NK cell rapid amplification method not limited by cell inoculation concentration

Abstract

The invention provides an NK (Natural Killer) cell rapid amplification method not limited by cell inoculation concentration, which belongs to the technical field of cell culture and comprises the following steps: resuspending cord blood source mononuclear cells by using an activation culture medium, supplementing the activation culture medium for culture, and then supplementing a proliferation culture medium for continuous culture until amplification is finished; the activation culture medium is prepared by adding IL-15 and IL-12 into a proliferation culture medium; the proliferation culture medium is prepared by adding IL-2 (Interleukin-2), exosome, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide and human AB serum 100-512 into a GT.T551 H3 culture medium. According to the technical scheme provided by the invention, the purposes that the initial concentration of the NK cells does not need to be specially controlled, the operation is simple, and the amplification speed is high are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for rapid proliferation of NK cells that is not limited by cell inoculation concentration. Background Art

[0002] Natural killer cells (NK) are the third largest type of lymphocytes besides T cells and B cells. They can spontaneously identify abnormal cells in the body and quickly eliminate them through cytotoxicity. At the same time, they produce various pro-inflammatory factors and chemokines, recruit and activate other immune cells to initiate adaptive immune responses.

[0003] There are many methods for NK cell in vitro expansion, including cytokine expansion, feeder cell expansion, etc., but there are requirements for the initial cell seeding density. It is generally believed that 1-2*10^6 cells / mL is the most suitable expansion concentration for NK cells. For example, publication number CN117866893A, entitled "In vitro expansion method for NK cells", discloses an in vitro expansion method for NK cells: resuspend mononuclear cells in activation culture medium and adjust the cell density to 1.0×10 6 ~2.0×10 6 / mL, added to the CD16 and NKp46 antibody-coated cell culture plates at a volume of 2mL / well, and cultured in a carbon dioxide incubator for more than 21 days. The activation medium was used for culture from the 1st to the 4th day, and the expansion medium was used for rehydration culture from the 5th day. After that, the rehydration was performed every 2 days. During the rehydration, the NK cell density was adjusted to 1.0×10 6 ~1.2×10 6 / mL; by the 21st day, a large number of high-purity and high-killing ability NK cells were collected.

[0004] However, the above technical solution and the conventional in vitro expansion method in the prior art require that the density of inoculated mononuclear cells reach 1-2*10^6 / mL during the initial inoculation. The principle is that the cells are suspended in the culture medium and contact the cytokines and nutrients in the culture medium to promote their division and expansion. During the expansion process, cells will also secrete a variety of cytokines to promote each other's proliferation, but also inhibit each other's proliferation. Therefore, the cell concentration needs to be strictly controlled to achieve the best growth environment. However, too low a cell concentration will cause the cells to be unable to receive nutrients secreted by other cells in a timely manner, resulting in slow proliferation and unsatisfactory expansion multiples. Too high a cell concentration will also lead to cell-to-cell inhibition and proliferation stagnation, seriously affecting the final cell harvest. In order to maintain the cell concentration in the range of 1~2*10^6 cells / mL throughout the amplification process, operators need to count frequently to determine the cell amplification during the cell multiple growth period. When the cell concentration exceeds twice the range of 1~2*10^6 cells / mL, culture medium needs to be added in multiples to keep the cell concentration still in the range of 1~2*10^6 cells / mL. The operation is cumbersome and the risk of contamination is huge. The culture cycle is usually 15-20 days, which is also a long period. Summary of the invention

[0005] In view of this, the present invention provides a method for rapid expansion of NK cells that is not limited by cell inoculation concentration, thereby achieving the purpose of not requiring special control of the initial concentration of NK cells, that is, simple operation and fast expansion speed.

