CN106399231B - Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells - Google Patents
Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells Download PDFInfo
- Publication number
- CN106399231B CN106399231B CN201610817382.6A CN201610817382A CN106399231B CN 106399231 B CN106399231 B CN 106399231B CN 201610817382 A CN201610817382 A CN 201610817382A CN 106399231 B CN106399231 B CN 106399231B
- Authority
- CN
- China
- Prior art keywords
- graphene oxide
- embryonic stem
- stem cells
- self
- mouse embryonic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12N2500/05—Inorganic components
-
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/34—Sugars
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Biotechnology (AREA)
- Reproductive Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells. The graphene oxide can promote the self-renewal of embryonic stem cells and inhibit cell differentiation under the culture condition without adding Leukemia Inhibitory Factor (LIF). The graphene oxide has various functional groups on the surface, has the characteristics of good biocompatibility, low cytotoxicity and the like, is low in cost, convenient and fast in material synthesis and good in action effect, and brings wide application prospects for research of embryonic stem cells.
Description
Technical Field
The invention relates to application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic cells.
Background
Nanotechnology is an emerging and rapidly developing field in modern science. In the past years, nanotechnology has been used efficiently in biomedical research to provide new methods for cellular imaging, delivery of genes or other small molecules, and preparation of biological scaffolds in tissue engineering.
Embryonic Stem Cells (ESCs), which are cells isolated from the cell mass within the blastocyst stage of an embryo, are widely used in biomedical and regenerative medicine research because of their simultaneous unlimited proliferation capacity and multi-directional differentiation potential. To date, ESCs are increasingly used in the treatment of clinical conditions, such as spinal cord injury, parkinson syndrome, myocardial infarction, and pulmonary fibrosis. Therefore, precise regulation of ESCs proliferation or differentiation is the key to current research. Researches show that the combination of nanotechnology and stem cells can bring important breakthrough to the culture and regulation of the stem cells, and the previous researches mainly focus on the separation tracing of the stem cells and the delivery of drugs and genes. The research scope is gradually expanded to the regulation of stem cell self-renewal, differentiation and cell fate by nano materials. Relevant researches show that the nano Layered Double Hydroxide (LDH) has the functions of replacing leukemia inhibitory factor LIF, maintaining self-renewal of mouse embryonic stem cells and inhibiting differentiation. The nano layered double hydroxide is an anionic inorganic sheet layered material, and is widely applied to biomedical research due to good biocompatibility and low toxicity.
Graphene Oxide (GO) is a lamellar material, has electrical conductivity, good biocompatibility and surface modifiability, and is increasingly used in biological research. Relevant researches show that the graphene oxide has a regulating effect on proliferation and differentiation of various stem cells.
Disclosure of Invention
The invention aims to provide application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic cells.
The application of the nano graphene oxide in promoting the culture and self-renewal of mouse embryonic cells provided by the invention comprises the following specific steps:
(1) removing feeder layer cells from the normally cultured mouse embryonic stem cells by a differential speed adherence method, and inoculating the feeder layer cells to a six-hole plate paved with 1% gelatin according to 20000 cells/hole;
(2) culturing the product obtained in the step (1) for 24 hours by adopting a complete culture medium, removing LIF factors, adding nano graphene oxide into the complete culture medium to maintain the final concentration of the nano graphene oxide at 10-40 mu g/mL, continuously culturing the mouse embryonic stem cells, and morphologically displaying the self-renewal capacity of the mouse embryonic stem cells after adding the nano graphene oxide for 48 hours; wherein: complete medium was made of high-sugar DMEM; 15% fetal bovine serum; 0.1 mmol/L non-essential amino acid; 2 mmol/L glutamine; 0.1 mmol/L beta-mercaptoethanol; 1000U/mL leukemia inhibitory factor.
The graphene oxide has the capacity of maintaining the self-renewal capacity of the mouse embryonic stem cells and inhibiting the differentiation of the mouse embryonic stem cells through alkaline phosphatase staining, Western Blot, fluorescent quantitative PCR (rt-PCR) and immunofluorescence staining.
