WO2022233080A1 - 一种脂类物质添加剂及其应用 - Google Patents

一种脂类物质添加剂及其应用 Download PDF

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WO2022233080A1
WO2022233080A1 PCT/CN2021/098712 CN2021098712W WO2022233080A1 WO 2022233080 A1 WO2022233080 A1 WO 2022233080A1 CN 2021098712 W CN2021098712 W CN 2021098712W WO 2022233080 A1 WO2022233080 A1 WO 2022233080A1
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acid
lipid
lipid substance
cells
cholesterol
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PCT/CN2021/098712
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French (fr)
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陈国凯
许发祥
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澳门大学
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    • 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
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N2500/36—Lipids

Definitions

  • the present disclosure relates to the technical field of cell culture, in particular, to a lipid substance additive and application thereof.
  • Human pluripotent stem cells have the characteristics of infinite self-replication and differentiation into all cell types in the human body, and are an important model system for studying human embryonic development.
  • the lipid concentration in the lipid substance additive that can be added to the culture medium provided on the market is a uniform concentration, and its concentration cannot correspond to its content ratio in bovine serum. If the lipid additive is used at the concentration recommended in the product instructions, the final lipid concentration is still far lower than the corresponding lipid concentration provided by bovine serum in the traditional medium, and further increasing the concentration of the lipid additive will cause stem cells to die.
  • One of the objectives of the present disclosure includes providing a lipid additive to improve or overcome the problems of existing lipid additives.
  • the second objective of the present disclosure includes providing a cell culture medium containing the above-mentioned lipid substance additive.
  • a third object of the present disclosure includes providing a method for culturing cells using the above-mentioned lipid substance additive or cell culture medium.
  • the fourth objective of the present disclosure includes providing an application of the above-mentioned lipid substance additive in a cell culture medium.
  • the present disclosure provides a lipid substance additive, which is obtained by dissolving a lipid substance in a solvent, and the lipid substance includes arachidonic acid, cholesterol, dl- ⁇ -tocopheryl acetate, linoleic acid , linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid, and at least Tween 80 is not included in the lipid additives.
  • the lipids corresponding to each milliliter of solvent include 0.7 ⁇ g-7mg arachidonic acid, 22 ⁇ g-220mg cholesterol, 7 ⁇ g-70mg dl- ⁇ -tocopheryl acetate, 4 ⁇ g-40mg of linoleic acid, 4 ⁇ g-40 mg of linolenic acid, 1 ⁇ g-10 mg of myristic acid, 4 ⁇ g-40 mg of oleic acid, 10 ⁇ g-100 mg of palmitic acid, 2 ⁇ g-20 mg of palmitoleic acid, and 16 ⁇ g-160 mg of stearic acid.
  • the lipid substances corresponding to each milliliter of solvent include 5 ⁇ g-5 mg of arachidonic acid, 100 ⁇ g-100 mg of cholesterol, 50 ⁇ g-50 mg of dl- ⁇ -tocopheryl acetate, 20 ⁇ g-20 mg of Linoleic acid, 20 ⁇ g-20 mg linolenic acid, 5 ⁇ g-5 mg myristic acid, 20 ⁇ g-20 mg oleic acid, 50 ⁇ g-50 mg palmitic acid, 10 ⁇ g-10 mg palmitoleic acid, and 80 ⁇ g-80 mg stearic acid.
  • the lipids corresponding to each milliliter of solvent may include 40 ⁇ g-4 mg of arachidonic acid, 800 ⁇ g-80 mg of cholesterol, 400 ⁇ g-40 mg of dl- ⁇ -tocopheryl acetate, 150 ⁇ g-15 mg linoleic acid, 150 ⁇ g-15mg linolenic acid, 40 ⁇ g-4mg myristic acid, 150 ⁇ g-15mg oleic acid, 400 ⁇ g-40mg palmitic acid, 80 ⁇ g-8mg palmitoleic acid and 500 ⁇ g-50mg stearic acid.
  • the lipids corresponding to each milliliter of solvent may include 100 ⁇ g-3mg of arachidonic acid, 2mg-60mg of cholesterol, 1mg-30mg of dl- ⁇ -tocopheryl acetate, 500 ⁇ g-15mg of linoleic acid, 500 ⁇ g-15mg of linolenic acid, 100 ⁇ g-3mg of myristic acid, 300 ⁇ g-10mg of oleic acid, 1mg-30mg of palmitic acid, 200 ⁇ g-6mg of palmitoleic acid and 1mg-30mg of stearic acid.
  • the lipid substance corresponding to each milliliter of solvent may include 200 ⁇ g-2 mg of arachidonic acid, 4 mg-40 mg of cholesterol, 2 mg-20 mg of dl- ⁇ -tocopheryl acetate, 1 mg-10 mg of linoleic acid, 1 mg-10 mg of linolenic acid, 200 ⁇ g-2 mg of myristic acid, 1 mg-10 mg of oleic acid, 2 mg-20 mg of palmitic acid, 400 ⁇ g-4 mg of palmitoleic acid, and 2 mg-20 mg of stearic acid.
  • the lipids corresponding to each milliliter of solvent may include 400 ⁇ g-1.2 mg of arachidonic acid, 10 mg-30 mg of cholesterol, 5 mg-15 mg of dl- ⁇ -tocopheryl acetate, 2 mg- 6mg linoleic acid, 2mg-6mg linolenic acid, 500 ⁇ g-1.5mg myristic acid, 2mg-6mg oleic acid, 5mg-15mg palmitic acid, 1mg-3mg palmitoleic acid and 6mg-18mg stearic acid acid.
  • the lipids corresponding to each milliliter of solvent include 0.7 mg of arachidonic acid, 22 mg of cholesterol, 7 mg of dl- ⁇ -tocopheryl acetate, 4 mg of linoleic acid, and 4 mg of flax acid, 1 mg of myristic acid, 4 mg of oleic acid, 10 mg of palmitic acid, 2 mg of palmitoleic acid, and 16 mg of stearic acid.
  • the lipid additive does not contain any Tween component.
  • the solvent is methanol or absolute ethanol, preferably absolute ethanol.
  • the present disclosure provides a cell culture medium comprising the lipid additive according to any of the preceding embodiments.
  • the present disclosure provides a cell culture method for culturing cells using the cell culture medium of the foregoing embodiments.
  • the cells are human embryonic stem cells.
  • the cells are human embryonic stem cells H1.
  • the present disclosure provides a use of the lipid additive according to any one of the preceding embodiments in a cell culture medium.
  • the lipid additive provided by the present disclosure does not contain the cytotoxic Tween 80 component in the formulation of the lipid additive on the market.
