Cell culture compositions and uses thereof

Abstract

The embodiment of the invention provides a cell culture composition and application thereof, the cell culture composition comprises a culture medium and mitochondria, and the cell culture composition can promote cell growth and improve functions of damaged or aged stem cells so as to improve the growth condition of the cells.

Description

[0001] This application is a divisional application of the patent application with the national application number 202180022924.X given by the Patent Office of the State Intellectual Property Office, the international application number PCT / CN2021 / 081854, the international application date of March 19, 2021, and the invention name of "Cell Culture Compositions and Their Uses". Technical Field

[0002] The present invention relates to a cell culture composition and its use, in particular to a cell culture composition containing mitochondria and its use. Background Art

[0003] Cell culture medium is a nutrient-based substance that simulates the growth environment of cells in animals and plants. It provides nutrients that cells cannot synthesize by themselves and maintains a suitable pH value and osmotic pressure, allowing cells to survive and proliferate in vitro. In the process of culturing cells, auxiliary culture additives are added to improve cell growth efficiency and cell function.

[0004] In a poor culture medium or under harsh culture conditions, cells grow slowly, and the grown cells may be fragile, difficult to survive, or even unable to grow. If the cultured cells are not healthy enough or grow too slowly, it will have an adverse effect on subsequent experiments or research. Summary of the invention

[0005] According to an embodiment of the present invention, the cell culture composition comprising mitochondria and culture medium can improve the growth condition of cells.

[0006] An embodiment of the present invention provides a cell culture composition, comprising a culture medium and mitochondria.

[0007] An embodiment of the present invention provides a use of a cell culture composition in promoting cell growth, wherein the cell culture composition comprises a culture medium and mitochondria.

[0008] An embodiment of the present invention provides a use of a cell culture composition in improving the function of damaged or aged stem cells, wherein the cell culture composition comprises a culture medium and mitochondria.

[0009] According to an embodiment of the present invention, by adding mitochondria to the cell culture medium, it is possible to help cell growth and increase the proliferation rate of cells. For damaged or aged cells, according to an embodiment of the present invention, it is also possible to improve the growth of damaged or aged cells and reduce the proportion of aged cells. In addition, according to an embodiment of the present invention, it is also possible to improve the function of damaged or aged cells. In terms of academics, the present invention ensures that the growth efficiency and stability of cells are helpful for subsequent research or experiments to lay the foundation for scientific development and biological research. In terms of industry, the present invention ensures that the growth efficiency and stability of cells are helpful for stabilizing the yield and yield, which can reduce the cost of cell culture and control product quality to maximize profits. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A and Figure 1B The cell proliferation rate of ARPE-19 cells cultured using the cell culture composition of the example is shown. Figure 1A The culture time is 24 hours. Figure 1B The culture time is 48 hours.

[0011] Figure 2 The cell proliferation rate of HepG2 cells cultured using the cell culture composition of the example is shown, and the culture time is 24 hours.

[0012] Figure 3 The cell proliferation rate of MDCK cells cultured using the cell culture composition of the example is shown, and the culture time is 24 hours.

[0013] Figure 4 The cell proliferation rate of human tenocytes cultured using the cell culture composition of the example is shown, and the culture time is 24 hours and 48 hours.

[0014] Figure 5 The cell proliferation rate of natural killer cells cultured using the cell culture composition of the example is shown, and the culture time is 24 hours and 48 hours.

[0015] Figure 6 The senescent cell ratio of damaged or aged ADSCs cultured using the cell culture composition of the Examples is shown.

[0016] Figure 7 The senescent cell ratio of damaged or aged AMSCs cultured using the cell culture composition of the Examples is shown.

[0017] Figure 8 The proliferation rate of CCD-996SK cells cultured with a cell culture composition containing only platelets as a control group is shown, and the culture time is 24 hours and 48 hours.

[0018] Fig. 9The proliferation rate of CCD-996SK cells cultured using the cell culture composition of the example containing only mitochondria is shown, and the culture time is 24 hours and 48 hours.

[0019] Fig.10 The proliferation rate of CCD-996SK cells cultured using the cell culture composition of the example containing mitochondria and platelets is shown, and the culture time is 24 hours and 48 hours.

[0020] Fig.11 The proliferation rate of ARPE-19 cells cultured using the cell culture composition of the example containing mitochondria and complement C3 is shown, and the culture time is 24 hours and 48 hours. DETAILED DESCRIPTION

[0021] The detailed features and advantages of the present invention are described in detail in the following embodiments, and the contents are sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly, and according to the contents disclosed in this specification, the claims and the drawings, any person familiar with the prior art can easily understand the relevant purposes and advantages of the present invention. The following examples further illustrate the viewpoints of the present invention in detail, but do not limit the scope of the present invention in any viewpoint.

