WO2021185364A1 - 细胞培养组合物及其用途 - Google Patents

细胞培养组合物及其用途 Download PDF

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WO2021185364A1
WO2021185364A1 PCT/CN2021/081854 CN2021081854W WO2021185364A1 WO 2021185364 A1 WO2021185364 A1 WO 2021185364A1 CN 2021081854 W CN2021081854 W CN 2021081854W WO 2021185364 A1 WO2021185364 A1 WO 2021185364A1
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cells
cell culture
cell
culture composition
mitochondria
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English (en)
French (fr)
Chinese (zh)
Inventor
郑汉中
许智凯
曾惠卿
杨舜杰
凃启堂
刘思廷
姚莉歆
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Taiwan Mitochondrion Application Technology Co Ltd
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Taiwan Mitochondrion Application Technology Co Ltd
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Priority to CN202411951276.8A priority Critical patent/CN119876009A/zh
Priority to CN202180022924.XA priority patent/CN115335065A/zh
Publication of WO2021185364A1 publication Critical patent/WO2021185364A1/zh
Priority to US17/947,994 priority patent/US20230023218A1/en
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Definitions

  • the present invention relates to a cell culture composition and its use, in particular to a cell culture composition containing mitochondria and its use.
  • Cell culture medium is a nutrient basic substance that mimics the growth environment of cells in animals and plants. It provides nutrients that cells cannot synthesize by themselves and maintains a suitable pH and osmotic pressure, so that cells can survive and survive in vitro. proliferation. In the process of culturing cells, in order to improve cell growth efficiency and cell function, auxiliary culture additives will be added.
  • the cell culture composition comprising mitochondria and a culture medium can improve the growth of cells.
  • the embodiment of the present invention provides a cell culture composition including a culture medium and mitochondria.
  • the embodiment of the present invention provides a use of a cell culture composition for promoting cell growth, wherein the cell culture composition includes a culture medium and mitochondria.
  • the embodiment of the present invention provides a use of a cell culture composition to improve the function of damaged or aged stem cells, wherein the cell culture composition includes a culture medium and mitochondria.
  • the embodiment of the present invention by adding mitochondria to the cell culture medium, it can help the growth of the cells and increase the proliferation rate of the cells.
  • the embodiments of the present invention can also improve the growth of damaged or aging cells and reduce the proportion of aging cells.
  • the function of damaged or aging cells can be improved.
  • the present invention ensures the growth efficiency and stability of cells, which is helpful for subsequent research or experiments to lay the foundation for scientific development and biological research.
  • the present invention ensures the growth efficiency and stability of cells, helps stabilize the yield and yield, reduces the cost of cell culture and controls the quality of products, so as to maximize benefits.
  • Fig. 1A and Fig. 1B show the cell proliferation rate of ARPE-19 cultured using the cell culture composition of the example.
  • the culture time of Fig. 1A is 24 hours, and the culture time of Fig. 1B is 48 hours.
  • Figure 2 shows the cell proliferation rate of HepG2 cultured with the cell culture composition of the example, and the culture time is 24 hours.
  • Figure 3 shows the cell proliferation rate of MDCK cultured using the cell culture composition of the example, and the culture time is 24 hours.
  • Figure 4 shows the cell proliferation rate of human tenocytes cultured using the cell culture composition of the example, and the culture time is 24 hours and 48 hours.
  • Figure 5 shows the cell proliferation rate of culturing natural killer cells using the cell culture composition of the example, and the culture time is 24 hours and 48 hours.
  • Fig. 6 shows the proportion of aged cells in which damaged or aged ADSCs were cultured using the cell culture composition of the example.
  • Fig. 7 shows the proportion of aged cells in which damaged or aged AMSCs were cultured using the cell culture composition of the example.
  • Fig. 8 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the control group containing only platelets, and the culture time is 24 hours and 48 hours.
  • Figure 9 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the example containing only mitochondria, and the culture time is 24 hours and 48 hours.