  • the lipid material additive can provide a lipid environment more similar to serum culture without changing the cell morphology and without affecting the pluripotency of stem cells. It is suitable for culturing cells, especially cells including human embryonic stem cells.
  • FIG. 1 shows the composition of CDL of Thermo Fisher; Composition of CDL; Fig. 3 shows the difference of lipid concentration in CDL composition and its difference in AlbuMAX; Fig. 4 shows the effect of different concentrations of CDL on cloning efficiency; Fig. 5 shows different concentrations (0, 1: 10000, 1:1000, 1:100) the effect of CDL on the proliferation index of cells.
  • Figures 6 to 8 show the cytotoxicity result chart of Tween 80 in CDL and the formula composition chart of hCDL provided in Example 2 of the present disclosure, wherein Figure 6 shows the effects of each component in the official formula CDL and CDL on the cell proliferation index ; Figure 7 shows the concentration of CDL-related lipids in AlbuMAX detected by liquid mass spectrometry; Figure 8 shows the formulation of the 1000 ⁇ new lipid additive (hCDL) after removing Tween 80 of the present disclosure.
  • hCDL new lipid additive
  • Figures 9 to 12 are graphs showing the effect of hCDL on human embryonic stem cell culture provided in Example 3 of the present disclosure, wherein Figure 9 shows the cells of human embryonic stem cells under the conditions of E8, Albumin, Albumin+CDL and Albumin+hCDL Morphology; Figure 10 shows changes in dry cell weight of human embryonic stem cells under E8, AlbuMAX, Albumin and Albumin+hCDL conditions; Figure 11 shows changes in E8, Albumin, Albumin+hCDL (0.1x), and Albumin+hCDL ( The effect of 1x), hCDL (0.1x) and hCDL (1x) conditions on the folding and expansion of human embryonic stem cells; Figure 12 shows lipids in cells cultured for 48 h with E8, Albumin, AlbuMAX, Albumin+CDL and Albumin+hCDL substance components.
  • Fig. 13 to Fig. 19 are graphs showing the effect of hCDL on the pluripotency and differentiation ability of human embryonic stem cells provided in Example 4 of the present disclosure, wherein Fig. 13 shows that in E8, Albumin, AlbuMAX, Albumin+CDL and Albumin+hCDL Expression of NANOG, OCT4 and SOX2 genes in cells cultured under conditions;
  • Figure 14 shows the expression of T, MIXL1 genes during spontaneous differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions;
  • Figure 15 shows Mock, AlbuMAX, Albumin And the expression of SOX17, GATA4 gene during spontaneous differentiation under the condition of Albumin+hCDL;
  • Figure 16 shows the expression of PAX6, SOX1 gene during spontaneous differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions;
  • Figure 17 shows Mock, AlbuMAX , Albumin and Albumin+hCDL under the condition of mesoderm differentiation to T, MIXL1 gene expression
  • the lipid substance additive provided by the present disclosure and its application will be specifically described below.
  • the present disclosure proposes a lipid substance additive, which is obtained by dissolving a lipid substance in a solvent, and the lipid substance includes arachidonic acid, cholesterol, dl- ⁇ -tocopheryl acetate, linoleic acid, linolenic acid, Myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid, and at least no Tween 80 in the lipid substance additives.
  • At least Tween 80 is not included in the lipid additive to reduce cytotoxicity. Further, the lipid substance additive may not contain any Tween component.
  • lipid additives that do not contain Tween 80 (the lipid additives contain arachidonic acid, cholesterol, dl-alpha-tocopheryl acetate, linoleic acid, linolenic acid, nutmeg acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid) are all within the scope of this disclosure.
  • the lipids corresponding to each milliliter of solvent include 0.7 ⁇ g-7mg arachidonic acid, 22 ⁇ g-220mg cholesterol, 7 ⁇ g-70mg dl- ⁇ -tocopheryl acetate, 4 ⁇ g-40mg of linoleic acid, 4 ⁇ g-40 mg of linolenic acid, 1 ⁇ g-10 mg of myristic acid, 4 ⁇ g-40 mg of oleic acid, 10 ⁇ g-100 mg of palmitic acid, 2 ⁇ g-20 mg of palmitoleic acid, and 16 ⁇ g-160 mg of stearic acid.
  • lipids corresponding to each milliliter of solvent may include 5 ⁇ g-5 mg of arachidonic acid, 100 ⁇ g-100 mg of cholesterol, 50 ⁇ g-50 mg of dl- ⁇ -tocopheryl acetate, 20 ⁇ g-20 mg of linoleic acid, 20 ⁇ g-20 mg of linolenic acid, 5 ⁇ g-5 mg of myristic acid, 20 ⁇ g-20 mg of oleic acid, 50 ⁇ g-50 mg of palmitic acid, 10 ⁇ g-10 mg of palmitoleic acid, and 80 ⁇ g-80 mg of stearic acid.
  • lipids corresponding to each milliliter of solvent may include 40 ⁇ g-4 mg of arachidonic acid, 800 ⁇ g-80 mg of cholesterol, 400 ⁇ g-40 mg of dl- ⁇ -tocopheryl acetate, and 150 ⁇ g-15 mg of linoleic acid.
  • the lipids corresponding to each milliliter of solvent may include 100 ⁇ g-3mg of arachidonic acid, 2mg-60mg of cholesterol, 1mg-30mg of dl- ⁇ -tocopheryl acetate, 500 ⁇ g-15mg of linoleic acid , 500 ⁇ g-15 mg of linolenic acid, 100 ⁇ g-3 mg of myristic acid, 300 ⁇ g-10 mg of oleic acid, 1 mg-30 mg of palmitic acid, 200 ⁇ g-6 mg of palmitoleic acid, and 1 mg-30 mg of stearic acid.
  • the lipid substances corresponding to each milliliter of solvent may include 200 ⁇ g-2 mg of arachidonic acid, 4 mg-40 mg of cholesterol, 2 mg-20 mg of dl- ⁇ -tocopheryl acetate, and 1 mg-10 mg of linoleic acid.
  • 1 mg-10 mg of linolenic acid 200 ⁇ g-2 mg of myristic acid, 1 mg-10 mg of oleic acid, 2 mg-20 mg of palmitic acid, 400 ⁇ g-4 mg of palmitoleic acid, and 2 mg-20 mg of stearic acid.
  • the lipids corresponding to each milliliter of solvent may include 400 ⁇ g-1.2mg of arachidonic acid, 10mg-30mg of cholesterol, 5mg-15mg of dl- ⁇ -tocopheryl acetate, and 2mg-6mg of linseed oil.
