CN111286488A - In vitro culture method of natural killer cells - Google Patents
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Abstract
The invention relates to the technical field of biological pharmacy, in particular to a natural killer cell in-vitro culture method, which comprises the steps of dividing the whole culture process into two stages of induction culture and proliferation culture, wherein hyaluronic acid and a CD16 antibody are used for pre-coating a culture bottle in the induction culture, vitamin E and quercetin are added in the culture process, sesamin and RetroNectin are used for pre-coating the culture bottle in the proliferation culture, glutathione and porphyrin- α are added in the culture process, the coating components and the added auxiliary components of the culture bottle are mutually matched with effective components in culture media in all stages, the activation and proliferation of NK cells are synergistically promoted, the amplification efficiency of the NK cells is improved, and the activity of the NK cells is improved.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a natural killer cell in-vitro culture method.
Background
The immune system of the human body is regulated by a complex mechanism, and when the immune system is abnormal, the immune system can be unbalanced, and various diseases which are difficult to treat, such as cancer, can occur. Therefore, the development of immune cell therapy, which is a method for treating immune-related diseases by alleviating the imbalance of the immune system and restoring the normal state, has been the focus of attention.
Natural killer cells (NK) are one of the human immune cells, derived from bone marrow lymphocytes. In humans, NK cells are mainly distributed in peripheral blood and spleen, accounting for approximately 5% -15% of peripheral blood lymphocytes; meanwhile, it is present in a small amount in other tissues such as lymph nodes. NK cells have their own unique properties relative to other types of lymphocytes. The most important property is that NK cells do not express specific antigen recognition receptors. Therefore, in the process of killing target cells, the NK cells do not need to identify the target cells through a specific mechanism, are the first defense line for the human body to rapidly resist the invasion of foreign substances, and can kill various pathogens in a broad spectrum. Meanwhile, the killing effect of the NK cells is not limited by an important histocompatibility complex, and the NK cells can rapidly kill and dissolve various tumor cells. In recent years, a great deal of clinical research shows that NK cells play a very important role in resisting tumor and virus infection.
Because the content of NK cells in peripheral blood lymphocytes is only about 10%, the NK cells in clinical application usually need to be expanded and cultured in vitro to meet the clinical dosage. In vitro culture conventional methods employ high doses of IL-2 to expand NK cells, only a few tens of times, and require the extraction of large amounts of peripheral blood from the patient to achieve the desired number of cells for treatment. Therefore, in recent years, many studies have been made to improve the efficiency of NK cell culture in vitro, including addition of inducing factors such as IL-5, IL-21, and GM-CSF and proliferation factors to the medium. Although these means for improving the medium improve the expansion efficiency of NK cells to some extent, injection of NK cells cultured in vitro, for example, in vivo, to exert their immune function also requires ensuring that NK cells have high activity. Therefore, how to improve the amplification efficiency of NK cells, prevent cell death and aging, and maintain the activity of cells has become a technical problem to be solved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an in vitro culture method of natural killer cells, which has high amplification efficiency and maintains the activity of the cells.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an in vitro culture method of natural killer cells comprises the following operation steps:
1) preparation of mononuclear cell suspension:
resuspending the peripheral blood mononuclear cells obtained by separation in an induction culture medium to obtain a mononuclear cell suspension;
2) and (3) induction culture:
adding hyaluronic acid and CD16 antibody into the culture flask to coat the culture flask, and using the culture flask as a culture flask for induction culture; inoculating the mononuclear cell suspension obtained in the step 1) into a culture bottle for induction culture, culturing for 24 hours, supplementing an induction culture medium in time during the culture process, and adding vitamin E and quercetin into the culture bottle every 6 hours to obtain an induction culture suspension;
3) and (3) proliferation culture:
inoculating the induced culture suspension obtained in the step 2) into the culture bottle for enrichment culture, culturing for 14 days in an enrichment culture medium environment, supplementing the enrichment culture medium in time in the culture process, and adding glutathione and porphyrin- α into the culture bottle every 2-3 days to finish the process;
wherein the inducing culture medium comprises a basic culture medium, IL-2, IL-5, autologous serum and amino acid; the proliferation culture medium comprises a basic culture medium, IL-15, GM-CSF, autologous serum and amino acid.
