Processing method of cell-containing sample and application thereof

Abstract

The invention relates to a treatment method of a cell-containing sample and application thereof, in particular to a treatment method of a low-purity NK cell-containing sample and application of the low-purity NK cell-containing sample in in-vitro amplification of NK cells. The specific treatment method is designed, samples such as blood can be rapidly treated, the purity of the NK cells in the samples can be improved, and the method has wide application prospects, for example, the method is applied to the process of carrying out in-vitro amplification on the NK cells from samples with low initial purity of the NK cells, the treated samples can be effectively amplified and prepared into qualified NK cells, and the application prospect is wide. And the method has the advantages of simplicity in operation, few steps, no need of special equipment, low cost, good batch stability and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a cell sample pretreatment method and its application, particularly a method for processing samples containing low-purity NK cells and its application in in vitro expansion of NK cells. Background Technology

[0002] Natural killer (NK) cells are the most important known immune cells capable of killing tumor cells that have undergone tumor immunomodulation, and are a crucial tool in clinical tumor cell immunotherapy. Clinical studies have shown that allogeneic and autologous NK cell immunotherapy is safe. However, NK cell therapy is not widely used in hospitals. In some early clinical trials, the efficacy of autologous NK cell therapy was not significant, primarily because a sufficient number and activity of NK immune cells could not be obtained. If the amount of NK cells reinfused is far less than the amount of tumor cells in vivo, the therapeutic effect will be weakened. Therefore, achieving large-scale, high-quality expansion and culture of NK cells in vitro is one of the key challenges in the field of NK cell therapy.

[0003] Currently, the common standard for large-scale in vitro expansion of NK cells is a final purity of 90% or higher. The starting cell sources for in vitro NK cell expansion include peripheral blood and umbilical cord blood. When the initial NK cell purity in umbilical cord blood or peripheral blood is low (<8%), it is often difficult for the final NK cell product to reach the 90% purity standard. Therefore, it is necessary to process the initial sample to improve the purity of the NK cells in the sample.

[0004] Among existing pretreatment techniques for initial samples of NK cell in vitro amplification, the two most commonly used methods are magnetic bead sorting and antibody-mediated adhesion removal (such as the Panning method). Magnetic bead sorting requires expensive high-purity magnetic beads, the sorting process may cause cell stress or phenotypic changes, and it is highly equipment-dependent, making it difficult to miniaturize and scale up. Antibody-mediated adhesion removal yields lower purity results than magnetic bead sorting, adhesion efficiency is greatly affected by antibody coating conditions, and batch-to-batch stability is poor.

[0005] In summary, there is an urgent need to develop a low-cost, simple-to-operate, and highly stable pretreatment method for initial samples of NK cell in vitro expansion, which can effectively improve the purity of NK cells and promote the development of the field of NK cell in vitro expansion. Summary of the Invention

[0006] In view of the shortcomings of existing technologies and practical needs, the present invention provides a method for processing cell-containing samples and its application, aiming to achieve rapid and efficient separation of cells from samples.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for processing cell-containing samples, the method comprising:

[0009] Take a cell-containing sample containing T cells, add T cell antigen antibody (primary antibody) to the cell-containing sample, then add the antibody of the T cell antigen antibody (secondary antibody), and centrifuge.

[0010] In this invention, a specific cell sample processing method is designed based on immunoagglutination centrifugation, which can effectively separate T cells from the sample and has the advantages of low antibody concentration, low cost, no need for special equipment, few steps, simple operation and good batch stability.

[0011] Preferably, the cell-containing sample also contains NK cells.

[0012] Preferably, the T-cell antigen antibody includes a CD3 antibody.

[0013] Preferably, the CD3 antibody comprises a murine OKT3 monoclonal antibody.

[0014] Preferably, the T-cell antigen antibody comprises goat anti-mouse antibody.

[0015] Preferably, the working concentration of the T-cell antigen antibody is 0.1~2 μg / mL, for example, it can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 1.2, 1.5, 1.6, 1.7, 1.8 or 1.9 μg / mL, and more preferably 0.5~1.5 μg / mL.

