Cellular in vitro propagation method

Abstract

The present application provides a method for in vitro proliferation of cells, comprising the following steps: adding a NKG2A conjugate to a first cell culture solution to form a second cell culture solution; and adding a cell to a cell culture container containing the second cell culture solution for a cell expansion culture to obtain an expanded cell. The method for in vitro proliferation of cells provided by the present application can efficiently obtain a large number of high-purity NK cells, and the expanded cells have a significant tumor killing ability.

Description

Technical Field

[0001] This invention relates to a method for in vitro cell proliferation, particularly a method for the in vitro expansion of natural killer cells (NK cells) using a culture medium containing NKG2A conjugates, which is applicable in the biomedical field. Background Technology

[0002] In recent years, immunocellular therapy has been widely used in clinical research and is considered a promising cancer treatment method. Most immunocellular therapies are T-cell based, including chimeric antigen receptor T-cell (CAR-T) therapy and cytokine-induced killer cell (CIK) therapy. However, the high cost of CAR-T therapy limits its accessibility and widespread application. Furthermore, T-cell-based therapies are prone to inducing acute graft-versus-host disease (GvHD). Therefore, natural killer cell (NK) cell therapy, due to its relative safety and potential advantages, is gradually becoming a promising cell therapy option.

[0003] Unlike T cells, NK cells can recognize tumor cells in real time without MHC matching and eliminate them by activating their surface receptors (such as NKG2D) to promote the release of cytotoxic molecules such as perforin and granolithase. However, NK cells constitute a relatively small proportion of the human body, accounting for only 10% of peripheral blood mononuclear cells (PBMCs). Even with large-scale proliferation of NK cells in the body, the side effects caused by NK cells are relatively lower compared to the rapid and large-scale proliferation of T cells.

[0004] Current technologies promote NK cell proliferation by introducing receptors that stimulate NK cell activity and using exogenous cytokines (such as IL-2 and IL-15), but these methods still have limitations in significantly increasing the number of NK cells proliferating. Furthermore, prolonged in vitro expansion processes can lead to NK cell aging or functional decline, thereby weakening their efficacy. Simultaneously, NK cells cultured using existing techniques are susceptible to T cell contamination, making it difficult to obtain high-purity NK cells from peripheral blood. More seriously, after multiple rounds of expansion, the activity of NK cells obtained through existing techniques significantly decreases, thus weakening their anti-tumor ability. Therefore, developing an in vitro proliferation technology that can efficiently obtain high-purity, highly active, and high-cell-count NK cells is crucial for clinical applications. Summary of the Invention

[0005] In view of this, the present invention provides a method for in vitro cell proliferation, comprising the following steps: adding an NKG2A conjugate to a first cell culture medium to form a second cell culture medium; and adding a cell to a cell culture container containing the second cell culture medium for cell amplification culture to obtain an amplified cell.

[0006] The cell was further identified as a peripheral blood mononuclear cell.

[0007] The volumetric molar concentration of the NKG2A conjugate ranges from 1 mM to 3 mM.

[0008] The NKG2A conjugate comprises a peptide chain, and an amino acid sequence of the peptide chain comprises PSSCIGVFRNSSHHPW (SEQ ID NO:1), ISIDNEEMKF (SEQ ID NO:2), CD94 LAFKHEIKDSEN (SEQ ID NO:3) and HLA-E QLNRLKSAQCGSSIIYHC (SEQ ID NO:4).

[0009] Of this, PSSCIGVFRNSSHHPW accounts for 25% to 35% of the NKG2A conjugate.

[0010] Of this, ISIDNEEMKF accounts for 15% to 25% of the NKG2A conjugate.

[0011] CD94 LAFKHEIKDSEN accounts for 20% to 30% of the NKG2A conjugate.

[0012] Of this, HLA-E QLNRLKSAQCGSSIIYHC accounts for 20% to 30% of the NKG2A conjugate.

[0013] The step of adding the cells to a cell culture container containing the second cell culture medium for cell expansion culture further includes the following sub-steps: at a first time point, culturing the cells in a first culture container containing the second cell culture medium of the NKG2A conjugate, and forming a first cell; at a second time point, transferring the cells to a second culture container containing the second cell culture medium of the NKG2A conjugate, and forming a second cell; at a third time point, removing the second culture medium from the second culture container, culturing the second cell in the first cell culture medium, and forming a cell slurry; and at a fourth time point, centrifuging and washing the cell slurry and removing the supernatant from the cell slurry to obtain the expanded cells, wherein the centrifugation speed is 300×g to 600×g, and the centrifugation time is 10 minutes to 15 minutes.