  • acid 2 mg-6 mg linolenic acid, 500 ⁇ g-1.5 mg myristic acid, 2 mg-6 mg oleic acid, 5 mg-15 mg palmitic acid, 1 mg-3 mg palmitoleic acid, and 6 mg-18 mg stearic acid.
  • lipid substances corresponding to each milliliter of solvent may include 0.7 mg of arachidonic acid, 22 mg of cholesterol, 7 mg of dl- ⁇ -tocopheryl acetate, 4 mg of linoleic acid, 4 mg of linolenic acid, 1 mg of of myristic acid, 4 mg of oleic acid, 10 mg of palmitic acid, 2 mg of palmitoleic acid, and 16 mg of stearic acid.
  • the solvent may be methanol or absolute ethanol, preferably absolute ethanol.
  • the content of lipid substances provided by the present disclosure is close to the ratio and concentration of lipid substances in the serum extract, on the one hand, it does not contain substances that are toxic to cells, and on the other hand, it can not change the cell shape and does not affect the Under the premise of pluripotency of stem cells, a lipid environment more similar to serum culture is provided to successfully complete the culture of cells.
  • the present disclosure also provides a cell culture medium containing the above-mentioned lipid substance additive.
  • the cell culture medium can be E8 medium, DMEM medium, DMEM/F12 medium, Nutristem XF/FF medium, StemPro medium, X-Vivo 10 medium, Neutrodoma-CS medium, HyClone HyCellStem Medium, StemFit medium, mTeSR1 medium, TeSR1 medium, TeSR2 and other stem cell or adult cell medium.
  • the components contained therein may also include FGF2 and other growth factors.
  • the present disclosure also provides the application of the above-mentioned lipid substance additive or cell culture medium, that is, for cell culture.
  • the present disclosure provides a cell culture method, which uses the above cell culture medium to culture cells.
  • the cells are human embryonic stem cells (including human pluripotent stem cells), such as human embryonic stem cells H1, and also human embryonic stem cells H9 and artificially induced pluripotent stem cells (iPSCs).
  • human embryonic stem cells including human pluripotent stem cells
  • H1 human embryonic stem cells
  • H9 human embryonic stem cells
  • iPSCs artificially induced pluripotent stem cells
  • Figure 1 shows the composition of ingredients disclosed in Thermo Fisher's CDL product information.
  • Figure 2 shows the composition of serum extracts AlbuMAX and CDL, which shows the composition of the serum extract AlbuMAX reported so far. Comparing Figure 1 and Figure 2, it can be seen that the types of lipid components contained in CDL are insufficient, and the concentration is also significantly lower. By mass spectrometry, the difference between the concentration of lipids in the CDL component and that in AlbuMAX is shown in Figure 3.
  • the human embryonic stem cell H1 cell line was the cell to be cultured, and the medium was E8.
  • the specific implementation is as follows: human embryonic stem cells H1 are cultured in E8 medium, fresh medium is replaced every day, and passage is performed when the cell density reaches 70-80%. First wash twice with DPBS-EDTA, then incubate for 5 min at room temperature, aspirate DPBS-EDTA a third time, and add E8 medium containing 10 ⁇ M ROCK inhibitor Y27632. After resuspending the cells, passage into Matrigel precoated cell culture plates at a density of 1:6 to 1:12.
  • the operation of the cell clone assay experiment is: pre-add 500 microliters of basal medium to a 12-well plate, digest the cells with TrypLE for 5 minutes, neutralize them with 9 times the volume of DMEM/F12, count the cells with a hemocytometer, centrifuge, wash And diluted to 5000 cells/ml and then added 100 ⁇ l to each well.
  • Cells were placed in a 37°C incubator with 5% oxygen, 10% carbon dioxide. Drugs were added every two days and the medium was changed. Six days later, the cells were stained with alkaline phosphatase and the number of clones was counted.
  • Figure 4 shows the effect of different concentrations of CDL on cloning efficiency
  • Figure 5 shows the effect of different concentrations (0, 1:10000, 1:1000, 1:100) CDL on cells effect on the proliferation index.
  • the cloning efficiency of cells decreases significantly with the increase of the concentration of CDL, that is, CDL has obvious cytotoxicity at the recommended concentration (1:100).
  • the proliferation index of cells treated with CDL at a concentration of 1:100 was significantly reduced compared with the control, that is, CDL at a concentration of 1:100 significantly inhibited the growth of H1 cells.
  • the commercially available lipid substance additive (chemically defined lipid concentrate, CDL) has the disadvantages of insufficient lipid concentration and cytotoxicity under the instructed use concentration.
  • the effect of hCDL on human embryonic stem cell culture was specifically tested.
  • the experimental operation was as follows: when the H1 cell density reached 60%, the cells were separated with EDTA/DPBS, and the cells were passaged into 12-well plates at a ratio of 1:12. Cell morphology was observed after culturing in medicated E8 medium for two days from the beginning of the second day; cells were passaged into 6-well plates at a ratio of 1:12, and cells were collected with TrypLE after culturing in medicated E8 medium for two days from the next day.
  • the number of cells was counted by flow cytometry, and dried at 50 degrees for three days to measure the dry weight of cells; the cells were passaged into a 24-well plate at a ratio of 1:20, the cells were collected by TrypLE the next day, and Day0 was calculated by flow cytometry.
  • Cells cultured in medicated E8 medium for three days, collected and counted daily to draw cell growth curves; cells were passaged into 6-well plates at a ratio of 1:12 and cultured in medicated E8 medium beginning the next day Two days later, the cells were collected with TrypLE, and the number of cells was counted by flow cytometry.
  • the lipids of the cells were extracted with chloroform-methanol-water system (2:1:1, v/v/v). After methylation treatment, the cells were determined by GC-MS. lipid content. All experiments were performed with daily medium changes, and the number of replicates for each treatment was 3.
  • FIGS. 9 to 12 The results are shown in FIGS. 9 to 12 .
  • Figure 9 shows the cell morphology of human embryonic stem cells under E8, Albumin, Albumin+CDL and Albumin+hCDL conditions. It can be seen from Figure 9 that hCDL does not affect the cell morphology of stem cells.
  • Figure 10 shows changes in dry cell weight of human embryonic stem cells under E8, AlbuMAX, Albumin and Albumin+hCDL conditions. As can be seen from Figure 10: hCDL increased cell dry weight.
  • Figure 11 shows the effect of E8, Albumin, Albumin+hCDL (0.1x), and Albumin+hCDL (1x), hCDL (0.1x) and hCDL (1x) conditions on the proliferation of human embryonic stem cells.