The invention has the advantages that; the invention provides a new application of nano Graphene Oxide (GO) in the field of stem cell culture, which can maintain the self-renewal capacity of mouse embryonic stem cells under the condition of removing feeder cells and having no Leukemia Inhibitory Factor (LIF). The embryonic stem cells are easy to differentiate in the long-term culture process, and in order to maintain the self-renewal capacity of the embryonic stem cells, the traditional culture of the embryonic stem cells needs to depend on feeder cells and leukocyte inhibitory factors, so that the culture cost of the embryonic stem cells is high, and the culture process is complicated.
Drawings
Fig. 1 is a transmission electron micrograph of graphene oxide.
Fig. 2 shows MTT detection results of toxicity generated to cells after different concentrations of graphene oxide are used for treating embryonic stem cells for 24 hours and 48 hours, respectively. Wherein: (a) 24 hours, (b) 48 hours.
FIG. 3 shows alkaline phosphatase staining of embryonic stem cells of mice after 4 days of graphene oxide treatment at different concentrations. Wherein: (a) adding LIF factor into the culture medium and (b) removing LIF factor to obtain control group. The final concentrations of the added graphene oxide were (c) 4mg/mL, (d) 8mg/mL, (e) 16mg/mL, (f) 32mg/mL, and (g) 40mg/mL, respectively.
FIG. 4 shows the expression of mouse embryonic stem cell pluripotency and self-renewal related protein detected by Western Blot.
FIG. 5 shows the expression of pluripotency genes detected by immunofluorescence staining. Wherein: (a) the graphs (b), (c) and (d) show the expression of the mouse embryonic stem cell self-renewal marker genes Nanog, Oct4, Sox2 and SSEA-1 after 4 days of culture without adding lif factors, respectively, and the graphs (e), (f), (g) and (h) show the expression of the mouse embryonic stem cell self-renewal marker genes Nanog, Oct4, Sox2 and SSEA-1 after 4 days of culture with adding graphene oxide under the condition of removing lif factors.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings.
Example 1: culture of mouse embryonic stem cells and action process operation of graphene oxide
Obtaining Mouse Embryo Fibroblast (MEF) primarily, culturing to 3-5 generations, treating with 80kV gamma-ray for 30min, and using as feeder layer cells within 1 week. The mouse embryonic stem cells adopt a complete culture medium: high-glucose DMEM, 15% fetal bovine serum, 0.1 mmol/L nonessential amino acids, 2 mmol/L glutamine, 0.1 mmol/L beta-mercaptoethanol, 1000U/ml0.1 leukemia inhibitory factor, after culturing normally for 2-3 days, adding trypsin (0.25%) -EDTA (0.02%) to digest the cells, neutralizing the pancreatin in the culture solution, and then placing the cells into a coating of 0.1% of the total amount of the culture dish was incubated for 30 minutes. After counting by pipetting unadhered mescs, the number was 2 × 104One/well was seeded on six-well plates coated with 1% gelatin.
And after culturing for 24 hours, removing LIF factors, adding a graphene oxide solution with the particle size of 200nm into the culture solution to enable the final concentration to be 10-40 mu g/mL, replacing the culture solution with the graphene oxide with the same concentration every day, and after continuously treating for 4 days, detecting the indexes related to the self-renewal capacity of the embryonic stem cells of the mice.
Example 2: MTT method for detecting cell survival rate
Digesting and blowing mESCs into single cells, removing feeder layer cells by differential adherence method, and then removing feeder layer cells according to 2 × 103The density of each well was plated on gelatin-coated 96-well plates at 200. mu.L per well and cultured overnight. The next day, the cell culture solution was replaced with a culture solution containing graphene oxide at concentrations of 0, 0.5, 1, 1.5, 2, 2.5, and 3 μ g/mL, respectively. The cells to which the graphene oxide culture medium was not added were replaced with a blank control. Both experimental and control groups were set up with 5 replicates. And (3) detecting the cells after the graphene oxide is treated for 24 hours and 48 hours respectively. Preparing 5 mg/mL brominated 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole [3- (4, 5-dimethylthiazole-2 yl) -2, 5-diphenylterazolium bromide, MTT]Solution, diluted with PBS, pH =7.4, 20 μ L per well. After 4 h incubation, the culture medium was aspirated from the wells, 150. mu.L DMSO was added to each well, and the wells were shaken horizontally for 10 min. Reading the light absorption value of each pore under the excitation wavelength of 570 nm by using a microplate reader, recording the result and calculating the cell survival rate according to a formula: survival% = a570 (experimental group)/a 570 (blank control group) × 100%.