[0022] Mitochondria are the sites in cells where oxidative phosphorylation reactions and adenosine triphosphate (ATP) synthesis occur. In addition to providing energy required for normal cell metabolism, they are also responsible for regulating functions such as intracellular oxidative stress processing and signal transmission. During cell culture, the function of mitochondria is closely related to cell growth. Cells with poor mitochondrial function are also less efficient in generating energy, resulting in slow or incomplete cell growth. Therefore, the inventors improved cell growth by using mitochondria as an additive in a cell culture composition.

[0023] According to one embodiment of the present invention, the cell culture composition comprises a culture medium and mitochondria. The cell culture composition can be in liquid form at room temperature and pressure, and can be stored at room temperature, preferably at 4°C.

[0024] The culture medium may include DMEM, F12, DMEM / F12, MEM, MEM-α, Keratinocyte SFM (1X), IMEM, RPMI 1640, M-199, Opti-MEM, Ham's F-10 Nutrition Mixture, Ham's F-12 Nutrition Mixture or IMDM, but is not limited thereto. The culture medium may be liquid or solid, and an appropriate culture medium may be selected according to the cells to be cultured and appropriate nutrient additives and water may be added.

[0025] Mitochondria may be mitochondria taken from animal cells or plant cells, but are not limited thereto. In some embodiments of the present invention, mitochondria of animal cells are added to the culture medium when animal cells are cultured. Animal cells providing mitochondria may include adipose stem cells, monocytes, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, CD34+ stem cells, bone marrow stem cells, skeletal muscle cells, liver cells, kidney cells, platelets, fibroblasts, endothelial cells and other cells with mitochondria. In the cell culture composition, each milliliter of the cell culture composition may contain 1 microgram to 100 micrograms of mitochondria, but are not limited thereto. In other embodiments, each milliliter of the cell culture composition may contain 5 micrograms to 80 micrograms of mitochondria. In other embodiments, each milliliter of the cell culture composition may contain 15 micrograms to 40 micrograms of mitochondria.

[0026] According to other embodiments of the present invention, the cell culture composition may include a culture medium, at least one nutrient additive, and mitochondria.

[0027] Nutritional additives may include, but are not limited to, fetal bovine serum (FBS), salts, amino acids, horse serum, human serum, vitamins, glucose, growth factors, proteins, animal extracts, carbohydrates, inositol, thioethanol, or folic acid. Salts include, but are not limited to, sodium salts, potassium salts, magnesium salts, calcium salts, chloride salts, carbonates, pyruvates, nitrates, phosphates, or inorganic salts. Appropriate nutritional additives may be selected based on the cells to be cultured.

[0028] According to other embodiments of the present invention, the cell culture composition may include culture medium, mitochondria and platelets.

[0029] The platelets may be platelets in animal blood, but are not limited thereto. In the cell culture composition, each milliliter of the cell culture composition may contain 1×10 6 Up to 1×10 8 In other embodiments, each milliliter of the cell culture composition may contain 1×10 8 Platelets.

[0030] According to other embodiments of the present invention, the cell culture composition may include a culture medium, mitochondria and complement C3.

[0031] In the cell culture composition, each milliliter of the cell culture composition may contain 0.1 microgram to 20 micrograms of complement C3, but is not limited thereto. In other embodiments, each milliliter of the cell culture composition may contain 10 micrograms of complement C3.

[0032] According to one embodiment of the present invention, the cell culture composition can promote cell growth and increase cell proliferation rate. The cells cultured using the cell culture composition of the embodiment of the present invention may include any somatic cells. Somatic cells may include retinal cells, liver cells, kidney cells, tendon cells, skin cells or immune cells. Immune cells may include natural killer cells, and natural killer cells may have at least one marker of CD56+, CD3-, CD94+, CD122+, CD127+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKp46+ and NKp80+.

[0033] According to one embodiment of the present invention, the cell culture composition can improve the aging degree of damaged or aged cells. The cells cultured using the cell culture composition of the embodiment of the present invention may include any somatic cells or any stem cells. The somatic cells may be retinal cells, liver cells, kidney cells, tendon cells, skin cells or immune cells. Stem cells may include mesenchymal stem cells, and mesenchymal stem cells may have at least one marker among CD44+, CD90+, CD105+, CD106+, CD166+, Stro-1+ and CD34-. Mesenchymal stem cells may be adipose stem cells, umbilical cord stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, skin stem cells, peripheral blood stem cells, endometrial stem cells, amniotic membrane stem cells, gingival stem cells or dental pulp stem cells.

[0034] The following describes how to prepare the cell culture composition according to the embodiments of the present invention.