  • Figure 12 shows lipid composition in cells cultured for 48 h with E8, Albumin, AlbuMAX, Albumin+CDL and Albumin+hCDL. It can be seen from Figure 12 that the structure of lipids in the H1 cells cultured with hCDL for 48 h was detected by GC-MS, and the structure of lipids in the cells was similar to that of the cells cultured with AlbuMAX.
  • the cells were passaged at 1:12 with EDTA/DPBS, and were cultured in E8 medium with medicated for 3 passages to collect and detect the expression level of pluripotency genes.
  • the number of replicates for each treatment group was 4, and the expression levels of pluripotency genes were compared with GAPDH and E8 group as control.
  • FIG. 13 shows the expression of NANOG, OCT4, and SOX2 genes in cells cultured under the conditions of E8, Albumin, AlbuMAX, Albumin+CDL, and Albumin+hCDL. It can be seen from Figure 13 that H1 cells cultured with hCDL for 3 generations still maintain the expression of pluripotency marker genes NANOG, OCT4 and SOX2, that is, hCDL does not affect the pluripotency of H1 cells.
  • the experimental operation of stem cell self-differentiation is as follows: when the cell density of H1 cells reaches about 30%, the cells are cultured in medicated E6 medium (E8 medium without FGF2 and TGF ⁇ ) for 12 days, and the medium is changed every day. Lineage marker genes were detected after 12 days.
  • FIGS. 14 to 16 show the expression of T and MIXL1 genes during spontaneous differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from Figure 14 that the mesoderm marker genes have the same trend as AlbuMAX when the hCDL-cultured cells differentiate spontaneously.
  • Figure 15 shows the expression of SOX17 and GATA4 genes during spontaneous differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from Fig. 15 that the endoderm marker gene has the same trend as AlbuMAX when the hCDL-cultured cells differentiate spontaneously.
  • Figure 16 shows the expression of PAX6 and SOX1 genes during spontaneous differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from FIG. 16 that the ectoderm marker genes are not affected by hCDL during spontaneous differentiation of hCDL-cultured cells.
  • hCDL had no effect on the ectoderm marker genes of cell self-differentiation, and had a similar effect on mesoderm and endoderm marker genes as AlbuMAX.
  • H1 cells were first cultured in medicated E8 medium for 48 hours, and then the mesoderm was differentiated into E8 medium with 20 ng/ml BMP4 added for two days. Detection of marker genes; endoderm differentiation was first cultured in E5 medium supplemented with 5 ⁇ M CHIR99021 (E8 medium removed FGF2, TGF ⁇ and insulin) for one day, and then cultured in E5 medium supplemented with 10 ng/ml Activin A for 3 Days; ectoderm differentiated into E6 medium with 10 ⁇ M SB431542 and 100 nM LDN193189 added for 4 days to detect the marker gene.
  • endoderm differentiation was first cultured in E5 medium supplemented with 5 ⁇ M CHIR99021 (E8 medium removed FGF2, TGF ⁇ and insulin) for one day, and then cultured in E5 medium supplemented with 10 ng/ml Activin A for 3 Days; ectoderm differentiated into E6 medium with 10 ⁇ M SB431542
  • FIGS. 17 to 19 show the expression of T and MIXL1 genes during mesoderm differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from FIG. 17 that when cells cultured with hCDL are guided to undergo mesoderm differentiation, the mesoderm marker genes are not affected by hCDL.
  • Figure 18 shows the expression of SOX17 and GATA4 genes during endoderm differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from Figure 18 that when cells cultured with hCDL are guided to undergo endoderm differentiation, the mesoderm marker genes are not affected by hCDL.
  • Figure 19 shows the expression of PAX6 and SOX1 genes during mesoderm differentiation under Mock, AlbuMAX, Albumin and Albumin+hCDL conditions. It can be seen from Fig. 19 that when the hCDL-cultured cells are guided to undergo mesoderm differentiation, the change trend of the ectoderm marker genes is consistent with that of AlbuMAX.
  • hCDL had no effect on directed differentiation.
  • the relevant lipid concentration in the lipid additive provided by the present disclosure is based on serum extract, and does not contain the cytotoxic Tween 80 component in the formulation of the lipid additive on the market.
  • the lipid material additive can provide a lipid environment more similar to serum culture without changing the cell morphology and without affecting the pluripotency of stem cells. It is suitable for culturing cells, especially cells including human embryonic stem cells.
  • the present disclosure provides a lipid substance additive, a cell culture medium containing the lipid substance additive, a method for culturing cells using the lipid substance additive or the cell culture medium, and the application of the lipid substance additive in the cell culture medium .
  • the lipid additive removes the cytotoxic Tween 80 component in the formula of the lipid additive in the market, and provides lipids that are more similar to serum culture without changing the cell shape or affecting the pluripotency of stem cells.
  • the environment is suitable for culturing cells, especially cells including human embryonic stem cells.

