High-function cell sorting method, application of high-function cell sorting method in preparation of therapeutic preparation and method for selecting cells with high cell proliferation capacity

Abstract

The invention relates to a high-function cell sorting method, which comprises the following steps of: dividing a plurality of target cells into four groups by a cell sorter according to mitochondrial activity and glycolytic activity of the target cells; in some embodiments, a plurality of mitochondrial activity and glycolytic activity are assigned to a first population having high mitochondrial activity and low glycolytic activity, a second population having high mitochondrial activity and high glycolytic activity, a third population having low mitochondrial activity and low glycolytic activity, and a fourth population having low mitochondrial activity and high glycolytic activity. When the target cells are stem cells, the target cells in the second group and the fourth group are high-function cells. When the target cells are immune cells, the target cells in the second population are high-function cells.

Description

Technical Field

[0001] The present invention provides a method for sorting high-function cells, application of the method in preparing therapeutic preparations, and a method for selecting cells with high cell proliferation ability. Background Art

[0002] According to statistics from the Ministry of Health and Welfare, the number of deaths in 2022 will be approximately 200,000, of which approximately 50,000 will be due to cancer, accounting for approximately 25% of the total. Traditionally, cancer treatments include surgical resection, radiotherapy, and chemotherapy. Recently, immune cell therapy has gained increasing attention due to its minimal impact on normal cells when treating cancer. This method involves extracting a patient's immune cells, purifying them, and then culturing, activating, inducing, and amplifying them in vitro. These cells are then injected back into the patient to fight cancer cells.

[0003] Currently, the most common method for purifying immune cells is to utilize highly specific pairings between antigens and immune cells to purify them. These highly purified immune cells are then activated, induced, and expanded. However, this method only yields immune cells that are specific to the antigen and cannot guarantee that all obtained immune cells possess normal cell function. Therefore, the problem of isolating large numbers of functional cells for subsequent activation, induction, and expansion has been extensively studied. Summary of the Invention

[0004] The present invention has been made in view of the above-mentioned problems, and its object is to provide a novel cell sorting method. Furthermore, the present invention also aims to provide uses of cells sorted using this cell sorting method.

[0005] The present invention provides a method for isolating high-function cells, comprising the following steps: using a cell sorter to sort a plurality of target cells into four populations, wherein the target cells are sorted, based on mitochondrial activity and glycolytic activity, into a first population with high mitochondrial activity and low glycolytic activity, a second population with high mitochondrial activity and high glycolytic activity, a third population with low mitochondrial activity and low glycolytic activity, and a fourth population with low mitochondrial activity and high glycolytic activity. When the target cells are stem cells, the target cells in the second and fourth populations are high-function cells. When the target cells are immune cells, the target cells in the second population are high-function cells.

[0006] The present invention also provides a use of stem cells sorted by the above method in preparing a preparation for promoting wound healing.

[0007] The present invention also provides a use of immune cells sorted by the above method in preparing a preparation for treating malignant tumors.

[0008] The present invention also provides a method for selecting cells with high cell proliferation capacity, comprising the following steps: using a cell sorter to sort a plurality of target cells into four populations, wherein the target cells are sorted, based on mitochondrial activity and glycolytic activity, into a first population with high mitochondrial activity and low glycolytic activity, a second population with high mitochondrial activity and high glycolytic activity, a third population with low mitochondrial activity and low glycolytic activity, and a fourth population with low mitochondrial activity and high glycolytic activity. When the target cells are stem cells, the target cells in the second and fourth populations are cells with high cell proliferation capacity. When the target cells are immune cells, the target cells in the second population are cells with high cell proliferation capacity.

[0009] The cell sorting method of the present invention allows for the initial screening of highly functional cell populations most suitable for subsequent treatments, followed by amplification and culture to yield a large number of highly functional cells. These highly functional cells exhibit superior cell function compared to cells sorted using conventional techniques in applications such as enhanced cell proliferation, enhanced cell differentiation, enhanced wound healing, or enhanced cytotoxicity.

