Application of gene recombination cell exosome in preparation of cell culture additive
Exosomes are prepared as core reagents by genetic recombination of IL21K562 cells, which solve the problems of scarcity of equipment used for trophoblasts in in vitro culture of NK cells, difficulty in controlling doses and waste of exosomes, and achieve safer and more effective NK cell expansion and activation.
Patent Information
- Application Number
- CN202510311549.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-06-06
AI Technical Summary
When using trophoblasts in NK cells in vitro culture, there are problems such as scarcity of equipment, difficulty in controlling doses and waste of exosomes, resulting in unstable results and inefficient efficiency.
Exosomes are prepared by genetically recombinant IL21K562 cell cells and used as core reagents in vitro culture of NK cells, replacing traditional trophoblasts or their empty shells.
This method significantly promotes the amplification and activation of NK cells, is safer and more effective than traditional methods, and can greatly reduce resource waste.
Smart Images
Figure BDA0005314681110000051 
Figure BDA0005314681110000061
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of genetically recombinant cell exosomes in the preparation of cell culture additives. Background Art
[0002] NK cells have multiple functions and effects, including immune surveillance, anti-tumor, anti-viral infection, immune regulation and cytokine secretion. NK cells are an immunotherapy product, but they need to be prepared in large quantities in vitro.
[0003] IL-21 is a multifunctional cytokine that has a significant effect on the expansion and activation of NK cells in vitro, and can enhance their killing activity through specific signaling pathways. IL-21 is an important agent in the in vitro culture system of NK cells. In the in vitro culture system of NK cells, using free IL-21 factors or K562 cells expressing IL-21 (IL21K562) as trophoblasts can expand NK cells in peripheral blood mononuclear cells (PBMC) by thousands of times, which has been widely used in the industry for many years; the effect of using IL21K562 trophoblasts is better than that of using free IL-21 factors.
[0004] However, trophoblasts must be inactivated before use to prevent them from contaminating NK cell products. The best way to inactivate trophoblasts is to irradiate them with cobalt 60. Actinomycin D is also used to kill trophoblasts during culture. However, the problem with the former is that the equipment is scarce and extremely inconvenient, and it is also difficult to determine the irradiation dose; the problem with the latter is that it is difficult to grasp the dose of the reagent and the culture time, so that the results are very unstable, and there is also the problem of actinomycin D residue.
[0005] IL-21 exosomes come from IL-21 trophoblasts. IL-21 trophoblasts are genetically recombinant IL21K562 cells that express IL-21 on the surface of K562 cells through genetic recombination methods. For many years, we have used IL21K562 cell shells with the cell nucleus removed as core reagents (NKC1) instead of trophoblasts to culture NK cells. The obtained NK cells have unique characteristics such as high quantity, high purity, and high activity. However, the process for preparing IL21K562 cell shells with the cell nucleus removed is relatively complicated, and it is usually difficult to remove the cell nucleus 100%, so the risk of contamination by K562 cell nuclei cannot be completely ruled out. In addition, in the process of preparing IL21K562 cell shells, we only used cells, and a large amount of supernatant rich in exosomes was discarded as waste, resulting in a great waste.
[0006] The present invention uses IL21K562 cell culture supernatant to prepare exosomes (IL15K562Exo), and then uses the exosomes as core reagents (NKC2) to amplify and activate NK cells in in vitro culture of NK cells. The novel core reagent (NKC2) of the present invention has a safer and more effective effect of promoting NK cell amplification and activation than traditional trophoblast cells and trophoblast empty shell core reagents (NKC1). Summary of the invention
[0007] In order to overcome at least one technical problem existing in the prior art, the present invention provides a method for preparing exosomes using genetically recombinant IL21K562 cells, and using the novel exosomes as core reagents in the in vitro culture of NK cells to expand and activate NK cells, thereby providing a large number of high-quality products for NK cell immunotherapy.
[0008] The technical solution of the present invention is as follows:
[0009] The present invention first provides an application of genetically recombinant cell exosomes in the preparation of cell culture additives, wherein the genetically recombinant cell exosomes are extracted from the culture supernatant of IL21K562 cells.
[0010] Preferably, the cell culture additive refers to a NK cell culture additive.
[0011] Preferably, the cell culture additive specifically refers to a cell culture additive used to promote the expansion and activation of NK cells.
[0012] Preferably, the cell culture additive specifically refers to a cell culture additive used to promote the expansion and activation of NK cells.
[0013] Preferably, the genetically recombinant cell exosomes are prepared by the following method:
[0014] (1) Take the IL21K562 cell culture supernatant and centrifuge to remove cell debris;
[0015] (2) Add treatment reagent, mix well and incubate for 6 to 20 hours;
[0016] (3) Centrifuging to obtain a precipitate, further suspending the precipitate in a buffer solution, and centrifuging again to obtain the re-centrifuged precipitate to obtain the genetically recombinant cell exosomes.
[0017] Preferably, the treatment reagent in step (2) is a TEI reagent.
[0018] Preferably, in step (2), the volume ratio of the treatment reagent to the IL21K562 cell culture supernatant is 0.3 to 1:1.
[0019] Preferably, in step (2), the volume ratio of the treatment reagent to the IL21K562 cell culture supernatant is 0.5:1.
[0020] Preferably, in step (2), a treatment reagent is added, mixed and incubated for 8 to 12 hours.
[0021] Preferably, the buffer solution in step (3) is a PBS solution.
[0022] Preferably, the pellet obtained by re-centrifugation is further suspended in a solution to obtain genetically recombinant cell exosomes dispersed in the solution.
[0023] Preferably, the solution is a PBS solution.
[0024] Beneficial effects: The present invention provides a recombinant cell exosome prepared by a new method; studies have shown that the recombinant cell exosome (NKC2) prepared from the IL21K562 cell culture supernatant by the method of the present invention has a significant effect of promoting NK cell proliferation and activation; its effect of promoting NK cell proliferation and activation is better than that of IL21K562 cell shells (IL21K562KK, or NKC1). Therefore, the recombinant cell exosomes described in the present invention are used as cell culture additives for the in vitro culture of NK cells, which has important application value. DETAILED DESCRIPTION
[0025] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.
[0026] Example 1 Preparation of IL-21 and K562 cell gene recombinant cells (IL21K562) and cell shells (IL21K562KK)
[0027] For detailed steps, please refer to our previous patents (ZL 201480017697, US9796960, WO 2015 / 103793). In short, the following steps are included.
[0028] (1) Isolation of human PBMC: Obtain peripheral blood from adults, separate mononuclear cells, and obtain PBMC;
[0029] (2) Gene cloning: Total RNA was extracted from PBMCs; the IL-21 gene in the total RNA of PBMCs was amplified by RT-PCR and cloned into the pDisplay eukaryotic expression vector to form the pDIL21 recombinant plasmid;
[0030] (3) Transfection of K562 cells: transfect the pDIL21 recombinant plasmid into K562 cells to obtain IL21K652 trophoblast cells;
[0031] (4) Preparation of IL21K562KK: IL21K562 cells were subjected to hypotonic treatment to swell and burst, and after centrifugation and washing, the cell nuclei were precipitated at the bottom of the centrifuge tube, and the supernatant contained IL21K562 cell shells (IL21K562KK). After high-speed centrifugation of the supernatant, the IL21K562 cell shells (IL21K562KK) sank to the bottom of the centrifuge tube; after suspension with physiological saline, the quantified and aliquoted NKC1 was obtained, and stored at low temperature for future use.
[0032] Example 2 Preparation of IL21K562 Cell Exosomes (IL21K562Exo)
[0033] The total exosome isolation (TEI) kit from Life Technology was used to enrich exosomes in the culture supernatant of IL15K562 cells according to the instructions.
[0034] (1) Collect 200 ml of IL21K562 cell culture supernatant and divide into two groups (4 tubes in each group) of 50 ml conical bottom centrifuge tubes, and centrifuge at 2000×g for 30 minutes to remove cell debris.
[0035] (2) Transfer the supernatant from each tube into two new centrifuge tubes, add 0.5 times the volume of TEI reagent, mix thoroughly, and incubate at 4 degrees overnight;
[0036] (3) The next day, centrifuge at 10,000×g for 1 hour; aspirate the supernatant and suspend each tube of precipitate in 5 ml of PBS; centrifuge at 10,000×g for 1 hour, aspirate the supernatant; take the precipitate to obtain IL21K562 cell exosomes (IL21K562Exo), i.e., the genetically recombinant cell exosomes of the present invention;
[0037] (4) All the precipitates of each group were suspended in 20 ml PBS to obtain 80-150 nm IL21K562 exosomes (IL21K562Exo), i.e. NKC2, and stored at -20 degrees for later use.
[0038] Example 3
[0039] We also found that the ratio of the optimized trophoblastic empty shell (NKC1) to PBMC is 1:1, and adding more trophoblastic empty shells is not good. However, the IL21K562Exo (NKC2) of the present invention is different. It can be increased multiple times based on the present experiment. When the dose is increased by 1 times, NKC2 will show a significant difference compared with NKC1. There is no adverse effect when the dose is increased to 10 times, but the effect will be better.
[0040] Example 5 Application of IL21K562Exo in in vitro culture of NK cells
[0041] Using our conventional NK cell in vitro culture system, we compared the effects of IL21K562KK (NKC1) and IL21K562 exosomes (abbreviated as IL21K562Exo or NKC2) on NK cell culture.
[0042] 1. NK cell culture medium: 2L of lymphocyte serum-free culture medium + 2 tubes of LCC1 (LC1) + appropriate amount of plasma (autologous plasma or AB plasma, 5% for the initial 50ml culture medium, and 1-2% for the rest of the culture medium).
[0043] 2. Blood collection and plasma preparation:
[0044] 2.1 Blood collection: Collect about 50 ml of fresh anticoagulated peripheral blood or umbilical cord blood.
[0045] 2.2 Separation of plasma and cells: Centrifuge at 600g for 15 minutes, aspirate the upper yellowish plasma into a 50ml centrifuge tube, add 0.5-1 times saline to the lower blood cell part, dilute and use it to separate MNC.
[0046] 2.3 Inactivated plasma: Inactivate plasma in a 56°C water bath for 30 minutes, centrifuge at 400g for 10 minutes after the plasma temperature drops below 37°C; transfer the upper plasma to a new 50ml centrifuge tube and store it in a 4°C refrigerator for the preparation of NK cell culture medium. It is recommended to centrifuge the stored plasma to remove the precipitate before the next use.
[0047] 3. Separation of MNC: Add 20 ml of human lymphocyte separation solution to a 50 ml centrifuge tube, add the cells diluted in step 2.2.2 to the lymphocyte separation solution, and be careful not to damage the liquid interface. Centrifuge at 600g for 15 minutes (it is recommended to adjust the centrifuge speed to the lowest). Pipette the middle white blood cell layer into a 50 ml centrifuge tube, wash twice with physiological saline, and count.
[0048] 4. NK cell in vitro culture enhancer is the core reagent in the kit. NKC1 is the original NK cell in vitro culture enhancer, and NKC2 is the NK cell in vitro culture enhancer prepared by the present invention. According to the number of MNCs to be cultured (per 40×10 6 MNC requires 1 NKC1). Take out NKC1 from the liquid nitrogen tank before use, immediately place it in a 37-40℃ water bath for quick dissolution, then centrifuge at 1300rpm (350g) for 5min, discard the supernatant, wash the precipitate twice with saline, and then gently suspend it with 3ml NK cell culture medium for later use. Take out IL21K562Exo (NKC2) from the low-temperature refrigerator.
[0049] Day 1: Culture in 2 replicates. Take approximately 40 × 10 6MNC + 50 ml of NK cell serum-free culture medium prepared in step 2.1 + 1 bottle of NKC1 or NKC2 core reagent treated in step 2.4, mixed in a T175 culture bottle, and cultured in an incubator (37° C., 5% CO2 concentration).
[0050] 5. Fluid replenishment on the 3rd day: add about 50 ml of prepared NK cell culture fluid.
[0051] 6. On the 5th day, add about 50 ml of NK cell culture medium. The amount of culture medium added should not exceed 1 times the existing volume, or adjust the cell concentration to 1×10 6 / ml.
[0052] 7. Rehydration on the 6th day: add about 50 ml of NK cell culture medium, or adjust the cell concentration to 1×10 6 / ml.
[0053] 8. On the 7th day, rehydration - adding NKC1 - sterility test: add about 50ml of NK cell culture medium, count the cells, and when the total number of cells is greater than 12X10 7 Add 1 NKC2 or 1 NKC1 treated as 2.5; if the total number of cells is 6-12X10 7 If the dose is too high, it is extended to the 8th day. Perform the first sterility test.
[0054] 9. On the 8th day, add fluid and transfer the bag: add about 200 ml of NK cell culture medium or adjust the cell concentration to 1×10 6 / ml. , transfer into a cell culture bag; try to remove air bubbles.
[0055] 10. Fluid replenishment on days 9, 10, and 11: add approximately 150 ml of NK cell culture fluid every day.
[0056] 11. Day 12 Fluid replenishment - quality control test: add 350ml of NK cell culture medium. Quality control test: comprehensive quality control test, and the culture can continue only when all indicators are qualified. We compared the results of this test on 2 parallel cultured cells.
[0057] 12. Results: The observation indicators are shown in the table below. Since we used the core reagent NKC1 to culture NK cells earlier, and then we had the NKC2 of the present invention, the comparison test between the two was to divide a certain amount of cultured IL21K562 gene recombinant cells into two parts, one of which was irradiated with cobalt 60 according to the previous process as a trophoblast (NKC1), and the other equal amount of cells was used to prepare the IL21K562Exo (NKC2) of the present invention. In addition, the ratio of PBMC to NKC1 or NKC2 in NK cell culture was optimized. Table 1 shows the results of culturing NK cells with NKC1 or NKC2 core reagents under such equivalent conditions. NKC2 is more effective than NKC1.
[0058] Table 1 Comparison of the effects of NKC1 and NKC2 in NK cell culture in vitro
[0059]
[0060]
[0061] We also found that the ratio of NKC1 to PBMC is 1:1, and adding more NKC1 is not good. However, the NKC2 of the present invention is different, and can be increased multiple times. A 1-fold increase in the dose will make NKC2 significantly different from NKC1, and there is no adverse effect when the dose is increased to 10 times, but the effect will be better.
[0062] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.
Claims
1. The use of genetically recombinant cell exosomes in the preparation of cell culture additives, characterized in that: The genetically recombinant cell exosomes are extracted from the culture supernatant of IL21K562 cells.
2. The use according to claim 1, wherein the cell culture additive is a NK cell culture additive.
3. The use according to claim 1, wherein the cell culture additive specifically refers to a cell culture additive for promoting NK cell proliferation.
4. The use according to claim 1, wherein the cell culture additive specifically refers to a cell culture additive for promoting NK cell activation.
5. The application according to claim 1, characterized in that: The genetically recombinant cell exosomes are prepared by the following method: (1) Take the IL21K562 cell culture supernatant and centrifuge to remove cell debris; (2) Add treatment reagent, mix well and incubate for 6 to 20 hours; (3) Centrifuging to obtain a precipitate, further suspending the precipitate in a buffer solution, and centrifuging again to obtain the re-centrifuged precipitate to obtain the genetically recombinant cell exosomes.
6. The use according to claim 5, characterized in that: The treatment reagent in step (2) is TEI reagent.
7. The use according to claim 5, characterized in that: In step (2), the volume ratio of the treatment reagent to the IL21K562 cell culture supernatant is 0.3 to 1:
1.
8. The use according to claim 5, characterized in that: In step (2), the volume ratio of the treatment reagent to the IL21K562 cell culture supernatant is 0.5:
1.
9. The use according to claim 5, characterized in that: In step (2), the treatment reagent is added, mixed and incubated for 8 to 12 hours.
10. The use according to claim 5, characterized in that: The buffer solution described in step (3) is PBS solution; Preferably, the pellet obtained by re-centrifugation is further suspended in a solution to obtain genetically recombinant cell exosomes dispersed in the solution.