CN111718899B - Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) - Google Patents
Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) Download PDFInfo
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Abstract
The invention relates to a culture method of in vitro induced NK cells after resuscitation of cryopreserved human PBMCs. Comprises three steps of cell recovery, NK cell induction and NK cell amplification; wherein, in the induction stage of NK cells, CD16 and thymosin are diluted by PBS solution and then are coated by a culture flask; adding IL-2, IL-12 and IL-15 into serum-free culture solution to prepare an induction culture medium; adding IL-2 and IL-7 into serum-free culture solution to prepare an activation culture medium; and adding IL-2 into the serum-free culture solution to prepare an amplification culture medium, and supplementing the culture medium with a half-changed solution by day3 for activation culture. The culture method is simple and efficient in vitro induction of NK cells after the recovery of the cryopreserved human PBMC, reduces the cell culture cost, improves the cell culture safety, can effectively overcome the difficulty of inducing the NK cells by the cryopreserved PBMC and the shortage of the amplification quantity, achieves the aim of cell treatment, and enhances the anti-tumor, anti-virus and anti-infection capacities of the PBMC.
Description
Technical Field
The invention belongs to the technical field of immune cell therapy, and relates to a culture method for in vitro induced NK cells after resuscitation of cryopreserved human PBMC (peripheral blood mononuclear cells).
Technical Field
Natural killer cells (NK cells), a population of large granular lymphocytes, are the first line of defense of the body against pathogens and tumor cells. NK cells are mainly distributed in peripheral blood and account for 5-10% of peripheral lymphocytes, and NK activity also exists in lymph nodes and bone marrow, but the level of the NK cells is lower compared with that of the peripheral blood.
Along with the deep research on the NK cells, the application of the NK cells in human bodies is more and more extensive, and the NK cells have better effects on the aspects of tumor resistance, anti-aging, sub-health treatment, disease prevention and the like at present. First, NK cells have a cytotoxic function, which can directly kill tumor cells. And secondly, the NK cells can be combined with receptors on the surfaces of aging or pathological cells to release perforin, granzyme and some cytokines, so that an apoptosis signal is generated, the aging or pathological cells are promoted to apoptosis, the balance of micro-environments in an organism is restored, and the inflammatory state is slowed down. Meanwhile, the NK cells can stimulate and recover the generation of new cells of the organism, improve the cell quality and improve the cell activity, so that the pathological changes of the cells are prevented and delayed, and the functions of the cells, organs and an immune system are recovered, thereby achieving the purposes of disease prevention, recovery and anti-aging.
When NK cell therapy is adopted for tumor patients, the treatment is generally carried out after surgical resection or used as an auxiliary treatment of radiotherapy and chemotherapy. However, after patients are treated by surgical excision or radiotherapy and chemotherapy, the immune function of the body is low, and it is not favorable for drawing blood and culturing NK cells in vitro, so that for patients in need of NK cell treatment, blood is drawn and stored before surgery or radiotherapy and chemotherapy. With the popularization of the concept of cell reserve, more people can freeze and store part of PBMC when the health state is good, and the PBMC can be used for treating sub-health and diseases in the future. Cryopreserved PBMC were selected primarily because NK cells are difficult to separate from blood. Secondly, the proportion of NK cells in peripheral blood is very small, which is too costly if we preserve the drawn blood directly. Again, PBMC are easier to separate from the blood and frozen PBMC still have the ability to induce NK cells with high efficiency.
For cell preservation, cell cryopreservation is one of the main methods, and is also the most common method at present. The cells can be stored in liquid nitrogen at the temperature of 196 ℃ below zero for low temperature by using a freezing technology, so that the cells are temporarily separated from a growth state but the characteristics of the cells are stored, and the cells are recovered for experiments when needed. At present, a programmed cooling method and a basic principle of slow freezing are mostly adopted for freezing and storing cells, so that the cell survival rate can be furthest stored. The cell is revived by adopting a rapid thawing method, so that the extracellular crystals can be thawed within a short time, and the damage to the cells caused by the water permeating into the cells to form intracellular recrystallization due to slow thawing can be avoided. Thus, the frozen cells after recovery have no obvious difference in form due to the preparation of the freezing solution and the influence of the freezing time, but have large loss in quantity, and are difficult to induce into NK cells. In addition, the current culture methods of peripheral blood NK cells mainly comprise three methods, namely a factor induction method, a trophoblast cell co-culture method and a flow sorting cell re-culture method; however, for PBMC after cryopreservation and recovery, the three methods have the defects of poor NK cell survival rate, unstable induction method and large dispute.
