can be rapidly improved and the passage time for the cell survival rate to recover to 80% can be shortened.

[0007] To achieve the above object, in the first aspect of the present invention, a resuscitation medium for ROBO1 CAR-NK92 cells is provided, which comprises a basal medium and an additive; wherein, the additive is composed of a cell growth factor and vitamin B group.

[0008] In the second aspect of the present invention, a kit for resuscitation culture of ROBO1 CAR-NK92 cells is provided, which comprises the resuscitation medium described in the first aspect of the present invention.

[0009] In the third aspect of the present invention, a method for resuscitation culture of ROBO1 CAR-NK92 cells is provided, which uses the resuscitation medium described in the first aspect of the present invention, or the kit described in the second aspect of the present invention to resuscitate and culture ROBO1 CAR-NK92 cells.

[0010] The present invention adopts the above technical solutions and has the following beneficial effects:

[0011] (1) The resuscitation medium for ROBO1 CAR-NK92 cells and the method for resuscitation culture of ROBO1 CAR-NK92 cells provided by the present invention can rapidly improve the resuscitation survival rate of ROBO1 CAR-NK92 cells and shorten the passage time for the cell survival rate to recover to 80%, and are easy to rapidly scale up the culture;

[0012] (2) The resuscitation medium for ROBO1 CAR-NK92 cells provided by the present invention does not contain animal-derived components. After culturing ROBO1 CAR-NK92 cells, the subsequent processes of cell harvesting, aliquoting and detection will not be too complicated; and it avoids the impact of the introduction of animal-derived components on human safety.

[0013] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. Brief Description of the Drawings

[0014] Figure 1 Shown is the standard flow chart for the resuscitation and culture of ROBO1 CAR-NK92 cells.

[0015] Figure 2 Shown is the effect of cytokines on the growth activity of ROBO1 CAR-NK92 cells.

[0016] Figure 3 Shown is the effect of cytokines on the killing activity of ROBO1 CAR-NK92 cells.

[0017] Figure 4 Shown is the effect of different trehalose concentrations on the density of ROBO1 CAR-NK92 cells.

[0018] Figure 5 Shown is the effect of different trehalose concentrations on the viability of ROBO1 CAR-NK92 cells.

[0019] Figure 6 Shown is the graph of the change in cell density during the resuscitation and culture of ROBO1 CAR-NK92 cells.

[0020] Figure 7 Shown is the graph of the change in cell viability during the resuscitation and culture of ROBO1 CAR-NK92 cells. Detailed Description of the Invention

[0021] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0022] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0023] In a first aspect of the present invention, a resuscitation medium for ROBO1 CAR-NK92 cells is provided, and the resuscitation medium includes a basal medium and an additive; wherein, the additive is composed of a cell growth factor and vitamin B.

[0024] To better improve the revival survival rate of ROBO1 CAR-NK92 cells and shorten the passage time for the cell survival rate to recover to 80%, one or more of the technical features can be further optimized.

[0025] In one example, the cell growth factor includes at least one of IL-2, IL-15, IL-18, IL-21, and GM-CSF.

[0026] In a preferred example, the cell growth factor is IL-2.

[0027] In one example, the B vitamins include at least one of nicotinamide (a derivative of vitamin B3), thiamine (vitamin B1), riboflavin (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B7), folic acid (vitamin B9), and vitamin B12.

[0028] In a preferred example, the B vitamin is nicotinamide.

[0029] In the present invention, both the cell growth factor and the B vitamins are of pharmaceutical grade. Adding pharmaceutical-grade additives to the revival medium can avoid introducing impurities or contaminants that may have an adverse effect on cell revival and ensure the drug safety of the cultured cell products.

[0030] The type of the basal medium is not specifically limited, and a basal medium commonly used in the art for reviving and culturing NK cells can be selected.

[0031] In a preferred example, the basal medium is HIPP TM -T009 (GMP grade, abbreviated as T009 medium), which is a serum-free medium.

