In-vitro culture method of NK (Natural Killer) cells
By designing and precisely controlling the non-bubbling oxygen transfer interface of the turbulent bioreactor, the problems of low yield and shear damage in NK cell culture were solved, enabling efficient large-scale expansion and high-activity culture.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-03-10
AI Technical Summary
In existing NK cell culture processes, cell yield is low and expansion efficiency is low. Traditional reactors have strong shear forces that cause cell damage, making it difficult to meet the needs of large-scale expansion.
NK cell culture was carried out using a turbulent bioreactor. The reactor tank with an inverted cone design and a rotating shaft unit generated horizontal circular motion, forming a non-bubbling oxygen transfer interface. Combined with precise control of pH and dissolved oxygen value, shear force was reduced and oxygen transfer efficiency was improved.
It significantly improves the efficiency of NK cell in vitro expansion, increases cell density by more than 20 times, and maintains cell viability above 80%, meeting the needs of clinical treatment and scientific research.
Smart Images
Figure CN121628828A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of cells, in particular to an in-vitro culture method of NK cells. BACKGROUND
[0002] NK cells are an important part of the immune system and are important cells for maintaining the body's normal cellular immunity. Scientists have found that NK cells have great effects on cell therapy, such as cancer, tumor, viral infection and other diseases. Researchers have found that NK cells can be cultured in vitro and expanded in vitro by using cytokines. However, due to the sensitivity of NK cells to shear force, slow cell growth rate, low yield and other characteristics, it is difficult to achieve large-scale expansion culture in actual expansion. Currently, artificial square bottles or culture bags are used for static culture and stirred reactors are used for culture. The manual static culture operation is complex, the volume is small, and the key parameters cannot be controlled in real time during the culture process, resulting in unstable cell yield, lack of expansion data, and large batch-to-batch differences. The traditional stirred reactor culture also has a large shear force generated by the stirring paddle and the bottom bubbling, which also makes the NK cell density not high, and cannot support the long-term research needs. Therefore, it is necessary to develop a new culture process to meet the market demand. SUMMARY
[0003] Therefore, it is necessary to provide an in-vitro culture method of NK cells.
[0004] The first aspect of the present application provides an in-vitro culture method of NK cells, comprising the following steps:
[0005] A culture bag containing a culture medium is placed in a reactor tank of a turbulent flow reactor for pre-culture, wherein the reactor tank is designed as an inverted cone and installed above an axial rotation unit, and during the pre-culture, the tank is driven by the axial rotation unit to move horizontally in a circular manner.
[0006] NK cells are inoculated into the culture medium obtained by the pre-culture to obtain a liquid culture system, and the liquid culture system generates a downward vortex under the condition of horizontal circular motion of the reactor tank to form a non-bubble type oxygen transfer interface, and the culture is expanded to obtain NK cells with a target density.
[0007] In some embodiments, the culture medium comprises one or more of fetal bovine serum, penicillin, interleukin-2, interleukin-15, anti-CD16 antibody, glutamine and sodium pyruvate.
[0008] In some embodiments, the concentration of the fetal bovine serum is 10% (w / v)~15% (w / v), the concentration of the penicillin is 1% (w / v)~5% (w / v), the concentration of the interleukin-2 is 100 IU / mL~150 IU / mL, the concentration of the interleukin-15 is 10 ng / mL~15 ng / mL, the concentration of the anti-CD16 antibody is 1 μg / mL~5 μg / mL, the concentration of the glutamine is 2 mM~4 mM, and the concentration of the sodium pyruvate is 1 mM~3 mM.
[0009] In some embodiments, the pre-culturing conditions satisfy one or more of the following conditions:
[0010] The temperature of the pre-culturing is 35℃~40℃;
[0011] The rotation speed of the shaft rotation unit during the pre-culturing is 35 rpm~40 rpm;
[0012] The surface-passing air flow rate of the pre-culturing is 20 mL / min~80 mL / min;
[0013] The pH value of the pre-culturing is 7~7.4;
[0014] The dissolved oxygen value of the pre-culturing is 40%~100%;
[0015] The time of the pre-culturing is 8 h~24 h.