Disclosure of Invention
The invention aims to provide a culture method for in vitro induced NK cells after resuscitation of cryopreserved human PBMCs; the efficiency, the quantity and the safety of the NK cells induced by the PBMC after cryopreservation recovery are promoted.
The purpose of the invention is realized by the following technical scheme:
a culture method of in vitro induced NK cells after recovery of cryopreserved human PBMCs comprises three steps of cell recovery, NK cell induction and NK cell amplification;
the NK cell induction step specifically comprises:
day0, diluting CD16 and thymosin with PBS, coating with a culture flask, mixing uniformly, standing at room temperature for 60min, and washing once with PBS;
day0 adding IL-2, IL-12 and IL-15 into serum-free culture solution to prepare induction culture medium, and centrifuging the incubated PBMC with the induction culture medium according to the density of 2.5-3.0 × 106Inoculating each/ml into coated bottle, adding 10% inactivated autologous plasma, standing at 37 deg.C with 5% CO2Culturing in an incubator;
day3, adding IL-2 and IL-7 into serum-free culture solution to prepare an activation culture medium; supplementing the solution by using the half-changed solution, centrifuging the cells, then re-suspending the cells by using the half-changed solution, inoculating the cells into a recovery bottle, and supplementing an equal amount of activation culture medium;
adding an activation medium into the Day5 and Day7 to maintain the cell density at 1.5-2 × 106One/ml, Day7 was transferred to a cell culture bag for expanded culture.
As a possible solution, it is possible to,the effective concentration range of the CD16 is 500ug/cm2～1500ug/cm2(ii) a The effective concentration range of the thymosin is 150ug/cm2～500ug/cm2。
As a further scheme, the effective concentration of the CD16 is 500ug/cm2(ii) a The effective concentration of the thymosin is 150ug/cm2。
As a possible proposal, the effective concentration ranges of the IL-2, the IL-12 and the IL-15 are 100IU/ml to 1000IU/ml, 10IU/m to 200IU/m and 10IU/ml to 50IU/m respectively.
As a further proposal, the effective concentration of the IL-2, the IL-12 and the IL-15 is 1000IU/ml, 50IU/ml and 20IU/ml respectively.
As a possible proposal, the effective concentration ranges of the IL-2 and the IL-7 are 100IU/ml to 1000IU/ml and 1IU/ml to 100IU/ml respectively.
As a further proposal, the effective concentration of the IL-2 and the IL-7 is 1000IU/ml and 100IU/ml respectively.
Further, the NK cell expansion step specifically comprises:
taking a serum-free culture solution, adding IL-2 to prepare an amplification culture medium, supplementing the amplification culture medium every two days, and maintaining the cell density at 1-1.5 multiplied by 106Each ml is stood at 37 ℃ with 5% CO2Culturing in an incubator;
d14 cells were harvested, washed, counted and detected.
As a possible proposal, the effective concentration range of the IL-2 is 100IU/ml to 1000IU/ml respectively.
As a further alternative, the effective concentration of IL-2 is 500 IU/ml.
Further, the cell recovery step specifically comprises:
(1) taking out the cells to be resuscitated from liquid nitrogen within 2 minutes, and immediately transferring the cells to a 37 ℃ water bath kettle for rapid dissolution;
(2) transferring the dissolved cell suspension to 5 times volume of serum-free culture solution preheated to 37 ℃, centrifuging at the normal temperature by 500Xg, and discarding the supernatant;
(3) adding serum-free culture solution containing 10% autologous plasma,by 1.0X106Resuspend cells at density per ml;
(4) after resuspending the cells, the cells were placed at 37 ℃ in 5% CO2The cells were incubated in the cell incubator for 60min and centrifuged to collect the cells.
Further, the culture method
(1) Taking out the cells to be resuscitated from the liquid nitrogen, immediately transferring the cells to a 37 ℃ water bath kettle, and quickly dissolving the cells, wherein the whole process cannot exceed 2 minutes;
(2) transferring the dissolved cell suspension into 5-volume ALyS505NK-EX culture medium preheated to 37 ℃, centrifuging at the normal temperature of 500Xg, and removing supernatant;
(3) adding ALyS505NK-EX culture medium containing 10% autologous plasma at a ratio of 1.0 × 106Resuspend cells at density per ml;
(4) after resuspending the cells, the cells were placed at 37 ℃ in 5% CO2The cells were incubated in the cell incubator for 60min and centrifuged to collect the cells.