[0032] In a preferred example, the additive includes IL-2 and nicotinamide;

[0033] In one example, the concentration of IL-2 is 10 - 1000 IU / mL, and for example, it can be 10 IU / mL, 100 IU / mL, 200 IU / mL, 300 IU / mL, 400 IU / mL, 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, or 1000 IU / mL.

[0034] In a preferred example, the concentration of IL-2 is 450 - 550 IU / mL.

[0035] In one example, the concentration of nicotinamide is 1 - 10 mM, and for example, it can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

[0036] In a preferred example, the concentration of nicotinamide is 4 - 6 mM.

[0037] In a preferred example, a ROBO1 CAR-NK92 cell resuscitation medium comprises HIPP TM -T009 (GMP grade), IL-2 with a concentration of 500 IU / mL, and nicotinamide with a concentration of 4 - 6 mM.

[0038] The inventors of the present invention have found through research that adding IL-2 and nicotinamide to the basic medium HIPP TM -T009, and adjusting the concentration of IL-2 to about 500 IU / mL and the concentration of nicotinamide to about 5 mM, the obtained resuscitation medium can significantly improve the resuscitation survival rate of ROBO1 CAR-NK92 cells and shorten the passage time for the cell survival rate to recover to 80%.

[0039] The inventors of the present invention have further found through research that although adding a variety of cell growth factors (such as IL-15, IL-18, IL-21, and GM-CSF) can also improve the resuscitation survival rate of ROBO1 CAR-NK92 cells, the effect is less different compared with the effect of adding IL-2 alone. Moreover, experimental verification has confirmed that the killing effect of ROBO1 CAR-NK92 cells resuscitated and cultured by adding IL-2 alone and adding other cytokines (IL-15, IL-18, and GM-CSF) on tumor cells is not much different.

[0040] The inventors of the present invention have also studied adding trehalose to the resuscitation medium. Some literature mentions that trehalose has a promoting effect on cell resuscitation and culture. For example: Jiang et al. believe that the bioprotective effect of trehalose on the cell freezing and resuscitation process is the result of the synergistic action of multiple protective mechanisms. On the one hand, trehalose can resist the effects of factors such as low osmotic pressure, chemical action, and hypoxia on cells through osmotic regulation. On the other hand, it can reduce or even avoid the excessive swelling and osmotic shock of cells caused by cryoprotectants during cell resuscitation (PMID: 24997279). At the same time, Fabbrocini et al. used 7% dimethyl sulfoxide, 7% glycerol, and 7% methanol combined with 0.04 mol / L trehalose to preserve sea urchin sperm at 4°C. After thawing, the proportion of rapidly motile sperm in the dimethyl sulfoxide combined with trehalose group was significantly higher than that in the other two groups, indicating that trehalose has an obvious protective effect on cells after cell freezing and thawing (PMID: 23329384). However, through experimental research, the present invention has found that adding trehalose has no promoting effect on the resuscitation of ROBO1 CAR-NK92 cells.

[0041] The second aspect of the present invention provides a kit for the resuscitation and culture of ROBO1 CAR-NK92 cells, which includes the resuscitation medium described in the first aspect of the present invention.

[0042] The kit for the resuscitation and culture of ROBO1 CAR-NK92 cells described in the second aspect of the present invention has the same advantages as the ROBO1 CAR-NK92 cell resuscitation medium described in the first aspect of the present invention, and will not be elaborated here.

[0043] The third aspect of the present invention provides a method for the resuscitation and culture of ROBO1 CAR-NK92 cells, which uses the resuscitation medium described in the first aspect of the present invention, or the kit described in the second aspect of the present invention to resuscitate and culture ROBO1 CAR-NK92 cells.

[0044] In one example, the method for the resuscitation and culture of ROBO1 CAR-NK92 cells includes the following steps:

[0045] (a) Cell thawing: Thaw the cryopreserved cells to obtain a cell suspension, then add pre-cooled washing solution and centrifuge to remove the supernatant to obtain a cell pellet;

[0046] (b) Cell seeding: Add a part of the pre-warmed resuscitation medium to the cell pellet in step (a), mix evenly and then seed it in the remaining resuscitation medium for culture;

[0047] (c) Medium replacement and culture: After culturing the cells in step (b) for 20 - 27 h, replace the fresh resuscitation medium and continue the culture;

[0048] Wherein, in step (a), the washing solution is a basal medium.

