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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PCT/CN2021/081854
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French (fr)
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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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台湾粒线体应用技术股份有限公司
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Priority to CN202180022924.XA priority Critical 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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Abstract

提供了一种细胞培养组合物及其用途。该细胞培养组合物包含培养基以及线粒体，可促进细胞生长、改善受损或老化的干细胞的功能，藉以改善细胞的生长状况。

Description

细胞培养组合物及其用途 技术领域
本发明是关于细胞培养组合物及其用途，尤其是关于包含线粒体的细胞培养组合物及其用途。
背景技术
细胞培养基(cell culture medium)是一种模拟动植物体内细胞的生长环境的营养基础物质，其提供细胞本身不能合成的营养物并维持适宜的pH值及渗透压，使细胞能在体外存活并增殖。在培养细胞的过程中，为了提高细胞的生长效率与细胞的功能，会添加辅助性的培养添加物。
在贫瘠的培养基中或是严苛的培养条件下，细胞成长的速率较慢，生长出来的细胞可能较脆弱、不易存活，甚至无法成长。培养出的细胞若是不够健康或是成长的速度过慢，对后续的实验或研究皆会造成不利的影响。
发明内容
根据本发明实施例，通过包含线粒体与培养基的细胞培养组合物，可改善细胞的生长状况。
本发明实施例提供一种细胞培养组合物，包含培养基以及线粒体。
本发明实施例提供一种细胞培养组合物在促进细胞生长的用途，其中该细胞培养组合物包含培养基以及线粒体。
本发明实施例提供一种细胞培养组合物在改善受损或老化的干细胞的功能的用途，其中该细胞培养组合物包含培养基以及线粒体。
根据本发明实施例，藉由在细胞培养基中添加线粒体，可帮助细胞生长、提高细胞的增生率。对于受损或老化的细胞而言，根据本发明实施例亦能改善受损或老化的细胞的生长、降低老化细胞比例。除此之外，根据本发明实施例还能改善受损或老化的细胞的功能。在学术方面，本发明确保细胞的生长效率及稳定性有助于进行后续的研究或实验，以奠定科学发展、生物学研究的基础。在产业方面，本发明确保细胞的生长效率及稳定性有助于稳定产率及良率，可降低细胞培养成本并控管产品质量，以达成利益最大化。
附图说明
图1A及图1B显示使用实施例的细胞培养组合物培养ARPE-19的细胞增生率，图1A的培养时间为24小时，图1B的培养时间为48小时。
图2显示使用实施例的细胞培养组合物培养HepG2的细胞增生率，培养时间为24小时。
图3显示使用实施例的细胞培养组合物培养MDCK的细胞增生率，培养时间为24小时。
图4显示使用实施例的细胞培养组合物培养人类肌腱细胞的细胞增生率，培养时间为24小时及48小时。
图5显示使用实施例的细胞培养组合物培养自然杀手细胞的细胞增生率，培养时间为24小时及48小时。
图6显示使用实施例的细胞培养组合物培养受损或老化的ADSC的老化细胞比例。
图7显示使用实施例的细胞培养组合物培养受损或老化的AMSC的老化细胞比例。
