WO2022065854A1 - A novel composition for stem cell culture - Google Patents

A novel composition for stem cell culture Download PDF

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WO2022065854A1
WO2022065854A1 PCT/KR2021/012901 KR2021012901W WO2022065854A1 WO 2022065854 A1 WO2022065854 A1 WO 2022065854A1 KR 2021012901 W KR2021012901 W KR 2021012901W WO 2022065854 A1 WO2022065854 A1 WO 2022065854A1
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stem cells
cells
medium
culturing
derived
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French (fr)
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Chaok YIM
TaeWoo GU
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Ts Bio Co., Ltd.
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

Definitions

  • the present invention relates to a novel composition for culturing stem cells, and more particularly, to a novel composition for culturing adipose-derived mesenchymal stem cells for culturing stem cells.
  • the present disclosure relates to a novel composition for culturing stem cells, and more particularly, to a novel composition for culturing stem cells for culturing adipose-derived mesenchymal stem cells.
  • Stem cells are progenitor cells with the ability to self-replicate and differentiate into various tissues by appropriate signals, which form the organs of the human body from the stages of development and play an important role in restoring the functions of organs and tissues after growth.
  • Stem cells include embryonic stem cells obtained from blastocysts in the early stages of development, and adult stem cells obtained from adults or placenta in which the development process is completed. The use of embryonic stem cells faces many ethical problems with regard to the use of living organisms, and therefore, their practical use is limited.
  • adult stem cells have the specificity to differentiate according to the characteristics of the organs after transplantation in vivo and the flexibility to cross-differentiate into different types of cells from the characteristics of original cells, and as it has been identified that adult stem cells have the potential to be differentiated into various cells, the cell therapy potential via adult stem cells has been increasing.
  • the method which is easiest and is able to obtain the most abundant amount is to isolate adipose stem cells derived from adipose tissue, and these stem cells are called adipose-derived stem cells (ASCs). Since it is easy to collect a large amount of tissue from adipose tissue, adipose tissue provides good conditions for obtaining stem cells.
  • ASCs adipose-derived stem cells
  • Korean Patent Application Publication No. 2020-0028865 disclose a medium for direct differentiation of mesenchymal stem cells derived from pluripotent stem cells; a method for preparing mesenchymal stem cells using the same, and the mesenchymal stem cells prepared thereby.
  • the present disclosure is designed to solve several disadvantages including those described above, and in accordance with an exemplary embodiment, the present invention disclosure provides a novel medium composition for culturing stem cells, in which inhibitory activity against oxidative stress is increased and stem cell proliferation ability is optimized.
  • these are exemplary purposes, and the scope of the present disclosure is not limited by the same.
  • a medium composition for culturing adipose-derived mesenchymal stem cells which contains: basal medium; 100-150 ⁇ g/mL of ascorbic-acid-2-phosphate (AA2P) and 0.5-2 ⁇ M vitamin E; 5-15 ⁇ g/mL of a tomato extract; 50-200 ⁇ g/mL of an extract of Ailanthus altissima leaves; 200-400 nM baicalein; 500-700 nM luteolin; 4-6 ⁇ M quercetin; 10-30 ⁇ M ascorbyl-2,6-dipalmitate; 5-15 ⁇ M vitamin D3; 1.5-3.5 ⁇ M gamma-glutamyl cystein ester; and 5-20 ⁇ M/mL of putrescine.
  • basal medium 100-150 ⁇ g/mL of ascorbic-acid-2-phosphate (AA2P) and 0.5-2 ⁇ M vitamin E
  • 5-15 ⁇ g/mL of a tomato extract 50-200 ⁇ g
  • a method for ex vivo culture of adipose-derived stem cells which includes a step of culturing adipose-derived stem cells isolated outside the body of a subject in the medium for culturing adipose-derived stem cells.
  • a novel composition for culturing stem cells of the present disclosure prepared as described above has improved resistance to cell proliferation and oxidative stress of stem cells, and thus, the composition can be used as a culture composition for optimizing stem cell ability of human adipose-derived stem cells (hAD-MSCs).
  • hAD-MSCs human adipose-derived stem cells
  • FIG. 1A to 1E shows the result of the addition of Vitamin E in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 1A is a schematic diagram showing an experimental process in the method.
  • FIG. 1B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 1C is a series of graphs showing the result of GSH analysis.
  • FIG. 1D is a histogram showing the result of the experiment.
  • FIG. 1E is a graph showing oxidative stress resistant capacity according to the concentration of treated Vitamin E.
  • FIGs. 2A to 2E shows the result of the addition of AA2P in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 2A is a schematic diagram showing an experimental process in the method.
  • FIG. 2B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 2C is a series of graphs showing the result of GSH analysis.
  • FIG. 2D is a histogram showing the result of the experiment.
  • FIG. 2E is a graph showing oxidative stress resistant capacity according to the concentration of treated AA2P.
  • FIGs. 3A to 3E shows the result of the addition of tomato extract in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 3A is a schematic diagram showing an experimental process in the method.
  • FIG. 3B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 3C is a series of graphs showing the result of GSH analysis.
  • FIG. 3D is a histogram showing the result of the experiment.
  • FIG. 3E is a graph showing oxidative stress resistant capacity according to the concentration of treated tomato extract.
  • FIGs. 4A to 4E shows the result of the addition of extract of Ailanthus altissima leaves in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 4A is a schematic diagram showing an experimental process in the method.
  • FIG. 4B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 4C is a series of graphs showing the result of GSH analysis.
  • FIG. 4D is a histogram showing the result of the experiment.
  • FIG. 4E is a graph showing oxidative stress resistant capacity according to the concentration of treated extract of Ailanthus altissima leaves.
  • FIGs. 5A to 5E shows the result of the addition of baicalein in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 5A is a schematic diagram showing an experimental process in the method.
  • FIG. 5B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 5C is a series of graphs showing the result of GSH analysis.
  • FIG. 5D is a histogram showing the result of the experiment.
  • FIG. 5E is a graph showing oxidative stress resistant capacity according to the concentration of treated baicalein.
  • FIGs. 6A to 6E shows the result of the addition of quercetin in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 6A is a schematic diagram showing an experimental process in the method.
  • FIG. 6B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 6C is a series of graphs showing the result of GSH analysis.
  • FIG. 6D is a histogram showing the result of the experiment.
  • FIG. 6E is a graph showing oxidative stress resistant capacity according to the concentration of treated quercetin.
  • FIGs. 7A to 7E shows the result of the addition of quercetin in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 7A is a schematic diagram showing an experimental process in the method.
  • FIG. 7B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 7C is a series of graphs showing the result of GSH analysis.
  • FIG. 7D is a histogram showing the result of the experiment.
  • FIG. 7E is a graph showing oxidative stress resistant capacity according to the concentration of treated quercetin.
  • FIGs. 8A to 8E shows the result of the addition of ascorbyl palmitate in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 8A is a schematic diagram showing an experimental process in the method.
  • FIG. 8B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 8C is a series of graphs showing the result of GSH analysis.
  • FIG. 8D is a histogram showing the result of the experiment.
  • FIG. 8E is a graph showing oxidative stress resistant capacity according to the concentration of treated ascorbyl palmitate.
  • FIGs. 9A to 9E shows the result of the addition of vitamin D3 in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells.
  • FIG. 9A is a schematic diagram showing an experimental process in the method.
  • FIG. 9B is a series of fluorescence microscope images showing the result of the experiment.
  • FIG. 9C is a series of graphs showing the result of GSH analysis.
  • FIG. 9D is a histogram showing the result of the experiment.
  • FIG. 9E is a graph showing oxidative stress resistant capacity according to the concentration of treated vitamin D3.
  • FIGs. 10A to 10C shows the result of the addition of antioxidants in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells(in which baicalein, quercetin, luteolin were used).
  • FIG. 10A is a schematic diagram showing an experimental process in the method.
  • FIG. 10B is a series of fluorescence microscope images showing the result of the experiment.
  • 10C is a graph showing oxidative stress resistant capacity according to the concentration of treated antioxidants.
  • FIGs. 11A to 11C shows the result of the addition of various components in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells(in which a tomato extract, an extract of Ailanthus altissima leaves, AA2P, ascorbyl palmitate, vitamin E and vitamin D3 were used).
  • FIG. 11A is a schematic diagram showing an experimental process in the method.
  • FIG. 11B is a series of fluorescence microscope images showing the result of the experiment.
  • 11C is a graph showing oxidative stress resistant capacity according to the concentration of treated various components.
  • FIGs. 12A to 12C shows the result of the addition of various components in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells(in which a tomato extract, an extract of Ailanthus altissima leaves, ascorbyl palmitate and vitamin D3 were used).
  • FIG. 12A is a schematic diagram showing an experimental process in the method.
  • FIG. 12B is a series of fluorescence microscope images showing the result of the experiment.
  • 12C is a graph showing oxidative stress resistant capacity according to the concentration of treated various components.
  • FIGs. 12D to 12F shows the result of the addition of various components in the method of screening a composition for culturing adipocyte-derived mesenchymal stem cells(in which baicalein, quercetin, luteolin, AA2P and vitamin E were used).
  • FIG. 12D is a schematic diagram showing an experimental process in the method.
  • FIG. 12E is a series of fluorescence microscope images showing the result of the experiment.
  • 12F is a graph showing oxidative stress resistant capacity according to the concentration of treated various components.
  • FIG. 13 is a graph analyzing the doubling time of human adipose-derived stem cells, in which the effect of improving antioxidant activity specific to human adipose-derived stem cells was confirmed through comparison with human umbilical cord blood-derived stem cells.
  • FIG. 14 is a graph analyzing the results of doubling time of human adipose-derived stem cells after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 15 is a graph analyzing the results of the cumulative population doubling level of human adipose-derived stem cells after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 16 is images observed under a microscope after culturing human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 17 is a graph analyzing the number of colonies after culturing human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 18 is a series of images in which colonies were observed after culturing human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 19 is graphs analyzing the GSH level of human adipose-derived stem cells after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 20 is graphs analyzing the GSH heterogeneity of human adipose-derived stem cells after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 21 is graphs analyzing the resistance of human adipose-derived stem cells to oxidative stress after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 22 is a graph analyzing the ability of human adipose-derived stem cells to inhibit T-cell proliferation after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • FIG. 23 is a graph analyzing the ability of human adipose-derived stem cells to differentiate into regulatory T-cells after culturing the human adipose-derived stem cells using three types of basal media (C1, C2, C3) of the present disclosure.
  • stem cell refers to an undifferentiated biological cell capable of differentiating into a specific cell, and a cell capable of proliferating in an undifferentiated state. These stem cells are characterized by having pluripotency to differentiate into various cells, and they can be classified into “embryonic stem cells (ESCs)”, which are derived from embryos and have pluripotency capable of differentiating into various cells, and “adult stem cells (ASC)”, which are derived from adults and have totipotency capable of forming individuals.
  • ESCs embryos and have pluripotency capable of differentiating into various cells
  • ASC adult stem cells
  • the fertilized egg which starts as a single cell, becomes a blastocyst consisting of several cells through cell division, and the cells inside the blastocyst are cells derived from the inner cell mass formed and these cells have the ability to differentiate into cells of all tissues in one individual, such as blood, bone, skin, and liver.
  • the “adult stem cells” refer to undifferentiated cells that can be amplified by somatic cell division to replace dead cells after development and regenerate damaged tissues, and these cells include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, etc.
  • adult stem cells Although these adult stem cells have a limited differentiation capability than the embryonic stem cells, they can also be differentiated into cells of a lineage different from that of the origin, which is called trasndifferentiation or plasticity. Since adult stem cells are stable in differentiation and there is no possibility of cancer cells, they have already reached a stage where they can be applied clinically. Additionally, unlike embryonic stem cells, adult stem cells have no destruction of the fertilized egg, thus not raising an ethical issue, and the number of stem cells that can be obtained is small, and adult stem cells have a disadvantage in that these cells are difficult to culture.
  • a medium composition for culturing adipose-derived mesenchymal stem cells which contains: basal medium; 100-150 ⁇ g/mL of ascorbic-acid-2-phosphate (AA2P) and 0.5-2 ⁇ M vitamin E; 5-15 ⁇ g/mL of a tomato extract; 50-200 ⁇ g/mL of an extract of Ailanthus altissima leaves; 200-400 nM baicalein; 500-700 nM luteolin; 4-6 ⁇ M quercetin; 10-30 ⁇ M ascorbyl-2,6-dipalmitate; 5-15 ⁇ M vitamin D3; 1.5-3.5 ⁇ M gamma-glutamyl cystein ester; and 5-20 ⁇ M/mL of putrescine.
  • basal medium 100-150 ⁇ g/mL of ascorbic-acid-2-phosphate (AA2P) and 0.5-2 ⁇ M vitamin E
  • 5-15 ⁇ g/mL of a tomato extract 50-200 ⁇ g
  • the medium composition may contain 120-130 ⁇ g/mL of AA2P and 0.8-1.5 ⁇ M vitamin E; 10-12 ⁇ g/mL of a tomato extract; 70-150 ⁇ g/mL of an extract of Ailanthus altissima leaves; 250-350 nM baicalein; 550-650 nM luteolin; 4.5-5.5 ⁇ M quercetin; 15-25 ⁇ M ascorbyl-2,6-dipalmitate; 7-13 ⁇ M vitamin D3; 1.7-3.2 ⁇ M gamma-glutamyl cystein ester; and 7-15 ⁇ M/mL of putrescine.
  • the basal medium may be ⁇ -MEM and the composition may further contain 5-15% fetal bovine serum (FBS) and 0.5-2% penicillin-streptomycin (PS).
  • FBS fetal bovine serum
  • PS penicillin-streptomycin
  • a method for ex vivo culture of adipose-derived stem cells which includes a step of culturing adipose-derived stem cells isolated outside the body of a subject in the medium for culturing adipose-derived stem cells.
  • the culture medium of the present disclosure may further contain mesenchymal stem cell growth factors that affect the growth of mesenchymal stem cells.
  • Growth factors of the mesenchymal stem cells are, for example, insulin, hydrocortisone, epidermal growth factor (EGF), leukemia inhibitory factor (LIF), granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), transforming growth factor (TGF), etc.
  • the present inventors performed a screening to discover substances that enhance antioxidant activity in human adipose-derived stem cells (hAD-MSC). Specifically, the cells were seeded with human adipose-derived stem cells in a well plate (4 ⁇ 10 3 cells/well) and were cultured for 24 hours after adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) to ⁇ -MEM, and the medium was replaced with a culture medium in which the materials for treatment were diluted at various concentrations and treated for 24 hours. Then, the cells were treated with an antioxidant and incubated for 2 hours, and subjected to Mito-FT staining and endpoint measurement. As the treatment materials, vitamin E, AA2P, a tomato extract, an extract of Ailanthus altissima leaves, baicalein, quercetin, luteolin, ascorbyl palmitate, and vitamin D3 were used.
  • hAD-MSCs human adipose-derived stem cells
  • the medium used at this time contained 10% fetal bovine serum and 1X penicillin-streptomycin in ⁇ -MEM. After removing the medium containing RSL3, Mito-FreSHtracer were added thereto and incubated at 37°C for 1.5 hours. The medium used at this time contained 10% fetal bovine serum and 1X penicillin-streptomycin in ⁇ -MEM. After removing the medium containing Mito-FreSHtracer, the cells were washed twice with 2 mL of DPBS. After adding 250 ⁇ L of TrypLE Express thereto, the cells were allowed to react at 37°C for 2 minutes and 30 seconds, an equal amount of DPBS containing 2% FBS was added thereto so as to detach the cells from the plate. The cells detached from the plate were transferred to a FACS tube and stored on ice, and the fluorescence value was measured using a flow cytometry device.