[0049] Step (a) Cell thawing

[0050] In one example, the cryopreserved cells are thawed in a 37 °C constant temperature water bath to obtain a cell suspension.

[0051] In one example, the pre-cooling temperature of the washing solution is 0 - 5 °C, preferably 4 °C.

[0052] In one example, the volume ratio of the washing solution to the cell suspension is (3 - 6):1, for example, it can be 3:1, 4:1, 5:1, 6:1, and the preferred volume ratio is 4:1.

[0053] In one example, the centrifugation conditions include: centripetal acceleration of 500 - 800 g and centrifugation time of 3 - 5 min.

[0054] In one specific example, the method for using the washing solution includes the following steps:

[0055] (1) Pre-cool the centrifuge tube to a temperature of 0 - 5°C.

[0056] (2) Slowly and completely transfer the thawed cell suspension into the pre-cooled centrifuge tube, then dropwise add the pre-cooled washing solution (pre-cooled to 0 - 5°C), cover the centrifuge tube cap, and gently invert the tube up and down to mix evenly.

[0057] Step (b) Cell seeding

[0058] In one example, the preheating temperature of the resuscitation medium is 35 - 38°C, preferably 37°C.

[0059] In one example, the seeding density is (1.5 - 2.5)×10 6 cells / mL.

[0060] In one example, the cell culture temperature is 37°C, the CO2 concentration is 5%, and the humidity is saturated.

[0061] Step (c) Medium change and culture

[0062] In one example, 20 - 27 hours after seeding, the cells need to be centrifuged and the medium changed (for example, the centrifugation conditions include 175g for 5 minutes), and fresh resuscitation medium of the original volume is added.

[0063] In one example, the cell culture temperature is 37°C, the CO2 concentration is 5%, and the humidity is saturated.

[0064] In one example, the conditions for changing the medium include:

[0065] (i) When the cell viability < 80% and the viable cell density < 1×10 6 cells / mL, change to fresh medium and keep the original volume unchanged;

[0066] (ii) When the cell viability < 80% and the viable cell density > 1×10 6 cells / mL, change to fresh medium and increase the volume of the liquid;

[0067] (iii) If the cell viability ≥ 80%, expand the culture at a viable cell seeding density of (2.5 - 3.5)×10 5 cells / mL.

[0068] In one example, if the culture system is greater than 20 mL, no niacinamide needs to be added. When the culture system is greater than 20 mL, the cell culture density is (2.5 - 3.5)×10 5cells / ml, viability ≥ 80%, and the cells enter the subsequent amplification and culture stage. At this stage, the cell viability generally has reached more than 80%, the cell resuscitation stage has been completed, and the cells enter the cell amplification stage, and no more nicotinamide needs to be added.

[0069] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0070] The present invention will be described in detail below with specific embodiments, and these embodiments are for understanding rather than limiting the present invention.

[0071] For the materials, reagents, etc. used in the following embodiments, refer to Tables 1 - 3. Unless otherwise specified, the materials or reagents not mentioned can be obtained from commercial sources.

[0072] Table 1 Cell Information

[0073]

[0074] Table 2 Main Reagent and Consumable Information

[0075]

[0076] Table 3 Main Equipment Information

[0077]

[0078]

[0079] Group A, Examples A1 - A14

[0080] Examples A1 - A14 and Comparative Examples DA1 - DA3 respectively provide ROBO1 CAR - NK92 cell resuscitation media, including the basal medium HIPP TM -T009 and additives. The specific components and concentrations of the additives are shown in Table 4.

[0081] Table 4

[0082]

[0083]

[0084] Using the resuscitation media provided by the above Examples A1 - A14 and Comparative Examples DA1 - DA3 for ROBO1 CAR - NK92 cell resuscitation and culture, specifically including the following steps:

[0085] 1. ROBO1 CAR-NK92 cell resuscitation

[0086] 1.2. Adjust the temperature of the water bath in the Class C clean area to 37°C and wait for the temperature to reach 37°C.