图8显示使用仅含血小板的对照组的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。
图9显示使用仅含线粒体的实施例的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。
图10显示使用含线粒体及血小板的实施例的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。
图11显示使用含线粒体及补体C3的实施例的细胞培养组合物培养ARPE-19的细胞增生率，培养时间为24小时及48小时。
具体实施方式
于以下实施方式中详细叙述本发明的详细特征及优点，其内容足以使任何熟习相关技艺者了解本发明的技术内容并据以实施，且根据本说明书所揭露的内容、申请专利范围及图式，任何熟习相关技艺者可轻易理解本发明相关的目的及优点。以下实施例系进一步详细说明本发明的观点，但非以任何观点限制本发明之范畴。
线粒体是细胞内进行氧化磷酸化反应及合成三磷酸腺苷(ATP)的场所，除了提供细胞正常代谢所需 的能量外，还负责调控细胞内氧化压力处理、讯号传递等功能。在细胞的培养过程中，线粒体的功能与细胞的生长有相当大的关联。线粒体功能较差的细胞在产生能量方面的效率亦较差，造成细胞生长缓慢或细胞生长不健全。因此，发明人藉由以线粒体作为细胞培养组合物中的添加物来改善细胞的生长情况。
根据本发明一实施例，细胞培养组合物包含培养基及线粒体。细胞培养组合物在常温常压下可呈液态，可保存于室温，以保存于4℃为佳。
培养基可包含DMEM、F12、DMEM/F12、MEM、MEM-α、Keratinocyte SFM(1X)、IMEM、RPMI 1640、M-199、Opti-MEM、Ham’s F-10 Nutrient Mixture、Ham’s F-12 Nutrient Mixture或IMDM，但不以此为限。培养基可为液体或固体，可依据所欲培养的细胞选择适当的培养基并添加适当的营养添加物及水。
线粒体可为取自动物细胞或植物细胞的线粒体，但不以此为限。在本发明部分实施例中，培养动物细胞时添加动物细胞的线粒体至培养基中。提供线粒体的动物细胞可包含脂肪干细胞、单核球细胞、胚胎干细胞、间质干细胞、造血干细胞、CD34+干细胞、骨髓干细胞、骨骼肌细胞、肝脏细胞、肾脏细胞、血小板、纤维母细胞、内皮细胞等具有线粒体的细胞。细胞培养组合物中，每一毫升的细胞培养组合物可包含1微克至100微克的线粒体，但不以此为限。在其他实施例中，每一毫升的细胞培养组合物可包含5微克至80微克的线粒体。在其他实施例中，每一毫升的细胞培养组合物可包含15微克至40微克的线粒体。
根据本发明其他实施例，细胞培养组合物可包含培养基、至少一种营养添加物及线粒体。
营养添加物可包含胎牛血清(Fetal Bovine Serum，FBS)、盐类、胺基酸、马血清、人类血清、维生素、葡萄糖、生长因子、蛋白质、动物体萃取物、碳水化合物、肌醇、巯乙醇或叶酸，但不以此为限。盐类包含钠盐、钾盐、镁盐、钙盐、氯盐、碳酸盐、丙酮酸盐、硝酸盐、磷酸盐或无机盐，但不以此为限。可依据所欲培养的细胞选择适当的营养添加物。
根据本发明其他实施例，细胞培养组合物可包含培养基、线粒体及血小板。
血小板可为动物血液中的血小板，但不以此为限。在细胞培养组合物中，每一毫升的细胞培养组合物可包含1×10 6至1×10 8个血小板，但不以此为限。在其他实施例中，每一毫升的细胞培养组合物可包含1×10 8个血小板。
根据本发明其他实施例，细胞培养组合物可包含培养基、线粒体及补体C3。
在细胞培养组合物中，每一毫升的细胞培养组合物可包含0.1微克至20微克的补体C3，但不以此为限。在其他实施例中，每一毫升的细胞培养组合物可包含10微克的补体C3。