[0016] In some embodiments, the inoculation of the NK cells into the liquid culture system obtained from the pre-cultured medium further comprises: obtaining the NK cells by subculture.
[0017] In some embodiments, the subculture comprises: culturing at 35℃~40℃, 4%~8% CO2.
[0018] In some embodiments, in the step of inoculating the NK cells into the liquid culture system obtained from the pre-cultured medium, the inoculation amount of the NK cells is 0.5×10 6 cells / mL~1.0×10 6 cells / mL.
[0019] In some embodiments, the temperature of the culture expansion is 35℃~40℃.
[0020] In some embodiments, the rotation speed of the shaft rotation unit during the culture expansion is 35 rpm~40 rpm.
[0021] In some embodiments, the surface-passing air flow rate of the culture expansion is 20 mL / min~80 mL / min.
[0022] In some embodiments, the pH value of the culture expansion is 7-7.4.
[0023] In some embodiments, the dissolved oxygen value of the culture expansion is greater than or equal to 50%.
[0024] In some embodiments, the time of the culture expansion is 6-7 days.
[0025] In some embodiments, the culture expansion further comprises: controlling the pH value by controlling the pH-associated alkali pump flow or controlling the CO2-associated flow, and / or controlling the dissolved oxygen value by controlling the N2-associated flow.
[0026] In some embodiments, the CO2-associated flow is 30-50 mL / min; the pH-associated alkali pump flow is 10-20 mL / min; and the N2-associated flow is 20-40 mL / min.
[0027] In some embodiments, the target density is greater than or equal to 10x10 6 cells / mL.
[0028] In some embodiments, during the culture expansion, further comprising monitoring and controlling the cell density and cell viability by regular sampling.
[0029] The foregoing in-vitro culture method of NK cells uses a jet flow bioreactor for culture. Since the jet flow bioreactor has no mechanical stirring and bottom aeration device, the shear force on the cells is small, which can increase the in-vitro culture expansion efficiency of NK cells, enhance the cell activity, and is simple to operate, and can meet the clinical treatment and scientific research. BRIEF DESCRIPTION OF DRAWINGS
[0030] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiment or example description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creating any inventive labor.
[0031] Figure 1 Figure 1 is a graph showing the change of cell density and cell viability of NK cells at different time points during the culture process in an embodiment of the present application;
[0032] Figure 2 Figure 2 is a comparison graph of NK cell growth conditions of Example 1 and Comparative Example 1. DETAILED DESCRIPTION
[0033] For the purposes of this application, the application will now be described in more detail with reference to the enclosed drawings. In the drawings, the preferred embodiments of the application are shown. The application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.
[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0035] In this application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "options" in a technical solution, if there is no special description, and there is no contradiction or mutual restriction, each "option" is independent.
[0036] In this application, "preferably", "better", "better", "as appropriate" only describe the better effect of the implementation or embodiment, and it should be understood that it does not constitute a limitation on the scope of protection of the application.
[0037] The terms "have", "contain", "include" and "comprise" used in this application are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members or features. Members or features such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions, timing, state, etc.
[0038] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.
[0039] In this application, the unit related to the data range, if only the right end point is followed by a unit, it means that the units of the left end point and the right end point are the same.
[0040] In the present application, unless otherwise explicitly stated, the execution of the steps involved in the method flow does not have strict order limitation, and can be executed in other order than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.
[0041] In the present application, the exemplary description involving "in some embodiments", "in an embodiment", etc. can cover but not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical schemes.
[0042] In the present application, in the terms "first aspect", "second aspect", "third aspect", "fourth aspect", "fifth aspect", etc., the terms "first", "second", "third", "fourth", "fifth" are only for description purpose, and cannot be understood as indicating or implying relative importance or quantity, nor can be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", "fifth" only play a non-exhaustive enumeration description purpose, and should be understood as not constituting a closed limitation on the quantity.
[0043] In the present application, in relation to the numerical interval (i.e. numerical range), if not specifically stated, the distribution of the optional values in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e. the minimum value and the maximum value) of the numerical interval, and every value between the two numerical endpoints. If not specifically stated, when the numerical interval only points to the integers in the numerical interval, including the two endpoint integers of the numerical range and every integer between the two endpoints, it is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise indicated, the numerical range disclosed herein should be understood to include any and all sub-ranges encompassed therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows to broadly include percentage intervals, ratio intervals, value intervals, etc.