The NK cell induction step specifically comprises:
day0, diluting with PBS using CD16 and thymosin, coating with a culture bottle, mixing uniformly, standing for 60min at room temperature, adding PBS, and washing once;
the Day0 takes ALyS505NK-EX and adds IL-2, IL-12 and IL-15 to prepare an induction culture medium, and the induction culture medium is used for centrifuging the incubated PBMC according to the density of 2.5-3.0 multiplied by 106Inoculating each/ml into coated bottle, adding 10% inactivated autologous plasma, standing at 37 deg.C with 5% CO2Culturing in an incubator;
day3, taking ALyS505NK-EX, adding IL-2 and IL-7 to prepare an activation medium; supplementing the solution by using the half-changed solution, centrifuging the cells, then re-suspending the cells by using the half-changed solution, inoculating the cells into a recovery bottle, and supplementing an equal amount of activation culture medium;
adding an activation medium into the Day5 and Day7 to maintain the cell density at 1.5-2 × 106One/ml, Day7 was transferred to a cell culture bag for expanded culture.
The NK cell expansion step specifically comprises:
taking ALyS505NK-EX, adding IL-2 to prepare an amplification culture medium, and supplementing the amplification culture medium every two daysThe cell density is maintained at 1-1.5 × 106Each ml is stood at 37 ℃ with 5% CO2Culturing in an incubator;
d14 cells were harvested and washed for counting, CD3 was detected by flow cytometry-CD56+The cell proportion is detected, and simultaneously, the cell activity detection, the tumor killing activity, the endotoxin detection, the fungus detection, the mycoplasma detection are carried out.
The invention has the following beneficial effects:
1. the invention relates to a culture method for in vitro induction of NK cells after resuscitation of cryopreserved human PBMCs, which utilizes a single antibody CD16 combined with a thymosin body to combine IL-2, IL-7, IL-12 and IL-15 for induction, activation and amplification, stimulates proliferation of the NK cells, secretion of cytokines, and regulates activation and differentiation of the NK cells, and can improve the induction efficiency of the NK cells and the number of the obtained NK cells.
2. The method for culturing the NK cells induced in vitro after the resuscitation of the cryopreserved human PBMCs comprises the steps of placing the resuscitated PBMCs in an incubator, incubating for 60min, then inducing the NK cells, recovering the cell state after the cells pass through a certain resuscitation period, and improving the cell amplification times.
3. According to the culture method for in vitro induced NK cells after resuscitation of cryopreserved human PBMCs, a serum-free culture medium is used, animal serum and heterogenous protein are not contained, cell pollution caused by viruses, mycoplasma, fungi and the like and influence caused by serum batch difference are avoided, and safety is improved.
4. The culture method provided by the invention is used for culturing the NK cells, the culture period is only 14 days, the high-purity, high-quantity and high-lethality NK cells can be obtained, and the culture period is short.
5. The culture method provided by the invention adopts medicines or factors of clinical treatment level, avoids the introduction of exogenous animal components, and has no toxic or side effect on cell culture.
In conclusion, the culture method of the cryopreserved human PBMC induced NK cells provided by the invention is simple and efficient, not only reduces the cell culture cost, but also improves the safety of cell culture, can effectively overcome the difficulties of inducing the NK cells by the cryopreserved PBMC and the shortage of amplification quantity, achieves the aim of cell treatment, and enhances the anti-tumor, anti-virus and anti-infection capabilities of the cells. Provides a high-quality culture scheme for the clinical wide application and research of cell therapy.
[ description of the drawings ]
FIG. 1 is a diagram showing the growth state of day14 cells in a control group;
FIG. 2 is a diagram showing the growth state of day14 cells in the experimental group;
FIG. 3NK cell growth profiles.
FIG. 4 is a graph showing the results of flow analysis and detection of control group Day14
FIG. 5 is a graph showing the results of flow analysis and detection of experimental group Day 14.
[ detailed description ] embodiments
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional reagent manufacturers.
The experimental environment, experimental materials and instrument equipment which need to be prompted and explained in the invention are as follows:
1. the experimental environment is as follows: operating in an ultra clean bench in a laboratory in a GMP environment.
2. Reagent: trypan blue staining solution (Beech Biotech Co., Ltd.), phosphate buffered saline PBS (Beech Biotech Co., Ltd.), ALyS505NK-AC (cell science institute CSTI), ALyS505NK-EX serum-free culture solution (cell science institute CSTI, Ltd.), CD16 (Beckman), (cell science institute CSTI, Ltd.), thymosin (Italy Petasin), human lymphocyte separation solution (Tianjin ocean), heparin sodium solution, IL-2(MACS), IL-7(MACS), IL-12(MACS), IL-15 (ABS), LaboBank 2 cell cryopreservation solution (BIOLO).