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Abstract

一种脂类物质添加剂、含有该脂类物质添加剂的细胞培养基及其应用。该脂类物质添加剂由脂类物质溶于溶剂中而得，脂类物质包括花生四稀酸、胆固醇、dl-α-生育酚乙酸酯、亚油酸、亚麻酸、肉豆蔻酸、油酸、棕榈酸、棕榈油酸以及硬脂酸，且脂类物质添加剂中至少不含吐温80。

Description

一种脂类物质添加剂及其应用
相关公开的交叉引用
本公开要求于2021年5月7日提交中国专利局的申请号为202110497193.6、名称为“一种脂类物质添加剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及细胞培养技术领域，具体而言，涉及一种脂类物质添加剂及其应用。
背景技术
人类多能干细胞(hPSC)具有无限自我复制及分化为人体内的所有细胞类型的特点，是研究人类胚胎发育的重要模型系统。
早期人多能干细胞培养于含有牛血清成分的培养基中，牛血清中含有大量脂类成分。随后干细胞培养基成分逐渐简化，牛血清及其脂类成分被移除。近年来多篇文献指出脂类成分对干细胞的多能性状态、代谢状态及表观遗传状态等均有显著影响。
目前市面上提供的可添加于培养基中的脂类物质添加剂中脂质的浓度为均一浓度，其浓度不能对应其在牛血清中的含量比例。若按产品说明推荐的浓度使用该脂类添加剂，脂类终浓度仍远低于传统培养基中牛血清提供的对应脂类浓度，而进一步提升该脂类添加剂的浓度会使干细胞死亡。
鉴于此，特提出本发明。
发明内容
本公开的目的之一包括提供一种脂类物质添加剂以改善或克服现有的脂类物质添加剂所存在的问题。
本公开的目的之二包括提供一种含有上述脂类物质添加剂的细胞培养基。
本公开的目的之三包括提供一种采用上述脂类物质添加剂或细胞培养基对细胞进行培养的方法。
本公开的目的之四包括提供一种上述脂类物质添加剂在细胞培养基中的应用。
本公开可这样实现：
第一方面，本公开提供一种脂类物质添加剂，其由脂类物质溶于溶剂中而得，脂类物质包括花生四稀酸、胆固醇、dl-α-生育酚乙酸酯、亚油酸、亚麻酸、肉豆蔻酸、油酸、棕 榈酸、棕榈油酸以及硬脂酸，且脂类物质添加剂中至少不含吐温80。
在可选的实施方式中，每毫升溶剂对应的脂类物质包括0.7μg-7mg的花生四稀酸、22μg-220mg的胆固醇、7μg-70mg的dl-α-生育酚乙酸酯、4μg-40mg的亚油酸、4μg-40mg的亚麻酸、1μg-10mg的肉豆蔻酸、4μg-40mg的油酸、10μg-100mg的棕榈酸、2μg-20mg的棕榈油酸以及16μg-160mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质包括5μg-5mg的花生四稀酸、100μg-100mg的胆固醇、50μg-50mg的dl-α-生育酚乙酸酯、20μg-20mg的亚油酸、20μg-20mg的亚麻酸、5μg-5mg的肉豆蔻酸、20μg-20mg的油酸、50μg-50mg的棕榈酸、10μg-10mg的棕榈油酸以及80μg-80mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质可包括40μg-4mg的花生四稀酸、800μg-80mg的胆固醇、400μg-40mg的dl-α-生育酚乙酸酯、150μg-15mg的亚油酸、150μg-15mg的亚麻酸、40μg-4mg的肉豆蔻酸、150μg-15mg的油酸、400μg-40mg的棕榈酸、80μg-8mg的棕榈油酸以及500μg-50mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质可包括100μg-3mg的花生四稀酸、2mg-60mg的胆固醇、1mg-30mg的dl-α-生育酚乙酸酯、500μg-15mg的亚油酸、500μg-15mg的亚麻酸、100μg-3mg的肉豆蔻酸、300μg-10mg的油酸、1mg-30mg的棕榈酸、200μg-6mg的棕榈油酸以及1mg-30mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质可包括200μg-2mg的花生四稀酸、4mg-40mg的胆固醇、2mg-20mg的dl-α-生育酚乙酸酯、1mg-10mg的亚油酸、1mg-10mg的亚麻酸、200μg-2mg的肉豆蔻酸、1mg-10mg的油酸、2mg-20mg的棕榈酸、400μg-4mg的棕榈油酸以及2mg-20mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质可包括400μg-1.2mg的花生四稀酸、10mg-30mg的胆固醇、5mg-15mg的dl-α-生育酚乙酸酯、2mg-6mg的亚油酸、2mg-6mg的亚麻酸、500μg-1.5mg的肉豆蔻酸、2mg-6mg的油酸、5mg-15mg的棕榈酸、1mg-3mg的棕榈油酸以及6mg-18mg的硬脂酸。
在可选的实施方式中，每毫升溶剂对应的脂类物质包括0.7mg的花生四稀酸、22mg的胆固醇、7mg的dl-α-生育酚乙酸酯、4mg的亚油酸、4mg的亚麻酸、1mg的肉豆蔻酸、4mg的油酸、10mg的棕榈酸、2mg的棕榈油酸以及16mg的硬脂酸。
在可选的实施方式中，脂类物质添加剂中不含任何吐温成分。
在可选的实施方式中，溶剂为甲醇或无水乙醇，优选为无水乙醇。
第二方面，本公开提供一种细胞培养基，其含有如前述实施方式任一项的脂类物质添加剂。
第三方面，本公开提供一种细胞培养方法，其采用如前述实施方式的细胞培养基对细胞进行培养。
在可选的实施方式中，细胞为人胚胎干细胞。
在可选的实施方式中，细胞为人胚胎干细胞H1。
第四方面，本公开提供一种如前述实施方式任一项的脂类物质添加剂在细胞培养基中的应用。
本公开的有益效果包括：
本公开提供的脂类物质添加剂不含市面脂类添加剂配方中具有细胞毒性的吐温80成分。该脂类物质添加剂能够在不改变细胞形态及不影响干细胞多能性的前提下提供更近似血清培养的脂类环境。其适用于对细胞尤其是人类胚胎干细胞在内的细胞进行培养。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1至图5为本公开实施例1提供的CDL的成分组成及细胞毒性结果图，其中，图1示出了赛默飞公司的CDL的成分组成；图2示出了血清提取物AlbuMAX与CDL的成分组成；图3示出CDL成分中脂类物质的浓度与其在AlbuMAX中的差异；图4示出CDL的不同浓度对克隆效率的影响；图5示出了不同浓度(0、1:10000、1:1000、1:100)CDL对细胞的增殖指数的影响。
图6至图8为本公开实施例2提供的CDL中吐温80的细胞毒性结果图及hCDL的配方组成图，其中，图6示出官方配方CDL中各成分以及CDL对细胞增殖指数的影响；图7示出经液相质谱检测的AlbuMAX中CDL相关脂类物质的浓度；图8示出了本公开的去除吐温80后的1000×的新脂类物质添加物(hCDL)的配方。