[0010] The above description of the present invention and the following description of the embodiments are intended to demonstrate and explain the spirit and principles of the present invention and to provide further explanation of the claims of the present invention. However, it will be apparent to those skilled in the art that these examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a flow chart of the high-function cell sorting method of the present invention;

[0012] Figure 2 A scatter plot showing that ADSC-MSCs are divided into four groups;

[0013] Figure 3 The figure shows the cell morphology of ADSC-MSC from Q1 to Q4;

[0014] Figures 4A to 4D The wound healing abilities of ADSC-MSCs Q1 to Q4 after 0 hours, 3 hours, and 6 hours are shown respectively;

[0015] Figure 5 A scatter plot showing that NK-92-MI cells are divided into four groups;

[0016] Figure 6A The figure shows the cell morphology of NK-92-MI cells from Q1 to Q4 after being cultured for three days;

[0017] Figure 6BThe figure shows the cell morphology from Q1 to Q4 of NK-92-MI cells cultured for six days;

[0018] Figure 7 The results of the control group and Q1 to Q4 and Mix of NK-92-MI cells cultured for six days were co-cultured with HepG2 / C3A TCSCs for three days;

[0019] Figure 8 The results of the control group and Q1 to Q4 and Mix of NK-92-MI cells cultured for six days were co-cultured with HepG2 / C3A TCSCs for three days;

[0020] Figure 9 The cell proliferation rates of ADSC-MSCs from Q1 to Q4 and Mix after 24 hours and 72 hours are shown;

[0021] Figure 10 The cell proliferation rates of NK-92-MI cells from Q1 to Q4 and Mix on the third and sixth days are shown. DETAILED DESCRIPTION

[0022] The following detailed description of the features and advantages of the present invention is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Furthermore, based on the disclosure of this specification, the claims, and the accompanying drawings, any person skilled in the art can easily understand the relevant objects and advantages of the present invention. The following examples further illustrate the concepts of the present invention but are not intended to limit the scope of the present invention in any way.

[0023] The stem cells described in the present invention can be any of various stem cells known to those skilled in the art. Here, mesenchymal stem cells derived from adipose tissue stem cells, which are readily available, are used as an example, but the stem cells described in the present invention are not limited thereto.

[0024] The immune cells described herein can be any of a variety of immune cells known to those skilled in the art. While natural killer cells (NK-92-MI cells), which can non-specifically and directly fight cancer cells, are used as an example, the immune cells described herein are not limited thereto.

[0025] The dye for detecting mitochondrial activity described in the present invention can be any dye known to those skilled in the art. Here, tetramethylrhodamine methyl ester (TMRM) is used as the dye, as it has little or almost negligible effect on target cell activity. However, the dye for detecting mitochondrial activity is not limited to this.

[0026] The dye for detecting glycolytic activity of the present invention can be any dye known to those skilled in the art. Here, 2-(N-(7-nitrobenzo-2- 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose (2-NBDG) is used as a dye, but the dye used to detect glycolytic activity is not limited thereto.

[0027] In the following experiments, the cells and culture medium used were all commercially available. Those skilled in the art can select appropriate culture medium and culture conditions based on the cells to be cultured.

[0028] [Cell sample preparation steps]

[0029] The cell sample preparation process used in the present invention, for example, includes the following steps: culturing target cells in a culture medium at a pH of 7.4 and a temperature of 37°C for 30 minutes; removing the culture medium and washing the target cells with phosphate buffered saline (PBS); adding a cell separation reagent (e.g., Accutase) to the target cells at 37°C to cover the cells and waiting for 5 minutes; centrifuging the target cells at 1200 rpm and 483 g for 8 minutes; removing the supernatant and washing the target cells with PBS; placing the target cells into two test tubes, adding 500 μL of culture medium and 1×10 5 Target cells are added to one tube, and the remaining target cells and a dye for detecting mitochondrial activity (e.g., TMRM) and a dye for detecting glycolytic activity (e.g., 2-NBDG) are added to another tube. The dyes and target cells are allowed to react at 37°C for 30 minutes. The target cells in both tubes are washed twice with PBS. 1 mL of EDTA (ethylenediaminetetraacetic acid)-PBS is added to each tube to prepare for subsequent cell sorting using a cell sorter.

[0030] [Cell Sorting Step]

[0031] Please refer to Figure 1 , Figure 1The flowchart of the method for sorting high-function cells according to the present invention is as follows: S1: Using a cell sorter, a plurality of target cells are sorted into four populations, wherein the target cells are sorted into a first population (Q1) with high mitochondrial activity and low glycolytic activity, a second population (Q2) with high mitochondrial activity and high glycolytic activity, a third population (Q3) with low mitochondrial activity and low glycolytic activity, and a fourth population (Q4) with low mitochondrial activity and high glycolytic activity, based on mitochondrial activity and glycolytic activity, and the cell numbers of the four populations are comparable. S2: When the target cells are stem cells, the target cells in the second and fourth populations are high-function cells; when the target cells are immune cells, the target cells in the second population are high-function cells.