[0044] In the present application, "greater than or equal to", "greater than or equal to" and "≥" have the same meaning and can be used interchangeably; "less than or equal to", "less than or equal to" and "≤" have the same meaning and can be used interchangeably; "greater than" can be equivalent to ">"; "less than" can be equivalent to "<". In the present application, unless otherwise stated, "greater than or equal to" and "≥" can be considered to provide both "greater than" and "equal to" options. In the present application, unless otherwise stated, "less than or equal to" and "≤" can be considered to provide both "less than" and "equal to" options.
[0045] Currently, there are some limitations in the traditional NK cell culture process, for example:
[0046] 1) Low cell yield, the cell harvest is not enough to support long-term anti-disease needs and research needs;
[0047] 2) Low expansion efficiency, the existing reactor has a long culture time when culturing NK cells, and the viability is low.
[0048] The inventors of the present application speculate that the strong shear force of the traditional NK cell culture process may cause damage or even death of NK cells, thereby affecting the proliferation and final yield of the cells. The inventors tried to reduce the stirring speed, reduce the aeration amount and other methods to reduce the shear force, but the effect was not ideal; either the shear force was still too strong, there was still obvious cell damage; or the shear force was too weak, which led to insufficient dissolved oxygen and uneven mixing of nutrients, limiting cell growth. Therefore, it is necessary to develop a special process to match the special sensitivity of NK cells to shear force.
[0049] Based on this, the present application at least provides an in vitro culture method of NK cells.
[0050] In some embodiments, a disposable jet flow bioreactor is used for in vitro culture. The reactor is a new type of bioreactor developed by Jin Yisheng, and its principle is a non-bubble type interface oxygen transfer mechanism. The device runs using a motor to generate kinetic energy, which is transmitted to the shaft rotating unit to generate horizontal circular motion through a belt drive. The loading tank is installed on the unit, and the culture medium generates periodic horizontal circular motion in the tank under the inertia. The tank is designed using an inverted cone design, which makes the culture medium generate a downward vortex when doing horizontal circular motion, increases the contact area of the liquid surface and the gas surface, and thus increases the gas mass transfer efficiency, achieving efficient oxygen transfer and gas exchange. There is no mechanical stirring and bottom aeration device in the reactor, so the shear force on the cells is small, and therefore the NK cells which are more sensitive to shear force are more suitable for being cultured using the reactor.
[0051] In the present application, "disposable" means that the core culture chamber (i.e. a disposable plastic bag) of the turbulent flow bioreactor and all fluid paths in contact with the culture are discarded after the completion of one production batch and are not reused.
[0052] In some embodiments, the matched disposable consumables are disposable plastic bags produced by Jinshengshi, which are well matched with the reactor and have good integrity. The disposable plastic bags use animal-free biological culture special film, which are products assembled by bag body, pipeline, connecting piece, interception clamp, filter, etc., and are specially used for mixing, sub-packaging, storage, transportation and cell culture of biological solution. The film material is 9101 film or Jinshengshi EB1596 film.
[0053] In the first aspect of the present application, a method for in vitro culture of NK cells is provided, comprising the following steps:
[0054] S100: placing a culture bag containing a culture medium in a reactor tank of a turbulent flow reactor for pre-culture, wherein the reactor tank is designed as an inverted cone and installed above an axial rotation unit, and in the pre-culture process, the tank is driven by the axial rotation unit to move horizontally in a circular manner;
[0055] S200: inoculating NK cells into the pre-cultured culture medium to obtain a liquid culture system, and culturing and expanding the NK cells to obtain NK cells with a target density under the condition that the liquid culture system generates a downward vortex in the reactor tank moving horizontally in a circular manner and forms a non-bubble type oxygen transfer interface.
[0056] In the present application, the "non-bubble type oxygen transfer interface" refers to a thin film layer that is continuously updated at the liquid-gas interface in the turbulent flow reactor by oscillation to repeatedly flush the inner surface of the culture bag, thereby realizing non-bubble, low shear and high efficiency oxygen transfer.
[0057] In some embodiments, the NK cells are derived from peripheral blood.