Further, the specific method for coating the culture bottle by adding hyaluronic acid and CD16 antibodies into the culture bottle in the step 2) comprises the steps of taking physiological saline, adding hyaluronic acid and CD16 antibodies to prepare a coating solution containing 3-6 mg/ml hyaluronic acid and 5-8 mug/ml CD16 antibodies, adding 5-10 ml PBS and 1-2 ml coating solution into the culture bottle, removing the coating solution after overnight at 4 ℃, and washing with PBS.
Further, the specific method for adding sesamin and RetroNectin into the culture medium in the step 3) to coat the culture bottle comprises the steps of taking physiological saline, adding sesamin and RetroNectin, preparing a coating solution containing 50-60 mu g/ml of sesamin and 5-8 mu g/ml of RetroNectin, adding 5-10 ml of PBS and 1-2 ml of coating solution into the culture bottle, removing the coating solution after overnight at 4 ℃, and washing with PBS to obtain the product.
Further, the specific composition of the induction medium is as follows: DMEM medium, 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acids, 500-750 IU/ml of IL-2 and 35-60 mg/ml of IL-5;
the specific composition of the proliferation medium is as follows: Cellgro-SCGM culture medium, 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acids, 25-50 ng/ml of IL-15 and 0.5-1 mu g/ml of GM-CSF.
Further, in the step 2), according to the volume of the culture medium in the culture bottle, the vitamin E is added into the culture bottle in an amount of 10-20 mg/ml, and the quercetin is added into the culture bottle in an amount of 15-20 mu g/ml.
Further, in the step 3), the amount of glutathione added into the culture flask is 5-10 mu g/ml and the amount of porphyrin- α added into the culture flask is 0.6-1 mu g/ml according to the volume of the culture medium in the culture flask.
Further, the specific method for preparing the mononuclear cell suspension in the step 1) comprises the steps of aseptically collecting peripheral blood, diluting the peripheral blood with a buffer solution, adding a Ficoll separating medium, centrifuging, washing to obtain peripheral blood mononuclear cells, and resuspending the peripheral blood mononuclear cells with an induction culture medium according to a certain concentration to obtain the mononuclear cell suspension.
The natural killer cell in-vitro culture method comprises the steps of dividing the whole culture process into two stages of induction culture and proliferation culture, pre-coating a culture bottle by hyaluronic acid and a CD16 antibody in the induction culture, adding vitamin E and quercetin in the culture process, pre-coating the culture bottle by sesamin and RetroNectin in the proliferation culture, adding glutathione and porphyrin- α in the culture process, and enabling coating components and added auxiliary components of the culture bottle to be mutually matched with effective components in culture media in all stages to synergistically promote activation and proliferation of NK cells, so that the activity of the NK cells is improved while the amplification efficiency of the NK cells is improved.
Detailed Description
The technical solution of the present invention will be described in detail by specific examples.