[0016] Preferably, the working concentration of the T-cell antigen antibody is 0.2~4 μg / mL, for example, it can be 0.3, 0.4, 0.5, 0.6, 0.8, 1, 1.2, 1.5, 2, 2.5, 3, 3.2, 3.5, 3.6, 3.7, 3.8 or 3.9 μg / mL, and more preferably 1.0~3.5 μg / mL.

[0017] The working concentration of the antibody in this invention is specifically designed to ensure efficient separation while avoiding antibody residue from affecting subsequent NK cell expansion.

[0018] Preferably, the cell-containing sample includes a blood sample.

[0019] Preferably, the blood sample includes peripheral blood or umbilical cord blood.

[0020] In this invention, the initial samples of umbilical cord blood or peripheral blood are pretreated to improve the initial purity of NK cells in the samples, thereby increasing the success rate of achieving the quality standards for the expanded NK cell products.

[0021] Preferably, the centrifugation method includes Ficoll density gradient centrifugation.

[0022] Preferably, the centrifugation conditions include: centrifugation at 600-800 g for 10-20 min.

[0023] Preferably, the addition of the antibody further includes an incubation step.

[0024] As a preferred technical solution, the method for processing the cell-containing sample includes:

[0025] Take a cell-containing sample containing T cells, add T cell antigen antibody to the cell-containing sample, perform a first incubation, add the antibody of the T cell antigen antibody, perform a second incubation, and then centrifuge.

[0026] Preferably, the temperature for the first incubation is 2~6℃ and the time is 20~60 min.

[0027] Preferably, the temperature of the second incubation is 20~30℃ and the time is 20~60 min.

[0028] Secondly, the present invention provides the application of the cell sample processing method described in the first aspect in the in vitro expansion of NK cells.

[0029] Thirdly, the present invention provides a method for in vitro expansion of NK cells, the method comprising:

[0030] A blood sample is processed using the cell-containing sample processing method described in the first aspect to collect mononuclear cells, and the mononuclear cells are cultured to obtain NK cells.

[0031] Preferably, the culture medium contains cells that help NK cell expansion, such as trophoblast cells.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] This invention designs a specific processing method that can quickly process blood and other samples to improve the purity of NK cells. It has broad application prospects, such as in the process of in vitro expansion of NK cells from samples with low initial NK cell purity. The processed sample can be effectively expanded to prepare qualified NK cells, significantly improving the success rate of achieving the quality standard of the expanded NK cell product. It also has the advantages of simple operation, few steps, no need for special equipment, low cost, and good batch stability. Attached Figure Description

[0034] Figure 1 This is a graph showing the purity results of NK cells in the sample processed in Example 1.

[0035] Figure 2 The figure shows the purity results of NK cells in the sample treated as Comparative Example 1.

[0036] Figure 3 This is a graph showing the purity results of NK cells in the amplified sample of Example 6.

[0037] Figure 4 This is a graph showing the purity results of NK cells in the amplified sample of Comparative Example 2. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0039] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0040] In a specific embodiment of the present invention, a mouse-derived OKT3 monoclonal antibody (antibody sourced from Linko Biotechnology, catalog number: F1100300) and a goat anti-mouse IgG secondary antibody (antibody sourced from Abcam, catalog number: ab6708) were used.

[0041] The method of this invention is verified using peripheral blood or umbilical cord blood as examples.

[0042] Example 1

[0043] This embodiment provides a method for processing cell samples.

[0044] Collect 30 mL of peripheral blood, centrifuge at 2000 rpm for 10 min to separate plasma, dilute the remaining blood cells to the original volume, add 1 μg / mL OKT3 monoclonal antibody (primary antibody), incubate at 4℃ for 40 min, gently mixing every 10 min; add 2 μg / mL goat anti-mouse IgG secondary antibody, incubate at room temperature for 30 min to promote cross-linking and formation of large agglutinin clusters, wash the centrifuged cells 4 times with PBS; then perform Ficoll density gradient centrifugation, slowly add 10-15 mL of Ficoll lymphocyte separation medium from the bottom to a 50 mL centrifuge tube. Add 1-2 times the volume of PBS to the washed cells, and mix by pipetting 3-5 times.