[0087] 1.3. Pipette 4 mL of T009 serum-free medium into a 15 mL centrifuge tube, mix well, and place it in a 4°C refrigerator for pre-cooling to obtain the washing solution (T009 serum-free medium). At the same time, pre-cool an empty 15 mL centrifuge tube.

[0088] 1.4. Pipette 5 mL of T009 serum-free medium into a cell culture flask, add 2.5 μL of IL-2 (final concentration 500 IU / mL) and 30.5 μL of nicotinamide (final concentration 5 mM), gently tap to mix well, and place it in a carbon dioxide incubator for pre-warming.

[0089] 1.5. Carefully take out the cell cryopreservation tube from the designated position in the liquid nitrogen tank with forceps (record the cryopreservation position, cryopreservation date and operator), quickly place it in a constant temperature water bath at 37°C, and gently shake it, paying attention to keeping the lid of the cryopreservation tube always above the water level.

[0090] 1.6. When the cells in the cryopreservation tube melt to the size of a soybean, quickly take out the cryopreservation tube, slowly transfer the cell suspension to a new pre-cooled 15 mL centrifuge tube with a pipette, pipette 100 μL of the cell suspension for cell counting and observe the cell status under an inverted microscope.

[0091] 1.7. Gently tap the cell suspension to mix well, pipette 40 μL with a pipette gun, mix it evenly with 40 μL of 0.2% trypan blue staining solution, pipette 20 μL of the mixed solution onto a cell counting plate, make three replicates, and use a cell counter to detect the viable cell density and cell viability of ROBO1 CAR-NK92.

[0092] 1.8. Slowly drip the pre-cooled medium (washing solution) into the above cell suspension, gently mix well, centrifuge at 745 g for 3 min.

[0093] 1.9. Discard all the supernatant, gently resuspend the cell pellet with the pre-warmed medium, mix well and transfer it into a T25 cell culture flask, place the culture flask in a 37°C, 5% carbon dioxide incubator for culture for 24 ± 3 h.

[0094] 2. ROBO1 CAR-NK92 cell culture

[0095] 2.1. According to the above standard operating procedure for cell resuscitation, 24 h after cell resuscitation, take out the resuscitated cells from the carbon dioxide incubator, observe the cell status under an inverted microscope, and count the cells at the same time.

[0096] 2.2. Prepare sterile 1.5 mL EP tubes and label them. Gently and evenly pipette the cell suspension in the culture flask with a 10 mL pipette, and aspirate approximately 0.2 mL of the cell suspension, then transfer it to the corresponding EP tube;

[0097] 2.3. After gently tapping and mixing the cell suspension, aspirate 40 μL with a pipette gun, mix it evenly with 0.2% trypan blue stain, aspirate 20 μL of the mixed solution onto a cell counting chamber, perform three replicates, and use a cell counter to detect the viable cell density and cell viability of ROBO1 CAR-NK92;

[0098] 2.4. Centrifuge at 175 g for 5 min. After centrifugation, discard the supernatant, add 5 mL of pre-warmed medium to resuspend the cell pellet, and then transfer all the cells to a new T25 culture flask;

[0099] 2.5. At an interval of every 2 - 3 days for cell processing, detect the viable cell density and cell viability in the T25 cell culture flask (such as on the 4th, 7th, 9th, 12th, and 15th days). Some data are recorded in Tables 5 and 6. Different treatments are carried out according to the viable cell density and cell viability:

[0100] (2.5.1) When the cell viability in the T25 cell culture flask < 80% and the viable cell density < 1×10 6 cells / mL, when changing the medium, keep the original volume unchanged, centrifuge the cells for medium change, add fresh medium, and keep the cell culture volume at 5 mL;

[0101] (2.5.2) When the cell viability in the T25 cell culture flask < 80% and the viable cell density > 1×10 6 cells / mL, centrifuge the cells for medium change, add fresh medium, and expand the cell culture volume to 10 mL;

[0102] (2.5.3) When the cell viability in the T25 cell culture flask ≥ 80%, centrifuge the cells for medium change treatment, add fresh medium, and expand the cell culture volume (expand the culture according to a seeding density of 3×10 5 cells / mL viable cells).