根据本发明一实施例，细胞培养组合物可促进细胞生长，提高细胞增生率。使用本发明实施例的细胞培养组合物所培养的细胞可包含任意体细胞。体细胞可包含视网膜细胞、肝脏细胞、肾脏细胞、肌腱细胞、皮肤细胞或免疫细胞。免疫细胞可包含自然杀手细胞，自然杀手细胞可具有CD56+、CD3-、CD94+、CD122+、CD127+、KIR+、NKG2A+、NKG2D+、NKp30+、NKp44+、NKp46+及NKp80+中的至少一种标记。
根据本发明一实施例，细胞培养组合物可改善受损或老化的细胞的老化程度。使用本发明实施例的细胞培养组合物所培养的细胞可包含任意体细胞或任意干细胞。体细胞可为视网膜细胞、肝脏细胞、肾脏细胞、肌腱细胞、皮肤细胞或免疫细胞。干细胞可包含间质干细胞，间质干细胞可具有CD44+、CD90+、CD105+、CD106+、CD166+、Stro-1+及CD34-中的至少一种标记。间质干细胞可为脂肪干细胞、脐带干细胞、胎盘干细胞、骨髓干细胞、羊水干细胞、皮肤干细胞、周边血干细胞、子宫内膜干细胞、羊膜干细胞、牙龈干细胞或牙髓干细胞。
以下说明如何制备本发明实施例的细胞培养组合物。
本发明一实施例所使用的线粒体取自人类脂肪干细胞(adipose-derived stem cell，ADSC)。干细胞培养液包含Keratinocyte SFM(1X)溶液(Gibco)、牛垂体提取物(bovine pituitary extract(BPE)，Gibco)、重量百分浓度10％的胎牛血清(HyClone)。首先，在培养皿中将人类脂肪干细胞培养至细胞数为1.5×10 8个细胞，再以杜氏磷酸盐缓冲液(DPBS)冲洗培养皿中的人类脂肪干细胞。接着，移除培养皿中的杜氏磷酸盐缓冲液后，在培养皿中加入细胞剥离用的胰蛋白酶(Trypsin)，并在37℃下反应3分钟后，再加入干细胞培养液以终止反应。接着，将人类脂肪干细胞自培养皿中冲洗下来后打散，以600g离心10分钟后，移除上清液。接着，将离心后留下的人类脂肪干细胞及80毫升的IBC-1缓冲液(225mM甘露醇、75mM蔗糖、0.1mM EDTA、30mM Tris-HCl pH 7.4)加入均质器中，并在冰上以均质器对人类脂肪干细胞进行研磨15次。接着，以1000g离心研磨后的人类脂肪干细胞15分钟，将上清液收集至另一离心管，再以9000g再次离心收集的上清液10分钟，移除再次离心后得到的上清液，获得的沉淀物即为线粒体。在线粒体沉淀物中加入1.5毫升的IBC-2缓冲液(225mM甘露醇、75mM蔗糖、30mM Tris-HCl pH 7.4)及蛋白质分解酶抑制剂，并置于4℃下保存。
进行细胞培养时，根据细胞种类选用适当的培养基，接着依期望浓度加入所需的营养添加物以及如上 所述的线粒体并混合均匀即可使用。细胞培养组合物以新鲜配制为佳，但不以此为限，亦可事先配制完成细胞培养组合物后保存于4℃，待进行细胞培养时再取用。
以下说明使用本发明实施例的细胞培养组合物进行细胞培养。
〔实验一，提高细胞增生率〕
在本实验中，使用实施例的细胞培养组合物作为培养液，来培养人类视网膜色素上皮细胞株(Human retinal pigment epithelium cell，ARPE-19)、肝脏细胞(HepG2)、肾脏上皮细胞(Madin-Darby Canine Kidney cell，MDCK)、人类肌腱细胞(Human tenocyte)及自然杀手细胞(Nature killer cell，NK92MI)。并且，通过阿尔玛蓝检测试剂来评估包含线粒体的细胞培养组合物对于细胞生长的影响，并以增生率(亦称为细胞增生率)来表示。
阿尔玛蓝(Alamar blue)是用于检测细胞活力的检测试剂。检测套组内的刃天青(resazurin)是一种氧化还原指示剂，其为无毒、可穿透细胞膜且低荧光性的深蓝色染料。当刃天青进入健康的细胞中，会因活细胞体内的还原环境而被还原成粉红色且具高荧光性的试卤灵(resorufin)。可藉由测量试卤灵的光吸收值或荧光值来评估细胞的增生率(cell proliferation)。试卤灵的光吸收值或荧光值越高，表示细胞量越多，细胞的增生率越高。细胞的增生率越高也表示细胞越健康、增生能力越强。因此，本实验使用阿尔玛蓝作为评估细胞增生率或细胞存活率的指标。