[0045] Using a pipette, draw diluted blood and slowly add it along the wall of a centrifuge tube containing Ficoll lymphocyte separation medium, close to the surface, creating two layers: blood and separation medium. After balancing, centrifuge at 700 g for 15 minutes with the centrifuge speed set to zero. After centrifugation, four distinct layers are visible: red blood cells at the bottom (deep red), separation medium in the middle layer (white to transparent), and physiological saline at the top (pale yellow). A thin, milky-white cell layer rich in immune cells lies between the separation medium and physiological saline. Aspirate the middle white cell layer and add it to a new 50 mL centrifuge tube to obtain mononuclear cells.

[0046] Example 2

[0047] This embodiment provides a method for processing cell samples.

[0048] Take 30 mL of peripheral blood, centrifuge at 2000 rpm for 10 min to separate plasma, dilute the remaining blood cells to the original volume, add 0.5 μg / mL OKT3 monoclonal antibody, incubate at 4℃ for 40 min, gently mix every 10 min; add 1 μg / mL goat anti-mouse IgG secondary antibody, incubate at room temperature for 30 min to promote cross-linking and formation of large agglutination groups, wash the centrifuged cells with PBS 4 times; then perform Ficoll density gradient centrifugation (refer to Example 1) to obtain mononuclear cells.

[0049] Example 3

[0050] This embodiment provides a method for processing cell samples.

[0051] Take 30 mL of peripheral blood, centrifuge at 2000 rpm for 10 min to separate plasma, dilute the remaining blood cells to the original volume, add 1.5 μg / mL OKT3 monoclonal antibody, incubate at 4℃ for 40 min, gently mix every 10 min; add 3.5 μg / mL goat anti-mouse IgG secondary antibody, incubate at room temperature for 30 min to promote cross-linking and formation of large agglutination groups, wash the centrifuged cells with PBS 4 times; then perform Ficoll density gradient centrifugation (refer to Example 1) to obtain mononuclear cells.

[0052] Example 4

[0053] This embodiment provides a method for processing cell samples.

[0054] Take 30 mL of peripheral blood, centrifuge at 2000 rpm for 10 min to separate plasma, dilute the remaining blood cells to the original volume, add 0.1 μg / mL OKT3 monoclonal antibody, incubate at 4℃ for 40 min, gently mix every 10 min; add 0.2 μg / mL goat anti-mouse IgG secondary antibody, incubate at room temperature for 30 min to promote cross-linking and formation of large agglutination groups, wash the centrifuged cells with PBS 4 times; then perform Ficoll density gradient centrifugation (refer to Example 1) to obtain mononuclear cells.

[0055] Example 5

[0056] This embodiment provides a method for processing cell samples.

[0057] Take 30 mL of peripheral blood, centrifuge at 2000 rpm for 10 min to separate plasma, dilute the remaining blood cells to the original volume, add 2 μg / mL OKT3 monoclonal antibody (primary antibody), incubate at 4℃ for 40 min, gently mix every 10 min; add 4 μg / mL goat anti-mouse IgG secondary antibody, incubate at room temperature for 30 min to promote cross-linking and formation of large agglutination groups, wash the centrifuged cells with PBS 4 times; then perform Ficoll density gradient centrifugation (refer to Example 1) to obtain mononuclear cells.

[0058] Example 6

[0059] This embodiment provides a method for in vitro expansion of NK cells.