Example 1
An in vitro culture method of natural killer cells comprises the following operation steps:
1) preparing a culture medium:
a: induction medium: taking a DMEM culture medium, adding 5% of autoserum, 1% of glutamine, 1% of non-essential amino acid, 600IU/ml of IL-2 and 45mg/ml of IL-5;
b: proliferation culture medium: adding 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acid, 35ng/ml of IL-15 and 0.7 mu g/ml of GM-CSF into Cellgro-SCGM culture medium;
wherein the optional amino acids are serine and alanine;
2) preparation of mononuclear cell suspension:
peripheral blood was collected aseptically, and the volume ratio was adjusted with PBS buffer according to 1: 2 dilution of peripheral blood; adding Ficoll separating medium into centrifuge tube, adding diluted peripheral blood into centrifuge tube, centrifuging, washing to obtain peripheral blood mononuclear cells, and processing the peripheral blood mononuclear cells into 5 × 105Resuspending the concentration in induction medium at a concentration of/ml;
3) and (3) induction culture:
a: coating of culture flask: adding hyaluronic acid and CD16 antibody into physiological saline to obtain coating solution containing hyaluronic acid 5mg/ml and CD16 antibody 7 μ g/ml, adding PBS 7ml and coating solution 1.5ml into culture flask, standing at 4 deg.C overnight, discarding the coating solution, and washing with PBS;
b: inoculating the mononuclear cell suspension prepared in the step 2) into a culture bottle which is completely coated, supplementing an induction culture medium in time in the culture process, adding vitamin E into the culture bottle until the concentration is 15mg/ml and quercetin is 18 mu g/ml every 6 hours, and culturing for 24 hours to obtain an induction culture suspension;
4) and (3) proliferation culture:
a: coating of culture flask: adding sesamin and RetroNectin into physiological saline to prepare a coating solution containing 55 mu g/ml of sesamin and 7 mu g/ml of RetroNectin, adding 8ml of PBS and 1.5ml of the coating solution into a culture flask, standing overnight at 4 ℃, discarding the coating solution, and washing with PBS;
b, inoculating the induced culture suspension prepared in the step 3) into a culture bottle which is completely coated, supplementing a proliferation culture medium in time during the culture process, adding glutathione into the culture medium until the concentration is 7 mu g/ml and porphyrin- α is 0.8 mu g/ml every 3 days, and culturing for 14 days for harvesting.
Example 2
An in vitro culture method of natural killer cells comprises the following operation steps:
1) preparing a culture medium:
a: induction medium: taking a DMEM culture medium, adding 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acid, 500IU/ml of IL-2 and 60mg/ml of IL-5;
b: proliferation culture medium: taking Cellgro-SCGM culture medium, adding 5% of autoserum, 1% of glutamine, 1% of non-essential amino acid, 25ng/ml of IL-15 and 1 mu g/ml of GM-CSF;
wherein the optional amino acids are serine and alanine;
2) preparation of mononuclear cell suspension:
peripheral blood was collected aseptically, and the volume ratio was adjusted with PBS buffer according to 1: 2 dilution of peripheral blood; adding Ficoll separating medium into centrifuge tube, adding diluted peripheral blood into centrifuge tube, centrifuging, washing to obtain peripheral blood mononuclear cells, and processing the peripheral blood mononuclear cells into 5 × 105Resuspending the concentration in induction medium at a concentration of/ml;
3) and (3) induction culture:
a: coating of culture flask: adding hyaluronic acid and CD16 antibody into physiological saline to obtain coating solution containing hyaluronic acid 3mg/ml and CD16 antibody 8 μ g/ml, adding PBS 10ml and coating solution 2ml into culture flask, standing at 4 deg.C overnight, discarding the coating solution, and washing with PBS;
b: inoculating the mononuclear cell suspension prepared in the step 2) into a culture bottle which is completely coated, supplementing an induction culture medium in time in the culture process, adding vitamin E into the culture bottle at an interval of 6 hours until the concentration is 10mg/ml and adding quercetin into the culture bottle at a concentration of 20 mu g/ml, and culturing for 24 hours to obtain an induction culture suspension;
4) and (3) proliferation culture:
a: coating of culture flask: adding sesamin and RetroNectin into physiological saline to prepare a coating solution containing 50 mu g/ml of sesamin and 8 mu g/ml of RetroNectin, adding 5ml of PBS and 1ml of the coating solution into a culture flask, standing overnight at 4 ℃, removing the coating solution, and washing with PBS;
b, inoculating the induced culture suspension prepared in the step 3) into a culture bottle which is completely coated, supplementing a proliferation culture medium in time during the culture process, adding glutathione into the culture medium until the concentration is 5 mu g/ml and porphyrin- α is 1 mu g/ml every 2 days, and culturing for 14 days for harvesting.