[0103] Figure 1 The standard flow chart for the resuscitation and culture of ROBO1 CAR-NK92 cells is shown. This standard process takes a total of 8 days, including the day when ROBO1 CAR-NK92 is resuscitated and inoculated and the subsequent 7 days of culturing until the cell viability is above 80%. The cell number and cell viability need to be detected each time the cells are processed.

[0104] Table 5

[0105]

[0106] In the above embodiments, Embodiment A8 - A14, the viable cell density and cell viability in the cell culture flask were detected at different times (e.g., day 0, day 2, day 4, and day 6), and the experimental results are as Figure 2 shown.

[0107] Test Example: Detect the killing activity of ROBO1 CAR - NK92 cells

[0108] The test method is as follows:

[0109] Detect the killing activity of ROBO1 CAR - NK92 cells obtained by resuscitation and culture in Embodiment A1, Embodiment A8 - A15, and Comparative Example DA3 above against tumor cells.

[0110] All methods for detecting cell killing activity involved in the present invention are detected by the RTCA (Real Time Cellular Analysis) method. The specific methods and operations include the following steps:

[0111] 1. Preparation of target cell suspension:

[0112] (1) In a laminar flow hood, under sterile conditions, aspirate the old culture medium in the culture flask;

[0113] (2) Wash twice with PBS solution. Add 0.5 ml (for T75 culture flask) of trypsin solution containing EDTA to the culture dish, incubate in a 37°C incubator for 1 min, and then observe the digestion of cells under an inverted microscope. If the cytoplasm retracts and the cell intervals increase, terminate the digestion;

[0114] (3) Add 10 ml of medium per culture flask. Gently pipette the adherent cells repeatedly to form a cell suspension. Transfer the cell suspension to a 15 - ml centrifuge tube, centrifuge at 800 g for 5 min, remove the supernatant, add 5 ml of fresh medium, pipette the cells evenly, centrifuge at 800 g for 5 min, remove the supernatant, and add 2 ml of fresh medium. Detect the cell density and cell viability with a cell counter, and then add medium to prepare a cell concentration of 5×10 5 cells / ml.

[0115] 2. Schedule setting:

[0116] Step Sweeps Interval Unit Total time Step 1 1 1 Minute 1 min Step 2 20 10 Hour 15

[0117] 3. Preparation of E - Plate 16 detection electrode plate and plating of target cells:

[0118] (1) Add 50 μl of medium to the wells of E - Plate 16;

[0119] (2) Place the E-Plate 16 on the xCELLigence RTCA Instrument;

[0120] (3) The RTCA system will automatically perform a scan ("Scan Plate") -> check for good contact (display "Connection OK" on the "Message" page);

[0121] (4) Start Step1, detect the baseline (Background), ensure that the selected wells are in normal contact, and the CellIndex of all wells is below 0.063;

[0122] (5) Remove the E-Plate 16, add 100 μl of well-mixed target cell suspension to the wells, so that the number of cells in each well is 50,000;

[0123] (6) Place the E-Plate 16 in the laminar flow hood at room temperature for 30 min;

[0124] (7) Place the E-Plate 16 on the xCELLigence RTCA Instrument in the incubator;

[0125] (8) The RTCA system will automatically perform a scan ("Scan Plate") -> check for good contact (display "Connection OK" on the "Message" page);

[0126] (9) Start Step2 (detect the cell proliferation curve). After 24 h, when the Cell Index of the target cells reaches 1 - 3, prepare to start the next operation.

