In vitro proliferation method for cells

Abstract

This 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 expansion culture to obtain expanded 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 cells possess significant tumor-killing ability.

Description

Technical Field

[0001] This invention relates to a method for in vitro cell proliferation, and more particularly to a method for the in vitro expansion of natural killer cells (NK cells) through a culture medium containing NKG2A conjugates, which is used in the biomedical field. Prior 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 instantly recognize tumor cells 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 significant NK cell proliferation 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, the long-term in vitro expansion process may lead to NK cell aging or functional decline, thereby weakening their efficacy. Simultaneously, NK cells cultured using existing techniques are easily contaminated by T cells, 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 expansion culture to obtain an expanded cell.

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

[0007] One of the NKG2A conjugates has a volume molar concentration ranging from 1 mM to 3 mM.

[0008] The NKG2A conjugate comprises a peptide chain, wherein one 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 which, PSSCIGVFRNSSHHPW (SEQ ID NO:1) accounts for 25% to 35% of the NKG2A conjugate.

[0010] Of which, ISIDNEEMKF (SEQ ID NO:2) accounts for 15% to 25% of the NKG2A conjugate.

[0011] Of which, CD94 LAFKHEIKDSEN (SEQ ID NO:3) accounts for 20% to 30% of the NKG2A conjugate.

[0012] HLA-E QLNRLKSAQCGSSIIYHC (SEQ ID NO:4) 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 one centrifugation speed is 300×g to 600×g, and one 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 NK cells, when cultured in vitro, are subject to various environmental limitations (e.g., cell self-regulation mechanisms, lack of specific growth factors, and immune function degradation due to long-term culture), limiting their proliferation capacity and typically making it difficult to reach the quantities required for clinical treatment or research. This invention, by optimizing culture conditions, effectively enhances the expansion capacity of NK cells, thereby supporting larger-scale clinical applications.

[0018] Furthermore, in clinical treatment, the high-purity NK cells generated through the in vitro cell proliferation method of this invention can more effectively exert their immune function, significantly enhancing the efficacy of cell therapy and ensuring 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), avoiding unnecessary immune responses that could harm patients. Moreover, the NK cells proliferated through this invention have higher activity, significantly enhancing their efficacy in the patient's body and achieving more ideal treatment outcomes. Simple Explanation of the Diagram Figure 1 is a flowchart illustrating the steps of an in vitro cell proliferation method according to a specific embodiment of the present invention. Figure 2 is a flowchart illustrating the steps of an in vitro cell proliferation method according to another specific embodiment of the present invention. Figure 3 shows the experimental results of NK cell expansion after several days of culture. Figure 4 shows the experimental results of NK cell purity analysis in expanded cells obtained using conventional techniques. Figure 5 shows the experimental results of NK cell purity analysis in the expanded cells obtained in this invention. Figure 6 shows the experimental results of NKG2A surface receptor expression levels in NK cells obtained using conventional techniques. Figure 7 shows the experimental results of NKG2A surface receptor expression levels in NK cells obtained by the present invention. Figure 8 shows the experimental results of the NKG2A / NKG2D ratio. Figure 9 shows the experimental results comparing the cell expansion fold between the present invention and the prior art. Figure 10 shows the experimental results of the tumor-killing ability of expanded cells using conventional techniques. Figure 11 shows the experimental results of the tumor-killing ability of the expanded cells obtained by the in vitro cell expansion method of the present invention. Implementation

[0019] 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 elements in the figures are only used to express 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.

[0020] Please refer to Figure 1, which 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, in practical applications, it is not limited to this. The in vitro cell proliferation method of the present invention can also be applied to the expansion of different types of cells.

[0021] In this specific embodiment, the NKG2A conjugate may contain four amino acid sequences: PSSCIGVFRNSSHHPW (SEQ ID NO:1), ISIDNEEMKF (SEQ ID NO:2), CD94 LAFKHEIKDSEN (SEQ ID NO:3), and HLA-E QLNRLKSAQCGSSIIYHC (SEQ ID NO:4). The proportions of each amino acid sequence in the NKG2A conjugate may vary: PSSCIGVFRNSSHHPW (SEQ ID NO:1) accounts for 25% to 35% of the NKG2A conjugate; ISIDNEEMKF (SEQ ID NO:2) accounts for 15% to 25% of the NKG2A conjugate; CD94 LAFKHEIKDSEN (SEQ ID NO:3) accounts for 20% to 30% of the NKG2A conjugate; and HLA-E QLNRLKSAQCGSSIIYHC (SEQ ID NO:4) accounts for 20% to 30% of the NKG2A conjugate. The preferred ratio is a combination of 30% PSSCIGVFRNSSHHPW (SEQ ID NO:1), 20% ISIDNEEMKF (SEQ ID NO:2), 25% CD94 LAFKHEIKDSEN (SEQ ID NO:3), and 25% HLA-E QLNRLKSAQCGSSIIYHC (SEQ ID NO:4). This composition ratio has been shown to significantly enhance the proliferation efficiency of NK cells, demonstrating excellent proliferative effects and application potential.