[0014] The first time point is day 0, the second time point is day 3, the third time point is day 10, and the fourth time point is day 14.

[0015] Among them, the expanded cells are further classified as natural killer cells (NK cells).

[0016] In summary, this invention involves adding an NKG2A conjugate at the initial stage of cell culture to temporarily inhibit NK cell activity, followed by cell culture. The NKG2A conjugate is then removed, and the cells are cultured for several more days to obtain a large number of expanded NK cells. The in vitro cell proliferation method provided by this invention can efficiently obtain a large number of high-purity NK cells, and the expanded NK cells exhibit a large number of NKG2A surface receptors and possess significant tumor-killing ability.

[0017] Furthermore, this invention provides an effective solution to the technical challenge of the inability to massively expand NK cells in vitro. Previous studies have shown that the proliferation capacity of NK cells during in vitro culture is limited by various environmental constraints (e.g., cell self-regulation mechanisms, lack of specific growth factors, and immune function degradation due to long-term culture), typically making it difficult to reach the quantities required for clinical treatment or research. This invention effectively enhances the expansion capacity of NK cells by optimizing culture conditions, thereby supporting larger-scale clinical applications.

[0018] Furthermore, in clinical treatment, the high-purity NK cells generated by the in vitro cell proliferation method of this invention can more effectively exert their immune function, significantly enhance the efficacy of cell therapy, and ensure the consistency and stability of therapeutic effects, thereby improving the predictability of treatment. In addition, the reduction in the number of other immune cells helps to lower the risk of acute graft-versus-host disease (GvHD) and avoid unnecessary immune reactions that could harm patients. Moreover, the NK cells proliferated using this invention have high activity, which can significantly enhance their efficacy in the patient's body, achieving more ideal treatment outcomes. Attached Figure Description

[0019] Figure 1 A flowchart illustrating the steps of a cell proliferation method in vitro according to a specific embodiment of the present invention is provided.

[0020] Figure 2 A flowchart illustrating the steps of an in vitro cell proliferation method according to another specific embodiment of the present invention is provided.

[0021] Figure 3 The results of an experiment to increase the number of NK cells cultured for several days.

[0022] Figure 4 The results of the NK cell purity analysis experiment obtained by conventional techniques in expanded cells.

[0023] Figure 5 The results are from the NK cell purity analysis experiment obtained in the expanded cells of this invention.

[0024] Figure 6 The results show the experimental findings on the expression level of the NKG2A surface receptor on NK cells obtained using conventional techniques.

[0025] Figure 7 The results show the experimental results of NKG2A surface receptor expression levels on NK cells obtained by this invention.

[0026] Figure 8 The experimental results show the ratio of NKG2A to NKG2D.

[0027] Figure 9 To compare the experimental results of cell expansion and growth fold between the present invention and the prior art.

[0028] Figure 10 The results of experiments demonstrating the tumor-killing ability of cells amplified using known techniques.

[0029] Figure 11 The results show the tumor-killing ability of the expanded cells obtained by the in vitro cell expansion method of the present invention.

[0030] In the attached figures, the following labels are used:

[0031] S1~S2, S21~S24: Steps Detailed Implementation

[0032] To make the advantages, spirit, and features of the present invention easier and clearer to understand, detailed descriptions and discussions will follow with reference to specific embodiments and the accompanying drawings. It should be noted that these specific embodiments are merely representative examples of the present invention, and the specific methods, apparatus, conditions, materials, etc., exemplified are not intended to limit the present invention or the corresponding specific embodiments. Furthermore, the components in the figures are only used to illustrate their relative positions and are not drawn to scale; the step numbers in the present invention are only for distinguishing different steps and do not represent the order of the steps, as will be stated previously.