[0060] Take the mononuclear cells obtained in Example 1, select 2-3E7 mononuclear cells, and culture them in Corning 581 medium (with 1-10% serum substitute, fetal bovine serum, or autologous plasma added). Add cells to help expand NK cells (natural killer (NK) cell expansion reagent, from Zhongying Biotechnology, ZY-NKZ-0104) at a ratio of 1:1. Culture conditions: 37℃, CO2 concentration 5%, T75 culture flask. Starting from day 3, replenish the medium every 24 hours to maintain the cell density at 2E6 / mL. On days 7-8, add cells to help expand NK cells again (natural killer (NK) cell expansion reagent, from Zhongying Biotechnology, ZY-NKZ-0104) for secondary activation at a ratio of 10:1. Afterward, fluid was added every 24 hours to maintain the cell density at 2E6 / mL. Cells were cultured for 13-14 days before being collected, and the corresponding parameters of NK cells (viability, purity) were detected.

[0061] Example 7

[0062] This embodiment provides a method for in vitro expansion of NK cells. Compared with Example 6, the only difference is that the mononuclear cells are replaced with an equal amount of mononuclear cells obtained in Example 2.

[0063] Example 8

[0064] This embodiment provides a method for in vitro expansion of NK cells. Compared with Example 6, the only difference is that the mononuclear cells are replaced with an equal amount of mononuclear cells obtained in Example 3.

[0065] Example 9

[0066] This embodiment provides a method for in vitro expansion of NK cells. Compared with Example 6, the only difference is that the mononuclear cells are replaced with an equal amount of mononuclear cells obtained in Example 4.

[0067] Example 10

[0068] This embodiment provides a method for in vitro expansion of NK cells. Compared with Example 6, the only difference is that the mononuclear cells are replaced with an equal amount of mononuclear cells obtained in Example 5.

[0069] Comparative Example 1

[0070] This embodiment uses a traditional separation method to process peripheral blood. 30 mL of peripheral blood is taken and centrifuged at 2000 rpm for 10 minutes to separate the plasma. The remaining blood cells are diluted to their original volume. 10-15 mL of Ficoll lymphocyte separation medium is slowly added from the bottom to a 50 mL centrifuge tube. Using a pipette, diluted blood is drawn and slowly added along the wall of the centrifuge tube containing the Ficoll lymphocyte separation medium, close to the liquid surface, forming two layers of blood and separation medium. After balancing, centrifuge at 750 g for 25 minutes with the centrifuge speed set to zero. After centrifugation, four distinct layers are visible: red blood cells at the bottom (deep red), separation medium in the middle layer (white to transparent), and physiological saline at the top (pale yellow). A thin, milky-white cell layer rich in immune cells is located between the separation medium and physiological saline. The middle white cell layer is aspirated and added to a new 50 mL centrifuge tube to obtain mononuclear cells.

[0071] Comparative Example 2

[0072] This comparative example uses mononuclear cells obtained from Comparative Example 1 to expand NK cells in vitro, and the specific method is as described in Example 6.

[0073] Test case

[0074] (1) Test the purity of NK cells in cell samples after treatment in Examples 1-5 and Comparative Example 1.

[0075] Detection method: Take 1 mL of cell suspension, and mix 20 μL of it with 20 μL of trypan blue. Wait about 30 s, then count the cells on the instrument. Centrifuge the remaining sample at 300 g for 3 min.

[0076] Based on the counting results, discard the supernatant, add an appropriate amount of PBS / physiological saline to resuspend the cells, and adjust the cell concentration to 1E7 cells / mL.

[0077] Take several 1.5 mL centrifuge tubes, add 2-5 μL of CD3 and CD56 antibodies to each tube, add 40 μL of the resuspended sample to each tube, and incubate at room temperature in the dark for 15 min.

[0078] After incubation, add 0.5 mL of PBS / physiological saline to each tube and centrifuge at 300 g for 3 min.

[0079] Discard the supernatant, resuspend in 0.5 mL of PBS / physiological saline, and then perform the analysis.