[0035] The mitochondria used in one embodiment of the present invention are obtained from human adipose-derived stem cells (ADSC). The stem cell culture medium includes Keratinocyte SFM (1X) solution (Gibco), bovine pituitary extract (BPE, Gibco), and fetal bovine serum (HyClone) with a weight percentage concentration of 10%. First, human adipose stem cells are cultured in a culture dish until the cell number is 1.5×10 8cells, and then rinsed the human adipose stem cells in the culture dish with Dulbecco's phosphate buffer (DPBS). Then, after removing the Dulbecco's phosphate buffer from the culture dish, trypsin for cell detachment was added to the culture dish, and after reacting at 37°C for 3 minutes, stem cell culture medium was added to terminate the reaction. Then, the human adipose stem cells were rinsed off from the culture dish and dispersed, and centrifuged at 600g for 10 minutes, and the supernatant was removed. Then, the human adipose stem cells left after centrifugation and 80 ml of IBC-1 buffer (225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4) were added to the homogenizer, and the human adipose stem cells were ground 15 times with the homogenizer on ice. Next, the ground human adipose stem cells were centrifuged at 1000g for 15 minutes, the supernatant was collected into another centrifuge tube, and the supernatant was centrifuged again at 9000g for 10 minutes. The supernatant obtained after the second centrifugation was removed, and the obtained precipitate was the mitochondria. 1.5 ml of IBC-2 buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl pH 7.4) and protease inhibitors were added to the mitochondrial precipitate and stored at 4°C.

[0036] When cell culture is performed, an appropriate culture medium is selected according to the cell type, and then the required nutrient additives and the mitochondria as described above are added at the desired concentration and mixed evenly before use. The cell culture composition is preferably freshly prepared, but not limited thereto. The cell culture composition can also be prepared in advance and stored at 4°C and then used when cell culture is performed.

[0037] The following describes cell culture using the cell culture composition according to the examples of the present invention.

[0038] [Experiment 1: Improving cell proliferation rate]

[0039] In this experiment, the cell culture composition of the embodiment was used as a culture medium to culture human retinal pigment epithelial cell line (Human retinal pigment epithelium cell, ARPE-19), liver cells (HepG2), kidney epithelial cells (Madin-Darby Canine Kidney cell, MDCK), human tenocytes (Human tenocyte) and natural killer cells (Nature killer cell, NK92MI). In addition, the effect of the cell culture composition containing mitochondria on cell growth was evaluated by an Alamar blue detection reagent, and expressed as a proliferation rate (also called cell proliferation rate).

[0040] Alamar blue is a detection reagent used to detect cell viability. Resazurin in the detection kit is a redox indicator, which is a non-toxic, cell membrane-penetrating, low-fluorescence dark blue dye. When resazurin enters healthy cells, it is reduced to pink and highly fluorescent resorufin due to the reducing environment in the living cells. The cell proliferation rate can be evaluated by measuring the light absorption value or fluorescence value of resorufin. The higher the light absorption value or fluorescence value of resorufin, the more cells there are and the higher the cell proliferation rate. The higher the cell proliferation rate also means that the cells are healthier and have a stronger proliferation ability. Therefore, this experiment uses Alamar blue as an indicator for evaluating cell proliferation rate or cell survival rate.

[0041] The detailed components of the cell culture compositions of various examples used in this experiment are shown in Tables 1 to 5.

[0042] The culture medium of ARPE-19 can be DMEM / F12 (Gibco), the nutrient additives can include 2.5mM glutamine, 15mM HEPES, 0.5mM sodium pyruvate, 1200 mg / L sodium bicarbonate and 10% fetal bovine serum by weight, and the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, 5μg / mL to 80μg / mL, 15μg / mL to 40μg / mL, or 15μg / mL or 40μg / mL.

[0043] The culture medium of HepG2 can be DMEM / F12, the nutrient additive can include 10% fetal bovine serum by weight, the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, can be 5μg / mL to 80μg / mL, can be 15μg / mL to 40μg / mL, or can be 15μg / mL or 40μg / mL.

[0044] The culture medium of MDCK can be MEM-α (Thermo Fisher Scientific) containing Earle's Balanced Salt, the nutrient supplement can include 5% fetal bovine serum by weight, and the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, can be 5μg / mL to 80μg / mL, can be 15μg / mL to 40μg / mL, or can be 15μg / mL or 40μg / mL.

[0045] The culture medium of human tenocytes may be DMEM / F12, the nutrient supplement may include 10% fetal bovine serum by weight, the mitochondrial concentration may be 1 microgram / milliliter (μg / mL) to 100 μg / mL, may be 5 μg / mL to 80 μg / mL, may be 15 μg / mL to 40 μg / mL, or may be 1 μg / mL, 15 μg / mL, 40 μg / mL or 100 μg / mL.