本实验所使用各实施例的细胞培养组合物的详细成分如表1至表5所示。
ARPE-19的培养基可为DMEM/F12(Gibco)，营养添加物可包含2.5mM麸酰胺酸、15mM HEPES、0.5mM丙酮酸钠、1200毫克/升(mg/L)碳酸氢钠及重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为15μg/mL或40μg/mL。
HepG2的培养基可为DMEM/F12，营养添加物可包含重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为15μg/mL或40μg/mL。
MDCK的培养基可为含有Earle’s Balanced Salt的MEM-α(Thermo Fisher Scientific)，营养添加物可包含重量百分浓度5％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为15μg/mL或40μg/mL。
人类肌腱细胞的培养基可为DMEM/F12，营养添加物可包含重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为1μg/mL、15μg/mL、40μg/mL或100μg/mL。
自然杀手细胞的培养基可为含有Earle’s Balanced Salt的MEM-α(Thermo Fisher Scientific)，营养添加物可包含0.02mM肌醇、0.1mM巯乙醇、0.02mM叶酸、重量百分浓度12.5％胎牛血清及重量百分浓度12.5％马血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为1μg/mL、15μg/mL、40μg/mL或100μg/mL。
〔表1〕
Figure PCTCN2021081854-appb-000001
〔表2〕
Figure PCTCN2021081854-appb-000002
〔表3〕
Figure PCTCN2021081854-appb-000003
〔表4〕
Figure PCTCN2021081854-appb-000004
〔表5〕
Figure PCTCN2021081854-appb-000005
以下说明各细胞的培养流程。首先，取培养代数为4至10代间的各细胞进行实验。使用不含线粒体的培养液，将各细胞培养至其体积为培养皿的八分满时，移除培养皿中的培养液并使用磷酸盐缓冲液(phosphate buffered saline，PBS)润洗细胞。接着，在培养皿中加入0.25％的胰蛋白酶并使胰蛋白酶在37℃下反应5分钟，再加入培养液以终止胰蛋白酶的反应。接者，将培养皿中的各细胞及培养液移至离心管中，以每分钟转速1000(Revolutions Per Minute，rpm)离心5分钟后，移除上清液。接着，再加入新的培养液至离心管中，进行细胞计数。接着，将ARPE-19以每孔2×10 4个细胞的密度于实施例1-1及1-2的细胞培养组合物中培养24或48小时。将HepG2以每孔5×10 4个细胞的密度于实施例2-1及2-2的细胞培养组合物中培养24小时。将MDCK以每孔5×10 4个细胞的密度于实施例3-1及3-2的细胞培养组合物中培养24小时。将人类肌腱细胞以每孔2×10 4个细胞的密度于实施例4-1至4-4的细胞培养组合物中培养24或48小时。将自然杀手细胞以每孔2×10 4个细胞的密度于实施例5-1至5-4的细胞培养组合物中培养24或48小时。培养完成后，使用磷酸盐缓冲液清洗各细胞，并将培养液更换为含有阿尔玛蓝的培养液，培养3小时。培养完成后，以OD530/595的波长量测荧光，计算各细胞的增生率。