[0032] Specifically, the target cells in the dye-containing test tube are first injected into a flow cytometer. Four flow tubes containing a culture medium containing 2% PBS are prepared. The flow cytometer then separates the target cells into four populations, each of which flows into the four flow tubes. The four populations consist of a first population with high mitochondrial activity and low glycolytic activity, a second population with high mitochondrial activity and high glycolytic activity, a third population with low mitochondrial activity and low glycolytic activity, and a fourth population with low mitochondrial activity and high glycolytic activity, with the cell numbers in each population being comparable. After the four flow tubes, each containing the four populations, are removed from the cell sorter, they can be used as populations of high-functioning cells for subsequent applications. For example, if the target cells are stem cells, the stem cells in the second and fourth populations are considered high-functioning cells; if the target cells are immune cells, the immune cells in the second population are considered high-functioning cells.

[0033] Next, the application of the high-function cells of the present invention in enhancing cell proliferation, enhancing cell differentiation, enhancing wound healing, or enhancing cytotoxicity is described.

[0034] [Example]

[0035] First Example: Stem Cells

[0036] The stem cells used in this embodiment are adipose-derived mesenchymal stem cells (ADSC-MSC), which are isolated according to the high-function cell isolation method of the present invention.

[0037] [Experimental Results]

[0038] Please refer to Figure 2 , Figure 2 This is a scatter plot showing that ADSC-MSC are divided into four groups according to the high-function cell sorting method of the present invention. Figure 3 , Figure 3The cell morphology of ADSC-MSC from Q1 to Q4 cultured at pH 7.4 and 37°C is shown. Figure 3 The results showed that the cells in Q2 and Q4 of ADSC-MSC were denser, and therefore the cell morphology of Q2 and Q4 was better and healthier. In other words, among the Q1 to Q4 ADSC-MSC populations, the cell morphology of Q2 and Q4 was the best and healthiest.

[0039] [Wound healing]

[0040] Please refer to Figures 4A to 4D , Figures 4A to 4D The results indicate that ADSC-MSCs cultured at pH 7.4 and 37°C for Q1 to Q4 can heal wounds.

[0041] Can be obtained from Figures 4A to 4D It was found that the wound healing speed of ADSC-MSC Q2 and Q4 was faster than that of Q1 and Q3. In other words, among the ADSC-MSC Q1 to Q4 populations, Q2 and Q4 had the fastest wound healing speed.

[0042] [in conclusion]

[0043] After sorting and culturing, it was found that compared with other groups, the cell morphology of Q2 and Q4 of the sorted ADSC-MSC was the best, so Q2 and Q4 caused the fastest wound healing speed.

[0044] Second Example: Immune Cells

[0045] The immune cells used in this embodiment are natural killer cells (NK-92-MI cells), which are isolated according to the high-function cell isolation method of the present invention.

[0046] [Experimental Results]

[0047] The results of NK-92-MI cells sorted by the high-function cell sorting method of the present invention can be found in Figure 5 , Figure 5 The figure is a scatter plot showing that NK-92-MI cells are divided into four groups according to the high-function cell sorting method of the present invention. Figure 6A The figure shows the cell morphology from Q1 to Q4 of NK-92-MI cells cultured at pH 7.4 and 37°C for three days. Figure 6B The cell morphology of NK-92-MI cells from Q1 to Q4 after being cultured for six days at a pH of 7.4 and a temperature of 37°C is shown. Figure 6A as well as Figure 6BThe morphology of Q2 NK-92-MI cells was found to be spherical and large, indicating that the cells in Q2 had a better morphology and were healthier. Specifically, among the Q1 to Q4 NK-92-MI cell populations, Q2 had the best morphology and was the healthiest.

[0048] [Cytotoxicity]

[0049] Please refer to Figure 7 、 Figure 8 , Figure 7 、 Figure 8 The results show that the control group and Q1 to Q4 and Mix of NK-92-MI cells cultured for six days were co-cultured with tumorigenic cancer stem cells (HepG2 / C3A TCSC) for three days, wherein Mix is ​​a mixture of Q1, Q2, Q3 and Q4, and the control group is a group without NK-92-MI cells added. When the NK-92-MI:TCSC (cell number ratio) is 1:1 or 2:1, the cytotoxic ability of Q2 of NK-92-MI cells is better than that of Q1, Q3, Q4, Mix and the control group. That is, after culturing under the above conditions, among the groups of Q1 to Q4, Mix and the control group of NK-92-MI cells, Q2 has the best cytotoxic ability. Figure 8 ***(p<0.001) and ****(p<0.0001) indicate significant differences.

[0050] [in conclusion]

[0051] After sorting and culturing, it was found that compared with other groups, the cell morphology of Q2 of NK-92-MI cells was the best, and therefore its Q2 had the best cytotoxic ability.