[0080] Figure 1 and Figure 2 To exemplify the detection results of Example 1 and Comparative Example 1, compared to traditional processing methods, the method designed in this invention increased the NK cell purity in the sample from 5.05% to 38.59%, an increase of 664%, while reducing the T cell percentage from 50.46% to 0.65%, removing 98.7% of the T cells. This demonstrates that the processing method designed in this invention can effectively improve the purity of NK cells in the sample.

[0081] Furthermore, the results of the samples treated in Examples 2-5 are shown in Table 1, indicating that the targeted design of the working concentrations of the primary and secondary antibodies in this invention can further improve the separation effect.

[0082] Table 1

[0083]

[0084] (2) Test the expanded cultured NK cells of Examples 6-10 and Comparative Example 2

[0085] Figure 3 and Figure 4To exemplify the detection results of Example 6 and Comparative Example 2, samples obtained using the conventional processing method in Comparative Example 2 were amplified and cultured. Due to the low initial NK cell ratio, the purity of the final amplified NK product was only 52.96%, failing to meet the 90% standard, and had to be discarded. However, samples obtained using the processing method designed in this invention achieved an NK cell purity of 92.63%, meeting the standard and qualifying as a high-purity product. This demonstrates that the sample processing method designed in this invention can be effectively applied to in vitro NK cell amplification, especially for samples with low initial NK cell purity, significantly improving the success rate of achieving the required quality standards for amplified NK cell products.

[0086] Furthermore, the results of the samples treated in Examples 7-10 are shown in Table 2, indicating that the targeted design of primary and secondary antibody concentrations in this invention can further improve the purity of NK cells obtained from subsequent amplification.

[0087] Table 2

[0088]

[0089] In summary, the present invention designs a specific processing method that can quickly process blood and other samples, improve the purity of NK cells, and has broad application prospects. For example, it can be applied to the process of in vitro expansion of NK cells from samples with low initial NK cell purity. The processed sample can be effectively expanded to prepare qualified NK cells, significantly improving the success rate of achieving the quality standard of the expanded NK cell product. It also has the advantages of simple operation, few steps, no need for special equipment, low cost, and good batch stability.

[0090] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for processing cell-containing samples, characterized in that, The processing method includes: Take a cell-containing sample containing T cells, add T cell antigen-antibody to the cell-containing sample, add the antibody of the T cell antigen-antibody, and centrifuge.

2. The method for processing cell-containing samples according to claim 1, characterized in that, The T-cell antigen antibody includes CD3 antibody; Preferably, the CD3 antibody comprises a murine OKT3 monoclonal antibody; Preferably, the antibody against the T-cell antigen includes a goat anti-mouse antibody; Preferably, the cell-containing sample also contains NK cells.

3. The method for processing cell-containing samples according to claim 1 or 2, characterized in that, The working concentration of the T-cell antigen antibody is 0.1~2 μg / mL, preferably 0.5~1.5 μg / mL.

4. The method for processing cell-containing samples according to any one of claims 1-3, characterized in that, The working concentration of the T-cell antigen antibody is 0.2~4 μg / mL, preferably 1~3.5 μg / mL.

5. The method for processing cell-containing samples according to any one of claims 1-4, characterized in that, The cell-containing sample includes a blood sample; Preferably, the blood sample includes peripheral blood or umbilical cord blood.

6. The method for processing cell-containing samples according to any one of claims 1-5, characterized in that, The centrifugation method includes the Ficoll density gradient centrifugation method.

7. The method for processing cell-containing samples according to any one of claims 1-6, characterized in that, The centrifugation conditions include: centrifugation at 600-800 g for 10-20 min.

8. The application of the cell sample processing method according to any one of claims 1-7 in the in vitro expansion of NK cells.

9. A method for in vitro expansion of NK cells, characterized in that, The method for in vitro expansion of NK cells includes: A blood sample is processed using the cell-containing sample processing method according to any one of claims 1-7, mononuclear cells are collected, and the mononuclear cells are cultured to obtain NK cells.

10. The method for in vitro expansion of NK cells according to claim 9, characterized in that, The culture medium contains cells that help NK cells expand.

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