3. Instruments and equipment: constant temperature automatic heating apparatus (Kyoto Biotechnology Co., Ltd.), centrifuge (Thermo, USA), and T25 suspension cultureBottle, T75 suspension culture bottle, T225 suspension culture bottle, CO2Incubator (SANYO, japan), clean bench (zhijing, china), 50ml centrifuge tube (naisi), pipette, blood cell counting plate (CSTI, cell science research institute, ltd.), and liquid nitrogen tank.
Example cell viability measurement and control assay
Collecting peripheral blood of healthy volunteers, separating peripheral blood mononuclear cells, simultaneously taking out cell samples at time points, performing cell recovery by using the cell recovery method in the culture method for in vitro induced NK cells after the recovery of cryopreserved human PBMCs, and calculating the cell survival rate.
1. PBMC preparation
Carefully pouring 20-30ml of blood on 15ml of lymphocyte separation solution; centrifuging at 800Xg for 20min at room temperature, slowly increasing and slowly decreasing, and if the blood is stored for more than 2 hours, increasing the centrifugation time to 30 min; after centrifugation, the blood was divided into 4 layers consisting of plasma (upper layer), a mononuclear cell layer (layer 2) between the plasma and the separation liquid, the separation liquid (layer 3) and a red blood cell layer (bottom layer); collecting the plasma on the upper layer by using a sterile pipette, collecting the plasma into a centrifuge tube, and not sucking the mononuclear cell layer by mistake; heating plasma at 56 deg.C for 30 min; centrifuging at 1200Xg for 10min at room temperature; collecting the supernatant with a straw to a new centrifuge tube, storing at 4 deg.C, and taking out before use; collecting the 2 nd layer of mononuclear cells to a new centrifuge tube by using a pipette; adding 35ml of PBS to dilute the cell suspension, and centrifuging for 10min at 500 Xg; removing supernatant, adding 35ml PBS to dilute cell suspension, mixing, counting 100ul suspension cells, and centrifuging the rest suspension at 500Xg for 10 min.
2. PBMC cryopreservation
Resuspending the centrifuged PBMC pellet with cell freezing medium, mixing well, adding LaboBank 2 cell freezing medium to dilute the cell density to 1.0X107About one/ml, and after being blown and evenly mixed, the cells are transferred into a freezing storage tube. Cell name, number, date are marked.
And (3) placing the freezing tube in a refrigerator at 4 ℃ for 10 minutes, transferring the freezing tube to a refrigerator at-80 ℃ for overnight, and transferring the freezing tube to a liquid nitrogen tank for storage the next day.
3. PBMC resuscitation
Experimental groups: respectively freezing and storing one cell for one month, three months, six months and one year; preparing 30ml of ALyS505NK-EX culture medium, and preheating to about 37 ℃; taking out the frozen cells from the liquid nitrogen tank, and quickly placing the cells in a constant-temperature automatic heating instrument preheated to 37 ℃ until ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2 minutes; transferring the dissolved cells into a centrifuge tube prepared in advance with 30ml of ALyS505NK-EX culture medium by using a gun head, centrifuging at the normal temperature at 500Xg for 10min, and removing the supernatant; adding ALyS505NK-EX culture medium containing 10% autologous plasma for cell resuspension, placing the cells at 37 deg.C and 5% CO2Incubating for 60min in the cell incubator; centrifuging at 500Xg for 10min at normal temperature, and discarding the supernatant; the cell viability was measured by trypan blue staining method for a small number of cells, and the cell viability was ═ total (total number of cells-number of stained cells)/total number of cells × 100%.
Control group: respectively freezing and storing one cell for one month, three months, six months and one year; taking out the frozen cells from the liquid nitrogen tank, and quickly placing the cells in a constant-temperature automatic heating instrument preheated to 37 ℃ until ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2 minutes; transferring the dissolved cells to a centrifuge tube prepared in advance with a 1640 culture medium containing 10% plasma by using a gun head, centrifuging at the normal temperature at 500Xg for 10min, and discarding the supernatant; the cell viability was measured by trypan blue staining method for a small number of cells, and the cell viability was ═ total (total number of cells-number of stained cells)/total number of cells × 100%.