[0046] The culture medium for natural killer cells can be MEM-α (ThermoFisher Scientific) containing Earle's Balanced Salt, the nutrient additives can include 0.02mM inositol, 0.1mM mercaptoethanol, 0.02mM folic acid, 12.5% ​​by weight fetal bovine serum and 12.5% ​​by weight horse serum, and the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, 5μg / mL to 80μg / mL, 15μg / mL to 40μg / mL, or 1μg / mL, 15μg / mL, 40μg / mL or 100μg / mL.

[0047] 〔Table 1〕

[0048]

[0049] 〔Table 2〕

[0050]

[0051] 〔Table 3〕

[0052]

[0053]

[0054] 〔Table 4〕

[0055]

[0056] 〔Table 5〕

[0057]

[0058] The following describes the culture process of each cell. First, take each cell with a culture generation number between 4 and 10 for the experiment. Use a culture medium that does not contain mitochondria to culture each cell until its volume is one-eighth of the culture dish. Remove the culture medium in the culture dish and rinse the cells with phosphate buffered saline (PBS). Next, add 0.25% trypsin to the culture dish and allow the trypsin to react at 37°C for 5 minutes. Then add the culture medium to terminate the trypsin reaction. Then, transfer each cell and culture medium in the culture dish to a centrifuge tube, centrifuge at 1000 revolutions per minute (rpm) for 5 minutes, and remove the supernatant. Next, add new culture medium to the centrifuge tube and count the cells. Next, add ARPE-19 at 2×10 per well. 4 HepG2 cells were cultured in the cell culture compositions of Examples 1-1 and 1-2 for 24 or 48 hours at a density of 5×10 4 MDCK cells were cultured in the cell culture compositions of Examples 2-1 and 2-2 for 24 hours at a density of 5×10 cells per well. 4 The human tendon cells were cultured in the cell culture compositions of Examples 3-1 and 3-2 for 24 hours at a density of 2×10 cells per well. 4 The cells were cultured in the cell culture compositions of Examples 4-1 to 4-4 for 24 or 48 hours at a density of 2×10 cells per well. 4 The cells were cultured in the cell culture compositions of Examples 5-1 to 5-4 for 24 or 48 hours at a density of 10 cells. After the culture was completed, each cell was washed with a phosphate buffer, and the culture medium was replaced with a culture medium containing Alamar Blue and cultured for 3 hours. After the culture was completed, the fluorescence was measured at a wavelength of OD530 / 595 to calculate the proliferation rate of each cell.

[0059] For the experimental results of ARPE-19, please refer to Table 6 and Figure 1A and Figure 1B , Figure 1A and Figure 1B The cell proliferation rate of ARPE-19 cells cultured using the cell culture composition of the example is shown. Figure 1A The culture time is 24 hours. Figure 1B The culture time is 48 hours. For the experimental results of HepG2, please refer to Table 7 and Figure 2 , Figure 2 The cell proliferation rate of HepG2 cells cultured using the cell culture composition of the embodiment is shown in Table 8 and Table 9 for the experimental results of MDCK. Figure 3 , Figure 3The cell proliferation rate of MDCK cells cultured using the cell culture composition of the embodiment is shown, and the culture time is 24 hours. The experimental results of human tendon cells are shown in Table 9 and Figure 4 , Figure 4 The cell proliferation rate of human tenocytes cultured using the cell culture composition of the embodiment is shown, and the culture time is 24 hours and 48 hours. The experimental results of natural killer cells are shown in Tables 10 and Figure 5 , Figure 5 The cell proliferation rate of natural killer cells cultured using the cell culture composition of the embodiment is shown, and the culture time is 24 hours and 48 hours. The initial control group is the number of cells when the cells are just cultured (hour 0), which is set to 1. The proliferation rate is the multiple of the number of cells after cell culture relative to the initial control group. The control group is a group without the addition of mitochondria. Figures 1A to 3 In the table, # indicates a significant difference relative to the initial control group (P<0.05), ## indicates a highly significant difference relative to the initial control group (P<0.01), and ### indicates a very significant difference relative to the initial control group (P<0.001). Figure 4 and Figure 5 In the table, # indicates significant difference compared with the control group (P<0.05).

[0060] 〔Table 6〕

[0061]

[0062] 〔Table 7〕

[0063]

[0064]

[0065] 〔Table 8〕

[0066]

[0067] 〔Table 9〕

[0068]

[0069] 〔Table 10〕

[0070]

[0071] The above experimental results show that using a cell culture composition containing mitochondria to culture cells can increase the proliferation rate of cells, and the proliferation rate increases with the increase in the concentration of mitochondria, indicating that the cell culture composition containing mitochondria does help the growth of cells. In addition, the increase in the proliferation rate becomes more obvious as the culture time increases, indicating that the cell culture composition containing mitochondria can stably help the growth of cells.