[0022] In practice, approximately 30 mL 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.

[0023] The cell proliferation method of the present invention may further include other specific embodiments. Please refer to Figure 2, which is a flowchart illustrating the steps of another specific embodiment of the cell proliferation method of the present invention. As shown in Figure 2, the difference between this specific embodiment and the above specific embodiments is that the 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 containing a second cell culture medium with NKG2A conjugate, forming first cells. Step S22: At a second time point, cells are transferred to a second culture container containing a second cell culture medium with NKG2A conjugate, forming second cells. 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 is removed to obtain expanded cells. The centrifugation speed is 300×g to 600×g, and the centrifugation time is 10 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 culture medium with NKG2A conjugate added. The first culture container can contain a T75 culture flask, and the second culture container can contain a T175 culture flask. However, in practice, this is not the only option; the culture medium and culture container can be selected according to the user's needs or experimental conditions.

[0024] 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 the 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 × 10⁷ cells to 3 × 10⁷ cells.

[0025] On day 3 of the actual procedure, the first cells were transferred from a T75 culture flask to a T175 culture flask, and 100-200 μL of NKG2A conjugate and 5%-10% plasma were added to form the second culture medium. The second culture medium was then mixed with the first cells to form the second cell line, which at this point had a cell count ranging from 5 × 10⁵ cells to 1 × 10⁶ cells. The second cells were cultured 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%-2.5% plasma was added to continue the expansion culture. On day 14, the cell culture 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.

[0026] Furthermore, experiments were designed using the in vitro cell proliferation method of this invention to demonstrate that a better NK cell proliferation effect can be obtained through this method. Please refer to Figures 3 to 11 in sequence; these figures illustrate the specific results of the cell proliferation experiment.

[0027] First, please refer to Figure 3, which shows the experimental results of NK cell proliferation after several days of culture. Figure 3 shows the proliferation of NK cells at various time points from day 0 to day 14 of culture. According to the data in Figure 3, the number of NK cells had expanded to 2.5 × 10⁸ cells by day 10 of culture, and further proliferated to approximately 1.8 × 10⁹ cells by day 14 of culture. This shows that the cell proliferation rate significantly increased during the later stages of culture, especially between day 10 and day 14, where the proliferation slope showed a significant upward trend, 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. However, in some embodiments, it was found that inhibiting NK cell activity with the NKG2A conjugate in the early stages of cell culture actually promoted the proliferation of NK cells.

[0028] Please refer to Figures 4 and 5. Figure 4 shows the results of NK cell purity analysis in expanded cells obtained using conventional techniques, and Figure 5 shows the results of NK cell purity analysis in expanded cells obtained using the present invention. Mononuclear cell populations were 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. As shown in Figures 4 and 5, the horizontal axis represents CD3 fluorescence expression level, and the vertical axis represents CD56 fluorescence expression level. The four quadrants in the figures 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 antigen; Q3 represents cells that do not express either CD3 or CD56 antigen; 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 the 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.

[0029] Please refer to Figures 6, 7, and 8. Figure 6 shows the experimental results of NKG2A surface receptor expression levels in NK cells obtained using conventional techniques. Figure 7 shows the experimental results of NKG2A surface receptor expression levels in NK cells obtained using the present invention. Figure 8 shows the experimental results of the NKG2A / NKG2D ratio. The purity of NK cells in the expanded cells was analyzed by flow cytometry. First, a mononuclear cell 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 NKG2A surface receptors was further analyzed. As shown in Figures 6 and 7, the four quadrants are Q1-1, Q2-1, Q3-1, and Q4-1, respectively. Among them, 56.3% of the NK cells expanded and cultured using conventional techniques expressed NKG2A surface receptors, while the NK cells obtained using the expansion method of the present invention expressed as much as 85.8% of NKG2A surface receptors, which is much higher than that of NK cells obtained using conventional techniques. Furthermore, as shown in Figure 8, the ratio of NKG2A / NKG2D expression in the NK cell population that does not express CD3 antigen but expresses CD56 antigen was plotted against the groups of the conventional technology and the group of the present invention. The ratio of NKG2A / NKG2D expression in the NK cells expanded by the present invention was significantly increased compared with the group of the conventional technology, further demonstrating the advantages of the present invention in promoting NK cell proliferation and NKG2A surface receptor expression.

[0030] Please refer to Figure 9, which shows the experimental results comparing the cell expansion fold growth between the present invention and the conventional technique. The sample size for both the present invention group and the conventional technique group was 4 (N=4), and all cells were statistically analyzed after 14 days of culture. The results showed that, compared to day 0, the average cell expansion fold of the conventional technique group was 579.8308, while the average cell expansion fold of 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 the production capacity of NK cells.