[0058] In some embodiments, in step S100, the culture medium also generates a downward vortex to form a non-bubble type oxygen transfer interface when the tank moves horizontally in a circular manner.
[0059] In some embodiments, the culture medium can be a conventional NK culture medium in the art, including one or more of fetal bovine serum, penicillin, interleukin-2, interleukin-15, anti-CD16 antibody, glutamine and sodium pyruvate.
[0060] In some embodiments, the concentration of fetal bovine serum in the culture medium is 10% (w / v) to 15% (w / v). Without limitation, the concentration of fetal bovine serum can be, but is not limited to, 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), 15% (w / v), or a value or a range between any two of the above values.
[0061] In some embodiments, the concentration of penicillin in the culture medium is 1% (w / v) to 5% (w / v). Without limitation, the concentration of penicillin can be, but is not limited to, 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), or a value or a range between any two of the above values.
[0062] In some embodiments, the concentration of interleukin-2 in the culture medium is 100 IU / mL to 150 IU / mL. Without limitation, the concentration of interleukin-2 can be, but is not limited to, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, or a value or a range between any two of the above values.
[0063] In some embodiments, the concentration of anti-CD16 antibody in the culture medium is 1 pg / mL to 5 pg / mL. Without limitation, the concentration of anti-CD16 antibody can be, but is not limited to, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, or a value or a range between any two of the above values.
[0064] In some embodiments, the concentration of sodium pyruvate in the culture medium is 1 mM to 3 mM. Without limitation, the concentration of sodium pyruvate can be, but is not limited to, 1 mM, 2 mM, 3 mM, or a value or a range between any two of the above values.
[0065] In some embodiments, in step S100, the pre-culturing conditions include temperature, rotation speed, pH, and dissolved oxygen value (DO value).
[0066] In some embodiments, in step S100, the temperature of pre-culturing is 35°C to 40°C. Without limitation, the temperature of pre-culturing can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or a value or a range between any two of the above values.
[0067] In some embodiments, in step S100, the rotation speed of the shaft rotation machine during the pre-culturing is 35 rpm to 40 rpm. Without limitation, the rotation speed of the shaft rotation machine during the pre-culturing can be 35 rpm, 36 rpm, 37 rpm, 38 rpm, 39 rpm, 40 rpm, or a value or a range between any two of the above values. By setting the rotation speed within the above range, the shear force can be minimized while providing necessary mixing and oxygen, thereby laying an optimal foundation for subsequent cell inoculation and high-density expansion.
[0068] In some embodiments, in step S100, the pH value during the pre-culturing is 7 to 7.4. Without limitation, the pH value during the pre-culturing can be 7, 7.1, 7.2, 7.3, 7.4, or a value or a range between any two of the above values.
[0069] In some embodiments, in step S100, the dissolved oxygen value during the pre-culturing is 40% to 100%. Without limitation, the dissolved oxygen value during the pre-culturing can be 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a value or a range between any two of the above values. It can be understood that if the dissolved oxygen value is too low, the cells will not be able to obtain sufficient oxygen, resulting in metabolic arrest, growth inhibition, or even mass death; if the dissolved oxygen value is too high, excessive oxygen free radicals will be generated, causing oxidative damage to the cells, which will also reduce cell activity and yield.
[0070] In some embodiments, in step S100, the surface aeration air flow rate during the pre-culturing is 20 mL / min to 80 mL / min. Without limitation, the surface aeration air flow rate can be 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, or a value or a range between any two of the above values.
[0071] In the present application, the "surface aeration air flow rate" refers to the volume of air introduced into the reactor per unit time, which is used to control the DO value during the culturing. The DO value (dissolved oxygen value) can be tested by conventional means in the art, such as online dissolved oxygen electrode monitoring.
[0072] In some embodiments, in step S100, the pre-culturing time is 8 h to 24 h. Without limitation, the pre-culturing time can be 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 22 h, 24 h, or a value or a range between any two of the above values.
[0073] In some embodiments, before step S100, the method further comprises: obtaining the NK cells by subculturing.
[0074] In some embodiments, the subculture culture comprises culturing at 35-40 °C under 4-8% CO2.