Example 3: effect of graphene oxide on self-renewal of mouse embryonic stem cells
(1) Alkaline phosphatase (ALP) activity assay. An important biological feature of undifferentiated embryonic stem cells is the ability to express high concentrations of Alkaline Phosphatase (AP) by which the differentiation status of the embryonic stem cells can be reflected. The mESC obtained from example 1 was fixed and then stained with alkaline phosphatase. As shown in fig. 3, morphologically, the mESC from which the LIF factor was removed was completely differentiated, the mESC to which the graphene oxide was added was maintained in an undifferentiated state, and the mESC to which the graphene oxide was added was strongly positive for alkaline phosphatase; the graphene oxide is proved to be capable of promoting the self-renewal capacity of the mESC.
(2) Western Blot detects the expression of mouse embryonic stem cell pluripotency and self-renewal related protein. The proteins of Nanog, Oct4, Sox2 and Rex-1 have important regulation and control functions on the self-renewal of ES cells. WesternBlot can detect the expression of these proteins and reflect the state of the cell. As shown in fig. 4, graphene oxide can up-regulate the expression levels of the four genes, and promote the self-renewal capacity of mouse embryonic stem cells.
(3) Immunofluorescence detects expression of pluripotency genes at the cellular level. Selecting Rabbit Anti-Nanog, Rabbit Anti-Oct4, Rabbit Anti-Sox2 and Mouse Anti-SSEA-1 as 1 Antibody, Fluorescein Conjugated coat Anti-Rabbit Secondary Antibody and Fluorescein Conjugated coat Anti-Mouse Secondary Antibody as 2 Antibody, DAPI for cell nucleus staining, and observing staining condition with a fluorescence microscope. As shown in fig. 5, graphene oxide was able to promote the expression of Nanog, Oct4, Sox2, and SSEA-1 at the cellular level, compared to the control group.
Claims (2)
1. The application of the nano graphene oxide in promoting the culture and self-renewal of mouse embryonic stem cells.
2. The application according to claim 1, characterized by the following specific steps:
(1) removing feeder layer cells from the normally cultured mouse embryonic stem cells by a differential speed adherence method, and inoculating the feeder layer cells to a six-hole plate paved with 1% gelatin according to 20000 cells/hole;
(2) culturing the product obtained in the step (1) for 24 hours by adopting a complete culture medium, removing LIF factors, adding nano graphene oxide into the complete culture medium to maintain the final concentration of the nano graphene oxide at 10-40 mu g/mL, continuously culturing the mouse embryonic stem cells, and morphologically displaying the self-renewal capacity of the mouse embryonic stem cells after adding the nano graphene oxide for 48 hours; wherein: complete medium was made of high-sugar DMEM; 15% fetal bovine serum; 0.1 mmol/L non-essential amino acid; 2 mmol/L glutamine; 0.1 mmol/L beta-mercaptoethanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610817382.6A CN106399231B (en) | 2016-09-13 | 2016-09-13 | Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610817382.6A CN106399231B (en) | 2016-09-13 | 2016-09-13 | Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106399231A CN106399231A (en) | 2017-02-15 |
| CN106399231B true CN106399231B (en) | 2019-12-27 |
Family
ID=57999860
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610817382.6A Active CN106399231B (en) | 2016-09-13 | 2016-09-13 | Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106399231B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022517A (en) * | 2017-05-25 | 2017-08-08 | 句容亿格纳米材料厂 | A kind of method for improving glutamine stability in stem cell media |
| CN107177542A (en) * | 2017-05-25 | 2017-09-19 | 句容亿格纳米材料厂 | A kind of optimization method for promoting culture medium GLN to dissolve |
| CN109055477A (en) * | 2018-08-20 | 2018-12-21 | 淮南师范学院 | A method of based on determination of immune cells GO biological effect |
| CN110106141B (en) * | 2019-04-24 | 2022-05-20 | 朗姿赛尔生物科技（广州）有限公司 | Method for scaling autologous stem cells |
| CN111808798B (en) * | 2020-09-03 | 2021-01-08 | 朗姿赛尔生物科技（广州）有限公司 | Culture medium for promoting stem cell expansion |
| CN111763655B (en) * | 2020-09-03 | 2021-01-08 | 朗姿赛尔生物科技（广州）有限公司 | Method for promoting stem cell expansion |