[0031] Please refer to Figures 10 and 11. Figure 10 shows the experimental results of the tumor-killing ability of expanded cells using conventional techniques. Figure 11 shows the experimental results of the tumor-killing ability of expanded cells using the in vitro cell expansion method of the present invention. In this experiment, immune cells and tumor cells were mixed at different ratios (ET Ratio), and the percentage of tumor cell killing by immune cells was observed to evaluate the tumor-killing ability of immune cells. As shown in Figure 10, the NK cell toxicity of the conventional technique group at ET Ratios of 0.3, 1, 3, and 10 was 7.7%, 28.8%, 60.3%, and 86.0%, respectively. As shown in Figure 11, the NK cell toxicity of the present invention group at ET Ratios of 0.3, 1, 3, and 10 was 36.8%, 83.8%, 95.5%, and 98.2%, respectively. The results show that compared to the conventional technique group, the NK cell toxicity of the present invention group 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 NK cell population ultimately expanded by this invention expresses more NKG2A receptors compared to conventional techniques, these NK cells still exhibit higher expansion capacity and stronger cancer cell killing ability, demonstrating the significant advantages of this invention in enhancing the potential of NK cell immunotherapy.

[0032] 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.

[0033] 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 NK cells, when cultured in vitro, are subject to various environmental limitations (e.g., cell self-regulation mechanisms, lack of specific growth factors, and immune function degradation due to long-term culture), limiting their proliferation capacity and typically making it difficult to reach the quantities required for clinical treatment or research. This invention, by optimizing culture conditions, effectively enhances the expansion capacity of NK cells, thereby supporting larger-scale clinical applications.

[0034] Furthermore, in clinical treatment, the high-purity NK cells generated through the in vitro cell proliferation method of this invention can more effectively exert their immune function, significantly enhancing the efficacy of cell therapy and ensuring 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), avoiding unnecessary immune responses that could harm patients. Moreover, the NK cells proliferated through this invention have higher activity, significantly enhancing their efficacy in the patient's body and achieving more ideal treatment outcomes.

[0035] 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.

[0036] As used herein, "peptide chain" refers to a short-chain polymer composed of amino acid residues, which may have local or overall three-dimensional configurations. 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.

[0037] 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.

[0038] 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 patent claims made by this invention. Therefore, the scope of the patent claims made by 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. S1~S2, S21~S24: Steps

Claims

1. 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, wherein the NKG2A conjugate has a volume molar concentration ranging from 1 mM to 3 mM; and adding a cell to a cell culture vessel containing the second cell culture medium for cell expansion culture to obtain expanded cells; wherein, The cell is further defined as a peripheral blood mononuclear cell (PBMC) with a cell number ranging from 1×10⁷ to 3×10⁷. The expanded cell is further defined as a natural killer cell (NK cell). The NKG2A conjugate contains a peptide chain for binding to NKG2A expressed on the cell surface of the cell. One amino acid sequence of the peptide chain includes PSSCIGVFRNSSHHPW (SEQ ID NO: 1), ISIDNEEMKF (SEQ ID NO: 2), LAFKHEIKDSEN (SEQ ID NO: 3), and QLNRLKSAQCGSSIIYHC (SEQ ID NO: 4).

2. The in vitro cell proliferation method as described in claim 1, wherein the PSSCIGVFRNSSHHPW (SEQ ID NO: 1) accounts for 25% to 35% of the NKG2A conjugate.

3. The in vitro cell proliferation method as described in claim 1, wherein the ISIDNEEMKF (SEQ ID NO: 2) accounts for 15% to 25% of the NKG2A conjugate.

4. The cell proliferation method as described in claim 1, wherein the LAFKHEIKDSEN (SEQ ID NO: 3) accounts for 20% to 30% of the NKG2A conjugate.

5. The cell proliferation method as described in claim 1, wherein the QLNRLKSAQCGSSIIYHC (SEQ ID NO: 4) accounts for 20% to 30% of the NKG2A conjugate.

6. The method for in vitro cell proliferation as described in claim 1, wherein the step of adding the cells to the cell culture container containing the second cell culture medium for cell amplification culture further comprises the following sub-steps: at a first time point, culturing the cells in a first culture container, wherein the first culture container contains the second cell culture medium containing the NKG2A conjugate, and forming a first cell; at a second time point, transferring the cells to a second culture container, wherein the second culture container contains the second cell culture medium containing 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 a supernatant from the cell slurry to obtain the amplified cells, wherein one centrifugation speed is 300×g to 600×g, and one centrifugation time is 10 minutes to 15 minutes.

7. The in vitro cell proliferation method as described in claim 6, 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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