[0072] [Experiment 2: Improving the aging of damaged or aged stem cells]

[0073] In this experiment, advanced glycation end product (AGE) was used to induce stem cell damage or aging, and the cell culture composition of the embodiment was used as a culture medium to culture the damaged or aged stem cells. In addition, the SA-β-gal kit was used to evaluate the effect of the cell culture composition containing mitochondria on damaged or aged stem cells, and the degree of aging was expressed as the proportion of aged cells (%). The stem cells used in this experiment include adipose-derived stem cells (ADSC) and amniotic membrane stem cells (AMSC).

[0074] Senescence-associated beta-galactosidase (SA-β-gal) is overexpressed in aged cells, so SA-β-gal can be used as a marker of cell senescence. This experiment uses the SA-β-gal kit (Senescenceβ-Galactosidase Staining Kit #9860, Cell Signaling technology) to assess the aging state of stem cells.

[0075] The detailed components of the cell culture composition used in this experiment are shown in Tables 11 and 12.

[0076] The culture medium of ADSC can be Keratinocyte SFM (1X) (Catalog number: 17005042, Life Technologies), the nutrient supplement can include 10% fetal bovine serum by weight, the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, can be 5μg / mL to 80μg / mL, can be 15μg / mL to 40μg / mL, or can be 1μg / mL, 15μg / mL or 40μg / mL.

[0077] The culture medium of AMSC can be DMEM / F12 (Gibco), the nutrient supplement can include 10% fetal bovine serum by weight, the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, can be 5μg / mL to 80μg / mL, can be 15μg / mL to 40μg / mL, or can be 1μg / mL, 15μg / mL or 40μg / mL.

[0078] [Table 11]

[0079]

[0080] [Table 12]

[0081]

[0082]

[0083] The following describes the stem cell culture process. First, take stem cells of the 10th generation for the experiment. Use a culture medium that does not contain mitochondria to culture the stem cells until their volume is one-eighth of the culture dish. Remove the culture medium in the culture dish and rinse the stem cells with phosphate buffered saline (PBS). Next, add 0.25% trypsin to the culture dish and allow the trypsin to react at 37°C for 5 minutes, then add the culture medium to terminate the trypsin reaction. Then, transfer each cell and culture medium in the culture dish to a centrifuge tube, centrifuge at 1000 revolutions per minute (rpm) for 5 minutes, and remove the supernatant. Next, add new culture medium to the centrifuge tube and count the cells. Next, place the stem cells at 1.5×10 per well. 4 The cells were cultured at a density of 100 μg / mL for 24 hours. Next, AGEs were added to the well plate, and the stem cells were cultured in a culture medium containing AGEs at a concentration of 400 μg / mL for 4 hours. Next, the cell culture medium containing AGEs was removed, and the cell culture composition of the embodiment was added, and the stem cells were cultured in a cell culture composition containing different mitochondrial concentrations for 24 hours. The concentration of mitochondria in the cell culture composition was 0, 1 μg / mL, 15 μg / mL or 40 μg / mL. After the culture was completed, each cell was washed with phosphate buffer and the SA-β-gal kit was used to evaluate cell aging.

[0084] The experimental results of ADSC are shown in Table 13 and Figure 6 , Figure 6 The aging cell ratio of damaged or aged ADSCs cultured using the cell culture composition of the embodiment is shown. The experimental results of AMSCs are shown in Tables 14 and Figure 7 , Figure 7 The aging cell ratio of damaged or aged AMSCs cultured using the cell culture composition of the embodiment is shown. The control group is a culture composition to which AGEs are added and which does not contain mitochondria. The control group is a group to which AGEs and mitochondria are not added. The aging cell ratio is the percentage of the number of aging cells in a unit area to the number of all cells, to represent the degree of aging. Figure 6 to Figure 7In the figure, the vertical axis is the proportion of senescent cells (%), # indicates a significant difference relative to the control group (P<0.05), ## indicates a highly significant difference relative to the control group (P<0.01), and ### indicates a very significant difference relative to the control group (P<0.001).

[0085] 〔Table 13〕

[0086]

[0087]

[0088] 〔Table 14〕

[0089]

[0090] The above experimental results show that using a cell culture composition containing mitochondria to culture damaged or aged stem cells can reduce the proportion of aged cells, and the proportion of aged cells decreases as the concentration of mitochondria increases, indicating that the cell culture composition containing mitochondria does help improve the growth of damaged or aged stem cells, and also helps improve the function of damaged or aged stem cells.

[0091] [Experiment 3: Cell culture composition containing platelets]

[0092] In this experiment, platelets were further added to the cell culture composition, and the fibroblast CCD-996SK was cultured using the cell culture composition containing platelets as a culture medium. In addition, the effect of the cell culture composition containing mitochondria on cell growth was evaluated using an Alamar Blue detection reagent and expressed as a proliferation rate (also called cell proliferation rate).