[0127] 4. Add effector cells:

[0128] (1) Take the effector cells and prepare an appropriate effector cell density according to the cell viability and the number of viable cells;

[0129] (2) Terminate Step2, remove the E-Plate 16, and place it in the laminar flow hood;

[0130] (3) Add 80 μl of effector cell suspension (or an equal volume of medium: blank control) to the wells of the culture plate at an effector-to-target ratio of 5:1;

[0131] (4) Place the E-Plate 16 on the xCELLigence RTCA Instrument;

[0132] (5) The RTCA system will automatically perform a scan ("Scan Plate") -> check for good contact (display "Connection OK" on the "Message" page);

[0133] (6) Continue to start Step 2, observe the effect of effector cells on the growth of target cells, and record the killing activity of effector cells at 2 h of effector-target interaction time.

[0134] The experimental results are shown in Figure 3 , from Figure 3 It can be seen that the killing effect on tumor cells is not much different between adding cytokine IL-2 alone and adding other cytokines (IL-15, IL-18, and GM-CSF), and there is no obvious difference among groups. In the present invention, adding cytokine IL-2 and nicotinamide alone can rapidly increase the resuscitation survival rate of ROBO1 CAR-NK92 cells and shorten the passage time for the cell survival rate to recover to 80%, and it will not affect the killing effect of ROBO1 CAR-NK92 cells on tumor cells.

[0135] The ROBO1 CAR-NK92 cells cultured in vitro by the resuscitation culture method provided by the present invention have good activity in killing tumor cells, significantly enhanced in vitro proliferation ability (the highest viable cell density), the cell survival rate remains above 90%, is easy for subsequent in vitro amplification and large-scale culture, has low production cost and is easy for later large-scale production.

[0136] Group B, Examples B1 - B5

[0137] Examples B1 - B5 respectively provide resuscitation culture methods for ROBO1 CAR-NK92 cells, and all use the resuscitation culture medium provided in Example A1.

[0138] Example B1

[0139] The method of Example B1 is the same as the resuscitation and culture methods of ROBO1 CAR-NK92 cells in the Examples of Group A.

[0140] Example B2

[0141] The difference from Example B1 is that the volume ratio of the washing solution to the cell suspension is 1:1.

[0142] Example B3

[0143] The difference from Example B1 is that the washing solution is not pre-cooled.

[0144] Example B4

[0145] The difference from Example B1 is that the resuscitation culture medium is not pre-heated.

[0146] Example B5

[0147] It is different from Example B1 in that the inoculation density of the inoculation is 5×10 6 cells / mL.

[0148] In the above Examples B1 - B5, the viable cell density and cell viability in the T25 cell culture flask were detected respectively (for example, on the 4th, 7th, 9th, 12th, and 15th days), and some data were recorded in Table 6.

[0149] Table 6

[0150]

[0151] It can be seen from the results in Table 6 that the cell resuscitation method and specific operation steps provided by the present invention play an important promoting role in improving the resuscitation viability and viable cell density of ROBO1CAR-NK92 cells.

[0152] Trehalose addition experiment in Group C Examples

[0153] Examples C1 - C5 Add trehalose alone

[0154] It is different from Example A1 in that trehalose with a final concentration of 1250 mM, 625 mM, 312.5 mM, 156.3 mM, and 0 mM is added to the cleaning solution in step 1.3, and in addition to containing 500 IU / ml IL-2 and 5 mM nicotinamide in the resuscitation medium, trehalose with a final concentration of 1250 mM, 625 mM, 312.5 mM, 156.3 mM, and 0 mM is added respectively to further improve the resuscitation effect of ROBO1 CARNK92 cells.