ARPE-19的实验结果请参考表6及图1A及图1B，图1A及图1B显示使用实施例的细胞培养组合物培养ARPE-19的细胞增生率，图1A的培养时间为24小时，图1B的培养时间为48小时。HepG2的实验结果请参考表7及图2，图2显示使用实施例之细胞培养组合物培养HepG2的细胞增生率，培养时间为24小时。MDCK的实验结果请参考表8及图3，图3显示使用实施例的细胞培养组合物培养MDCK的细胞增生率，培养时间为24小时。人类肌腱细胞的实验结果请参考表9及图4，图4显示使用实施例的细胞培养组合物培养人类肌腱细胞的细胞增生率，培养时间为24小时及48小时。自然杀手细胞的实验结果请参考表10及图5，图5显示使用实施例的细胞培养组合物培养自然杀手细胞的细胞增生率，培养时间为24小时及48小时。初始对照组为细胞刚开始培养时(第0小时)的细胞数，将其定为1。增生率为细胞培养后的细胞数相对于初始对照组的倍数。对照组为未添加线粒体的组别。图1A至图3中，#表示相对初始对照组具有显著差异(P<0.05)，##表示相对初始对照组具有高显著差异(P<0.01)，###表示相对初始对照组具有非常显著差异(P<0.001)。图4及图5中，#表示相对于对照组具有显著差异(P<0.05)。
〔表6〕
Figure PCTCN2021081854-appb-000006
Figure PCTCN2021081854-appb-000007
〔表7〕
Figure PCTCN2021081854-appb-000008
〔表8〕
Figure PCTCN2021081854-appb-000009
〔表9〕
Figure PCTCN2021081854-appb-000010
〔表10〕
Figure PCTCN2021081854-appb-000011
上述实验结果显示，使用含有线粒体的细胞培养组合物来培养细胞，可使细胞具有提高的增生率，且增生率随着线粒体的浓度增加而提升，表示含有线粒体的细胞培养组合物确实有助于细胞的生长。并且，增生率的提高随培养时间增长而更加明显，表示含有线粒体的细胞培养组合物可稳定帮助细胞生长。
〔实验二，改善受损或老化的干细胞的老化程度〕
在本实验中，使用醣化终产物(advanced glycation end product，AGE)诱导干细胞损伤或老化，再使用实施例的细胞培养组合物作为培养液来培养受损或老化的干细胞。并且，通过SA-β-gal套组来评估包含线粒体的细胞培养组合物对于受损或老化的干细胞的影响，并以老化细胞比例(％)来表示老化程度。本实验使用的干细胞包含脂肪干细胞(Adipose-derived stem cell，ADSC)及羊膜干细胞(Amniotic membrane stem cell，AMSC)。
在老化的细胞中，老化相关-β-半乳糖苷酶(Senescence-associated beta-galactosidase，SA-β-gal)会被过度表达，因此SA-β-gal可作为细胞衰老的标记之一。本实验使用SA-β-gal套组(Senescenceβ-Galactosidase Staining Kit#9860，Cell Signaling technology)来评估干细胞的老化状态。
本实验所使用的细胞培养组合物的详细成分如表11及表12所示。
ADSC的培养基可为Keratinocyte SFM(1X)(Catalog number:17005042，Life Technologies)，营养添加物可包含重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为1μg/mL、15μg/mL或40μg/mL。
AMSC的培养基可为DMEM/F12(Gibco)，营养添加物可包含重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或 者可为1μg/mL、15μg/mL或40μg/mL。
[表11]
Figure PCTCN2021081854-appb-000012
[表12]
Figure PCTCN2021081854-appb-000013