[0033] Please see Figure 1 , Figure 1 This is a flowchart illustrating the steps of an in vitro cell proliferation method according to a specific embodiment of the present invention. The in vitro cell proliferation method of the present invention includes the following steps: Step S1: Adding the NKG2A conjugate to a first cell culture medium to form a second cell culture medium; Step S2: Adding cells to a cell culture container containing the second cell culture medium for cell expansion culture to obtain expanded cells. In this specific embodiment, the cells may include peripheral blood mononuclear cells. The expanded cells may be natural killer cells (NK cells). However, the method is not limited to this in practical applications. The in vitro cell proliferation method of the present invention can also be applied to the expansion of different types of cells.

[0034] In this specific embodiment, the NKG2A conjugate may contain four amino acid sequences: PSSCIGVFRNSSHHPW, ISIDNEEMKF, CD94 LAFKHEIKDSEN, and HLA-E QLNRLKSAQCGSSIIYHC. The proportions of each amino acid sequence in the NKG2A conjugate may vary: PSSCIGVFRNSSHHPW accounts for 25% to 35% of the NKG2A conjugate; ISIDNEEMKF accounts for 15% to 25%; CD94 LAFKHEIKDSEN accounts for 20% to 30%; and HLA-E QLNRLKSAQCGSSIIYHC accounts for 20% to 30%. The optimal ratio is a combination of 30% PSSCIGVFRNSSHHPW, 20% ISIDNEEMKF, 25% CD94 LAFKHEIKDSEN, and 25% HLA-EQLNRLKSAQCGSSIIYHC. This composition ratio has been shown to significantly enhance the proliferation efficiency of NK cells, demonstrating excellent proliferative effects and application potential.

[0035] In practice, approximately 30 to 50 mL of peripheral blood is drawn from the individual and centrifuged to separate the plasma. The centrifugation speed is 300 × g to 600 × g, and the centrifugation time is 10 to 15 minutes. Subsequently, the separated plasma is heated to 55°C to 60°C for deactivation treatment, effectively reducing the activity of pathogens and inhibiting the reproduction of viruses, bacteria, and other microorganisms, ensuring its safety in clinical applications. Further, the deactivated plasma is centrifuged using the Ficoll-Paque density gradient centrifugation method to separate the individual's peripheral blood mononuclear cells. The centrifugation speed is also 300 × g to 600 × g, and the centrifugation time is 10 to 15 minutes. In practical applications, the individuals mentioned can include primates (such as humans and non-human primates), mice, and rats. In practical applications, the steps, conditions, and procedures for separating peripheral blood mononuclear cells can also be adjusted according to user needs and experimental design.

[0036] The in vitro cell proliferation method of the present invention may further include other specific embodiments. Please refer to [link / reference]. Figure 2 , Figure 2 A flowchart illustrating the steps of an in vitro cell proliferation method according to another specific embodiment of the present invention is provided. Figure 2 As shown, this specific embodiment differs from the above specific embodiment in that the in vitro cell proliferation method of this specific embodiment further includes steps S21 to S24, which are performed after step S1. Step S21: At a first time point, cells are cultured in a first culture container, wherein the first culture container contains a second cell culture medium containing the NKG2A conjugate, and first cells are formed; Step S22: At a second time point, the cells are transferred to a second culture container, wherein the second culture container contains a second cell culture medium containing the NKG2A conjugate, and second cells are formed; Step S23: At a third time point, the second culture medium in the second culture container is removed, and the second cells are cultured in the first cell culture medium, forming cell slurry; Step S24: At a fourth time point, the cell slurry is centrifuged and washed, and the supernatant in the cell slurry is removed to obtain expanded cells, wherein the centrifugation speed is 300×g to 600×g, and the centrifugation time is 10 minutes to 15 minutes. In this specific embodiment, the first culture medium can be SCGM (Stem Cell Growth Medium), and the second culture medium is SCGM medium with NKG2A conjugate added. The first culture container can include a T75 culture flask, and the second culture container can include a T175 culture flask. However, in practice, it is not limited to these, and the culture medium and culture container can be selected according to the user's needs or experimental conditions.

[0037] In this specific embodiment, the time points are defined as follows: the first time point is day 0, the second time point is day 3, the third time point is day 10, and the fourth time point is day 14. Day 0 represents the first day of the culture operation, i.e., the day the cells are placed in the incubator, and subsequent time points are calculated accordingly. On day 0 of culture, 250 μL to 500 μL of NKG2A conjugate and 5% to 10% plasma are added to 20 mL of the first culture medium to form the second culture medium, which is then mixed with peripheral blood mononuclear cells to form the first cell type. The first cells are cultured in T75 culture flasks and placed in an incubator at 37°C and 5% CO2. The cell number of peripheral blood mononuclear cells can range from 1 × 10⁻⁶ cells / year. 7 cells up to 3×10 7 cells.