[0075] In some embodiments, in step S200, the inoculation amount of the NK cells is 0.5 x 10 6 cells / mL-1.0 x 10 6 cells / mL. Non-limitingly, the inoculation amount of the NK cells can be, but is not limited to, 0.5 x 10 6 cells / mL, 0.6 x 10 6 cells / mL, 0.7 x 10 6 cells / mL, 0.8 x 10 6 cells / mL, 0.9 x 10 6 cells / mL, 1.0 x 10 6 cells / mL, or a value or a range between any two of the above values.
[0076] In some embodiments, in step S200, the culture expansion condition comprises temperature, rotation speed, pH, dissolved oxygen value, and nitrogen aeration amount.
[0077] In some embodiments, the temperature of the culture expansion is 35-40 °C. Non- limitingly, the temperature of the culture expansion can be 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, or a value or a range between any two of the above values.
[0078] In some embodiments, the rotation speed of the shaft rotation machine during the culture expansion is 35-40 rpm. Non-limitingly, the rotation speed of the shaft rotation machine during the culture expansion can be 35 rpm, 36 rpm, 37 rpm, 38 rpm, 39 rpm, 40 rpm, or a value or a range between any two of the above values.
[0079] In some embodiments, the surface aeration air flow rate of the culture expansion is 20-80 mL / min. Non-limitingly, the surface aeration air flow rate can be 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, or a value or a range between any two of the above values.
[0080] In some embodiments, the pH value of the culture expansion is 7-7.4. Non- limitingly, the pH value of the culture expansion can be 7, 7.1, 7.2, 7.3, 7.4, or a value or a range between any two of the above values.
[0081] In some embodiments, the dissolved oxygen value of the culture expansion is greater than or equal to 50%. Without limitation, the dissolved oxygen value of the culture expansion can be 50%, 60%, 70%, 80%, 90%, 100%, or a value or range between any two of the aforementioned values.
[0082] In some embodiments, the time of the culture expansion is 6 days to 7 days. Without limitation, the time of the culture expansion can be 6 days, 6.5 days, 7 days, or a value or range between any two of the aforementioned values.
[0083] In some embodiments, the NK cell culture parameters provided by the embodiments of the present application are compared with the culture parameters of other animal cells (such as CHO cells) as shown in Table 1 below:
[0084] Table 1
[0085]
[0086] In the NK cell culture process of the present application, nitrogen is introduced to precisely lower and stabilize the dissolved oxygen level in the culture solution. Unlike ordinary engineering cells (such as CHO cells) that generally rely on the metabolic oxygen consumption of the cells to naturally regulate the DO value, the reactor of the present application may, during the NK cell expansion process, especially during the high cell density stage, cause over-saturation of dissolved oxygen due to its efficient non-bubbling oxygen transfer mechanism. An excessively high dissolved oxygen environment can induce oxidative stress in NK cells, thereby inhibiting their proliferation and damaging their activity. Therefore, by adjusting the nitrogen ventilation amount, the DO value is actively suppressed and stabilized in the optimal lower range for NK cells.
[0087] In the present application, the NK cells are cultured under the culture conditions provided above, which can significantly improve the in vitro expansion efficiency of the NK cells. The NK cell density at the time of harvest is more than 10 x 10 6 cells / mL, which is more than 20 times higher than the inoculation density, and the cell activity is always excellent. During the entire culture process, the effective cell viability is always maintained at more than 80%; even when the cell density is expanded by 20 times, the viability can still be stably maintained at a high level of more than 90%.
[0088] In some embodiments, in step S200, the concentration of glutamine is also monitored and controlled to be 2 mM to 4 mM by regular sampling. By monitoring and controlling the concentration of glutamine, a stable and suitable growth environment can be provided for NK cell proliferation, thereby improving the yield and survival rate of NK cells.