  • Figure 10 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the example containing mitochondria and platelets, and the culture time is 24 hours and 48 hours.
  • Figure 11 shows the cell proliferation rate of ARPE-19 cultured using the cell culture composition of the example containing mitochondria and complement C3, and the culture time is 24 hours and 48 hours.
  • Mitochondria are the sites for oxidative phosphorylation and the synthesis of adenosine triphosphate (ATP) in cells.
  • mitochondria are also responsible for regulating intracellular oxidative stress processing, signal transmission and other functions.
  • the function of mitochondria is closely related to cell growth. Cells with poor mitochondrial function are also less efficient in producing energy, resulting in slow cell growth or unsound cell growth. Therefore, the inventors used mitochondria as an additive in cell culture compositions to improve cell growth.
  • the cell culture composition includes a culture medium and mitochondria.
  • the cell culture composition can be liquid under normal temperature and pressure, and can be stored at room temperature, preferably at 4°C.
  • the culture medium may include DMEM, F12, DMEM/F12, MEM, MEM- ⁇ , Keratinocyte SFM (1X), IMEM, RPMI 1640, M-199, Opti-MEM, Ham's F-10 Nutrient Mixture, Ham's F-12 Nutrient Mixture Or IMDM, but not limited to this.
  • the medium can be liquid or solid, and an appropriate medium can be selected according to the cells to be cultured, and appropriate nutrient additives and water can be added.
  • the mitochondria can be mitochondria derived from animal cells or plant cells, but not limited to this.
  • mitochondria of animal cells are added to the culture medium when animal cells are cultured.
  • Animal cells that provide 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, etc. Cells with mitochondria.
  • each milliliter of the cell culture composition may contain 1 microgram to 100 micrograms of mitochondria, but it is not limited thereto. In other embodiments, each milliliter of cell culture composition may contain 5 micrograms to 80 micrograms of mitochondria. In other embodiments, each milliliter of cell culture composition may contain 15 micrograms to 40 micrograms of mitochondria.
  • the cell culture composition may include a culture medium, at least one nutritional supplement, and mitochondria.
  • Nutritional supplements may include Fetal Bovine Serum (FBS), salts, amino acids, horse serum, human serum, vitamins, glucose, growth factors, proteins, animal body extracts, carbohydrates, inositol, sulfhydryl Ethanol or folic acid, but not limited to this.
  • Salts include sodium salt, potassium salt, magnesium salt, calcium salt, chloride salt, carbonate, pyruvate, nitrate, phosphate or inorganic salt, but not limited to this.
  • Appropriate nutritional supplements can be selected according to the cells to be cultured.
  • the cell culture composition may include culture medium, mitochondria, and platelets.
  • the platelet may be the platelet in the blood of an animal, but it is not limited to this.
  • each milliliter of the cell culture composition may contain 1 ⁇ 10 6 to 1 ⁇ 10 8 platelets, but it is not limited to this. In other embodiments, each milliliter of cell culture composition may contain 1 ⁇ 10 8 platelets.
  • the cell culture composition may include a culture medium, mitochondria, and complement C3.
  • each milliliter of the cell culture composition may contain 0.1 micrograms to 20 micrograms of complement C3, but it is not limited to this. In other embodiments, each milliliter of cell culture composition may contain 10 micrograms of complement C3.
  • the cell culture composition can promote cell growth and increase the 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.
  • the immune cells may include natural killer cells, and the natural killer cells may have at least one marker of CD56+, CD3-, CD94+, CD122+, CD127+, KIR+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKp46+, and NKp80+.
  • the cell culture composition can improve the aging degree of damaged or aging cells.
  • the cells cultured using the cell culture composition of the embodiment of the present invention may include any somatic cell or any stem cell. Somatic cells can be retinal cells, liver cells, kidney cells, tendon cells, skin cells, or immune cells.
  • the stem cells may include mesenchymal stem cells, and the mesenchymal stem cells may have at least one marker of CD44+, CD90+, CD105+, CD106+, CD166+, Stro-1+, and CD34-.