图9至图12为本公开实施例3提供的hCDL对人胚胎干细胞培养的影响结果图，其中，图9示出在E8、Albumin、Albumin+CDL以及Albumin+hCDL条件下的人胚胎干细胞的细胞形态；图10示出在E8、AlbuMAX、Albumin以及Albumin+hCDL条件下的人胚胎干细胞的细胞干重变化；图11示出在E8、Albumin、Albumin+hCDL(0.1x)、及Albumin+hCDL(1x)、hCDL(0.1x)以及hCDL(1x)条件对人胚胎干细胞的折叠膨胀的影响；图12示出经E8、Albumin、AlbuMAX、Albumin+CDL以及Albumin+hCDL培养48h后的细胞中的脂类物质成分。
图13至图19为本公开实施例4提供的hCDL对人胚胎干细胞的多能性及分化能力的影响结果图，其中，图13示出在E8、Albumin、AlbuMAX、Albumin+CDL以及Albumin+hCDL条件下培养的细胞对NANOG、OCT4及SOX2基因的表达；图14示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对T、MIXL1基因的表达；图15示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对SOX17、GATA4基因的表达；图16示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对PAX6、SOX1基因的表达；图17示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下中胚层分化时对T、MIXL1基因的表达；图18示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下内胚层分化时对SOX17、GATA4基因的表达；图19示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下中胚层分化时对对PAX6、SOX1基因的表达。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开提供的脂类物质添加剂及其应用进行具体说明。
本公开提出一种脂类物质添加剂，其由脂类物质溶于溶剂中而得，脂类物质包括花生四稀酸、胆固醇、dl-α-生育酚乙酸酯、亚油酸、亚麻酸、肉豆蔻酸、油酸、棕榈酸、棕榈油酸以及硬脂酸，且脂类物质添加剂中至少不含吐温80。
脂类物质添加剂中至少不含吐温80，以降低细胞毒性。进一步地，脂类物质添加剂中还可不含任何吐温成分。
值得强调的是，其它所有不含吐温80的脂类物质添加剂(该脂类物质添加剂含有花生四稀酸、胆固醇、dl-α-生育酚乙酸酯、亚油酸、亚麻酸、肉豆蔻酸、油酸、棕榈酸、棕榈油酸以及硬脂酸)均在本公开的保护范围内。
在可选的实施方式中，每毫升溶剂对应的脂类物质包括0.7μg-7mg的花生四稀酸、22μg-220mg的胆固醇、7μg-70mg的dl-α-生育酚乙酸酯、4μg-40mg的亚油酸、4μg-40mg的亚麻酸、1μg-10mg的肉豆蔻酸、4μg-40mg的油酸、10μg-100mg的棕榈酸、2μg-20mg的棕榈油酸以及16μg-160mg的硬脂酸。
进一步地，每毫升溶剂对应的脂类物质可包括5μg-5mg的花生四稀酸、100μg-100mg的胆固醇、50μg-50mg的dl-α-生育酚乙酸酯、20μg-20mg的亚油酸、20μg-20mg的亚麻酸、5μg-5mg的肉豆蔻酸、20μg-20mg的油酸、50μg-50mg的棕榈酸、10μg-10mg的棕榈油酸以 及80μg-80mg的硬脂酸。
更进一步地，每毫升溶剂对应的脂类物质可包括40μg-4mg的花生四稀酸、800μg-80mg的胆固醇、400μg-40mg的dl-α-生育酚乙酸酯、150μg-15mg的亚油酸、150μg-15mg的亚麻酸、40μg-4mg的肉豆蔻酸、150μg-15mg的油酸、400μg-40mg的棕榈酸、80μg-8mg的棕榈油酸以及500μg-50mg的硬脂酸。
更进一步地，每毫升溶剂对应的脂类物质可包括100μg-3mg的花生四稀酸、2mg-60mg的胆固醇、1mg-30mg的dl-α-生育酚乙酸酯、500μg-15mg的亚油酸、500μg-15mg的亚麻酸、100μg-3mg的肉豆蔻酸、300μg-10mg的油酸、1mg-30mg的棕榈酸、200μg-6mg的棕榈油酸以及1mg-30mg的硬脂酸。
更进一步地，每毫升溶剂对应的脂类物质可包括200μg-2mg的花生四稀酸、4mg-40mg的胆固醇、2mg-20mg的dl-α-生育酚乙酸酯、1mg-10mg的亚油酸、1mg-10mg的亚麻酸、200μg-2mg的肉豆蔻酸、1mg-10mg的油酸、2mg-20mg的棕榈酸、400μg-4mg的棕榈油酸以及2mg-20mg的硬脂酸。
更进一步地，每毫升溶剂对应的脂类物质可包括400μg-1.2mg的花生四稀酸、10mg-30mg的胆固醇、5mg-15mg的dl-α-生育酚乙酸酯、2mg-6mg的亚油酸、2mg-6mg的亚麻酸、500μg-1.5mg的肉豆蔻酸、2mg-6mg的油酸、5mg-15mg的棕榈酸、1mg-3mg的棕榈油酸以及6mg-18mg的硬脂酸。
更进一步地，每毫升溶剂对应的脂类物质可包括0.7mg的花生四稀酸、22mg的胆固醇、7mg的dl-α-生育酚乙酸酯、4mg的亚油酸、4mg的亚麻酸、1mg的肉豆蔻酸、4mg的油酸、10mg的棕榈酸、2mg的棕榈油酸以及16mg的硬脂酸。
在可选的实施方式中，溶剂例如可以为甲醇或无水乙醇，优选为无水乙醇。
承上，本公开所提供的脂类物质的含量与血清提取物中脂质物质的比例及浓度接近，一方面不含对细胞有毒性的物质，另一方面能够在不改变细胞形态及不影响干细胞多能性的前提下提供更近似血清培养的脂类环境，以成功完成对细胞的培养。
此外，本公开还提供一种细胞培养基，其含有上述的脂类物质添加剂。
可参考地，该细胞培养基可以为E8培养基、DMEM培养基、DMEM/F12培养基、Nutristem XF/FF培养基、StemPro培养基、X-Vivo 10培养基、Neutrodoma-CS培养基、HyClone HyCellStem培养基、StemFit培养基、mTeSR1培养基、TeSR1培养基、TeSR2等干细胞或成体细胞培养基。其所含成分除上述脂类物质添加剂以外，还可包括FGF2及其他生长因子等。
此外，本公开还提供了上述脂类物质添加剂或细胞培养基的应用，即用于进行细胞培养。
对应地，本公开提供了一种细胞培养方法，其采用上述细胞培养基对细胞进行培养。
在可选的实施方式中，细胞为人胚胎干细胞(包括人类多能干细胞)，例如可以为人胚胎干细胞H1，此外还可以为人胚胎干细胞H9及人工诱导多能干细胞(iPSC)等。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