[0038] On day 3 of the actual culture process, the first cells were transferred from a T75 culture flask to a T175 culture flask, and 100-200 μL of NKG2A conjugate was added and mixed with 5%-10% plasma to form a second culture medium. The second culture medium was then mixed with the first cells to form the second cell line, at which point the number of the first cells was approximately 5 × 10⁻⁶. 5 cells up to 1×10 6 Cells were cultured in a second cell culture medium at 37°C in a 5% CO2 incubator. On day 10, the second culture medium was removed from the T175 culture flask, and the first culture medium containing 1% to 2.5% plasma was added to continue expansion culture. On day 14, the cell culture fluid in the T175 culture flask was centrifuged at a speed ranging from 300×g to 600×g for 10 to 15 minutes. After removing the supernatant, the cells were washed with physiological saline and centrifuged again, for a total of two washings. After this treatment, the expanded cells were isolated.

[0039] Furthermore, experiments were designed using the in vitro cell proliferation method of the present invention to demonstrate that the in vitro cell proliferation method of the present invention can achieve better NK cell proliferation effects. Please refer to the following in sequence. Figures 3 to 11 These accompanying figures illustrate the specific results of the cell proliferation experiments.

[0040] First, please refer to Figure 3 , Figure 3 The results of an experiment to increase the number of NK cells cultured for several days. Figure 3 This shows the proliferation of NK cells at various time points from day 0 to day 14 of culture. Based on... Figure 3 Data showed that on day 10 of culture, the number of NK cells had increased to 2.5 × 10⁻⁶. 8 The cells further multiplied to approximately 1.8 × 10⁻⁶ on day 14 of culture. 9This shows that the cell proliferation rate significantly increased during the later stages of culture, especially between days 10 and 14, with a significant upward trend in the proliferation slope, further demonstrating that the in vitro cell proliferation method of the present invention has a good promoting effect on NK cell proliferation. This may be related to the mechanism of action of the NKG2A conjugate. The NKG2A conjugate targets and binds to the NKG2A inhibitory receptor on the surface of NK cells, inhibiting NK cell activity and reducing the release of cytokines such as perforin, thereby weakening their cytotoxic ability.

[0041] However, in some embodiments, it has been found that inhibiting NK cell activity with NKG2A conjugates in the early stages of cell culture actually promotes the proliferation of NK cells.

[0042] Please see Figure 4 as well as Figure 5 , Figure 4 The results of the NK cell purity analysis experiment obtained from expanded cells using conventional techniques. Figure 5 This presents the experimental results of NK cell purity analysis in the expanded cells obtained in this invention. A mononuclear cell population was screened using flow cytometry, and the purity of NK cells in the expanded cells was further identified and analyzed based on the expression characteristics of cell surface antigens. For example... Figure 4 as well as Figure 5 As shown, the horizontal axis represents CD3 fluorescence expression level, and the vertical axis represents CD56 fluorescence expression level. The four quadrants in the figure are Q1, Q2, Q3, and Q4. Specifically, Q1 represents NK cells that do not express CD3 antigen but express CD56 antigen; Q2 represents NKT cells that express both CD3 and CD56 antigens; Q3 represents cells that do not express either CD3 or CD56 antigens; and Q4 represents T cells that express CD3 antigen but not CD56 antigen. Experimental results show that the NK cell population obtained by this invention, which does not express CD3 antigen but expresses CD56 antigen, accounts for 90.75% of the total monocytes. Compared with expanded cells obtained by conventional techniques, the purity of NK cells is significantly improved. Therefore, the in vitro cell proliferation method provided by this invention can effectively obtain high-purity NK cells, providing a reliable cell source for clinical applications.