  • the 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.
  • the mitochondria used in an embodiment of the present invention are derived from human adipose-derived stem cells (ADSC).
  • the stem cell culture medium contains Keratinocyte SFM (1X) solution (Gibco), bovine pituitary extract (BPE) (Gibco), and 10% fetal bovine serum (HyClone).
  • Keratinocyte SFM (1X) solution Gibco
  • BPE bovine pituitary extract
  • HyClone 10% fetal bovine serum
  • the human adipose stem cells were cultured in a petri dish to a cell number of 1.5 ⁇ 10 8 cells, and then the human adipose stem cells in the petri dish were washed with Dulbecco's Phosphate Buffered Saline (DPBS).
  • DPBS Dulbecco's Phosphate Buffered Saline
  • the human adipose stem cells were washed from the petri dish and then dispersed, centrifuged at 600g for 10 minutes, and then 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 placed on ice.
  • IBC-1 buffer 225mM mannitol, 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4
  • the homogenizer grinds human adipose stem cells 15 times. Then, the ground human adipose stem cells were centrifuged at 1000 g for 15 minutes, the supernatant was collected in another centrifuge tube, and the collected supernatant was centrifuged again at 9000 g for 10 minutes, and the supernatant obtained after the centrifugation was removed to obtain The precipitate is mitochondria. 1.5 ml of IBC-2 buffer (225mM mannitol, 75mM sucrose, 30mM Tris-HCl pH 7.4) and proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and stored at 4°C.
  • IBC-2 buffer 225mM mannitol, 75mM sucrose, 30mM Tris-HCl pH 7.4
  • proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and stored at 4°C.
  • the cell culture composition is preferably prepared fresh, but it is not limited to this.
  • the cell culture composition can also be pre-prepared and stored at 4°C, and then used when cell culture is performed.
  • the cell culture composition of the example was used as the culture medium to cultivate human retinal pigment epithelial cell lines (Human retinal pigment epithelium cell, ARPE-19), liver cells (HepG2), and kidney epithelial cells (Madin-Darby).
  • human retinal pigment epithelial cell lines Human retinal pigment epithelium cell, ARPE-19
  • liver cells HepG2
  • kidney epithelial cells Medin-Darby
  • MDCK Human Kidney cell
  • Human tenocyte Human tenocyte
  • NK92MI Natural killer cell
  • the effect of the cell culture composition containing mitochondria on cell growth is evaluated by the Alma Blue detection reagent, and expressed in terms of proliferation rate (also called cell proliferation rate).
  • Alamar blue is a detection reagent used to detect cell viability.
  • the resazurin in the detection kit is a redox indicator, which is a non-toxic, dark blue dye that can penetrate cell membranes and has low fluorescence. When resazurin enters healthy cells, it will be reduced to pink and highly fluorescent resorufin due to the reducing environment in living cells.
  • the cell proliferation can be evaluated by measuring the light absorption or fluorescence value of resorufin. The higher the light absorption value or fluorescence value of resorufin, the higher the cell mass and the higher the cell proliferation rate. The higher the proliferation rate of the cells, the healthier the cells and the stronger the proliferation ability. Therefore, this experiment uses Alma Blue as an indicator to evaluate the cell proliferation rate or cell survival rate.
  • the medium of ARPE-19 can be DMEM/F12 (Gibco), and the nutrient supplement can include 2.5mM glutamic acid, 15mM HEPES, 0.5mM sodium pyruvate, 1200 milligrams/liter (mg/L) sodium bicarbonate and weight percent.
  • the concentration of 10% fetal bovine serum, the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, can be 5 ⁇ g/mL to 80 ⁇ g/mL, can be 15 ⁇ g/mL to 40 ⁇ g/mL, or can be 15 ⁇ g /mL or 40 ⁇ g/mL.