  • hAD-MSCs human adipose-derived stem cells
  • the medium used at this time contained 10% fetal bovine serum and 1X penicillin-streptomycin in ⁇ -MEM. After removing the medium containing RSL3, 100 ⁇ L each of 15 ⁇ M Mito-FreSHtracer was added thereto and incubated at 37°C for 1 hour.
  • the medium used at this time contained Hanks’balanced salt solution (HBSS) containing 10 mM HEPES. In order to remove Mito-FreSHtracer in the medium before measurement, the medium was replaced with HBSS containing 10 mM HEPES, and then the fluorescence image was measured using Operetta, a confocal imaging device.
  • HBSS Hanks’balanced salt solution
  • the value obtained by dividing the F510 value by the F580 value was obtained as the F510/F580 ratio value, which means the average value of intracellular GSH.
  • the prism 5 program the F510/F580 ratio value of each cell is shown as the X-axis, and the percentage(%) amount of cells corresponding to the F510/F580 ratio value is shown on the Y-axis as a histogram.
  • Alexa 430/PE (F510/F580) parameters were analyzed in all samples using Flowjo software for analyzing flow cytometry, GSH High (right peak) and Low cell (left peak) were divided so as to indicate the percentage of the corresponding cells based on the point where the histogram showing the distribution of F510/F580 was divided into two peaks.
  • vitamin E has cytotoxicity from 25 ⁇ M, AA2P from 250 ⁇ g/mL, the tomato extract from 10 ⁇ g/mL, the extract of Ailanthus altissima leaves from 200 ⁇ g/mL, baicalein from 0.625 ⁇ M, quercetin from 10 ⁇ M, luteolin from 1.25 ⁇ M, ascorbyl palmitate from 50 ⁇ M, and vitamin D3 from 25 ⁇ M (FIGs. 1A to 9E).
  • the present inventors have confirmed the effect of enhancing antioxidant activity specific to human adipose-derived stem cells through comparison with human umbilical cord-derived stem cells (hUC-MSCs) and human cord blood-derived stem cells (hUCB-MSCs).
  • the experimental method was performed under the same conditions as in Example 1 above.
  • antioxidant activity-enhancing substances discovered according to an embodiment of the present disclosure were specific to human adipose-derived stem cells(FIGs. 10A to 13).
  • a basal medium C1 was prepared by adding 120 ⁇ g/mL of ascorbic-acid-2-phosphate (AA2P) and 1 ⁇ M vitamin E to a medium, which contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in ⁇ -MEM.
  • a basal medium C2 was prepared by adding 10 ⁇ g/mL of a tomato extract, 100 ⁇ g/mL of an extract of Ailanthus altissima leaves, 300 nM baicalein, 600 nM luteolin, and 5 ⁇ M quercetin to the above medium.
  • a medium C3 was prepared by adding all of the materials contained in C1 and C2 to the above medium, and further adding 20 ⁇ M ascorbyl 2,6-dipalmitate, 10 ⁇ M vitamin D3, 2.5 ⁇ M ⁇ -glutamyl cystein ester, and 10 ⁇ M/mL of putrescine to the above medium.
  • Example 7 Thawing and culturing of cells
  • a frozen vial stored under refrigeration was placed in a water bath for 1 minute and then the thawed solution in the vial was mixed with 4 mL of a culture medium in a 50 mL conical tube. Thereafter, the mixture was centrifuged at 4°C and at 1,700 rpm for 5 minutes, and the supernatant was removed after confirming the presence of a pellet by the naked eye. After suspending the cells by adding 5 mL of a culture medium to the pellet, a sample for measuring the number of cells was collected, and the culture medium and cells were mixed and placed into a culture vessel so as to measure the number of cells (based on one T-75 flask, 5.0 ⁇ 10 6 cells/12 mL).