[0052] Example 3: Cell Proliferation

[0053] The ADSC-MSC and NK-92-MI cells used in this example were isolated according to the high-function cell isolation method of the present invention.

[0054] Please refer to Figure 9 , Figure 9 The cell proliferation rates of Q1 to Q4 and Mix of ADSC-MSCs cultured at pH 7.4 and 37°C for 24 hours and 72 hours are described, wherein Mix is ​​a mixture of Q1, Q2, Q3 and Q4. Figure 9 It was found that the cell proliferation rates of Q2 and Q4 of ADSC-MSC were higher than those of Q1, Q3 and Mix. That is, after culture under the above conditions, the cell proliferation rates of Q2 and Q4 were the highest among the groups of Q1 to Q4 and Mix of ADSC-MSC. Figure 9In the table, **(p<0.01) and ****(p<0.0001) indicate significant differences.

[0055] Please refer to Figure 10 , Figure 10 The cell proliferation rate of NK-92-MI cells cultured for three and six days at a pH of 7.4 and a temperature of 37°C is shown, wherein Mix is ​​a mixture of Q1, Q2, Q3 and Q4. Figure 10 It was found that the cell proliferation rate of Q2 of NK-92-MI cells was higher than that of Q1, Q3, Q4 and Mix. That is, among the populations of Q1 to Q4 and Mix of NK-92-MI cells, the cell proliferation rate of Q2 was the highest. Figure 10 In the table, **(p<0.01) and ****(p<0.0001) indicate significant differences.

[0056] The cell sorting method of the present invention allows for the initial screening of highly functional cell populations most suitable for subsequent treatments, followed by amplification and culture to yield a large number of highly functional cells. These highly functional cells exhibit superior cell function compared to cells sorted using conventional techniques in applications such as enhanced cell proliferation, enhanced cell differentiation, enhanced wound healing, or enhanced cytotoxicity.

[0057]

Explanation of symbols

[0058] S1, S2: steps.

Claims

1. A method for sorting high-function cells, characterized in that: The following steps are involved: Using a cell sorter, the plurality of target cells are divided into four groups, wherein the plurality of target cells are divided into a first group having high mitochondrial activity and low glycolytic activity, a second group having high mitochondrial activity and high glycolytic activity, a third group having low mitochondrial activity and low glycolytic activity, and a fourth group having low mitochondrial activity and high glycolytic activity based on mitochondrial activity and glycolytic activity; and When the plurality of target cells are stem cells, the plurality of target cells in the second population and the fourth population are high-function cells, When the plurality of target cells are immune cells, the plurality of target cells in the second population are high-function cells.

2. The method according to claim 1, wherein The cell sorter is a flow cytometer.

3. The method according to claim 1, wherein Before the step of dividing the plurality of target cells into four groups using a cell sorter, the method further includes adding a dye for detecting mitochondrial activity into the plurality of target cells.

4. The method according to claim 1, wherein Before the step of dividing the plurality of target cells into four groups using a cell sorter, the method further includes adding a dye for detecting glycolytic activity to the plurality of target cells.

5. The method according to claim 1, wherein The cell number ratio of each of the first population, the second population, the third population, and the fourth population is 25% of the number of the plurality of target cells.

6. The method according to claim 1, wherein When the plurality of target cells are stem cells, the number of the second population and the fourth population of the plurality of target cells is 14.6% to 21.0% of the number of the plurality of target cells.

7. The method according to claim 1, wherein When the plurality of target cells are immune cells, the number of the second population of the plurality of target cells is 19.8% to 22.5% of the number of the plurality of target cells.

8. A stem cell separated by the method according to claim 1, characterized in that: Use in preparing a preparation for promoting wound healing.

9. An immune cell sorted according to the method of claim 1, characterized in that: Use in preparing preparations for treating malignant tumors.

10. A method for selecting cells with high cell proliferation ability, characterized in that: The following steps are involved: Using a cell sorter, the plurality of target cells are divided into four groups, wherein the plurality of target cells are divided into a first group having high mitochondrial activity and low glycolytic activity, a second group having high mitochondrial activity and high glycolytic activity, a third group having low mitochondrial activity and low glycolytic activity, and a fourth group having low mitochondrial activity and high glycolytic activity based on mitochondrial activity and glycolytic activity; and When the plurality of target cells are stem cells, the plurality of target cells in the second population and the fourth population are cells with high cell proliferation ability, When the plurality of target cells are immune cells, the plurality of target cells in the second population are cells with high cell proliferation ability.

Images