  • the medium of HepG2 can be DMEM/F12
  • the nutritional supplement can contain 10% fetal bovine serum by weight
  • the concentration of mitochondria can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, and can be 5 ⁇ g/mL to 80 ⁇ g /mL, may be 15 ⁇ g/mL to 40 ⁇ g/mL, or may be 15 ⁇ g/mL or 40 ⁇ g/mL.
  • the medium of MDCK can be MEM- ⁇ (Thermo Fisher Scientific) containing Earle's Balanced Salt
  • the nutritional supplement can contain 5% by weight fetal bovine serum
  • the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g /mL, may be 5 ⁇ g/mL to 80 ⁇ g/mL, may be 15 ⁇ g/mL to 40 ⁇ g/mL, or may be 15 ⁇ g/mL or 40 ⁇ g/mL.
  • the culture medium for human tendon cells can be DMEM/F12
  • the nutritional supplement can contain 10% fetal bovine serum by weight
  • the concentration of mitochondria can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, which can be 5 ⁇ g/mL To 80 ⁇ g/mL, may be 15 ⁇ g/mL to 40 ⁇ g/mL, or may be 1 ⁇ g/mL, 15 ⁇ g/mL, 40 ⁇ g/mL, or 100 ⁇ g/mL.
  • the medium for natural killer cells can be MEM- ⁇ (Thermo Fisher Scientific) containing Earle's Balanced Salt
  • the nutritional supplement can include 0.02mM inositol, 0.1mM mercaptoethanol, 0.02mM folic acid, and 12.5% fetal bovine serum by weight.
  • the weight percentage concentration of 12.5% horse serum, the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, can be 5 ⁇ g/mL to 80 ⁇ g/mL, can be 15 ⁇ g/mL to 40 ⁇ g/mL, or It is 1 ⁇ g/mL, 15 ⁇ g/mL, 40 ⁇ g/mL or 100 ⁇ g/mL.
  • each cell takes each cell between 4 to 10 generations for experiment. Using a culture medium without mitochondria, culture each cell until its volume is eighth full of the culture dish, remove the culture medium in the culture dish and rinse the cells with phosphate buffered saline (PBS). Then, 0.25% trypsin was added to the petri dish and the trypsin was allowed to react at 37°C for 5 minutes, and then the culture medium was added to terminate the trypsin reaction. Then, transfer each cell and culture solution in the culture dish to a centrifuge tube, centrifuge at 1000 (Revolutions Per Minute, rpm) for 5 minutes, and then remove the supernatant.
  • PBS phosphate buffered saline
  • ARPE-19 was cultured in the cell culture compositions of Examples 1-1 and 1-2 at a density of 2 ⁇ 10 4 cells per well for 24 or 48 hours.
  • HepG2 was cultured in the cell culture compositions of Examples 2-1 and 2-2 at a density of 5 ⁇ 10 4 cells per well for 24 hours.
  • MDCK was cultured in the cell culture compositions of Examples 3-1 and 3-2 at a density of 5 ⁇ 10 4 cells per well for 24 hours.
  • Human tenocytes were cultured in the cell culture compositions of Examples 4-1 to 4-4 at a density of 2 ⁇ 10 4 cells per well for 24 or 48 hours.
  • the natural killer cells were cultured in the cell culture compositions of Examples 5-1 to 5-4 at a density of 2 ⁇ 10 4 cells per well for 24 or 48 hours. After the culture is completed, wash the cells with phosphate buffer solution, replace the culture medium with a culture medium containing Alma Blue, and culture for 3 hours. After the culture is completed, the fluorescence is measured at the wavelength of OD530/595, and the proliferation rate of each cell is calculated.
  • Figures 1A and 1B show the cell proliferation rate of ARPE-19 cultured with the cell culture composition of the example.
  • the culture time of Figure 1A is 24 hours.
  • the incubation time for 1B is 48 hours.
  • Figure 2 shows the cell proliferation rate of HepG2 cultured with the cell culture composition of the example, and the culture time is 24 hours.
  • Table 8 and Figure 3 for the experimental results of MDCK.