  • the culture vessel was placed in a 5% CO 2 incubator at 37°C, and the culture medium was replaced on Day 2 from the day of culture initiation, and cultured to a confluency of 80% or more of the culture vessel area (Day 3 from the day of culture initiation).
  • the culture medium in the culture vessel was removed, and 5 mL of DPBS was added to wash the bottom of the culture vessel and then removed. Then, 1.5 to 2 mL of the cell treatment solution was added thereto, and the cells were placed in a 5% CO 2 incubator at 37°C for 3 minutes, and whether the cells became single cells was checked under a microscope. Thereafter, 5 mL of the culture medium was added, and the cells were harvested and transferred to a 50 mL conical tube. Then, the culture medium was added into the culture vessel to a total volume of 10 mL and the cells were collected once again.
  • the resultant was centrifuged at 4°C at 1,700 rpm conditions for 5 minutes, and the supernatant was removed after confirming the presence of a pellet by the naked eye.
  • a sample for measuring the number of cells was collected and the number of cells was counted, and the culture medium and cells were mixed and placed into a culture vessel (based on one T-75 flask, 5.0 ⁇ 10 6 cells/12 mL).
  • the culture vessel was placed in a 5% CO 2 incubator at 37°C, and the culture medium was replaced on Day 2 from the day of culture initiation, and cultured to a confluency of 80% or more of the culture vessel area (Day 3 from the day of culture initiation).
  • the sample for measurement of cell number was collected using a 20 ⁇ L pipette and diluted in 20 ⁇ L of Trypan blue in a 1:1 ratio. The resultant was placed into a hemocytometer, and the number of cells was measured by checking under a microscope. The number of cells was calculated by Equation 1 below, and the viability was calculated by Equation 2 below.
  • the present inventors analyzed the CPDL of human adipose-derived stem cells(BA200220-1, BA200220-2, and BA200220-3) using three types of antioxidant-added basal media(C1, C2, and C3), a basal medium, and a commercialized medium for comparison of the present disclosure. Specifically, after thawing the three types of p1 cell stock, each of the 4 passage subcultures in the types of media was seeded into 5E+5/75T flasks, and each culture medium was replaced every two days. The analysis of results was averaged without individual results for the three types of cells, and expressed as a comparison value between media.
  • the values of C1, C3, and comparison media were shown to be lower than those of the basal medium and C2 medium(FIG. 14).
  • the CPDL values were shown to be high in the order of cells cultured in the medium for comparison, C1 and C3 media, basal medium, and C1 medium (FIGs. 15 and 16).
  • CFU Colony-forming unit
  • the present inventors compared and analyzed the number of colonies after culturing for 8 days using the cells of Example 4-1 at both conditions of 10 cells/cm 2 and 20 cells/cm 2 . As a result, it was found that the cells cultured in C1, C3, and comparison media formed a greater number of colonies compared to the cells cultured in the basal medium and C2 medium, and that the size of the colonies in the comparative media was formed to be relatively larger than that in the C1 and C3 media(FIGs. 17 and 18).
  • the present inventors seeded human adipose-derived stem cells in well plates(6 ⁇ 10 3 cells/well). After culturing for 24 hours, the cells were treated with an antioxidant and cultured for 2 hours, and Mito-FT staining and endpoint measurement were performed.
  • the present inventors seeded human adipose-derived stem cells in well plates(6 ⁇ 10 3 cells/well).
  • the present inventors seeded human peripheral blood mononuclear cells (hPBMCs) labeled with CFSE(i.e., a fluorescence material) which indicates the degree of cell division, into a U-bottom well plate(1 ⁇ 10 5 cells/well), and stimulated for 5 days with PHA, which promotes T-cell activation. Then, the activity level was compared and analyzed using a flow cytometer.
  • CFSE i.e., a fluorescence material

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