[0093] The following is a description of the platelet preparation process used in this experiment. First, blood is drawn into a blood collection tube or centrifuge tube containing an anticoagulant, such as a CPT tube (BD CPT TMCell Preparation Tube, REF362761). Next, the blood is centrifuged to separate into layers, for example, at 1500 g for 10 minutes to separate into a red blood cell layer, a colloid layer, a buffy coat, and a plasma layer, and the buffy coat is collected in a new centrifuge tube, wherein the buffy coat contains monocytes and platelets. Next, the buffy coat is uniformly mixed with HEP buffer (140 mM NaCl, 2.7 mM KCl, 3.8 mM HEPES, 5 mM EGTA, pH 7.4) at a ratio of 1:1, and Prostaglandin E1 is added to a concentration of 1 μM to prevent platelet activation. Next, the buffy coat is centrifuged at 100 g for 15 to 20 minutes to precipitate the white blood cells and residual red blood cells, and the supernatant containing platelets is collected in a new centrifuge tube. Next, the supernatant containing platelets is centrifuged at 800 g for 15 to 20 minutes to precipitate the platelets. Remove the supernatant and rinse the precipitated platelets twice with wash buffer (10mM sodium citrate, 150mM NaCl, 1mM EDTA, 1% (w / v) dextrose). Then, use Tyrode's buffer (134mM NaCl, 12mM NaHCO3, 2.9mM KCl, 0.34mM Na2HPO4, 1mM MgCl2, 10mM HEPES, pH 7.4) to dissolve the platelets and count the platelets with a hemacytometer. The prepared platelets can be stored at 20°C to 24°C and added to the cell culture composition when culturing cells, but it is best to use them immediately after preparation.

[0094] The detailed components of the cell culture composition used in this experiment are shown in Tables 15 and 16.

[0095] The culture medium of CCD-996SK can be DMEM (Gibco; 10566016) containing 2 mM glutamine, the nutrient supplement can include 10% fetal bovine serum by weight, the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100 μg / mL, can be 5 μg / mL to 80 μg / mL, can be 15 μg / mL to 40 μg / mL, or can be 1 μg / mL, 15 μg / mL, 40 μg / mL, and the platelet concentration can be 1×10 6 Up to 1×10 8 / mL or 1×10 6 pcs, 1×10 7 or 1×10 8 Pieces / mL.

[0096] 〔Table 15〕

[0097]

[0098] 〔Table 16〕

[0099]

[0100] Please refer to Experiment 1 for the cell culture process of this experiment. When counting cells, place CCD-996SK at 1.5×10 per well. 4 The cells were cultured in the control group, the control group, and the cell culture compositions of Examples 8-1 to 8-4 at a density of 100 cells / mL for 24 hours. After the culture was completed, each cell was washed with a phosphate buffer, and the culture medium was replaced with a culture medium containing Alamar Blue and cultured for 3 hours. After the culture was completed, the fluorescence was measured at a wavelength of OD530 / 595 to calculate the proliferation rate of each cell.

[0101] Please refer to Table 17 and Table 17 for the experimental results of CCD-996SK. Figures 8 to 10 . Figure 8 The proliferation rate of CCD-996SK cells cultured with a cell culture composition containing only platelets as a control group is shown, and the culture time is 24 hours and 48 hours. Fig. 9 The proliferation rate of CCD-996SK cells cultured using the cell culture composition of the example containing only mitochondria is shown, and the culture time is 24 hours and 48 hours. Fig.10 The cell proliferation rate of CCD-996SK cultured using the cell culture composition of the embodiment containing mitochondria and platelets is shown, and the culture time is 24 hours and 48 hours. The initial control group is the number of cells when the cells are just started to be cultured (hour 0), which is set to 1. The proliferation rate is the multiple of the number of cells after cell culture relative to the initial control group. The control group is a group without the addition of mitochondria. The control group is a group without the addition of mitochondria and platelets. Figures 8 to 10 In the table, # indicates a significant difference compared with the control group (P<0.05), ## indicates a highly significant difference compared with the control group (P<0.01), and ### indicates a very significant difference compared with the control group (P<0.001).