4. Example results
TABLE 1 comparison of cell viability
| Freezing time | Control group | Experimental group |
| One month | 92.33％ | 97.52％ |
| Three months old | 86.89％ | 98.71％ |
| Six months old | 68.68％ | 95.08％ |
| One year | 67.56％ | 93.36％ |
Example two
Collecting 40ml of peripheral blood of a healthy volunteer, separating peripheral blood mononuclear cells, inducing NK cells (an experimental group for short) by using the culture method of the in vitro induced NK cells after the recovery of the cryopreserved human PBMC, observing cell forms at different time points, taking out cell samples for counting, detecting cell phenotypes, and observing whether the induced NK cells can meet the requirements of clinical treatment.
1. PBMC preparation
Carefully pouring 20-30ml of blood on 15ml of lymphocyte separation solution; centrifuging at 800Xg for 20min at room temperature, slowly increasing and slowly decreasing, and if the blood is stored for more than 2 hours, increasing the centrifugation time to 30 min; after centrifugation, the blood was divided into 4 layers consisting of plasma (upper layer), mononuclear cells between plasma and the separation liquid (layer 2), separation liquid (layer 3), and red blood cell layer (bottom layer); collecting the plasma on the upper layer by using a sterile pipette, collecting the plasma into a centrifuge tube, and not sucking the mononuclear cell layer by mistake; heating plasma at 56 deg.C for 30 min; centrifuging at 1200Xg for 10min at room temperature; collecting the supernatant with a straw to a new centrifuge tube, storing at 4 deg.C, and taking out before use; collecting the 2 nd layer of mononuclear cells to a new centrifuge tube by using a pipette; adding 35ml of PBS to dilute the cell suspension, and centrifuging for 10min at 500 Xg; removing supernatant, adding 35ml PBS to dilute cell suspension, mixing, counting 100ul suspension cells, and centrifuging the rest suspension at 500Xg for 10 min.
2. PBMC cryopreservation
Resuspending the centrifuged PBMC pellet with cell freezing medium, mixing well, adding LaboBank cell freezing medium to dilute the cell density to 1.0x107About one/ml, and after being blown and evenly mixed, the cells are transferred into a freezing storage tube. Marking the name, the number and the date of the cells; and (3) placing the freezing tube in a refrigerator at 4 ℃ for 10 minutes, transferring the freezing tube to a refrigerator at-80 ℃ for overnight, and transferring the freezing tube to a liquid nitrogen tank for storage the next day.
3. PBMC resuscitation
Preheating a constant temperature automatic heating instrument to 37 ℃ (37 ℃ water bath can also be adopted), preparing a 50ml centrifuge tube, and adding 30ml of ALyS505NK-EX culture medium preheated to about 37 ℃; taking out the frozen cells from the liquid nitrogen tank, and quickly placing the cells in a constant-temperature automatic heating instrument until ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2 minutes; transferring the dissolved cells to a centrifuge tube prepared in advance with 30ml of ALyS505NK-EX culture medium by using a gun head, centrifuging at the normal temperature for 10min at 500Xg, and removing the supernatant; by 1.0X106Adding 10% autologous plasma-containing ALyS505NK-EX culture medium to resuspend cells at a density of each ml (in order to simulate the temperature environment of the cells in the body and better start cell growth, the ALyS505NK-EX serum-free cell culture medium is recommended to be subjected to temperature return treatment before use, the temperature return condition in the embodiment is 35-37 ℃ for 30min), placing the cells at 37 ℃ and 5% CO2The cells were incubated in the incubator for 60min, centrifuged at 400Xg for 10min, and the supernatant was discarded.
4. Antibody coating of culture bottle
1ml of PBS, CD16 antibody and thymosin are added into a T-25 suspension cell culture flask; slightly shaking to diffuse the solution at the bottom of the culture flask and fully spreading the solution at the bottom of the flask; standing for 1h at room temperature; the coating solution was removed and the bottom of the flask was washed 1 time with 4ml PBS and the washed flask was used immediately.
The coating solution can be prepared by using CD16 antibody and thymosin, CD16 monoclonal antibodyActivating NK cell antibody-dependent cell-mediated cytotoxicity, with effective concentration range of 500ug/cm2～1500ug/cm2(ii) a The thymosin can improve activity of NK cells, and has an effective concentration range of 150ug/cm2～500ug/cm2. The concentration of the CD16 monoclonal antibody and the thymosin in the embodiment of the invention is 500ug/cm of the CD16 antibody2And thymosin 150ug/cm2The components are preferably used in clinical grade.
5. NK cell proliferation Induction
day 0: adding 5ml of ALyS505NK-EX serum-free culture medium into the washed culture bottle, and adding IL-2, IL-12 and IL-15; adjusting the cell density to 2.5-3.0 × 106Placing the mixture at 37 ℃ and 5% CO2The cell culture box of (1) is kept still for culture.