目前市售适用于细胞培养的脂类物质添加物种类很少，我们选择了赛默飞公司的chemically defined lipid concentrate(目录编号：11905031)，以下简称CDL作为参考。
赛默飞公司的CDL产品信息中公开的成分组成如图1所示。图2示出了血清提取物AlbuMAX与CDL的成分组成，其中示出了目前报道的血清提取物AlbuMAX中的成分组成。通过图1及图2相比，可以看出：CDL中所含的脂类成分种类不足，浓度也明显偏低。经质谱检测，CDL成分中脂类物质的浓度与其在AlbuMAX中的差异如图3所示。
进一步地，进行以下实验：
实验中人胚胎干细胞H1细胞系为待培养细胞，培养基为E8。具体实现如下：在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，第三次抽吸DPBS-EDTA，并添加含有10μM ROCK抑制剂Y27632的E8培养基。重悬细胞后，以1:6至1:12的密度传代至预先包被Matrigel的细胞培养板中。
细胞克隆测定实验的操作是：预先在12孔板中加入500微升基础培养基，细胞经TrypLE消化5分钟后以9倍体积DMEM/F12中和，以血球计数板数细胞数，离心，清洗并稀释到5000细胞/毫升后再于每个孔加入100微升。细胞置于5％氧气，10％二氧化碳的37℃培养箱中。每一或两天加药换液，六天后以碱性磷酸酶染色并数克隆数。
其结果如图4和图5所示，图4示出CDL的不同浓度对克隆效率的影响；图5示出了不同浓度(0、1:10000、1:1000、1:100)CDL对细胞的增殖指数的影响。由图4可以看出：细胞的克隆效率随着CDL的浓度增加而显著降低，也即，CDL在建议使用浓度(1:100)下具有明显的细胞毒性。由图5可以看出，1:100浓度的CDL处理的细胞与对照相比，细胞的增殖指数明显降低，也即1:100浓度的CDL明显抑制了H1细胞的生长。
也即说明，市售脂类物质添加物(chemically defined lipid concentrate，CDL)在指导使用浓度下存在脂类浓度不足且具有细胞毒性的缺点。
实施例2
本公开脂类物质添加剂(简称hCDL)配方的构建。
根据CDL官方给出的配方，我们测试了每种成分对细胞生长的影响。其结果如图6所示，图6示出官方配方CDL中各成分以及CDL对细胞增殖指数的影响。由图6可以看出， 含吐温80的CDL以及吐温80处理72h后的细胞的增殖指数显然降低，其中吐温80处理72h后的细胞的增殖指数降低最多，由此可推断，吐温80是造成细胞毒性的主要原因。
进一步地，我们以LC-MS检测了AlbuMAX中CDL相关脂类物质的浓度，其结果如图7所示。随后，计算出每克AlbuMAX中含有各脂类物质的多少。基于AlbuMAX中脂类物质的含量，我们做出了去除吐温80后的1000×的新脂类物质添加物(hCDL)的配方(具体请参照图8)。
实施例3
hCDL在人胚胎干细胞培养中的作用。
具体测试了hCDL在人胚胎干细胞培养中的影响，其实验操作为：在H1细胞密度达到60％时以EDTA/DPBS分离细胞，以1:12的比例将细胞传代至12孔板中，第二天开始以加药的E8培养基培养两天后观察细胞形态；以1:12的比例将细胞传代至6孔板中，第二天开始以加药的E8培养基培养两天后用TrypLE收集细胞，用流式细胞仪计算细胞数，50度烘干三天以测量细胞干重；以1:20的比例将细胞传代至24孔板中，第二天以TrypLE收集细胞并用流式细胞仪计算Day0细胞，以加药的E8培养基培养三天，每天收集并计数以绘制细胞生长曲线；以1:12的比例将细胞传代至6孔板中，第二天开始以加药的E8培养基培养两天后用TrypLE收集细胞，用流式细胞仪计算细胞数，用氯仿甲醇水体系(2:1:1，v/v/v)提取细胞脂类物质，甲基化处理后以GC-MS测定脂类物质含量。所有实验均为每天更换培养基，每组处理的重复数为3。
其结果如图9至图12所示。图9示出在E8、Albumin、Albumin+CDL以及Albumin+hCDL条件下的人胚胎干细胞的细胞形态。由图9可以看出：hCDL不影响干细胞的细胞形态。图10示出在E8、AlbuMAX、Albumin以及Albumin+hCDL条件下的人胚胎干细胞的细胞干重变化。由图10可以看出：hCDL增加了细胞干重。图11示出在E8、Albumin、Albumin+hCDL(0.1x)、及Albumin+hCDL(1x)、hCDL(0.1x)以及hCDL(1x)条件对人胚胎干细胞的增殖的影响。由图11可以看出：与1％血清白蛋白albumin联用时，hCDL对细胞生长影响不大。图12示出经E8、Albumin、AlbuMAX、Albumin+CDL以及Albumin+hCDL培养48h后的细胞中的脂类物质成分。由图12可以看出：经hCDL培养48h的H1细胞经GC-MS检测，其细胞中脂类物质的结构与经AlbuMAX培养的细胞相似。
实施例4
hCDL对人胚胎干细胞的多能性及分化能力的影响。
(一)、对人胚胎干细胞的多能性的影响：
在H1细胞的细胞密度达到60％左右时以EDTA/DPBS将细胞以1:12传代，经加药的E8培养基培养3代后用收集并检测多能性基因的表达水平。每组处理的重复数为4，多能 性基因的表达水平以GAPDH作为参照并以E8组作为对照。
其结果如图13所示，图13示出在E8、Albumin、AlbuMAX、Albumin+CDL以及Albumin+hCDL条件下培养的细胞对NANOG、OCT4及SOX2基因的表达。由图13可以看出：经hCDL培养3代的H1细胞仍维持多能性标识基因NANOG，OCT4及SOX2的表达，也即hCDL不影响H1细胞的多能性。
(二)、对人胚胎干细胞的分化能力的影响：
干细胞自分化的实验操作为：在H1细胞的细胞密度到30％左右时以加药的E6培养基(E8培养基去除FGF2和TGFβ)培养12天，每天换液。12天后检测谱系标识基因。
其结果如图14至16所示。图14示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对T、MIXL1基因的表达。由图14可以看出：hCDL培养的细胞在自发分化时，中胚层标识基因具有与AlbuMAX相同的趋势。图15示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对SOX17、GATA4基因的表达。由图15可以看出：hCDL培养的细胞在自发分化时，内胚层标识基因具有与AlbuMAX相同的趋势。图16示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下自发分化时对PAX6、SOX1基因的表达。由图16可以看出：hCDL培养的细胞在自发分化时，外胚层标识基因不受hCDL影响。