[0006] To achieve the above-mentioned object, the present invention provides a method for rapid expansion of NK cells that is not limited by cell inoculation concentration, comprising the following steps: using an activation medium to resuspend umbilical cord blood-derived mononuclear cells, supplementing the activation medium for culturing, and then supplementing the proliferation medium to continue culturing until the expansion is completed; the activation medium is a proliferation medium supplemented with IL-15 and IL-12; the proliferation medium is a GT.T551 H3 medium supplemented with IL-2, exosomes, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide, and human AB serum 100-512.

[0007] Optionally, the concentration of IL-15 in the activation medium is 40-60 ng / mL, and the concentration of IL-12 is 90-110 μg / mL.

[0008] Optionally, the concentration of IL-15 in the activation medium is 50 ng / mL, and the concentration of IL-12 is 100 μg / mL.

[0009] Optionally, the IL-2 content in the proliferation medium is 400-600 IU / mL, and the exosome content is 1-2×10^9 indivual / L, the content of Wharton's jelly in the umbilical cord is 4.5~5.5g / L, and the content of nicotinamide adenine dinucleotide is 20~30mol / L.

[0010] Optionally, the IL-2 content in the proliferation medium is 500 IU / mL, the exosome content is 1×10^9 cells / L, the umbilical cord Wharton's jelly content is 5 g / L, and the nicotinamide adenine dinucleotide content is 25 mol / L.

[0011] Optionally, the volume of human AB serum 100-512 accounts for 4-6% of the total volume of the proliferation medium.

[0012] Optionally, the volume of human AB serum 100-512 accounts for 5% of the total volume of the proliferation medium.

[0013] Optionally, a total of 500 ml of activation medium is used, 40 ml of activation medium is used to resuspend the cord blood-derived mononuclear cells, the resuspended cells are added to a cell culture bag, and then 460 ml of activation medium is added to the cell culture bag for culture.

[0014] Optionally, after supplementing with 460 ml of activation medium, the cell concentration in the cell culture bag is 8~12×10^4 cells / mL.

[0015] Optionally, the cells are cultured in activation medium for 4 to 5 days and in proliferation medium for 4 to 5 days.

[0016] The above technical solution of the present invention includes at least the following beneficial effects: The NK cell expansion method provided by the present invention is simple to operate and is not limited by cell concentration during the culture process. The exosomes in the culture medium of the present invention actively transmit cytokines, proteins, and nucleic acid substances, and can quickly activate and induce NK cell expansion. Nicotinamide adenine dinucleotide directly transports nicotinamide mononucleotide into cells under the action of exocytosis of exosomes, and plays a role in generating NAD, activating cell acetylase, continuously maintaining cell telomere length, enhancing cell viability, and making the cells in the logarithmic proliferation stage throughout the culture cycle. Wharton's jelly is a viscous substance containing a large amount of mucopolysaccharides, which can increase the consistency of the culture medium. When used in combination with exosomes and nicotinamide adenine dinucleotide, it can participate in physiological processes such as cell signal recognition and signal transduction, promote the transmission of substances such as intercellular factors and proteins, and quickly activate cell proliferation. At the same time, the operation steps of frequently adding activation factors and replenishing fluids are reduced, so that NK cells can be quickly expanded, the culture efficiency is greatly improved, the production cost is effectively reduced, and the risk of contamination caused by human operation is reduced. The obtained NK cells have high purity, high expansion multiples, and fast cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the phenotype ratio of cord blood mononuclear cells in Example 1 of the present invention; Figure 2 is the percentage of cryopreserved cell phenotypes in Example 1 of the present invention; Figure 3 is the phenotype ratio of cord blood mononuclear cells in Comparative Example 1 of the present invention; Figure 4 is the percentage of cryopreserved cell phenotypes in Comparative Example 1 of the present invention; Figure 5 is the phenotype ratio of cord blood mononuclear cells in Comparative Example 2 of the present invention; Figure 6 is the percentage of frozen cell phenotypes in Comparative Example 2 of the present invention; Figure 7 is the phenotype proportion of cord blood mononuclear cells in Comparative Example 3 of the present invention; Figure 8 is the percentage of cryopreserved cell phenotypes in Comparative Example 3 of the present invention; Fig. 9 is the phenotype proportion of cord blood mononuclear cells in Comparative Example 4 of the present invention; Fig.10 is the percentage of cryopreserved cell phenotypes in Comparative Example 4 of the present invention; Fig.11 The cell proliferation curves of Example 1 and Comparative Examples 1 to 4 of the present invention are shown.