[0155] Examples C6 - C9

[0156] It is different from Example A1 in that the composition concentration of the additives in the resuscitation medium or the components of the cleaning solution are different. The specific components are shown in Table 7:

[0157] Table 7

[0158]

[0159] In the above Examples C1 - C9, the viable cell density and cell viability were detected at different times (for example, on the 0th, 1st, 4th, 7th, 9th, and 12th days). Among them, the results of Examples C1 - C5 Figure 4 and Figure 5 shown, the results of Examples C6 - C9 Figure 6 and Figure 7 shown

[0160] Figures 4 - 7 The results show that adding trehalose not only fails to promote the recovery of ROBO1 CAR-NK92 cells, but instead affects the viable cell density and cell viability of ROBO1 CAR-NK92 cells, which is somewhat different from the role reported in the literature during the cell thawing and recovery process. Analyzing the reasons, on the one hand, it may be due to different targeted cells. ROBO1 CAR-NK92 cells are relatively sensitive to the transient increase in osmotic pressure. On the other hand, excessive trehalose has a certain toxic effect on ROBO1 CAR-NK92 cells, leading to cell apoptosis. The specific reasons need to be further analyzed later.

[0161] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A ROBO1 CAR-NK92 cell resuscitation medium, characterized in that, It includes a basal medium and additives; wherein, the additives are composed of a cell growth factor and vitamin B complex; The cell growth factor includes at least one of IL-2, IL-15, IL-18, and GM-CSF; The vitamin B complex is nicotinamide; The concentration of IL-2 is 10 - 1000 IU / mL; the concentration of IL-15 is 50 ng / mL; the concentration of IL-18 is 5 ng / mL; the concentration of GM-CSF is 10 ng / mL; The concentration of nicotinamide is 1 - 10 mM.

2. The resuscitation medium according to claim 1, characterized in that, The cell growth factor is IL-2.

3. The resuscitation medium according to claim 1, wherein The basal medium is HIPP TM -T009.

4. The resuscitation medium according to claim 1, wherein The additives include IL-2 and nicotinamide.

5. The resuscitation medium according to claim 4, characterized in that, The concentration of IL-2 is 450 - 550 IU / mL; and / or, the concentration of nicotinamide is 4 - 6 mM.

6. A kit for the resuscitation and culture of ROBO1 CAR-NK92 cells, characterized in that, It includes the resuscitation medium according to any one of claims 1 - 5.

7. A method for resuscitating and culturing ROBO1 CAR-NK92 cells, characterized in that, Using the resuscitation medium according to any one of claims 1 - 5, or using the kit according to claim 6 to resuscitate and culture ROBO1 CAR-NK92 cells.

8. The resuscitation culture method according to claim 7, wherein, It includes the following steps: (a) Cell thawing: Thaw the cryopreserved cells to obtain a cell suspension, then add a pre-cooled washing solution and centrifuge to remove the supernatant to obtain a cell pellet; (b) Cell seeding: Add a part of the pre-warmed resuscitation medium to the cell pellet in step (a), mix evenly and seed it in the remaining resuscitation medium for culture; (c) Medium replacement and culture: After culturing the cells in step (b) for 20 - 27 h, replace them with fresh resuscitation medium and continue the culture; Wherein, in step (a), the washing solution is the basal medium.

9. The resuscitation culture method according to claim 8, characterized in that, In step (a), the volume ratio of the washing solution to the cell suspension is (3 - 6):

1.

10. The resuscitation culture method according to claim 9, characterized in that, The pre-cooling temperature of the washing solution is 0 - 5 °C.

11. The resuscitation culture method according to claim 9, wherein The conditions of the centrifugation include: the centripetal acceleration is 500 - 800 g, and the centrifugation time is 3 - 5 min.

12. The resuscitation culture method according to claim 8, characterized in that, In step (b), the pre-warming temperature of the resuscitation medium is 35 - 38 °C.

13. The resuscitation culture method according to claim 12, characterized in that, The inoculation density of the inoculation is (1.5 - 2.5)×10 6 CFU / mL.

14. The resuscitation culture method according to claim 8, wherein, In step (c), the conditions for medium replacement include: (i) When the cell viability < 80% and the viable cell density < 1×10 6 cells / mL, replace with fresh medium and maintain the original volume unchanged; (ii) When the cell viability < 80% and the viable cell density > 1×10 6 cells / mL, change to fresh medium and expand the liquid volume to 8 - 12 mL; (iii) When the cell viability is ≥ 80%, expand the culture at a viable cell seeding density of (2.5 - 3.5) × 10 5 cells / mL.

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