Example 3
An in vitro culture method of natural killer cells comprises the following operation steps:
1) preparing a culture medium:
a: induction medium: taking a DMEM culture medium, adding 5% of autoserum, 1% of glutamine, 1% of non-essential amino acid, 750IU/ml of IL-2 and 35mg/ml of IL-5;
b: proliferation culture medium: adding 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acid, 50ng/ml of IL-15 and 0.5 mu g/ml of GM-CSF into Cellgro-SCGM culture medium;
wherein the optional amino acids are serine and alanine;
2) preparation of mononuclear cell suspension:
peripheral blood was collected aseptically, and the volume ratio was adjusted with PBS buffer according to 1: 2 dilution of peripheral blood; adding Ficoll separating medium into centrifuge tube, adding diluted peripheral blood into centrifuge tube, centrifuging, washing to obtain peripheral blood mononuclear cells, and processing the peripheral blood mononuclear cells into 5 × 105Resuspending the concentration in induction medium at a concentration of/ml;
3) and (3) induction culture:
a: coating of culture flask: adding hyaluronic acid and CD16 antibody into physiological saline to obtain coating solution containing hyaluronic acid 6mg/ml and CD16 antibody 5 μ g/ml, adding PBS 5ml and coating solution 1.5ml into culture flask, standing at 4 deg.C overnight, discarding the coating solution, and washing with PBS;
b: inoculating the mononuclear cell suspension prepared in the step 2) into a culture bottle which is completely coated, supplementing an induction culture medium in time in the culture process, adding vitamin E into the culture bottle until the concentration is 20mg/ml and quercetin is 15 mu g/ml every 6 hours, and culturing for 24 hours to obtain an induction culture suspension;
4) and (3) proliferation culture:
a: coating of culture flask: adding sesamin and RetroNectin into physiological saline to prepare a coating solution containing 60 mu g/ml of sesamin and 5 mu g/ml of RetroNectin, adding 10ml of PBS and 2ml of the coating solution into a culture flask, standing overnight at 4 ℃, removing the coating solution, and washing with PBS;
b, inoculating the induced culture suspension prepared in the step 3) into a culture bottle which is completely coated, supplementing a proliferation culture medium in time during the culture process, adding glutathione into the culture medium until the concentration is 10 mu g/ml and porphyrin- α is 0.6 mu g/ml every 3 days, and culturing for 14 days for harvesting.
Comparative example 1
This comparative example is different from example 1 in that a culture flask for induction culture was coated with sesamin and RetroNectin, and a culture flask for proliferation culture was coated with hyaluronic acid and a CD16 antibody, and the other examples are the same as example 1.
Comparative example 2
The comparative example is different from example 1 in that glutathione and porphyrin- α are added in place of vitamin E and quercetin during the induction culture process, and vitamin E and quercetin are added in place of glutathione and porphyrin- α during the proliferation culture process.
Comparative example 3
This comparative example is different from example 1 in that hyaluronic acid was not used in the procedure of coating the culture flask for induction culture, and the procedure was otherwise the same as example 1.
Comparative example 4
This comparative example is different from example 1 in that sesamin is not used in the procedure of coating the growth culture flask, and an equal amount of sesamin is added to the growth medium, and the procedure is otherwise the same as example 1.
Test example 1 comparison of Total cell proliferation amount
Preparing trypan blue solution with pH = 7.0-7.2 and mass concentration of 0.4%, taking NK cell suspensions after proliferation culture of examples 1-3, comparative examples 1-4 and 14 days respectively, adding equal volume of trypan blue for mixing, after cell staining, counting the total number of cells by a cell counting plate, and the result shows that example 1 is about example 2, example 3 is about example 3, comparative example 4 is about comparative example 1 > 2, and the total number of cells after proliferation culture of example 14 days is 5 times of that of comparative example 1 and 10 times of that of comparative example 2.
Test example 2 comparison of cell Activity
A96-well flat-bottom culture plate is provided with 3 experimental wells, single effector cell wells and single target cell wells respectively, blank wells are arranged, and K562 is added into the experimental wells and the single target cell wells. NK cells cultured for 14 days in proliferation according to example 1 were added to each of the experimental wells and the single effector cell wells, and the effective-to-target ratio was set to 40: 1, culturing for 24 hours, adding 20 mul of WST, continuously culturing for 4 hours, detecting an OD value by an enzyme-labeling instrument, and multiplying killing activity (%) = [1- (experiment hole OD value-simple effector cell group OD value) × simple target cell OD value ] × 100%;
according to the same method, the killing activity of the NK cells on K562 after proliferation culture for 14 days in comparative examples 1-4 was calculated.