[0018] Fig.11 The example in the figure represents Example 1. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described in combination with the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0020] Example 1 Prepare proliferation medium: including 2L GT.T551 H3 medium with IL-2, exosomes, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide, and human AB serum (Human AB Serum) 100-512; the IL-2 content is 500IU / mL and the exosome content is 1×10^9 indivual / L, umbilical cord Wharton's jelly content of 5g / L, nicotinamide adenine dinucleotide content of 25mol / L, human AB serum (Human AB Serum) 100-512 volume accounts for 5% of the total volume of the proliferation medium and is set aside.

[0021] Prepare activation medium: take 500 ml of proliferation medium, add IL-15 and IL-12; the concentration of IL-15 is 50 ng / mL, and the concentration of IL-12 is 100 μg / mL, and set aside.

[0022] NK cell activation stage: Take 4~6×10^7 umbilical cord blood-derived mononuclear cells, take a small amount of cells for cell phenotype detection, use 40ml activation medium to resuspend the umbilical cord blood-derived mononuclear cells, add them to the cell culture bag after resuspension, and then add 460ml activation medium to the cell culture bag for culture. At this time, the cell concentration in the culture bag is 8~12×10^4 / mL. Place the culture bag in an incubator and culture for 5 days. Count at a fixed time every day, record the number of cells, and calculate the cell expansion curve.

[0023] NK cell proliferation stage: After 6 days of culture, 1.5 L of proliferation medium was added to the NK cell culture bag, and the culture bag was placed in the incubator for further culture for 5 days. After the culture was completed, the cells were harvested and frozen.

[0024] Example 2 Prepare proliferation medium: including 2L GT.T551 H3 medium supplemented with IL-2, exosomes, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide, and human AB serum (Human AB Serum) 100-512; the IL-2 content in the proliferation medium is 400IU / mL, the exosome content is 2×10^ / L, the umbilical cord Wharton's jelly content is 4.5g / L, the nicotinamide adenine dinucleotide content is 30mol / L, and the volume of human AB serum (Human AB Serum) 100-512 accounts for 4% of the total volume of the proliferation medium, and set aside.

[0025] Prepare activation medium: take 500 ml of proliferation medium, add IL-15 and IL-12; the concentration of IL-15 in the activation medium is 60 ng / mL, and the concentration of IL-12 is 90 μg / mL, and set aside.

[0026] NK cell activation stage: Take 4~6×10^7 umbilical cord blood-derived mononuclear cells, take a small amount of cells for cell phenotype detection, use 40ml activation medium to resuspend the umbilical cord blood-derived mononuclear cells, add them to the cell culture bag after resuspension, and then add 460ml activation medium to the cell culture bag for culture. At this time, the cell concentration in the culture bag is 8~12×10^4 / mL, and the culture bag is placed in an incubator for 5 days.

[0027] NK cell proliferation stage: After 6 days of culture, 1.5 L of proliferation medium was added to the NK cell culture bag, and the culture bag was placed in the incubator for further culture for 4 days. After the culture was completed, the cells were harvested and frozen.

[0028] Example 3 Prepare proliferation medium: including 2L GT.T551 H3 medium supplemented with IL-2, exosomes, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide, and human AB serum (Human AB Serum) 100-512; the IL-2 content in the proliferation medium is 600IU / mL, the exosome content is 1×10^9 cells / L, the umbilical cord Wharton's jelly content is 5.5g / L, the nicotinamide adenine dinucleotide content is 20mol / L, and the volume of human AB serum (Human AB Serum) 100-512 accounts for 6% of the total volume of the proliferation medium, and set aside.