  • Figure 3 shows the cell proliferation rate of MDCK cultured with the cell culture composition of the example, and the culture time is 24 hours.
  • Table 9 and Figure 4 for the experimental results of human tenocytes.
  • Figure 4 shows the cell proliferation rate of human tenocytes cultured using the cell culture composition of the example.
  • the culture time is 24 hours and 48 hours.
  • Figure 5 shows the cell proliferation rate of natural killer cells cultured using the cell culture composition of the example.
  • the culture time is 24 hours and 48 hours.
  • the initial control group is the number of cells at the beginning of the cell culture (the 0th hour), which is set to 1.
  • the proliferation rate is a multiple of the number of cells after cell culture relative to the initial control group.
  • the control group was the group without mitochondria added.
  • # indicates a significant difference relative to the initial control group (P ⁇ 0.05)
  • ## indicates a high significant difference relative to the initial control group (P ⁇ 0.01)
  • ### indicates a very significant difference relative to the initial control group Difference (P ⁇ 0.001).
  • # indicates a significant difference from the control group (P ⁇ 0.05).
  • 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 of the concentration of mitochondria, indicating that the cell culture composition containing mitochondria does help Cell growth. Moreover, the increase in the proliferation rate becomes more obvious with the increase of the culture time, indicating that the cell culture composition containing mitochondria can stably help the cell growth.
  • AGE advanced glycation end products
  • SA- ⁇ -gal set is used to evaluate the effect of the cell culture composition containing mitochondria on damaged or aging stem cells, and the aging degree is expressed as the percentage of aging cells (%).
  • the stem cells used in this experiment include Adipose-derived Stem Cell (ADSC) and Amniotic Membrane Stem Cell (AMSC).
  • SA- ⁇ -gal Senescence-associated beta-galactosidase
  • This experiment uses the SA- ⁇ -gal kit (Senescence ⁇ -Galactosidase Staining Kit#9860, Cell Signaling technology) to assess the aging status of stem cells.
  • the ADSC medium can be Keratinocyte SFM (1X) (Catalog number: 1705042, Life Technologies), the nutritional supplement can include 10% fetal bovine serum by weight, and the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, may be 5 ⁇ g/mL to 80 ⁇ g/mL, may be 15 ⁇ g/mL to 40 ⁇ g/mL, or may be 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL.
  • ⁇ g/mL microgram/ml
  • the medium of AMSC can be DMEM/F12 (Gibco)
  • the nutritional supplement can contain 10% fetal bovine serum by weight
  • the concentration of mitochondria can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, which can be 5 ⁇ g/ mL to 80 ⁇ g/mL, may be 15 ⁇ g/mL to 40 ⁇ g/mL, or may be 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL.
  • the process of culturing stem cells is explained below. First, take the 10th generation stem cells for experiment. Using a culture medium without mitochondria, when the stem cells are cultured until their volume is eighth full of the petri dish, the culture fluid in the petri dish is removed and the stem cells are rinsed with phosphate buffered saline (PBS). Then, 0.25% trypsin was added to the petri dish and the trypsin was allowed to react at 37°C for 5 minutes, and then the culture medium was added to terminate the trypsin reaction. Then, transfer each cell and culture solution in the culture dish to a centrifuge tube, centrifuge at 1000 (Revolutions Per Minute, rpm) for 5 minutes, and then remove the supernatant.
  • PBS phosphate buffered saline
  • the stem cells were cultured at a density of 1.5 ⁇ 10 4 cells per well for 24 hours.
  • AGE was added to the well plate, and the stem cells were cultured in a culture medium containing AGE at a concentration of 400 ⁇ g/mL for 4 hours.
  • the cell culture solution containing AGE was removed, and the cell culture composition of the example was added, and the stem cells were cultured in the cell culture composition containing different mitochondrial concentrations for 24 hours.
  • the concentration of mitochondria in the cell culture composition is 0, 1 ⁇ g/mL, 15 ⁇ g/mL, or 40 ⁇ g/mL.