以下说明干细胞的培养流程。首先，取培养代数为第10代的干细胞进行实验。使用不含线粒体的培养液，将干细胞培养至其体积为培养皿的八分满时，移除培养皿中的培养液并使用磷酸盐缓冲液(phosphate buffered saline，PBS)润洗干细胞。接着，在培养皿中加入0.25％的胰蛋白酶并使胰蛋白酶在37℃下反应5分钟，再加入培养液以终止胰蛋白酶的反应。接者，将培养皿中的各细胞及培养液移至离心管中，以每分钟转速1000(Revolutions Per Minute，rpm)离心5分钟后，移除上清液。接着，再加入新的培养液至离心管中，进行细胞计数。接着，将干细胞以每孔1.5×10 4个细胞的密度培养24小时。接着，于孔盘中加入AGE，使干细胞在含有浓度为400μg/mL的AGE的培养液中培养4小时。接着，将含有AGE的细胞培养液移除，加入实施例的细胞培养组合物，使干细胞在含有不同线粒体浓度的细胞培养组合物中培养24小时。细胞培养组合物中线粒体的浓度为0、1μg/mL、15μg/mL或40μg/mL。培养完成后，使用磷酸盐缓冲液清洗各细胞并使用SA-β-gal套组进行细胞老化的评估。
ADSC的实验结果请参考表13及图6，图6显示使用实施例的细胞培养组合物培养受损或老化的ADSC的老化细胞比例。AMSC的实验结果请参考表14及图7，图7显示使用实施例的细胞培养组合物培养受损或老化的AMSC的老化细胞比例。对照组为添加AGE且不包含线粒体的培养组合物。控制组为未添加AGE且未添加线粒体的组别。老化细胞比例为在单位面积中老化细胞的数量占所有细胞的数量的百分比，以代表老化程度。图6至图7中，纵轴为老化细胞比例(％)，#表示相对对照组具有显著差异(P<0.05)，##表示相对对照组具有高显著差异(P<0.01)，###表示相对对照组具有非常显著差异(P<0.001)。
〔表13〕
Figure PCTCN2021081854-appb-000014
〔表14〕
Figure PCTCN2021081854-appb-000015
上述实验结果显示，使用含有线粒体的细胞培养组合物来培养受损或老化的干细胞，可降低老化细胞比例，且老化细胞比例随着线粒体的浓度增加而降低，表示含有线粒体的细胞培养组合物确实有助于改善受损或老化的干细胞的生长，同时有助于改善受损或老化的干细胞的功能。
〔实验三，细胞培养组合物包含血小板〕
在本实验中，进一步在细胞培养组合物中添加血小板，以包含血小板的细胞培养组合物作为培养液来培养纤维母细胞CCD-996SK。并且，通过阿尔玛蓝检测试剂来评估包含线粒体的细胞培养组合物对于细胞生长的影响，并以增生率(亦称为细胞增生率)来表示。
以下说明本实验所使用的血小板的制备流程。首先，将血液抽取至含有抗凝血剂的采血管或离心管中，采血管为例如CPT管(BD
Figure PCTCN2021081854-appb-000016
CPT TM Cell Preparation Tube，REF362761)。接着，将血液离心使血液分层，例如以1500g离心10分钟使血液分层为红血球层、胶体层、白细胞层(buffy coat)及血浆层，将白细胞层收集至新的离心管中，其中白细胞层包含单核球细胞及血小板。接着，将白细胞层与HEP buffer(140mM NaCl、2.7mM KCl、3.8mM HEPES、5mM EGTA，pH 7.4)以1：1的比例均匀混合，再加入Prostaglandin E1使其浓度为1μM以防止血小板活化。接着，将白细胞层以100g离心15至20分钟使白血球及残余的红血球沉淀，将含有血小板的上清液收集至新的离心管中。接着，将含有血小板的上清液以800g离心15至20分钟使血小板沉淀。移除上清液，使用wash buffer(10mM柠檬酸钠、150mM NaCl、1mM EDTA、1％(w/v)dextrose)将所沉淀的血小板润洗两次。接着，使用Tyrode’s buffer(134mM NaCl、12mM NaHCO 3、2.9mM KCl、0.34mM Na 2HPO 4、1mM MgCl 2、10mM HEPES，pH 7.4)进行回溶，以血球计数器进行血小板计数。制备完成的血小板可保存于20℃至24℃，待培养细胞时再加入细胞培养组合物中，但以配制后立即使用为佳。