也即，hCDL对细胞自分化的外胚层标识基因无影响，对中胚层和内胚层标识基因有和AlbuMAX类似的影响。
H1的三个胚层的定向分化实验操作分别为：H1细胞先在加药的E8培养基中培养48小时，之后的中胚层分化为，以加入20纳克/毫升BMP4的E8培养基培养两天后检测标识基因；内胚层分化为，首先以加入5μM的CHIR99021的E5培养基(E8培养基去除FGF2，TGFβ及胰岛素)培养一天，然后以加入10纳克/毫升的Activin A的E5培养基培养3天；外胚层分化为，以加入10μM SB431542和100nM LDN193189的E6培养基培养4天后检测标识基因。
其结果如图17至19所示。图17示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下中胚层分化时对T、MIXL1基因的表达。由图17可以看出：引导hCDL培养的细胞进行中胚层分化时，中胚层标识基因不受hCDL影响。；图18示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下内胚层分化时对SOX17、GATA4基因的表达。由图18可以看出：引导hCDL培养的细胞进行内胚层分化时，中胚层标识基因不受hCDL影响。图19示出Mock、AlbuMAX、Albumin以及Albumin+hCDL条件下中胚层分化时对对PAX6、SOX1基因的表达。由图19可以看出：引导hCDL培养的细胞进行中胚层分化时，外胚层标识基因的改变趋势与AlbuMAX一致。
也即，hCDL对定向分化无影响。
综上所述，本公开提供的脂类物质添加剂中相关脂类浓度基于血清提取物，不含市面脂类物质添加剂配方中具有细胞毒性的吐温80成分。该脂类物质添加剂能够在不改变细胞形态及不影响干细胞多能性的前提下提供更近似血清培养的脂类环境。其适用于对细胞尤其是人类胚胎干细胞在内的细胞进行培养。
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
工业实用性
本公开提供一种脂类物质添加剂、含有该脂类物质添加剂的细胞培养基、采用该脂类物质添加剂或细胞培养基对细胞进行培养的方法以及该脂类物质添加剂在细胞培养基中的应用。该脂类物质添加剂中去除了存在于市面脂类物质添加剂配方中具有细胞毒性的吐温80成分，在不改变细胞形态,不影响干细胞多能性的前提下提供了更近似血清培养的脂类环境，适用于对细胞尤其是人类胚胎干细胞在内的细胞进行培养。

Claims (15)

  1. 一种脂类物质添加剂，其特征在于，由脂类物质溶于溶剂中而得，所述脂类物质包括花生四稀酸、胆固醇、dl-α-生育酚乙酸酯、亚油酸、亚麻酸、肉豆蔻酸、油酸、棕榈酸、棕榈油酸以及硬脂酸，且所述脂类物质添加剂中至少不含吐温80。
  2. 根据权利要求1所述的脂类物质添加剂，其特征在于，每毫升所述溶剂对应的所述脂类物质包括0.7μg-7mg的所述花生四稀酸、22μg-220mg的所述胆固醇、7μg-70mg的所述dl-α-生育酚乙酸酯、4μg-40mg的所述亚油酸、4μg-40mg的所述亚麻酸、1μg-10mg的所述肉豆蔻酸、4μg-40mg的所述油酸、10μg-100mg的所述棕榈酸、2μg-20mg的所述棕榈油酸以及16μg-160mg的所述硬脂酸。
  3. 根据权利要求1或2所述的脂类物质添加剂，其特征在于，每毫升所述溶剂对应的所述脂类物质包括5μg-5mg的花生四稀酸、100μg-100mg的胆固醇、50μg-50mg的dl-α-生育酚乙酸酯、20μg-20mg的亚油酸、20μg-20mg的亚麻酸、5μg-5mg的肉豆蔻酸、20μg-20mg的油酸、50μg-50mg的棕榈酸、10μg-10mg的棕榈油酸以及80μg-80mg的硬脂酸。
  4. 根据权利要求1-3中任一项所述的脂类物质添加剂，其特征在于，每毫升溶剂对应的脂类物质可包括40μg-4mg的花生四稀酸、800μg-80mg的胆固醇、400μg-40mg的dl-α-生育酚乙酸酯、150μg-15mg的亚油酸、150μg-15mg的亚麻酸、40μg-4mg的肉豆蔻酸、150μg-15mg的油酸、400μg-40mg的棕榈酸、80μg-8mg的棕榈油酸以及500μg-50mg的硬脂酸。
  5. 根据权利要求1-4中任一项所述的脂类物质添加剂，其特征在于，每毫升溶剂对应的脂类物质可包括100μg-3mg的花生四稀酸、2mg-60mg的胆固醇、1mg-30mg的dl-α-生育酚乙酸酯、500μg-15mg的亚油酸、500μg-15mg的亚麻酸、100μg-3mg的肉豆蔻酸、300μg-10mg的油酸、1mg-30mg的棕榈酸、200μg-6mg的棕榈油酸以及1mg-30mg的硬脂酸。
  6. 根据权利要求1-5中任一项所述的脂类物质添加剂，其特征在于，每毫升溶剂对应的脂类物质可包括200μg-2mg的花生四稀酸、4mg-40mg的胆固醇、2mg-20mg的dl-α-生育酚乙酸酯、1mg-10mg的亚油酸、1mg-10mg的亚麻酸、200μg-2mg的肉豆蔻酸、1mg-10mg的油酸、2mg-20mg的棕榈酸、400μg-4mg的棕榈油酸以及2mg-20mg的硬脂酸。
  7. 根据权利要求1-6中任一项所述的脂类物质添加剂，其特征在于，每毫升溶剂对应的脂类物质可包括400μg-1.2mg的花生四稀酸、10mg-30mg的胆固醇、5mg-15mg的dl-α-生育酚乙酸酯、2mg-6mg的亚油酸、2mg-6mg的亚麻酸、500μg-1.5mg的肉豆蔻酸、2mg-6mg的油酸、5mg-15mg的棕榈酸、1mg-3mg的棕榈油酸以及6mg-18mg的硬脂酸。
  8. 根据权利要求1-7中任一项所述的脂类物质添加剂，其特征在于，每毫升所述溶剂对应的所述脂类物质包括0.7mg的所述花生四稀酸、22mg的所述胆固醇、7mg的所述dl-α- 生育酚乙酸酯、4mg的所述亚油酸、4mg的所述亚麻酸、1mg的所述肉豆蔻酸、4mg的所述油酸、10mg的所述棕榈酸、2mg的所述棕榈油酸以及16mg的所述硬脂酸。
  9. 根据权利要求1-8中任一项所述的脂类物质添加剂，其特征在于，所述脂类物质添加剂中不含任何吐温成分。
  10. 根据权利要求1-9中任一项所述的脂类物质添加剂，其特征在于，所述溶剂为甲醇或无水乙醇，优选为无水乙醇。
  11. 一种细胞培养基，其特征在于，所述细胞培养基中含有如权利要求1-10中任一项所述的脂类物质添加剂。
  12. 一种细胞培养方法，其特征在于，采用如权利要求11所述的细胞培养基对细胞进行培养。
  13. 根据权利要求12所述的细胞培养方法，其特征在于，所述细胞为人胚胎干细胞。
  14. 根据权利要求12或13所述的细胞培养方法，其特征在于，所述细胞为人胚胎干细胞H1。
  15. 如权利要求1-10中任一项所述的脂类添加剂在细胞培养基中的应用。
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