[0029] Prepare activation medium: take 500 ml of proliferation medium, add IL-15 and IL-12; the concentration of IL-15 in the activation medium is 40 ng / mL, and the concentration of IL-12 is 110 μg / mL, and set aside.

[0030] NK cell activation stage: Take 4~6×10^7 umbilical cord blood-derived mononuclear cells, take a small amount of cells for cell phenotype detection, use 40ml activation medium to resuspend the umbilical cord blood-derived mononuclear cells, add them to the cell culture bag after resuspension, and then add 460ml activation medium to the cell culture bag for culture. At this time, the cell concentration in the culture bag is 8~12×10^4 / mL, and the culture bag is placed in an incubator for 4 days.

[0031] NK cell proliferation stage: After 5 days of culture, 1.5 L of proliferation medium was added to the NK cell culture bag, and the culture bag was placed in the incubator for further culture for 5 days. After the culture was completed, the cells were harvested and frozen.

[0032] Comparative Example 1 The only difference from Example 1 is that no exosomes are added to the proliferation medium, the activation medium is cultured for 6 days, and the proliferation medium is cultured for 8 days. The remaining steps and components are the same as in Example 1.

[0033] Comparative Example 2 The only difference from Example 1 is that no Wharton's jelly of umbilical cord is added to the proliferation medium, the activation medium is cultured for 6 days, and the proliferation medium is cultured for 8 days. The remaining steps and components are the same as in Example 1.

[0034] Comparative Example 3 The only difference from Example 1 is that nicotinamide adenine dinucleotide is not added to the proliferation medium, the activation medium is cultured for 6 days, and the proliferation medium is cultured for 8 days. The remaining steps and components are the same as in Example 1.

[0035] Comparative Example 4 The NK cell expansion and culture method commonly used in the prior art was used for comparison: the NK cell kit was from Tongli Haiyuan.

[0036] Culture steps: One day before culture: prepare complete culture medium: 2L GT.T551 H3, add 100IU IL-2, take a T75 suspension culture bottle, coat it with coating solution, and place it in a refrigerator away from light overnight.

[0037] Day 0: Take out the culture flask from the refrigerator, aspirate the coating solution, take 4~6×10^7 mononuclear cells from cord blood, take a small amount of cells for phenotypic detection, add about 30mL complete culture medium to resuspend, adjust the cell concentration to 2~2.3×10^6 / mL, add the cell suspension to the culture flask, and avoid damaging the coating layer at the bottom of the flask during the addition process. Add the B factor and 5% human AB serum (Human AB Serum) 100-512 in the kit.

[0038] On day 3, add 45 mL of complete medium, factor C, and 5% human AB serum (Human AB Serum) 100-512 to a total volume of 75 mL.

[0039] On the 5th day, transfer the cells: blow off the cell suspension and aspirate it into a T182 suspension culture flask, add 175 mL of complete medium, D factor and 5% human AB serum (Human AB Serum 100-512), and the total volume is 250 mL.

[0040] On the 8th day, the cells in the culture flask were blown off and then aspirated into the culture bag, and 350 mL of complete culture medium and 5% human AB serum (Human AB Serum) 100-512 were added to a total volume of 600 mL.

[0041] On day 10, add 600 mL of complete medium and 5% human AB serum (Human AB Serum) 100-512 to a total volume of 1200 mL.

[0042] On day 13, add 800 mL of complete medium and 5% human AB serum (Human AB Serum) 100-512 to a total volume of 2000 mL.

[0043] On day 15, cells were harvested and frozen.

[0044] The supernatant of the frozen cells of Example 1 and Comparative Examples 1 to 4 was used for mycoplasma detection, and a small amount of cell suspension was used for counting, sterility detection, and endotoxin detection to obtain Table 1.

[0045] The phenotype test of the cord blood mononuclear cells of Example 1 and Comparative Examples 1 to 4 was performed respectively. Figure 1 , 3 , 5, 7, 9; respectively, the phenotype test was performed on the frozen cells of Example 1 and Comparative Examples 1 to 4. Figure 2 , 4 , 6, 8, 10.

[0046] The NK cells of Example 1 and Comparative Examples 1 to 4 were cultured and counted at a fixed time every day to obtain a cell proliferation curve. Fig.11 .

[0047] Table 1 Test results of Example 1 and Comparative Examples 1 to 4

[0048] As shown in Table 1, the number and viability of cord blood mononuclear cells in Example 1 and Comparative Examples 1 to 4 are similar, with the number of cells being between 4 and 5*10 7 Within the range of cells, the cell viability was >90%, which excluded the differences caused by mononuclear cells from different sources.

[0049] Depend on Figure 1 , 3 , 5, 7, 9 combined with Table 1, it can be seen that the phenotypes of cord blood mononuclear cells, CD3-, CD16+, and CD56+, are surface markers of NK cells. The NK cell ratio refers to the proportion of NK cells in the total number of mononuclear cells. The phenotype ratio is in the range of 5%-15%, which is within the normal range.

[0050] The number and viability of frozen cells refer to the number and viability of cells collected and counted after a certain number of days of culture. As can be seen from Table 1, Example 1 has the shortest culture time, the largest number of harvested cells, and the highest cell viability. Comparative Example 1 lacks exosomes, and Comparative Example 2 lacks Wharton's jelly, which prevents the cells from fully contacting cytokines, proteins and other substances, so the amplification results fail, and the amplification multiples are only 53 times and 91 times. Comparative Example 3 has an amplification multiple of 663 times due to the combined effect of exosomes and Wharton's jelly, but lacks the effect of nicotinamide adenine dinucleotide to extend cell telomerase, and the cells have a low density, which makes the cell viability very low. Comparative Example 4 is a prior art that uses ordinary factors sold on the market to amplify NK cells, with a long culture time and an unsatisfactory amplification multiple.

[0051] Depend on Fig.11 Combined with Table 1, it can be seen that the NK cell expansion speed of Example 1 is very fast, efficient, and continuously in the logarithmic growth stage. Comparative Examples 1 and 2 cannot deliver cytokines due to the lack of the combined effect of exosomes and Wharton's jelly, and the cell expansion is not obvious, and the experiment fails. Comparative Example 3 has exosomes and Wharton's jelly. The cells are in a stable period from the 12th day and no longer expand logarithmically. The expansion multiple is close to that of Comparative Example 4, but not as ideal as Example 1. Comparative Example 4 only has a short logarithmic expansion period on the 13th-14th day, the expansion multiple is not ideal, and the operation is not as simple as Example 1.

[0052] That is, in the technical solution of the present invention, exosomes, Wharton's jelly and nicotinamide adenine dinucleotide work together to enhance the amplification effect and reduce the density requirement of cell inoculation during amplification.

[0053] The cryopreserved cell phenotype is the phenotype of cells collected and counted after a certain number of days of culture. CD3-, CD16+, and CD56+ are surface markers of NK cells. The NK cell ratio refers to the ratio of NK cells to the total number of cells harvested, which also indicates the purity of NK cells. Figure 2 , 4 , 6, 8, and 10 combined with Table 1 show that the NK cell phenotype in the cryopreserved cells of Example 1 is the highest, the cell purity is the best, and it is very suitable for clinical use. In comparison, the cryopreserved cells of Comparative Examples 1 to 4 are not pure enough, and the cells contain a large number of other miscellaneous cells, which cannot play an effective role of NK cells.