[0043] Please see Figure 6 , Figure 7 as well as Figure 8 , Figure 6 The results show the experimental findings on the expression level of the NKG2A surface receptor on NK cells obtained using conventional techniques. Figure 7 The results show the experimental results of NKG2A surface receptor expression levels on NK cells obtained by this invention. Figure 8The results show the NKG2A / NKG2D ratio. The purity of NK cells in the expanded cells was analyzed by flow cytometry. First, a monocyte population was screened, and NK cells were isolated from the expanded cells based on the expression characteristics of cell surface antigens. Then, the number of NK cells expressing the NKG2A surface receptor was further analyzed. Figure 6 as well as Figure 7 As shown in the figure, the four quadrants are Q1-1, Q2-1, Q3-1, and Q4-1, respectively. Among them, 56.3% of NK cells amplified and cultured using conventional techniques express the NKG2A surface receptor, while the NK cells obtained using the amplification method of this invention express up to 85.8% of the NKG2A surface receptor, significantly higher than those obtained using conventional techniques. Furthermore, as... Figure 8 As shown, in a population of NK cells that do not express CD3 antigen but express CD56 antigen, the ratio of NKG2A / NKG2D expression levels was plotted against the groups of the conventional technique and the group of the present invention. The ratio of NKG2A / NKG2D expression levels in the NK cells amplified by the present invention was significantly increased compared with the group of the conventional technique, further demonstrating the advantages of the present invention in promoting NK cell proliferation and NKG2A surface receptor expression.

[0044] Please see Figure 9 , Figure 9 To compare the cell expansion fold growth results between the present invention and conventional techniques, the sample size for both the present invention group and the conventional technique group was 4 (N=4). All cells were statistically analyzed after 14 days of culture. The results showed that, compared to day 0, the average cell expansion fold in the conventional technique group was 579.8308, while the average cell expansion fold in the present invention group was 1207.559. ANOVA analysis showed a p-value of 0.01717, indicating a significant difference between the two groups. The in vitro cell expansion method provided by the present invention can significantly increase NK cell production capacity.

[0045] Please see Figure 10 as well as Figure 11 , Figure 10 The results of experiments demonstrating the tumor-killing ability of cells amplified using known techniques. Figure 11 This presents the experimental results of the tumor-killing ability of the expanded cells obtained using the in vitro cell expansion method of the present invention. Specifically, this experiment involved mixing immune cells and tumor cells at different ratios (ET Ratio) and observing the percentage of tumor cell cytotoxicity by the immune cells to assess the tumor-killing ability of the immune cells. Figure 10 As shown, the conventional technology group exhibited NK cell cytotoxicity of 7.7%, 28.8%, 60.3%, and 86.0% at ET Ratios of 0.3, 1, 3, and 10, respectively. Figure 11As shown, the NK cell cytotoxicity of the groups of the present invention at ET Ratios of 0.3, 1, 3, and 10 were 36.8%, 83.8%, 95.5%, and 98.2%, respectively. The results indicate that, compared to the conventional technology groups, the NK cell cytotoxicity of the groups of the present invention increased by 55% at an ET Ratio of 1, and the ability of NK cells to kill tumor cells approached 100% at ET Ratios of 3 and 10. Furthermore, although the final expanded NK cell population of the present invention expressed more NKG2A receptors than that of the conventional technology, these NK cells still exhibited higher expansion capacity and stronger cancer cell killing ability, demonstrating the significant advantages of the present invention in enhancing the potential of NK cell immunotherapy.

[0046] In summary, this invention involves adding an NKG2A conjugate at the initial stage of cell culture to temporarily inhibit NK cell activity, followed by cell culture. The NKG2A conjugate is then removed, and the cells are cultured for several more days to obtain a large number of expanded NK cells. The in vitro cell proliferation method provided by this invention can efficiently obtain a large number of high-purity NK cells, and the expanded NK cells exhibit a large number of NKG2A surface receptors and possess significant tumor-killing ability.

[0047] Furthermore, this invention provides an effective solution to the technical challenge of the inability to massively expand NK cells in vitro. Previous studies have shown that the proliferation capacity of NK cells during in vitro culture is limited by various environmental constraints (e.g., cell self-regulation mechanisms, lack of specific growth factors, and immune function degradation due to long-term culture), typically making it difficult to reach the quantities required for clinical treatment or research. This invention effectively enhances the expansion capacity of NK cells by optimizing culture conditions, thereby supporting larger-scale clinical applications.