NK cells are considered to be matured in bone marrow at present, IL-12 plays a role in the development process, and the expression of NKG2D and NKp46 can be obviously up-regulated by IL-12, so that target cells can be more easily identified by the NK cells. While the combined use of IL-2 and IL-15 is a classical scheme for the in vitro expansion of NK cells, IL-2 is an important cytokine for inducing NK cell proliferation; IL-15 can up-regulate the expression of NK cell surface receptors (such as CD16 and NKG2D), and simultaneously generate a large amount of IFN-gamma, thereby promoting the expansion of CD56+ cells.
IL-2, IL-12 and IL-15 added on the 0 th day are important immune function regulating factors and can improve the expression of killing effect factors of NK cells, and the effective concentration ranges of the IL-2, the IL-12 and the IL-15 are 100 IU/ml-1000 IU/ml, 10 IU/ml-200 IU/ml and 10 IU/ml-50 IU/ml respectively. In the embodiment of the invention, the concentrations of IL-2, IL-12 and IL-15 are respectively 1000IU/ml, 50IU/ml and 20IU/ml, and the components are preferably in clinical medication level.
day 3: adding IL-2 and IL-7 into an ALyS505NK-EX culture medium to prepare an activation culture medium; for activation of Day 3-Day 7 cells; liquid supplement is carried out according to a half-conversion mode: mixing the cells uniformly and counting, taking 2.5ml of cell suspension, adding 2.5ml of activation medium, centrifuging the taken cell suspension for 10min at the speed of 500Xg, and removing supernatant; resuspending the cells at 1.5-2X 106Inoculating the strain in a recovery bottle at the density of each ml; placing at 37 deg.C and 5% CO2And (5) standing and culturing in a cell culture box.
NK cells are currently considered to be mature in the bone marrow, and IL-2 is an important cytokine for inducing NK cell proliferation; in recent years, IL-7 has been found to regulate the activation and differentiation of NK cells; the effective concentration ranges of the IL-2 and the IL-7 are 100IU/ml to 1000IU/ml and 1IU/ml to 50IU/ml respectively. In the embodiment of the invention, the concentrations of IL-2 and IL-7 are respectively 1000IU/ml and 100IU/ml, and the components are preferably in clinical medication level.
day 5: transferring into T-175 cell culture flask, supplementing with 12ml of activating medium to maintain cell density at 1.0 × 106Adding 5% L autologous plasma per ml, mixing culture medium, placing culture flask at 37 deg.C and 5% CO2And continuing culturing in the cell culture box.
day 7: 42.5ml of the fluid replacement activation medium was added to maintain the cell density at 1.0X106Adding 5% autologous plasma per ml, mixing culture medium, placing culture flask at 37 deg.C and 5% CO2And continuing culturing in the cell culture box.
7. Expanding culture
ALyS505NK-EX medium was supplemented with IL-2 to prepare EXPM medium. The IL-2 has the functions of promoting the proliferation and the effector function of NK cells, can ensure that the NK cells are greatly amplified, and has the effective concentration ranges of 100IU/ml to 1000IU/ml respectively. In the embodiment of the invention, the concentration of IL-2 is 500IU/ml, and the components are all preferably in clinical medication level. In vitro studies show that IL-2 is an important cytokine for inducing the proliferation of NK cells and can promote the activation and proliferation of NK cells and T cells.
day 9: transferring into T225 cell culture bottle, supplementing EXPM culture medium 87.5ml, mixing, placing at 37 deg.C and 5% CO2A cell culture box.
day 11: 100ml of supplementing EXPM culture medium, mixing, placing at 37 deg.C and 5% CO2And continuing culturing in the cell culture box.
day 14: cells were collected, counted and detected. During detection, the phenotype detection index is CD3-CD56 +; reagents and instruments used by other detection items are not specially required, and detection is carried out according to actual conditions and skilled techniques of operators. The growth pattern at day14 of the experimental group is shown in FIG. 2.