| CN111979182B (en) * | 2020-09-07 | 2023-08-15 | 上海市同济医院 | Application of magnesium-iron layered double hydroxide nano material in mouse embryonic stem cell subculture and cryopreservation |
| CN112779220B (en) * | 2021-04-01 | 2023-10-10 | 新疆赛尔托马斯生物科技有限公司 | Culture medium for neural stem cell expansion |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820387A (en) * | 2014-03-11 | 2014-05-28 | 中国科学院上海微***与信息技术研究所 | Application of germanium-based graphene in osteogenesis promotion |
| WO2015157647A2 (en) * | 2014-04-10 | 2015-10-15 | Rutgers, The State University Of New Jersey | Guiding stem cell differentiation using graphene-nanofiber hybrid scaffolds |
| CN105062964A (en) * | 2015-07-17 | 2015-11-18 | 山东大学 | Inducing liquid for improving osteogenic differentiation efficiency in stem cells and application of inducing liquid |
-
2016
- 2016-09-13 CN CN201610817382.6A patent/CN106399231B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820387A (en) * | 2014-03-11 | 2014-05-28 | 中国科学院上海微***与信息技术研究所 | Application of germanium-based graphene in osteogenesis promotion |
| WO2015157647A2 (en) * | 2014-04-10 | 2015-10-15 | Rutgers, The State University Of New Jersey | Guiding stem cell differentiation using graphene-nanofiber hybrid scaffolds |
| CN105062964A (en) * | 2015-07-17 | 2015-11-18 | 山东大学 | Inducing liquid for improving osteogenic differentiation efficiency in stem cells and application of inducing liquid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106399231A (en) | 2017-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106399231B (en) | Application of nano graphene oxide in promoting culture and self-renewal of mouse embryonic stem cells | |
| Zhang et al. | A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells | |
| Rodin et al. | Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions | |
| Arnhold et al. | Amniotic‐Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD‐117‐Based Selection Procedure | |
| Wu et al. | Acoustic assembly of cell spheroids in disposable capillaries | |
| Elliott et al. | NKX2-5 eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes | |
| Xu et al. | Feeder-free growth of undifferentiated human embryonic stem cells | |
| CN103966160B (en) | Application of inorganic nano-material layered double hydroxides (LDHs) in mouse embryonic stem cell culture | |
| Moosazadeh Moghaddam et al. | Engineered substrates with imprinted cell-like topographies induce direct differentiation of adipose-derived mesenchymal stem cells into Schwann cells | |
| JP6860539B2 (en) | Adhesive signature-based method for isolating stem cells and cells derived from stem cells | |
| CN111484970B (en) | Serum-free and feeder-layer-free embryo and pluripotent stem cell culture medium with low protein content | |
| CN113846060B (en) | Application of nanomaterial in promoting differentiation of embryonic stem cells to neural precursor cells | |
| WO2016052657A1 (en) | Method for culturing pluripotent stem cells | |
| Jing et al. | Suspended graphene oxide nanosheets maintain the self-renewal of mouse embryonic stem cells via down-regulating the expression of Vinculin | |
| CN113811316A (en) | Method for purifying neural crest cells or corneal epithelial cells | |
| JP2008306987A (en) | Carrier for cell culture and method for cell culture | |
| JP2017085963A (en) | Cell culture method using culture medium containing laminin fragments | |
| KR101490671B1 (en) | A screening method for the optimal surface structure of embryonic stem cell culture using a cell culture plate comprising gradient nanopattern | |
| Muduli et al. | Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides | |
| Yukawa et al. | Embryonic body formation using the tapered soft stencil for cluster culture device | |
| EP2821789A1 (en) | Microtissues | |
| Jing et al. | Layer-number-dependent effects of graphene oxide on the pluripotency of mouse embryonic stem cells through the regulation of the interaction between the extracellular matrix and integrins | |
| Hwang et al. | In vitro direct osteogenesis of murine embryonic stem cells without embryoid body formation | |
| Ribeiro et al. | Assessing the combined effect of surface topography and substrate rigidity in human bone marrow stem cell cultures | |
| Perugini et al. | Hyperbranched poly (ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |