A method for improving the efficiency of virus infection of immune cells and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a method and application for improving the efficiency of virus infection of immune cells. The method provided by the present invention comprises: S01: activating immune cells; S02: adding the activated immune cells to a freezing solution; S03: rapidly freezing the immune cells after adding the freezing solution at ‑80°C to ‑70°C; S04: rapidly thawing the frozen immune cells in a 35°C‑37°C water bath. The method provided by the present invention can obtain immune cells with improved virus infection efficiency. In addition, this method not only does not destroy the normal killing function of immune cells, but also the virus infection positive rate of the treated immune cells is more persistent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for improving the efficiency of virus infection of immune cells and its application. Background Art

[0002] In recent years, immunotherapy has shown great potential in the treatment of various cancers and chronic diseases. Among them, T cells and natural killer cells (NK cells) play a vital role as key effector cells. T cells perform the core function of cellular immunity by specifically identifying and attacking cancer cells; NK cells can quickly respond and eliminate cancerous or infected cells without prior recognition of specific antigens. Given their unique positioning and attack mechanisms, these two immune cells have become ideal immunotherapy tools.

[0003] At present, there are still obstacles to the widespread application of immune cells. One of the main challenges is the low efficiency of gene modification, which in turn affects the transfection efficiency. Although the traditional virus-mediated gene transduction method has certain results, it has limitations in modification efficiency and will increase the cost of subsequent immune cell therapy. At present, there are also infection reagents being developed to improve the efficiency of viral infection of immune cells. For example, the patent with publication number CN117721155A and invention name "An infection reagent and infection method for improving the efficiency of lentiviral infection of immune cells" contains protamine and polyether non-ionic surfactant (poloxamer). The infection reagent is added when the target cells are co-cultured with lentiviruses, or when CAR-T or NK cells are cultured, which can increase the positive rate of viral infection. However, this patent treats the target cells by adding chemical reagents, which may affect the biological functions of the target cells.

[0004] Therefore, it is necessary to develop a method that can improve the efficiency of viral infection of immune cells without affecting the biological functions of immune cells. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a simple method and application for improving the efficiency of viral infection of immune cells, to partially solve or alleviate the above-mentioned deficiencies in the prior art, and the present invention specifically adopts the following technical solutions.

[0006] A method for improving the efficiency of viral infection of immune cells, the method being a physical method, comprising the following steps:

[0007] S01: Activated immune cells;

[0008] S02: centrifuging the activated immune cells at 500-1500 rpm, for example, for 2-5 minutes, discarding the supernatant, and adding the immune cells collected after centrifugation into a freezing solution; the freezing solution comprises CS10 cell freezing solution and 2-10% by mass of human serum albumin (HSA);

[0009] S03: Mix the immune cells after adding the freezing solution, and then quickly freeze them at -80°C to -70°C, control the temperature so that the immune cells are completely frozen within 3 minutes, and continue to freeze for 3-24 hours after the immune cells are frozen;

[0010] S04: Place the frozen immune cells in a 35-37°C water bath for rapid thawing, and control the temperature so that the cells are completely thawed within 1 minute;

[0011] S05: Add the completely thawed cells into the pre-warmed culture medium (in advance) and resuspend them to obtain immune cells with improved virus infection efficiency.

[0012] Furthermore, the immune cells include T cells or NK cells.

[0013] As a preferred embodiment, the rapid freezing temperature is -80°C.

[0014] As a preferred embodiment, the rapid thawing temperature is 37°C.

[0015] As a preferred embodiment, the temperature of the rapid freezing and the rapid thawing is not fixed, and the temperature is adjusted for the purpose of completely freezing and completely thawing the cells within a specified time.

[0016] As a preferred method, after the immune cells are frozen solid, they are kept frozen at this temperature for 24 hours.

[0017] Furthermore, the preheated culture medium includes T cell complete culture medium, and its components include X-VIVO serum-free culture medium and 200-800 U / mL of human interleukin 2.

[0018] Furthermore, the preheated culture medium includes NK cell complete culture medium, and its components include X-VIVO serum-free culture medium and 200-800 U / mL of human interleukin 15.

[0019] Furthermore, the step S01 also includes detecting the expression rate of characteristic molecules on the surface of activated immune cells to determine whether the immune cells have been fully activated.

[0020] Use of immune cells with improved virus infection efficiency prepared by the above method in preparing an immune cell virus infection kit.

[0021] Furthermore, the kit also includes lentivirus and lentivirus infection reagents.

[0022] Beneficial technical effects:

[0023] (1) The present invention provides a simple and easy-to-operate pure physical method to treat immune cells to obtain immune cells with improved virus infection efficiency. The method of the present invention is not only applicable to T cells, but also to NK cells. In addition, the immune cells treated by the pure physical method provided by the present invention not only do not destroy the normal killing function of the immune cells, but also the virus infection positive rate of the immune cells treated by the method is more persistent.

[0024] (2) Specifically, the purely physical means provided by the present invention is a technical means of "fast freezing and fast thawing", which is achieved by regulating the freezing and thawing temperatures and matching a specific cryopreservation solution formula. In the traditional cell cryopreservation process, "slow freezing" and "fast recovery" are generally used, the purpose of which is to maximize the protection of cell survival rate and maintain cell function. Slow freezing is to prevent the formation of large ice crystals within and between cells, which will destroy the cell membrane structure and cause cell death. However, the present invention adopts a rapid freezing method to completely freeze the cells in a very short time. It has been verified that this method not only does not damage the function of the cells, but also improves the virus infection efficiency and the durability of the infection efficiency of the cells. It is speculated that this may be related to the time of rapid freezing and the protection of a specific cryopreservation solution. In addition, the present invention also found that under the protection of the cryopreservation solution, the cell thawing time is also faster.

[0025] In summary, the method of improving the efficiency of viral infection of immune cells of the present invention is simple, convenient, easy to operate, and does not require the introduction of other chemical reagents and genetic modification methods. The cells modified by the method of the present invention can be efficiently used in subsequent scientific research or clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is the flow cytometry result of T cell activation in one of the embodiments of the present invention;

[0028] Figure 2 This is the flow cytometry result of NK cell activation in one of the embodiments of the present invention;

[0029] Figure 3 This is a T cell infection efficiency test in one of the embodiments of the present invention;

[0030] Figure 4 It is the NK cell infection efficiency detection in one of the embodiments of the present invention;

[0031] Figure 5 This is a test of the killing activity of each group of T cells against SK-OV-3 in one of the embodiments of the present invention;

[0032] Figure 6 This is a test for the persistence of T cell infection efficiency in one embodiment of the present invention;

[0033] Figure 7 Schematic diagram of the operation of the method of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0037] As used in this specification, the term "about" typically means + / -5% of the stated value, more typically + / -4% of the stated value, more typically + / -3% of the stated value, more typically + / -2% of the stated value, even more typically + / -1% of the stated value, and even more typically + / -0.5% of the stated value.

[0038] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0039] The "improving the efficiency of virus infection of immune cells" mentioned in the present invention includes the positive rate of virus-infected immune cells and the persistence of the positive rate of virus-infected immune cells cultured in vitro.

[0040] The "immune cells with improved virus infection efficiency" mentioned in the present invention refers to the virus infection efficiency relative to that of immune cells that have not been treated in any way.

[0041] Example 1

[0042] This embodiment provides an overview of a method for improving the efficiency of viral infection of immune cells based on physical means.

[0043] S01: Activate immune cells and detect the expression rate of surface antigens of activated immune cells to determine whether the immune cells used have been fully activated.

[0044] S02: Centrifuge the activated immune cells at 500-1500 rpm for 2-5 minutes, discard the supernatant, and add the immune cells collected after centrifugation into a freezing solution; the freezing solution comprises CS10 cell freezing solution and 2-10% human serum albumin by mass; after the immune cells are frozen solid, continue to freeze for 3-24 hours.

[0045] S03: Mix the immune cells after adding the freezing solution, and then quickly freeze them at -80°C~-70°C. Control the temperature so that the immune cells are completely frozen within 3 minutes. After the immune cells are frozen, continue to freeze for 3-24 hours.

[0046] S04: Place the frozen immune cells in a 35℃~37℃ water bath for rapid thawing, and control the temperature so that the cells are completely thawed within 1 minute.

[0047] S05: Add the completely thawed cells to the pre-heated complete culture medium and resuspend them. Figure 7 .

[0048] Example 2

[0049] This embodiment provides a specific example of a method for improving the efficiency of viral infection of immune cells.

[0050] Cell pretreatment:

[0051] (1) Count mononuclear cells using a cell counter, adjust the cell concentration (for example, to 1-5 *10^6 cells / mL), and add a commercial reagent for T cell expansion and activation to the T cell suspension, wherein the commercial reagent includes microbeads or magnetic beads. Mix the T cells and magnetic beads at 1*10^6 cells / 1µL magnetic beads to activate the cells. Then transfer the mixture containing T cells and magnetic beads to a cell culture plate, culture at 37°C, 5% CO2 for 2-3 days to activate the T cells, and wash off the magnetic beads. The magnetic beads selected in this embodiment are inert superparamagnetic beads with a uniform diameter of 4.5µm, which are similar in size to antigen presenting cells, and are covalently coupled to anti-CD3 antibodies and anti-CD28 antibodies. It is understandable that magnetic beads or microbeads of other sizes or properties are also used to achieve the purpose of cell activation.

[0052] (2) The T cells treated in step (1) were cultured for 3-5 days, and the expression rate of CD3 on the surface of T cells was detected by flow cytometry to determine whether the T cells were fully activated. When the CD3 expression on the surface of T cells reached 90%, the T cells were considered to be fully activated. Figure 1 As shown, the CD3 positivity rate has reached 99.5%.

[0053] It is understandable that similar methods and means can also be used in this embodiment to pre-treat NK cells.

[0054] NK cell activation:

[0055] (1) Isolating and obtaining mononuclear cells from peripheral blood: mixing peripheral blood with NK sorting reagent at a volume ratio of 25:1, incubating at room temperature in the dark for 30 minutes; obtaining mononuclear cells from the peripheral blood after centrifugation and washing.

[0056] (2) resuspending the mononuclear cells with NK cell activation medium and incubating them; adding NK cell expansion medium on the third day of culture, and then adding the NK cell expansion medium every other day, and culturing until the 21st day to obtain the high-purity NK cells.

[0057] The NK cell activation medium is prepared by adding 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL CD16 antibody, 50 ng / mL CD137 antibody and 2-10% plasma to the serum-free NK cell basal medium.

[0058] The NK cell expansion medium is based on the serum-free NK cell basal medium, and is supplemented with IL-2 with a final concentration of 1000 IU / mL, IL-15 with 50 ng / mL, and 2-10% plasma.

[0059] It is understandable that the NK cell basal medium is a commercially available reagent.

[0060] (3) Culture NK cells for 3-5 days, and detect the expression rate of CD3 and CD56 on the surface of NK cells by flow cytometry to determine whether NK activation is sufficient. When the expression of CD3 and CD56 on the surface of NK cells reaches 65%, it is determined that NK cells are fully activated. Figure 2 As shown, the proportion of CD56 positive and CD3 negative has reached 77.85%.

[0061] Cell freeze-thaw treatment:

[0062] This embodiment uses a rapid freezing-rapid thawing (rapid freezing and rapid melting) method to process immune cells, as follows.

[0063] (1) The activated T cells and NK cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in a freezing solution at 1*10^7 / mL. After gentle mixing, the cells were transferred into a cell freezing tube, marked and frozen, and the cells were completely frozen within 3 minutes. In this example, the cells were directly frozen at -80°C.

[0064] The basis for judging whether the cells are frozen solid is to observe the color of the cells and whether they contain ice crystals. Completely frozen cells appear as milky white ice crystals and have no liquid.

[0065] The cryopreservation solution used in this example is CS10 cell cryopreservation solution, and 10% human serum albumin (HSA) is additionally added.

[0066] (2) The cells frozen at -80°C for 4 hours and 24 hours were placed in a water bath for rapid thawing, respectively, and the cells were controlled to be completely thawed within 1 minute. In this example, the cells were thawed in a 37°C water bath.

[0067] The basis for the complete thawing of cells is to observe the color, state and presence of ice crystals in the cells. Completely thawed cells are transparent and round in shape, and the cryopreservation solution has completely turned into liquid.

[0068] (3) After the cells are completely thawed, add the pre-warmed complete culture medium (T cell complete culture medium or NK cell complete culture medium should be selected according to the actual cells to be cultured) into a centrifuge tube at a ratio of 1:1, mix gently, centrifuge at 1000 rpm for 3 minutes, and resuspend in complete culture medium to obtain cells that have completed the rapid freezing and rapid thawing process.

[0069] Viral Infections:

[0070] (1) Calculate the amount of virus according to the virus titer, remove the virus concentrate, thaw on ice, resuspend the virus concentrate in T cell complete medium, the final volume is 1mL, mix the virus with T cells at an MOI of 5-10, add 2*10^6 cells to each well, and the final volume is 2mL. Resuspend the virus concentrate in T cell complete medium. Centrifuge at 32°C and 1000g for 2 hours in a plate centrifuge.

[0071] (2) After centrifugation, gently remove the cell culture plate and move it steadily to the cell culture incubator to continue culturing at 37°C and 5% CO2.

[0072] It can be understood that the present embodiment adopts the same method and means to virus-infect NK cells.

[0073] In this embodiment, the virus used is a lentivirus.

[0074] In this example, the components of the T cell complete culture medium used include X-VIVO serum-free culture medium and 200-800 U / mL human IL-2.

[0075] In this example, the components of the NK cell complete culture medium used include X-VIVO serum-free culture medium and 200-800 U / mL human IL-15.

[0076] This example verifies the key technical means of cell freeze-thaw treatment.

[0077] Table 1 Freezing temperature

[0078]

[0079] Table 2 Thawing temperature

[0080]

[0081] Table 3 Effect of cryopreservation medium on thawing time

[0082]

[0083] Example 3

[0084] This example provides the detection of infection efficiency of virus-infected T cells and NK cells in Example 2.

[0085] (1) 72 hours after lentivirus infection, the GFP positivity rates of fresh T cells infected with lentivirus, T cells thawed after 4 hours of freezing, and T cells thawed after 24 hours of freezing were measured.

[0086] (2) Experimental grouping: 5*10^5 cells were collected from each group and placed in a 5 mL cell flow cytometry tube. The cells to be tested were centrifuged at 1500 rpm for 3 minutes, the culture medium was discarded, and then the cells were washed twice with 1 mL PBS, and then 500 μL PBS was used to resuspend the cells of each experimental group.

[0087] (3) Flow cytometry was used to detect the GFP expression of cells in each experimental group. Figure 3 As shown ( Figure 3 The "fresh" in the sentence represents fresh T cells).

[0088] The results showed that the efficiency of lentiviral infection of T cells after rapid freezing and thawing was higher. The lentiviral infection efficiency of T cells thawed after 4 hours of freezing was about 4.5% higher than that of fresh T cells in the control group; the lentiviral infection efficiency of T cells thawed after 24 hours of freezing was about 13.8% higher than that of fresh T cells in the control group; the freezing time was proportional to the infection efficiency.

[0089] The GFP positive rate of fresh NK cells infected with lentivirus and NK cells thawed after 24h freezing was determined by the same method. The results are shown in Figure 4 shown.

[0090] The results showed that the lentiviral infection efficiency of NK cells thawed after 24 hours of freezing was increased by about 29.5% compared with the fresh NK cells in the control group.

[0091] The above results prove that the method of the present invention also has the same effect on NK cells that are extremely difficult to infect by viruses.

[0092] Example 4

[0093] This example provides verification of the killing activity of the immune cells treated in Example 2.

[0094] (1) Take well-growing human ovarian cancer cells (SK-OV-3 cells), digest them with trypsin and count them, and resuspend them in X-VIVO serum-free medium to a final concentration of 5*10^5 cells / mL.

[0095] (2) Take the virus-infected T cells, gently blow them apart and count them, centrifuge them at 1000 rpm for 3 minutes, discard the supernatant, and resuspend them in X-VIVO serum-free medium to a final concentration of 1*10^6 cells / mL.

[0096] (3) Set different effector-target ratios (effector cells: target cells / E:T) of 1:1, 2.5:1, and 5:1, where T cells are effector cells and SK-OV-3 cells are target cells. SK-OV-3 cells are calculated at 1*10^4 per well, and each effector-target ratio is set to the following 4 groups: Fresh T + Tumor (control group), 3h T + Tumor (frozen and thawed for 3 hours), 9h T + Tumor (frozen and thawed for 9 hours), and 24h T + Tumor (frozen and thawed for 24 hours). Each group is set to 3 replica wells, and the final volume of each well is 200μL.

[0097] (4) After 24 hours of co-culture with target cells, remove the 96-well cell culture plate, add 20 μL of Lysis Solution (10X) to each well, and incubate at 37°C for 45 minutes. After the incubation, centrifuge at 250g for 4 minutes at room temperature, and take 50 μL of supernatant from each well to a new 96-well plate for subsequent LDH release detection. Add 50 μL of LDH detection reaction solution to each well and incubate at room temperature in the dark for 30 minutes. After the incubation is complete, add 50 μL of stop solution to each well. After the reaction is terminated, use an ELISA reader to read the absorbance at 490 nm within 1 hour.

[0098] Killing activity Figure 5 As shown, the results show that the T cells treated with the rapid freezing and thawing method of the present invention have no significant effect on the killing activity of SK-VO-3, proving that the rapid freezing and thawing method of the present invention for treating immune cells will not affect the biological function of immune cells.

[0099] Example 5

[0100] This example provides the infection efficiency detection of the virus-infected T cells in Example 2 after in vitro culture.

[0101] 72 hours after lentivirus infection, fresh T cells infected with lentivirus and T cells thawed after 24 hours of freezing were cultured in vitro for three days, and their GFP positive rates were detected by flow cytometry according to the method of Example 3. The results are as follows: Figure 6 shown.

[0102] The results showed that compared with fresh T cells infected with the virus, the infection efficiency of T cells thawed after 24 hours of freezing was more persistent. After 3 days of in vitro culture, the GFP positive rate of fresh T cells infected with the virus was only 48.99%, while the GFP positive rate of T cells thawed after 24 hours of freezing and freezing infected with the virus was still 75.99%. This proves that the quick freezing and thawing method of the present invention can also treat immune cells with a more persistent positive rate.

[0103] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0104] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for improving the efficiency of viral infection of immune cells, characterized in that: The method is a physical method, comprising the following steps: S01: Activate immune cells, wherein the immune cells are T cells or NK cells; S02: centrifuging the activated immune cells at 500-1500 rpm, discarding the supernatant, and adding the immune cells collected after centrifugation into a freezing solution; the freezing solution comprises CS10 cell freezing solution and 2-10% by mass of human serum albumin; S03: Mix the immune cells after adding the freezing solution, and then quickly freeze them at -80°C to -70°C, control the temperature so that the immune cells are completely frozen within 3 minutes, and continue to freeze for 3-24 hours after the immune cells are frozen; S04: Place the frozen immune cells in a 35-37°C water bath for rapid thawing, and control the temperature so that the cells are completely thawed within 1 minute; S05: Add the completely thawed cells to the preheated culture medium and resuspend them to obtain immune cells with improved viral infection efficiency; the preheated culture medium is a complete T cell culture medium, and its components include X-VIVO serum-free culture medium and 200-800U / mL of human interleukin 2; or, the preheated culture medium is a complete NK cell culture medium, and its components include X-VIVO serum-free culture medium and 200-800U / mL of human interleukin 15.

2. The method according to claim 1, characterized in that The rapid freezing temperature is -80°C.

3. The method according to claim 1, characterized in that The temperature of the rapid thawing is 37°C.

4. The method according to claim 1, characterized in that The amount of immune cells added to the freezing solution is 1×10^7 / mL.

5. The method according to claim 1, characterized in that The S01 also includes detecting the expression rate of characteristic molecules on the surface of activated immune cells to determine whether the immune cells are fully activated.

6. Use of immune cells with improved virus infection efficiency prepared by the method according to any one of claims 1 to 5 in preparing an immune cell virus infection kit.

7. The use according to claim 6, characterized in that The kit also includes a lentivirus and a lentivirus infection reagent.

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