Comparing the killing activity of NK cells obtained by different culture methods, the result shows that example 1 is more than comparative example 3, comparative example 4 is more than comparative example 1, and the result shows that the killing activity of NK cells is more than comparative example 2.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. An in vitro culture method of natural killer cells is characterized by comprising the following operation steps:
1) preparation of mononuclear cell suspension:
resuspending the peripheral blood mononuclear cells obtained by separation in an induction culture medium to obtain a mononuclear cell suspension;
2) and (3) induction culture:
adding hyaluronic acid and CD16 antibody into the culture flask to coat the culture flask, and using the culture flask as a culture flask for induction culture; inoculating the mononuclear cell suspension obtained in the step 1) into a culture bottle for induction culture, culturing for 24 hours, supplementing an induction culture medium in time during the culture process, and adding vitamin E and quercetin into the culture bottle every 6 hours to obtain an induction culture suspension;
3) and (3) proliferation culture:
inoculating the induced culture suspension obtained in the step 2) into the culture bottle for enrichment culture, culturing for 14 days in an enrichment culture medium environment, supplementing the enrichment culture medium in time in the culture process, and adding glutathione and porphyrin- α into the culture bottle every 2-3 days to finish the process;
wherein the inducing culture medium comprises a basic culture medium, IL-2, IL-5, autologous serum and amino acid; the proliferation culture medium comprises a basic culture medium, IL-15, GM-CSF, autologous serum and amino acid.
2. The in vitro natural killer cell culture method of claim 1, wherein the step 2) of adding hyaluronic acid and CD16 antibodies to the culture flask to coat the culture flask comprises taking physiological saline, adding hyaluronic acid and CD16 antibodies to the culture flask to prepare a coating solution containing 3-6 mg/ml hyaluronic acid and 5-8 μ g/ml CD16 antibodies, adding 5-10 ml PBS and 1-2 ml coating solution to the culture flask, removing the coating solution after overnight at 4 ℃, and washing with PBS.
3. The in vitro natural killer cell culture method of claim 1, wherein the step 3) of adding sesamin and RetroNectin to the culture medium comprises the steps of collecting physiological saline, adding sesamin and RetroNectin, preparing a coating solution containing 50-60 μ g/ml sesamin and 5-8 μ g/ml RetroNectin, adding 5-10 ml PBS and 1-2 ml coating solution to the culture flask, removing the coating solution after overnight at 4 ℃, and washing with PBS.
4. The in vitro natural killer cell culture method of claim 1, wherein the induction medium comprises: DMEM medium, 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acids, 500-750 IU/ml of IL-2 and 35-60 mg/ml of IL-5.
5. The in vitro natural killer cell culture method according to claim 4, wherein the propagation medium is composed of: Cellgro-SCGM culture medium, 5% of autologous serum, 1% of glutamine, 1% of non-essential amino acids, 25-50 ng/ml of IL-15 and 0.5-1 mu g/ml of GM-CSF.
6. The in vitro natural killer cell culturing method according to claim 1, wherein in step 2), vitamin E is added in an amount of 10-20 mg/ml and quercetin is added in an amount of 15-20 μ g/ml, depending on the volume of the medium in the culture flask.
7. The in vitro culture method of natural killer cells according to claim 1, wherein in step 3), glutathione is added in an amount of 5 to 10 μ g/ml and porphyrin- α is added in an amount of 0.6 to 1 μ g/ml, depending on the volume of the medium in the culture flask.
8. The in vitro natural killer cell culture method of claim 1, wherein the specific method for preparing the mononuclear cell suspension in step 1) comprises the steps of aseptically collecting peripheral blood, diluting the peripheral blood with a buffer solution, adding a Ficoll separating medium, centrifuging and washing to obtain peripheral blood mononuclear cells, and resuspending the peripheral blood mononuclear cells with an induction medium according to a certain concentration.
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