[0054] Compared with Example 1, in Comparative Example 4, culture medium must be added according to the number of mononuclear cells to ensure that the cell density is between 1 and 2*10 6 The cell density must be within this range so that the cells can promote each other's proliferation. Otherwise, there will be a risk of poor proliferation or even culture failure. Usually, the number of single nuclei in a cord blood sample is between 6 and 10*10 7 Therefore, only 30-50 ml of culture medium can be added on the 0th day of culture. At this time, due to the small volume of culture medium, T75 culture flasks need to be used for inoculation and culture, and then the culture medium is added according to the cell growth situation. Because the cell density must be strictly controlled, the cell suspension in the bottle needs to be counted before each rehydration, and the culture medium is added in proportion to the number of cells to ensure that the cell density after rehydration remains at 1-2*10 6 / mL range, and each time before adding culture medium, the culture medium needs to be rewarmed to room temperature to avoid low temperature affecting normal cell proliferation. The operation is cumbersome and time-consuming. When the volume is greater than 100 ml, it needs to be transferred to a large bottle, that is, from a T75 culture bottle to a T182 culture bottle. When the total volume is greater than 400 ml, it can be transferred to a cell culture bag for culture. Similarly, culture medium needs to be added almost every other day, and cell counts must be performed before each addition of culture medium to ensure the cell density. The number of times of adding culture medium and counting is as high as more than 6 times until the final culture volume is 2L. The prior art requires frequent cell handling, so the risk of contamination is high, the operation time is long, the steps are cumbersome, the cells are highly sensitive to density, and the culture is easily failed, and the expansion is not ideal.

[0055] In summary, the technical solution of the present application simplifies the steps compared to the prior art, reduces the requirements of cell seeding density for amplification culture, and provides a NK cell amplification method with high amplification multiple, high cell purity and simple process.

[0056] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid expansion of NK cells that is not limited by cell seeding concentration, characterized in that: The following steps are involved: The umbilical cord blood-derived mononuclear cells are resuspended in activation medium, supplemented with activation medium for culture, and then supplemented with proliferation medium to continue culture until the end of amplification; the activation medium is proliferation medium supplemented with IL-15 and IL-12; the proliferation medium is GT.T551 H3 medium supplemented with IL-2, exosomes, umbilical cord Wharton's jelly, nicotinamide adenine dinucleotide, and human AB serum 100-512.

2. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 1, characterized in that: The IL-15 concentration in the activation culture medium is 40-60 ng / mL, and the IL-12 concentration is 90-110 μg / mL.

3. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 2, characterized in that: The concentration of IL-15 in the activation medium is 50 ng / mL, and the concentration of IL-12 is 100 μg / mL.

4. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 1, characterized in that: The proliferation medium contains 400-600 IU / mL IL-2, 1-2×10^9 exosomes / L, 4.5-5.5 g / L umbilical cord Wharton's jelly, and 20-30 mol / L nicotinamide adenine dinucleotide.

5. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 4, characterized in that: The IL-2 content in the proliferation medium is 500 IU / mL, and the exosome content is 1×10^9 indivual / L, the content of Wharton's jelly in the umbilical cord is 5g / L, and the content of nicotinamide adenine dinucleotide is 25mol / L.

6. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 1, characterized in that: The volume of the human AB serum 100-512 accounts for 4-6% of the total volume of the proliferation medium.

7. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 6, characterized in that: The volume of human AB serum 100-512 was 5% of the total volume of the proliferation medium.

8. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 1, characterized in that: A total of 500 ml of activation medium was used, and 40 ml of activation medium was used to resuspend the mononuclear cells from umbilical cord blood. The resuspended cells were added to the cell culture bag, and then 460 ml of activation medium was added to the cell culture bag for culture.

9. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 8, characterized in that: After adding 460 ml of activation medium, the cell concentration in the cell culture bag was 8~12×10^4 cells / mL.

10. The method for rapid expansion of NK cells not limited by cell seeding concentration according to claim 1, characterized in that: The cells were cultured in activation medium for 4 to 5 days and in proliferation medium for 4 to 5 days.

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