[0089] In some embodiments, the pH value is controlled by controlling the pH-associated alkali pump flow rate or the CO2-associated flow rate, and / or the dissolved oxygen value (DO value) is controlled by controlling the N2-associated flow rate. Illustratively, the CO2-associated flow rate is 30 mL / min ~ 50 mL / min; the pH-associated alkali pump flow rate is 10 mL / min ~ 20 mL / min; and the N2-associated flow rate is 20 mL / min ~ 40 mL / min. Non-limitatively, the CO2-associated flow rate can be 30 mL / min, 40 mL / min, 50 mL / min, or a value or a range between any two of the above values; the pH-associated alkali pump flow rate can be 10 mL / min, 15 mL / min, 20 mL / min, or a value or a range between any two of the above values; and the N2-associated flow rate can be 20 mL / min, 30 mL / min, 40 mL / min, or a value or a range between any two of the above values.
[0090] In the present application, the "pH-associated alkali pump flow rate" refers to the flow rate of the alkali pump (i.e., the addition rate of the alkali) in the reactor, which is automatically adjusted by real-time monitoring of the pH value of the culture solution, so as to maintain the pH value in the target range.
[0091] In the present application, the "CO2-associated flow rate" refers to the flow rate of the CO2 gas in the reactor, which is automatically adjusted by real-time monitoring of the pH value of the culture solution.
[0092] In the present application, the "N2-associated flow rate" refers to the flow rate of the nitrogen gas in the reactor, which is automatically adjusted by real-time monitoring of the DO value of the culture solution, so as to reduce or maintain the DO value.
[0093] In some embodiments, in step S200, the culture is terminated when the cell density is expanded by 20 times, i.e., the target density is greater than or equal to 10 x 10 6 cells / mL. Non-limitatively, the target density can be, but is not limited to, 10 x 10 6 cells / mL, 12 x 10 6 cells / mL, 14 x 10 6 cells / mL, 16 x 10 6 cells / mL, 18 x 10 6 cells / mL, or 20 x 10 6 cells / mL.
[0094] In some embodiments, in step S200, it further includes monitoring and controlling the cell density and the cell viability by periodic sampling. The cell density and the cell viability can be detected by conventional means in the art, such as using a fully-automatic cell counter, etc.
[0095] Some embodiments are provided below.
[0096] The embodiments of the present application will be described in detail below with reference to the embodiments. It should be understood that these embodiments are only used to illustrate but not to limit the scope of the present application. The experimental methods in the following examples without specified conditions, priority is given to the guidance given in the present application, but also can be in accordance with the experimental manual or conventional conditions in the art, but also in accordance with the conditions suggested by the manufacturer, or reference to the known experimental methods in the art.
[0097] In the following examples, the amount of raw material components involved in the parameter, such as no special mention, there may be a slight deviation in the range of weighing accuracy. Involving temperature and time parameters, allow the instrument test accuracy or operation accuracy caused by acceptable deviation.
[0098] Example 1
[0099] I. Extraction of NK cells
[0100] Reagents and materials
[0101] Culture medium: RPMI-1640; 10% FBS; 1% penicillin.
[0102] Cytokines: interleukin-2 (100 IU / mL), interleukin-15 (10 ng / mL).
[0103] Separation reagent: Ficoll-paque plus (lymphocyte separation medium).
[0104] NK cell sorting reagent: CD56+ magnetic bead sorting kit.
[0105] Auxiliary reagent: PBS.
[0106] Consumables: centrifuge tube, pipette, cell culture bottle T125, T25.
[0107] Equipment: centrifuge, cell counter, CO2 incubator, biological safety cabinet.
[0108] Mononuclear cells were extracted from the peripheral blood of healthy donors, and the target cells were separated by density gradient centrifugation, and then the NK cells were sorted by magnetic beads.
[0109] Dilute blood: mix the anticoagulant with PBS at a volume ratio of 1:1.
[0110] Layering: add 5 mL separation reagent to a 15 mL centrifuge tube, and slowly add the diluted blood to the surface of the separation reagent along the wall of the tube.
[0111] Centrifugation: 400g, 20min.
[0112] Collection: pipette the target cells out and transfer them to other pipelines.
[0113] Washing: 300g centrifugation for 10min after adding 10ml PBS, and discard the supernatant. Repeat twice.
[0114] II. Passage culture of NK cells
[0115] 1. The extracted NK cells are inoculated into a T25 flask and cultured. Cytokines, activators, and metabolic regulators are added to the culture medium to promote normal proliferation and activation of the cells.
[0116] Cytokines: interleukin-2 (100 IU / mL), interleukin-15 (10 ng / mL).