本实验所使用的细胞培养组合物的详细成分如表15及表16所示。
CCD-996SK的培养基可为含有2mM麸酰胺酸的DMEM(Gibco；10566016)，营养添加物可包含重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为1μg/mL、15μg/mL、40μg/mL，血小板可为1×10 6至1×10 8个/mL或者可为1×10 6个、1×10 7个或1×10 8个/mL。
〔表15〕
Figure PCTCN2021081854-appb-000017
〔表16〕
Figure PCTCN2021081854-appb-000018
本实验的细胞培养流程请参考实验一，进行细胞计数时，将CCD-996SK以每孔1.5×10 4个细胞的密度培养于控制组、对照组及实施例8-1至8-4的细胞培养组合物中培养24小时。培养完成后，使用磷酸盐缓冲液清洗各细胞，并将培养液更换为含有阿尔玛蓝的培养液，培养3小时。培养完成后，以OD530/595的波长量测荧光，计算各细胞的增生率。
CCD-996SK的实验结果请参考表17及图8至图10。图8显示使用仅含血小板的对照组的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。图9显示使用仅含线粒体的实施例的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。图10显示使用含线粒体及血小板的实施例的细胞培养组合物培养CCD-996SK的细胞增生率，培养时间为24小时及48小时。初始对照组为细胞刚开始培养时(第0小时)的细胞数，将其定为1。增生率为细胞培养后的细胞数相对于初始对照组的倍数。对照组为未添加线粒体的组别。控制组为未添加线粒体且未添加血小板的组别。图8至图10中，#表示相较于控制组具有显著差异(P<0.05)，##表示相较于控制组具有高显著差异(P<0.01)， ###表示相较于控制组具有非常显著差异(P<0.001)。
〔表17〕
Figure PCTCN2021081854-appb-000019
根据上述实验结果，由对照组可知，在细胞培养基中添加血小板可提高细胞增生率，且增生率随着血小板的数量增加而提升。由实施例8-1至8-3可知，使用含有线粒体的细胞培养组合物来培养细胞，可使细胞具有提高的增生率，且增生率随着线粒体的浓度增加而提升，表示含有线粒体的细胞培养组合物确实有助于细胞的生长。由对照组8-1、实施例8-3及实施例8-4可知，使用含有线粒体及血小板的细胞培养组合物来培养细胞，可进一步提升细胞的增生率。实施例8-3及实施例8-4证实在线粒体浓度皆为40μg/mL的情况下，添加1×10 6个/mL的血小板可使细胞增生率进一步提升。实施例8-4及对照组8-1证实在血小板数量皆为1×10 6的情况下，添加40μg/mL的线粒体可使细胞增生率超过血小板数量为1×10 8(对照组8-3)始能达到的效果。因此，相较于仅添加血小板或仅添加线粒体的细胞培养组合物，同时含有线粒体及血小板的细胞培养组合物在提升细胞增生率上可具有加乘的效果。并且，增生率的提高随培养时间增长而更加明显，表示含有线粒体的细胞培养组合物可稳定帮助细胞生长。于此，特别说明，实施例8-4的48小时增生率略低于实施例8-3的48小时增生率，原因在于细胞已生长至饱和，细胞生长至饱和时细胞总数应略为相等，故细胞生增生率的差异不明显。
〔实验四，细胞培养组合物包含补体C3〕
在本实验中，进一步在细胞培养组合物中添加补体C3(Sigma-Aldrich，204885)，以包含补体C3的细胞培养组合物作为培养液来培养ARPE-19。并且，通过阿尔玛蓝检测试剂来评估包含线粒体的细胞培养组合物对于细胞生长的影响，并以增生率(亦称为细胞增生率)来表示。