[0102] 〔Table 17〕

[0103]

[0104] According to the above experimental results, it can be seen from the control group that adding platelets to the cell culture medium can increase the cell proliferation rate, and the proliferation rate increases with the increase in the number of platelets. From Examples 8-1 to 8-3, it can be seen that using a cell culture composition containing mitochondria to culture cells can increase the proliferation rate of cells, and the proliferation rate increases with the increase in the concentration of mitochondria, indicating that the cell culture composition containing mitochondria does help cell growth. From the control group 8-1, Example 8-3 and Example 8-4, it can be seen that using a cell culture composition containing mitochondria and platelets to culture cells can further increase the proliferation rate of cells. Example 8-3 and Example 8-4 confirm that when the mitochondrial concentration is 40μg / mL, adding 1×10 6 The platelet count of 1×10 / mL can further increase the cell proliferation rate. Example 8-4 and control group 8-1 confirmed that the platelet count was 1×10 6 In the case of 1×10 8 (Control Group 8-3) can only achieve the effect. Therefore, compared with the cell culture composition with only platelets or only mitochondria added, the cell culture composition containing both mitochondria and platelets can have a multiplying effect on improving the cell proliferation rate. Moreover, the increase in proliferation rate becomes more obvious with the increase of culture time, indicating that the cell culture composition containing mitochondria can stably help cell growth. Here, it is particularly noted that the 48-hour proliferation rate of Example 8-4 is slightly lower than the 48-hour proliferation rate of Example 8-3. The reason is that the cells have grown to saturation. When the cells grow to saturation, the total number of cells should be roughly equal, so the difference in cell proliferation rate is not obvious.

[0105] [Experiment 4, cell culture composition containing complement C3]

[0106] In this experiment, complement C3 (Sigma-Aldrich, 204885) was further added to the cell culture composition, and ARPE-19 was cultured using the cell culture composition containing complement C3 as a culture medium. In addition, the effect of the cell culture composition containing mitochondria on cell growth was evaluated using an Alamar Blue detection reagent and expressed as a proliferation rate (also called cell proliferation rate).

[0107] The detailed components of the cell culture composition used in this experiment are shown in Table 18.

[0108] The culture medium of ARPE-19 can be DMEM / F12 (Gibco), the nutrient supplement can include 2.5mM glutamine, 15mM HEPES, 0.5mM sodium pyruvate, 1200mg / L sodium bicarbonate and 10% fetal bovine serum by weight, the mitochondrial concentration can be 1 microgram / milliliter (μg / mL) to 100μg / mL, can be 5μg / mL to 80μg / mL, can be 15μg / mL to 40μg / mL, or can be 15μg / mL. The complement C3 concentration can be 0.1μg / mL to 20μg / mL, or can be 10μg / mL.

[0109] [Table 18]

[0110]

[0111]

[0112] Please refer to Experiment 1 for the cell culture process of this experiment. When counting cells, ARPE-19 was cultured at 2×10 4 The density of cells was cultured in the cell culture composition of the embodiment for 24 or 48 hours. The concentration of mitochondria in the well plate was 15 μg / mL. The concentration of complement C3 in the well plate was 10 μg / mL. After the culture was completed, each cell was washed with phosphate buffer, and the culture medium was replaced with a culture medium containing Alamar blue and cultured for 3 hours. After the culture was completed, the fluorescence was measured at a wavelength of OD530 / 595 to calculate the proliferation rate of each cell.

[0113] For the experimental results of ARPE-19, please refer to Table 19 and Fig.11 , Fig.11 The cell proliferation rate of ARPE-19 cells cultured using the cell culture composition of the embodiment containing mitochondria and complement C3 is shown, and the culture time is 24 hours and 48 hours. The control group is a group without mitochondria and complement C3, which is set to 1. The proliferation rate is the multiple of the number of cells after cell culture relative to the control group.

[0114] 〔Table 19〕

[0115]

[0116] The above experimental results show that using a cell culture composition containing mitochondria and complement C3 to culture cells can further increase the proliferation rate of cells. In addition, the increase in proliferation rate becomes more obvious as the culture time increases, indicating that the cell culture composition containing mitochondria can stably help cells grow.

[0117] According to an embodiment of the present invention, by adding mitochondria to the cell culture medium, it is possible to help cell growth and increase the proliferation rate of cells. For damaged or aged cells, according to an embodiment of the present invention, it is also possible to improve the growth of damaged or aged cells and reduce the proportion of aged cells. In addition, according to an embodiment of the present invention, it is also possible to improve the function of damaged or aged cells. In terms of academics, the present invention ensures that the growth efficiency and stability of cells are helpful for subsequent research or experiments to lay the foundation for scientific development and biological research. In terms of industry, the present invention ensures that the growth efficiency and stability of cells are helpful for stabilizing the yield and yield, which can reduce the cost of cell culture and control product quality to maximize profits.

Claims

1. A method for improving damaged or aged cells, characterized in that: It includes the following steps: Step a: preparing a cell culture composition and adding it to a culture medium, wherein the cell culture composition mainly comprises a mitochondria; Step b: Cultivating a damaged or aged cell in a culture medium supplemented with the cell culture composition for at least 24 hours, so that the proportion of damaged or aged cells in the culture medium after the cultivation is completed is reduced.

2. The method for improving damaged or aged cells according to claim 1, characterized in that: The cell culture composition consists of a mitochondria and a platelet or a mitochondria and a complement C3.

3. The method for improving damaged or aged cells according to claim 1, characterized in that: The damaged or aged cell is a stem cell or an immune cell.

4. The method for improving damaged or aged cells according to claim 1, 2 or 3, characterized in that: The mitochondria are taken directly from a cell.

5. The method for improving damaged or aged cells according to claim 1, 2 or 3, characterized in that: Each milliliter of the culture medium contains 1 microgram to 100 micrograms of mitochondria.

6. The method for improving damaged or aged cells according to claim 2, characterized in that: Each ml of this culture medium contains 1×10 6 Up to 1×10 8 Platelets.

7. The method for improving damaged or aged cells according to claim 2, characterized in that: Each milliliter of the culture medium contains 0.1 microgram to 20 micrograms of complement C3.

8. The method for improving damaged or aged cells according to claim 7, characterized in that: The concentration ratio of the mitochondria to the complement C3 is 3:

2.

9. The method for improving damaged or aged cells according to claim 3, characterized in that: The damaged or aged cell is a mesenchymal stem cell, and the mesenchymal stem cell has at least one of the following markers: CD44+, CD90+, CD105+, CD106+, CD166+, Stro-1+ and CD34-.

10. The method for improving damaged or aged cells according to claim 9, characterized in that: The damaged or aged cells are adipose stem cells, umbilical cord stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, skin stem cells, peripheral blood stem cells, and endometrial stem cells.

11. The method for improving damaged or aged cells according to claim 3, wherein: The immune cell is a natural killer cell, and the natural killer cell has at least one of the following markers: CD56+, CD3-, CD94+, CD122+, CD127+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKp46+ and NKp80+.

12. The method for improving damaged or aged cells according to claim 1, 2 or 3, characterized in that: In step b, the culturing time is 24-48 hours.

13. The method for improving damaged or aged cells according to claim 1, 2 or 3, characterized in that: The culture medium contains at least one nutrient additive.

14. The method for improving damaged or aged cells according to claim 13, characterized in that: The nutritional supplement is glutamic acid, fetal bovine serum, HEPES, sodium pyruvate, sodium bicarbonate, inositol, thioethanol, folic acid, horse serum or a combination of at least any two of the above.

15. A cell culture fluid composition, characterized in that: The composition is used for culturing a stem cell or an immune cell and comprises: a culture medium; and a mitochondria, wherein each milliliter of the stem cell culture medium composition may contain 1 microgram to 100 micrograms of mitochondria.

16. The cell culture fluid composition according to claim 15, characterized in that The invention is a stem cell culture composition, further comprising a platelet or a complement C3.

17. The cell culture fluid composition according to claim 15, characterized in that: The culture medium is DMEM, F12, DMEM / F12, MEM, MEM-α, Keratinocyte SFM (1X), IMEM, RPMI 1640, M-199, Opti-MEM, Ham's F-10 Nutrition Mixture, Ham's F-12 Nutrition Mixture or IMDM.

18. The cell culture fluid composition according to claim 16, characterized in that: Each milliliter of the cell culture fluid composition may contain 0.1 microgram to 20 micrograms of complement C3.

19. The cell culture fluid composition according to claim 16, characterized in that: Each milliliter of the cell culture fluid composition may contain 1×10 6 Up to 1×10 8 Platelets.

20. The cell culture fluid composition according to claim 15, 16 or 17, characterized in that: The mitochondria are isolated from a stem cell, a monocyte, a skeletal muscle cell, a liver cell, a kidney cell, a platelet, a fibroblast or an endothelial cell.

21. The cell culture fluid composition according to claim 15, 16 or 17, characterized in that: Used to culture mesenchymal stem cells, and the mesenchymal stem cells are adipose stem cells, umbilical cord stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, skin stem cells, peripheral blood stem cells, endometrial stem cells, amniotic membrane stem cells, gum stem cells or dental pulp stem cells.

22. The cell culture fluid composition according to claim 15, 16 or 17, wherein: Used to culture mesenchymal stem cells, and the mesenchymal stem cells have at least one of the following markers: CD44+, CD90+, CD105+, CD106+, CD166+, Stro-1+ and CD34-.

23. The cell culture fluid composition according to claim 15 or 17, characterized in that: The invention is an immune cell culture composition, further comprising a nutrient additive, wherein the nutrient additive is inositol, thioethanol, folic acid, fetal bovine serum, horse serum or a combination of at least any two of the above.

24. A method for increasing the proliferation rate of immune cells, characterized in that: An immune cell is cultured with the cell culture fluid composition as claimed in claim 26 for at least 24 hours.

25. The method for increasing the proliferation rate of immune cells according to claim 24, characterized in that: The immune cell is a natural killer cell, and the natural killer cell has at least one of the following markers: CD56+, CD3-, CD94+, CD122+, CD127+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKp46+ and NKp80+.

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