[0117] Activators: anti-CD16 antibody (1 μg / mL).
[0118] Metabolic regulators: glutamine (2 mM), sodium pyruvate (1 mM).
[0119] 2. Inoculation
[0120] The sorted NK cells are inoculated into a T25 flask at a density of 1 x 10 6 cells / mL, and then placed in a CO2 incubator after adding NK culture medium.
[0121] 3. Culture conditions: 37°C, 5% CO2. Replace the medium every 48 hours and sample to detect cell density, cell viability, and other data.
[0122] 4. Harvest: collect when the cells have expanded 50-100 times.
[0123] III. Expansion culture of NK cells in a perfusion reactor
[0124] Install the disposable culture bag that is part of the perfusion reactor, inflate the bag to fit the reactor tank. Inflate at room temperature and maintain pressure, and after the pressure fluctuation is less than 10% for 30 minutes, add the culture medium for pre-culture. The culture medium is pumped into the tank through the disposable bag's reserved supplement pipeline using the reactor's supplement pump. After weighing and stabilizing, set the parameters as shown in Table 1.
[0125] After 8-24 hours of pre-culture, sample to detect whether there is bacterial contamination, and offline detect and calibrate the culture medium pH (the deviation between offline and online values is ≥0.1). Confirm that the culture medium is not contaminated, inoculate at a density of 0.5 x 10 6 cells / mL. The harvested cells in the shake flask are inoculated into the perfusion reactor through the bag's reserved deep inoculation tube, and the parameters are set (see Table 2 below) for culture for 6 days or until the cell density is greater than 10 x 10 6 cells / mL.
[0126] The reactor model selected: Jin Yishengshi CUR2.5 (one-time torrential reaction reactor 2.5 liters).
[0127] The maximum culture volume of the reactor: 2.5L.
[0128] The pre-culture volume of the medium: 500mL. The culture volume: 1.5L.
[0129] The cell inoculation density: 0.5×10 6 cells / mL.
[0130] Operation steps
[0131] A, bagging: The culture bag is installed one day in advance, and is inflated to fit the reactor tank body. After installing the disposable electrode, pressure test is performed. After the pressure test passes (30min pressure fluctuation less than 10%), the medium is pumped into the tank body through the medium supplementing pipeline reserved in the reactor.
[0132] B, pre-culture: After the medium is pumped in, the pre-culture parameters are set to start pre-culture, and the pre-culture volume is 500mL. After 8-24 hours without pollution, the cells are inoculated.
[0133] C, dilute the cells harvested from the square bottle to 3.75×10 6 cells / mL, the total volume is 200mL, and then transfer to the inoculation bottle with a pipette gun. The cell suspension is pumped into the bag body through the inoculation pipeline reserved in the bag, and the culture volume is set to 1.5L.
[0134] D, add 500mL of medium to the culture for 48 hours, and set the culture volume to 2L.
[0135] E, count and detect each operation parameter every day.
[0136] F, after 6 days of culture, the tank is unloaded, and the cell density, viability and effectiveness are detected.
[0137] Table 2
[0138]
[0139] Four, culture results
[0140] The cell density and viability of NK cells were detected during the culture period, and the results are shown in Figure 1 and Table 3, it can be seen that the NK cells cultured by the method provided in the application grow well, the cell density is expanded by 24 times in 6 days, the cell viability is maintained above 80%, and the cell density is maintained above 90% in the first 5 days. It can fully meet the expansion demand, can realize large-scale expansion of NK cells, and maintain high viability.
[0141] Table 3
[0142]
[0143] Comparative Example 1
[0144] Compared to Example 1, this comparative example uses a stirred bioreactor for NK cell culture. The parameters of the stirred bioreactor are as follows:
[0145] Temperature: 37℃
[0146] Dissolved oxygen: ≥50%
[0147] pH: 7.2±0.2
[0148] Bottom air passage: 10 mL / min
[0149] Culture volume: 2L
[0150] Speed: 30 rpm
[0151] CO2 associated flow rate: 15 mL / min.
[0152] The cell density and viability of NK cells cultured in a stirred bioreactor are as follows: Figure 2 As shown in Table 4, "Stirred VCD" represents the live cell density in the stirred bioreactor culture system, and "Turbulent VCD" represents the live cell density in the culture system of Example 1 of this application. It can be seen that after the third day of cell growth in the stirred bioreactor, the cells are more susceptible to shear force due to the increased density, which leads to a slower cell proliferation rate. Finally, the cell density at harvest is lower than the harvest density of Example 1 of this application.
[0153] Table 4
[0154]
[0155] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0156] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. A method for in vitro culturing of NK cells, characterized by, The method comprises the following steps: placing a culture bag containing a culture medium in a reactor tank of a jet flow reactor for pre-culturing, wherein the reactor tank is installed above an axial rotation machine unit in an inverted conical design, and during the pre-culturing, the axial rotation machine unit drives the tank to move horizontally in a circular manner; inoculating NK cells into the culture medium after the pre-culturing to obtain a liquid culture system, and making the liquid culture system generate a downward vortex under the condition of horizontal circular movement of the reactor tank to form a non-bubble oxygen transfer interface, and culturing and expanding to obtain NK cells with a target density.
2. The in vitro culturing method of claim 1, wherein, The culture medium comprises one or more of fetal bovine serum, penicillin, interleukin-2, interleukin-15, anti-CD16 antibody, glutamine and sodium pyruvate; Optionally, the concentration of the fetal bovine serum is 10% (w / v) to 15% (w / v), the concentration of the penicillin is 1% (w / v) to 5% (w / v), the concentration of the interleukin-2 is 100 IU / mL to 150 IU / mL, the concentration of the interleukin-15 is 10 ng / mL to 15 ng / mL, the concentration of the anti-CD16 antibody is 1 μg / mL to 5 μg / mL, the concentration of the glutamine is 2 mM to 4 mM, and the concentration of the sodium pyruvate is 1 mM to 3 mM.
3. The in vitro culturing method of claim 1, wherein, The pre-culturing conditions satisfy one or more of the following conditions: The pre-culturing temperature is 35°C to 40°C; The rotation speed of the axial rotation machine unit during the pre-culturing is 35 rpm to 40 rpm; The surface-passing air flow rate during the pre-culturing is 20 mL / min to 80 mL / min; The pre-culturing pH value is 7 to 7.4; The pre-culturing dissolved oxygen value is 40% to 100%; The pre-culturing time is 8 h to 24 h.
4. The in vitro culturing method of claim 1, wherein, Before inoculating the NK cells into the culture medium after the pre-culturing to obtain the liquid culture system, the NK cells are obtained by subculture.
5. The in vitro culturing method of claim 4, wherein, The subculture comprises culturing at 35°C to 40°C and 4% to 8% CO2.
6. The in-vitro culturing method according to claim 1, wherein In the step of inoculating the NK cells into the culture medium after the pre-culturing to obtain the liquid culture system, The inoculation amount of the NK cells is 0.5 x 10 6 cells / mL ~ 1.0 x 10 6 cells / mL.
7. The in vitro culturing method of claim 6, wherein, satisfying one or more of the following conditions: The culturing and expanding temperature is 35°C to 40°C; The rotation speed of the axial rotation machine unit during the culturing and expanding is 35 rpm to 40 rpm; The surface-passing air flow rate during the culturing and expanding is 20 mL / min to 80 mL / min; The culturing and expanding pH value is 7 to 7.4; The culturing and expanding dissolved oxygen value is greater than or equal to 50%; The culturing and expanding time is 6 days to 7 days.
8. The in vitro culturing method of claim 7, wherein, The culturing and expanding further comprises controlling the pH value by controlling a pH-associated alkali pump flow or controlling a CO2-associated flow, and / or controlling the dissolved oxygen value by controlling an N2-associated flow; Optionally, the CO2-associated flow is 30 mL / min to 50 mL / min, the pH-associated alkali pump flow is 10 mL / min to 20 mL / min, and the N2-associated flow is 20 mL / min to 40 mL / min.
9. The in vitro culture method according to any one of claims 1 to 8, wherein The target density is greater than or equal to 10 x 10 6 cells / mL.
10. The in vitro culture method according to any one of claims 1 to 8, wherein During the culture expansion, the cell density and cell viability are also monitored and controlled by periodic sampling.