本实验所使用的细胞培养组合物的详细成分如表18所示。
ARPE-19的培养基可为DMEM/F12(Gibco)，营养添加物可包含2.5mM麸酰胺酸、15mM HEPES、0.5mM丙酮酸钠、1200毫克/升(mg/L)碳酸氢钠及重量百分浓度10％胎牛血清，线粒体浓度可为1微克/毫升(μg/mL)至100μg/mL，可为5μg/mL至80μg/mL，可为15μg/mL至40μg/mL，或者可为15μg/mL。补体C3浓度可为0.1μg/mL至20μg/mL，或者可为10μg/mL。
〔表18〕
Figure PCTCN2021081854-appb-000020
本实验的细胞培养流程请参考实验一，进行细胞计数时，将ARPE-19以每孔2×10 4个细胞的密度于实施例的细胞培养组合物中培养24或48小时。孔盘中线粒体的浓度为15μg/mL。孔盘中补体C3的浓度为10μg/mL。培养完成后，使用磷酸盐缓冲液清洗各细胞，并将培养液更换为含有阿尔玛蓝的培养液，培养3小时。培养完成后，以OD530/595的波长量测荧光，计算各细胞的增生率。
ARPE-19的实验结果请参考表19及图11，图11显示使用含线粒体及补体C3的实施例的细胞培养组合物培养ARPE-19的细胞增生率，培养时间为24小时及48小时。控制组为未添加线粒体且未添加补体 C3的组别，将其定为1。增生率为细胞培养后的细胞数相对于控制组的倍数。
〔表19〕
Figure PCTCN2021081854-appb-000021
上述实验结果显示，使用含有线粒体及补体C3的细胞培养组合物来培养细胞，可进一步提升细胞的增生率。并且，增生率的提高随培养时间增长而更加明显，表示含有线粒体的细胞培养组合物可稳定帮助细胞生长。
根据本发明实施例，藉由在细胞培养基中添加线粒体，可帮助细胞生长、提高细胞的增生率。对于受损或老化的细胞而言，根据本发明实施例亦能改善受损或老化的细胞的生长、降低老化细胞比例。除此之外，根据本发明实施例还能改善受损或老化的细胞的功能。在学术方面，本发明确保细胞的生长效率及稳定性有助于进行后续的研究或实验，以奠定科学发展、生物学研究的基础。在产业方面，本发明确保细胞的生长效率及稳定性有助于稳定产率及良率，可降低细胞培养成本并控管产品质量，以达成利益最大化。

Claims (12)

  1. 一种细胞培养组合物，包含：
    培养基；以及
    线粒体。
  2. 如权利要求1所述的细胞培养组合物，其中，每一毫升的所述细胞培养组合物包含1微克至100微克的线粒体。
  3. 如权利要求1或2所述的细胞培养组合物，进一步包含血小板，其中，每一毫升的所述细胞培养组合物包含1×10 6至1×10 8个血小板。
  4. 如权利要求1或2所述的细胞培养组合物，进一步包含补体C3，其中，每一毫升的所述细胞培养组合物包含0.1微克至20微克的补体C3。
  5. 一种细胞培养组合物在促进细胞生长中的用途，其中，所述细胞培养组合物包含培养基以及线粒体。
  6. 如权利要求5所述的用途，其中，每一毫升的所述细胞培养组合物包含1微克至100微克的线粒体。
  7. 如权利要求5或6所述的用途，其中，所述细胞包含视网膜细胞、肝脏细胞、肾脏细胞、肌腱细胞、皮肤细胞或免疫细胞。
  8. 如权利要求7所述的用途，其中，所述免疫细胞为具有CD56+及CD3-标记的自然杀手细胞。
  9. 一种细胞培养组合物在改善受损或老化的干细胞的功能中的用途，其中，所述细胞培养组合物包含培养基以及线粒体。
  10. 如权利要求9所述的用途，其中，每一毫升的所述细胞培养组合物包含1微克至100微克的线粒体。
  11. 如权利要求9或10所述的用途，其中，使用所述细胞培养组合物培养的干细胞为间质干细胞。
  12. 如权利要求9或10所述的用途，其中，使用所述细胞培养组合物培养的干细胞具有CD90+及CD34-标记。
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