  • FIG. 6 shows the proportion of aged cells in the damaged or aged ADSC cultured using the cell culture composition of the example.
  • FIG. 7 shows the proportion of aged cells of damaged or aged AMSC cultured using the cell culture composition of the example.
  • the control group was a culture composition supplemented with AGE and not containing mitochondria.
  • the control group was the group without AGE and mitochondria added.
  • the ratio of aging cells is the percentage of the number of aging cells to the number of all cells in a unit area to represent the degree of aging.
  • the vertical axis is the proportion of aging cells (%), # indicates a significant difference from the control group (P ⁇ 0.05), ## indicates a high significant difference from the control group (P ⁇ 0.01), ### Indicates a very significant difference compared to the control group (P ⁇ 0.001).
  • platelets were further added to the cell culture composition, and the fibroblast CCD-996SK was cultured with the cell culture composition containing platelets as the culture medium.
  • the effect of the cell culture composition containing mitochondria on cell growth is evaluated by the Alma Blue detection reagent, and expressed in terms of proliferation rate (also called cell proliferation rate).
  • the blood is drawn into a blood collection tube or centrifuge tube containing an anticoagulant.
  • the blood collection tube is, for example, a CPT tube (BD CPT TM Cell Preparation Tube, REF362761).
  • centrifuge the blood to separate the blood. For example, centrifuge at 1500g for 10 minutes to separate the blood into a red blood cell layer, a colloid layer, a white blood cell layer (buffy coat), and a plasma layer.
  • the white blood cell layer is collected into a new centrifuge tube.
  • the layer contains monocytes and platelets.
  • the white blood cell layer and HEP buffer 140mM NaCl, 2.7mM KCl, 3.8mM HEPES, 5mM EGTA, pH 7.4 were uniformly mixed at a ratio of 1:1, and Prostaglandin E1 was added to make the concentration to 1 ⁇ M to prevent platelet activation.
  • the white blood cell layer is centrifuged at 100 g for 15 to 20 minutes to precipitate the white blood cells and the remaining red blood cells, and the supernatant containing the platelets is collected in a new centrifuge tube.
  • the supernatant containing the platelets is centrifuged at 800 g for 15 to 20 minutes to precipitate the platelets.
  • the supernatant was removed, and the precipitated platelets were rinsed twice with wash buffer (10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% (w/v) dextrose). Next, use Tyrode's buffer (134mM NaCl, 12mM NaHCO 3 , 2.9 mM KCl, 0.34 mM Na 2 HPO 4 , 1 mM MgCl 2 , 10 mM HEPES, pH 7.4) for reconstitution, and count platelets with a hemocytometer.
  • the prepared platelets can be stored at 20°C to 24°C, and then added to the cell culture composition when the cells are cultured, but it is better to use them immediately after preparation.
  • the medium of CCD-996SK can be DMEM (Gibco; 10566016) containing 2mM glutamic acid
  • the nutritional supplement can contain 10% fetal bovine serum by weight
  • the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, can be 5 ⁇ g/mL to 80 ⁇ g/mL, can be 15 ⁇ g/mL to 40 ⁇ g/mL, or can be 1 ⁇ g/mL, 15 ⁇ g/mL, 40 ⁇ g/mL
  • platelets can be 1 ⁇ 10 6 to 1 ⁇ 10 8 pieces/mL or may be 1 ⁇ 10 6 pieces, 1 ⁇ 10 7 pieces or 1 ⁇ 10 8 pieces/mL.
  • CCD-996SK was cultured in the control group, the control group and the cells of Examples 8-1 to 8-4 at a density of 1.5 ⁇ 10 4 cells per well. Cultured in the culture composition for 24 hours. After the culture is completed, wash the cells with phosphate buffer solution, replace the culture medium with a culture medium containing Alma Blue, and culture for 3 hours. After the culture is completed, the fluorescence is measured at the wavelength of OD530/595, and the proliferation rate of each cell is calculated.