8. Treatment of the control group: taking out the frozen cells from the liquid nitrogen tank, and quickly placing the cells in a constant-temperature automatic heating instrument preheated to 37 ℃ until ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2 minutes; the lysed cells were transferred to a centrifuge tube prepared in advance with 1640 medium containing 10% plasma using a pipette tip, centrifuged at 500Xg for 10min at room temperature, and the supernatant was discarded. 1ml of PBS and 500ug/cm of CD16 antibody were added to a T-25 suspension cell culture flask2(ii) a Slightly shaking to diffuse the solution at the bottom of the culture flask and fully spreading the solution at the bottom of the flask; standing for 1h at room temperature; the coating solution was removed and the bottom of the flask was washed 1 time with 4ml PBS and the washed flask was used immediately. Adding 5ml of ALyS505NK-EX serum-free culture medium into the washed culture bottle, and adding 1000IU/ml of IL-2, 50IU/ml of IL-12 and 20IU/ml of IL-15; cells were adjusted to a density of 1.6X 106Placing the mixture at 37 ℃ and 5% CO2The cell culture box of (1) is kept still for culture. Day3 to Day11 at 1.0X106The density of each ml was not in the serum-free medium ALyS505NK-EX containing IL-21000 IU/ml. day 14: cells were collected, counted and detected. The growth pattern of the control group at day14 is shown in FIG. 1.
Results of the experiment
1. Cell counting and Activity detection
Detecting the cell survival rate of a small amount of cells by trypan blue staining method, wherein the cell survival rate is (total number of cells-number of stained cells)/total number of cells multiplied by 100%; the significant differences between the experimental and conventional groups described in the examples are seen in the cell growth curves shown in FIG. 3.
TABLE 2 cell viability rate
2、CD3 -CD56 +NK cell phenotype detection
The cultured Day14 cells were harvested and the washed cells were resuspended to a density of 1X 107Each per ml, two tubes were added with 100. mu.l of cell suspension, one tube withMu.l IgG1-APC and 10. mu.l IgG1-FITC as isotype controls, and 5. mu.l CD in another tube56APC and 10. mu.l CD3-FITC as sample detection; and (5) incubating for 15min in dark and detecting. The obvious difference between the experimental group and the control group in the examples can be seen from the attached figures 4 and 5, and the culture advantages of the NK cells of the reagent and the culture method of the invention are shown.
TABLE 3 cell phenotype
| Group of | CD3-CD56+Is (%) |
| Control group | 40 |
| Experimental group | 72 |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (11)
1. A culture method of in vitro induced NK cells after recovery of cryopreserved human PBMCs comprises three steps of cell recovery, NK cell induction and NK cell amplification; it is characterized in that the preparation method is characterized in that,
the cell recovery step specifically comprises:
(1) taking out the cells to be resuscitated from liquid nitrogen within 2 minutes, and immediately transferring the cells to a 37 ℃ water bath kettle for rapid dissolution;
(2) transferring the dissolved cell suspension into 5 times volume of serum-free culture solution preheated to 37 ℃, centrifuging at the normal temperature by 500Xg, and removing supernatant;
(3) adding serum-free culture medium containing 10% autologous plasma at a ratio of 1.0 × 106Resuspend cells at density per ml;
(4) after resuspending the cells, the cells were placed at 37 ℃ in 5% CO2Incubating for 60min in the cell incubator, and centrifuging to collect cells;
the NK cell induction step specifically comprises:
on day0, diluting CD16 and thymosin with PBS, coating with culture flask, mixing, standing at room temperature for 60min, and washing with PBS;
on day0, adding IL-2, IL-12 and IL-15 into the serum-free culture solution to prepare an induction culture medium, and centrifuging the incubated PBMC at a density of 2.5-3.0 × 10 by using the induction culture medium6Inoculating each/ml into coated bottle, adding 10% inactivated autologous plasma, standing at 37 deg.C and 5% CO2Culturing in an incubator;
on the 3 rd day, taking serum-free culture solution, adding IL-2 and IL-7 to prepare an activation culture medium; supplementing the solution by using the half-changed solution, centrifuging the cells, then re-suspending the cells by using the half-changed solution, inoculating the cells into a recovery bottle, and supplementing an equal amount of activation culture medium;
adding an activation culture medium on the 5 th day and the 7 th day to maintain the cell density at 1.5-2 multiplied by 106And (4) transferring to a cell culture bag on the 7 th day for expansion culture.
2. The culture method according to claim 1, wherein the effective concentration range of CD16 is 500 μ g/cm2~1500 μg/cm2(ii) a The effective concentration range of the thymosin is 150 mu g/cm2~500 μg/cm2。
3. The culture method according to claim 2, wherein the effective concentration of CD16 is 500. mu.g/cm2(ii) a The effective concentration of the thymosin is 150 mug/cm2。
4. The method according to claim 1, wherein the effective concentrations of IL-2, IL-12 and IL-15 in the induction medium are in the range of 100 to 1000IU/ml, 10 to 200IU/ml and 10 to 50IU/ml, respectively.