[0048] Furthermore, in clinical treatment, the high-purity NK cells generated by the in vitro cell proliferation method of this invention can more effectively exert their immune function, significantly enhance the efficacy of cell therapy, and ensure the consistency and stability of therapeutic effects, thereby improving the predictability of treatment. In addition, the reduction in the number of other immune cells helps to lower the risk of acute graft-versus-host disease (GvHD) and avoid unnecessary immune reactions that could harm patients. Moreover, the NK cells proliferated using this invention have high activity, which can significantly enhance their efficacy in the patient's body, achieving more ideal treatment outcomes.

[0049] It should be noted that relational terms in this document, such as “first” and “second”, are used only to distinguish an entity or operation from another entity or operation, without requiring or implying any actual relationship or order between these entities or operations. Furthermore, the words “including,” “having,” and “containing,” as well as other similar forms, are intended to be equivalent in meaning and are open-ended; one or more items following any of these words do not imply an exhaustive list of such items or that the list is limited to only one or more items.

[0050] As used herein, a "peptide chain" refers to a short-chain polymer composed of amino acid residues, which may possess a local or overall three-dimensional configuration. Peptide chains include, but are not limited to, short-chain molecules that are naturally or artificially synthesized, recombinant, or modified, such as hormones, antibiotics, and signaling molecules. Peptide chains may contain post-translational modifications, non-natural amino acids, prosthetic groups, and their analogues.

[0051] The range should be understood to include the value at the end of the range and all logical values ​​in between. For example, 5% to 10% should be understood to include 5% and 10% as well as all possible values ​​in between.

[0052] The detailed description of the preferred embodiments above is intended to more clearly illustrate the features and spirit of the present invention, and is not intended to limit the scope of the invention to the preferred embodiments disclosed above. Rather, the aim is to cover various modifications and equivalent arrangements within the scope of the claims to which this invention is sought. Therefore, the scope of protection of the claims of this invention should be interpreted in the broadest possible sense based on the foregoing description, so as to cover all possible modifications and equivalent arrangements.

Claims

1. A method for in vitro cell proliferation, comprising the following steps: An NKG2A conjugate is added to a first cell culture medium to form a second cell culture medium; and A cell is added to a cell culture container containing the second cell culture medium for cell amplification culture to obtain an amplified cell.

2. The in vitro cell proliferation method according to claim 1, wherein the cell is further a peripheral blood mononuclear cell (PBMC), and the expanded cell is further a natural killer cell (NK cell).

3. The in vitro cell proliferation method according to claim 1, wherein the volumetric molar concentration of the NKG2A conjugate ranges from 1 mM to 3 mM.

4. The in vitro cell proliferation method according to claim 1, wherein the NKG2A conjugate comprises a peptide chain, and an amino acid sequence of the peptide chain comprises PSSCIGVFRNSSHHPW, ISIDNEEMKF, CD94 LAFKHEIKDSEN, and HLA-E. QLNRLKSAQCGSSIIYHC.

5. The in vitro cell proliferation method according to claim 4, wherein the PSSCIGVFRNSSHHPW accounts for 25% to 35% of the NKG2A conjugate.

6. The in vitro cell proliferation method according to claim 4, wherein the ISIDNEEMKF accounts for 15% to 25% of the NKG2A conjugate.

7. The method for in vitro cell proliferation according to claim 4, wherein the CD94 LAFKHEIKDSEN accounts for 20% to 30% of the NKG2A conjugate.

8. The in vitro cell proliferation method according to claim 4, wherein the HLA-E QLNRLKSAQCGSSIIYHC accounts for 20% to 30% of the NKG2A conjugate.

9. The method for in vitro cell proliferation according to claim 1, wherein the step of adding the cells to the cell culture container containing the second cell culture medium for cell expansion culture further comprises the following sub-steps: At a first time point, the cell is cultured in a first culture vessel containing a second cell culture medium containing the NKG2A conjugate, and a first cell is formed. At a second time point, the cell is transferred to a second culture vessel for culture, wherein the second culture vessel contains the second cell culture medium containing the NKG2A conjugate, and a second cell is formed; At a third time point, the second culture medium is removed from the second culture vessel, and the second cells are cultured in the first cell culture medium to form a cell culture medium; and At a fourth time point, the cell solution is centrifuged and washed, and a supernatant is removed to obtain the expanded cells. The centrifugation speed is 300×g to 600×g, and the centrifugation time is 10 minutes to 15 minutes.

10. The in vitro cell proliferation method according to claim 1, wherein the first time point is day 0, the second time point is day 3, the third time point is day 10, and the fourth time point is day 14.

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