  • Fig. 8 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the control group containing only platelets, and the culture time is 24 hours and 48 hours.
  • Figure 9 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the example containing only mitochondria, and the culture time is 24 hours and 48 hours.
  • Figure 10 shows the cell proliferation rate of CCD-996SK cultured with the cell culture composition of the example containing mitochondria and platelets, and the culture time is 24 hours and 48 hours.
  • the initial control group is the number of cells at the beginning of the cell culture (the 0th hour), which is set to 1.
  • the proliferation rate is a multiple of the number of cells after cell culture relative to the initial control group.
  • the control group was the group without mitochondria added.
  • the control group was a group that did not add mitochondria and did not add platelets.
  • # indicates a significant difference compared to the control group (P ⁇ 0.05), ## indicates a high significant difference compared to the control group (P ⁇ 0.01), ### indicates a significant difference compared to the control group There is a very significant difference (P ⁇ 0.001).
  • Example 8-3 and Example 8-4 confirm that when the mitochondrial concentration is 40 ⁇ g/mL, the addition of 1 ⁇ 10 6 platelets/mL can further increase the cell proliferation rate.
  • Example 8-4 and the control group 8-1 confirmed that when the number of platelets is 1 ⁇ 10 6 , the addition of 40 ⁇ g/mL mitochondria can make the cell proliferation rate exceed the number of platelets by 1 ⁇ 10 8 (Control group 8-3 ) The effect that can only be achieved. Therefore, compared to a cell culture composition containing only platelets or only mitochondria, a cell culture composition containing both mitochondria and platelets can have a multiplier effect in increasing the rate of cell proliferation.
  • Example 8-4 the 48-hour proliferation rate of Example 8-4 is slightly lower than that of Example 8-3. The reason is that the cells have grown to saturation, and the total number of cells should be slightly equal when the cells grow to saturation. The difference in cell proliferation rate is not obvious.
  • complement C3 (Sigma-Aldrich, 204885) was further added to the cell culture composition, and the cell culture composition containing complement C3 was used as a culture medium to cultivate ARPE-19.
  • the effect of the cell culture composition containing mitochondria on cell growth is evaluated by the Alma Blue detection reagent, and expressed in terms of proliferation rate (also called cell proliferation rate).
  • the medium of ARPE-19 can be DMEM/F12 (Gibco), and the nutrient supplement can include 2.5mM glutamic acid, 15mM HEPES, 0.5mM sodium pyruvate, 1200 milligrams/liter (mg/L) sodium bicarbonate and weight percent.
  • the concentration of 10% fetal bovine serum, the mitochondrial concentration can be 1 microgram/ml ( ⁇ g/mL) to 100 ⁇ g/mL, can be 5 ⁇ g/mL to 80 ⁇ g/mL, can be 15 ⁇ g/mL to 40 ⁇ g/mL, or can be 15 ⁇ g /mL.
  • the complement C3 concentration may be 0.1 ⁇ g/mL to 20 ⁇ g/mL, or may be 10 ⁇ g/mL.
  • Figure 11 shows the cell proliferation rate of ARPE-19 cultured using the cell culture composition of the example containing mitochondria and complement C3.
  • the culture time is 24 hours and 48 hours.
  • the control group was the group that did not add mitochondria and did not add complement C3, and set it as 1.
  • the proliferation rate is the multiple of the number of cells after cell culture relative to the control group.
  • the embodiment of the present invention by adding mitochondria to the cell culture medium, it can help the growth of the cells and increase the proliferation rate of the cells.
  • the embodiments of the present invention can also improve the growth of damaged or aging cells and reduce the proportion of aging cells.
  • the function of damaged or aging cells can be improved.
  • the present invention ensures the growth efficiency and stability of cells, which is helpful for subsequent research or experiments to lay the foundation for scientific development and biological research.
  • the present invention ensures the growth efficiency and stability of cells, helps stabilize the yield and yield, reduces the cost of cell culture and controls the quality of products, so as to maximize benefits.

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