5. The method according to claim 4, wherein the effective concentrations of IL-2, IL-12 and IL-15 in the induction medium are 1000IU/ml, 50IU/ml and 20IU/ml, respectively.
6. The method according to claim 1, wherein the effective concentrations of IL-2 and IL-7 in the activation medium are in the range of 100 to 1000IU/ml and 1 to 100IU/ml, respectively.
7. The method according to claim 6, wherein the effective concentrations of IL-2 and IL-7 in the activation medium are 1000IU/ml and 100IU/ml, respectively.
8. The culture method according to claim 1, wherein the NK cell expansion step comprises:
taking a serum-free culture solution, adding IL-2 to prepare an amplification culture medium, supplementing the amplification culture medium every two days, and maintaining the cell density at 1-1.5 multiplied by 106Standing at 37 deg.C and 5% CO2Culturing in an incubator;
cells were harvested on day14, washed, counted and tested.
9. The culture method according to claim 8, wherein the effective concentration range of IL-2 in the amplification medium is 100IU/ml to 1000 IU/ml.
10. The culture method according to claim 9, wherein the effective concentration of IL-2 in the amplification medium is 500 IU/ml.
11. The culture method according to any one of claims 1 to 10,
the cell recovery step specifically comprises:
(1) taking out the cells to be resuscitated from the liquid nitrogen, immediately transferring the cells to a 37 ℃ water bath kettle, and quickly dissolving the cells, wherein the whole process cannot exceed 2 minutes;
(2) transferring the dissolved cell suspension into 5-volume ALyS505NK-EX culture medium preheated to 37 ℃, centrifuging at the normal temperature of 500Xg, and removing supernatant;
(3) adding ALyS505NK-EX culture medium containing 10% autologous plasma at a ratio of 1.0 × 106Resuspend cells at density per ml;
(4) after resuspending the cells, the cells were placed at 37 ℃ in 5% CO2Incubating for 60min in the cell incubator, and centrifuging to collect cells;
the NK cell induction step specifically comprises:
on day0, diluting CD16 and thymosin with PBS, performing culture bottle coating, mixing, standing at room temperature for 60min, adding PBS, and washing once;
on day0, taking ALyS505NK-EX, adding IL-2, IL-12 and IL-15 to prepare an induction culture medium, and centrifuging the incubated PBMC at a density of 2.5-3.0 × 10 by using the induction culture medium6Inoculating each/ml into coated bottle, adding 10% inactivated autologous plasma, standing at 37 deg.C and 5% CO2Culturing in an incubator;
on the 3 rd day, ALyS505NK-EX is taken and added with IL-2 and IL-7 to prepare an activation medium; supplementing the solution by using the half-changed solution, centrifuging the cells, then re-suspending the cells by using the half-changed solution, inoculating the cells into a recovery bottle, and supplementing an equal amount of activation culture medium;
adding an activation culture medium on the 5 th day and the 7 th day to maintain the cell density at 1.5-2 multiplied by 106Culturing the cells per ml in a cell culture bag for expansion on day 7;
the NK cell expansion step specifically comprises:
taking ALyS505NK-EX, adding IL-2 to prepare an amplification culture medium, supplementing the amplification culture medium every two days, and maintaining the cell density at 1-1.5 multiplied by 106Standing at 37 deg.C and 5% CO2Culturing in an incubator;
cells were harvested on day14 and washed for counting, and CD3 was detected by flow cytometry-CD56+The proportion of the cells, and simultaneously carrying out cell activity detection, tumor killing activity, endotoxin, fungus bacteria and mycoplasma detection.
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| CN113528439A (en) * | 2021-09-03 | 2021-10-22 | 东莞再立健生物科技有限公司 | NK cell culture method |
| CN114058583B (en) * | 2021-11-10 | 2023-10-13 | 厦门大学 | Method for in vitro induction of peripheral blood mononuclear cell amplification NK cells |
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| CN114736859B (en) * | 2022-06-13 | 2022-08-19 | 广东先康达生物科技有限公司 | Culture solution and culture method for cord blood NK cells |
| CN116218781A (en) * | 2022-12-30 | 2023-06-06 | 四川阿思科力生物科技有限公司 | ROBO1CAR-NK92 cell resuscitating medium, kit and resuscitating culture method |
| CN116254233A (en) * | 2022-12-30 | 2023-06-13 | 四川阿思科力生物科技有限公司 | Resuscitation and culture method of ROBO1CAR NK cells |
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