CN111909894B - Aminated graphene culture medium and preparation and application thereof in regulating and controlling induced pluripotent stem cells - Google Patents
Aminated graphene culture medium and preparation and application thereof in regulating and controlling induced pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses an aminated graphene culture medium and application of the aminated graphene culture medium in preparation and regulation and control of induced pluripotent stem cells, and belongs to the field of biomedical engineering. The preparation method comprises the following steps: dissolving aminated graphene in water, and uniformly dispersing to obtain an aminated graphene dispersion liquid; carrying out vacuum filtration on the aminated graphene dispersion liquid to the surface of the microporous filter membrane, drying, and then forming an aminated graphene membrane loaded on the surface of the microporous filter membrane by the aminated graphene dispersion liquid; carrying out gravity extrusion on the microporous filter membrane to transfer the aminated graphene membrane to the support layer, namely attaching the aminated graphene membrane to the surface of the support layer; and (3) dissolving the microporous filter membrane, washing to remove the residual solvent, and drying to obtain the aminated graphene culture medium. The culture substrate provided by the invention has a unique positive charge amino group, regulates and controls induced pluripotent stem cells (iPS) to direct myocardial differentiation, and can effectively stimulate the maturation of myocardial precursor cells.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to an aminated graphene culture medium and application of the aminated graphene culture medium in preparation and regulation and control of induced pluripotent stem cells.
Background
Heart disease remains a leading cause of death worldwide, and the infarcted area of an injured heart is difficult to recover due to the lack of mature cardiomyocytes. The emergence of induced pluripotent stem cells offers the possibility of a cell source with a high similarity to embryonic stem cells in morphology, self-renewal and pluripotency, but without ethical problems.
Induced pluripotent stem cells differentiate from the initial pluripotent stage to the final, culture medium plays a crucial role in their fate. The most commonly used matrix for maintenance and differentiation of induced pluripotent stem cells is currently a matrix-coated culture dish. However, in a differentiation microenvironment coated with a matrix, cardiomyocytes derived from induced pluripotent stem cells are often immature and difficult to use in vivo.
More and more biomaterial-mediated culture matrices are designed to regulate the induction of myocardial differentiation of pluripotent stem cells. Graphene and its derivatives are designed as good materials for inducing cardiac muscle differentiation due to their unique electrical conductivity, excellent mechanical properties, convenient modification and suitable biocompatibility. The present invention therefore solves the above problems by a thin film prepared by amination of graphene.
Disclosure of Invention
The invention solves the technical problem that the induced pluripotent stem cells are differentiated into the myocardial cells to be immature so that the immature myocardial cells are difficult to apply in vivo in the prior art. The invention aims to design a novel culture substrate by applying a graphene material which can provide positive amino charges and has good biocompatibility, and the novel culture substrate is used for regulating and controlling the maturity of induced pluripotent stem cell derived cardiomyocytes.
According to a first aspect of the present invention, there is provided a preparation method of an aminated graphene culture substrate, comprising the following steps:
(1) dissolving aminated graphene in water, and uniformly dispersing to obtain an aminated graphene dispersion liquid;
(2) carrying out vacuum filtration on the aminated graphene dispersion liquid obtained in the step (1) to the surface of the microporous filter membrane, and drying to obtain the aminated graphene dispersion liquid, wherein an aminated graphene membrane is formed and loaded on the surface of the microporous filter membrane;
(3) performing gravity extrusion on the microporous filter membrane in the step (2) to transfer the aminated graphene membrane onto a support layer, namely attaching the aminated graphene membrane onto the surface of the support layer;
(4) and (4) soaking the three-layer superposed structure of the microporous filter membrane, the aminated graphene membrane and the support layer obtained in the step (3) in a solvent to dissolve the microporous filter membrane, washing away the residual solvent, and drying to obtain the aminated graphene culture medium.
Preferably, in the aminated graphene dispersion liquid in the step (1), the content of aminated graphene is 0.5-2 mg/ml.
Preferably, the microporous filter membrane in the step (2) is a cellulose water system filter membrane.
Preferably, the gravity of the gravity extrusion in the step (3) is 10N-50N, and the extrusion time is 8h-12 h.
Preferably, the support layer in step (3) is a cover glass, a silica gel sheet or a gelatin film.
Preferably, the solvent in step (4) is acetone or ethyl acetate.
According to another aspect of the invention, an aminated graphene culture substrate prepared by any one of the methods is provided.
According to another aspect of the invention, the application of the aminated graphene culture medium for promoting the induction of the maturation of pluripotent stem cell-derived cardiomyocytes is provided.
Preferably, a layer of matrigel is laid on the aminated graphene culture matrix, the matrigel is used for promoting cell adhesion, then induced pluripotent stem cells are inoculated for cell proliferation culture, when the cell density of the induced pluripotent stem cells reaches 85% -95%, a proliferation culture medium is changed into a myocardial differentiation culture medium for myocardial cell differentiation, and the aminated graphene culture matrix can provide a positive-charge microenvironment to achieve the effect of promoting maturation of the derived myocardial cells; and after the cardiac muscle cells are differentiated, replacing a cardiac muscle maintaining culture medium for continuous culture to obtain mature cardiac muscle cells.
Preferably, the cardiomyocytes are differentiated between 8 days and 14 days.
Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) the aminated graphene culture matrix has good biocompatibility and can support the adhesion, proliferation and differentiation of cells. Because the aminated graphene is functionalized graphene, the amino ions of the aminated graphene can enable the surface of the material to have active functional groups, so that the cell and biological reaction activity of the culture substrate is improved. The matrix can realize the integrated culture of differentiation from the induced pluripotent stem cells to the myocardial cells, thereby providing mature myocardial cells for cardiac tissue engineering treatment, disease model research and the like.
(2) In the contact process of the aminated graphene culture matrix and cells, the amino group of the aminated graphene culture matrix provides a positive charge microenvironment, so that the expression of PDGFRB genes is up-regulated. PDGFRB is an extracellular matrix receptor playing a key role in the maturation process of myocardial cells, so that the aminated graphene improves the remodeling effect of a PDGFRB signal channel on the extracellular matrix in the myocardial differentiation process, regulates and controls induced pluripotent stem cells (iPS) to direct myocardial differentiation, and can effectively stimulate the maturation of myocardial precursor cells.
(3) The preparation method of the aminated graphene culture medium is simple, and a feasible method is provided for large-scale production of mature myocardial cells. According to the method, aminated graphene dispersion liquid with a certain concentration is subjected to vacuum filtration to enable aminated graphene to be adsorbed on a microporous filter membrane to form an aminated graphene film, the aminated graphene film is transferred to a support layer from the microporous filter membrane in a gravity extrusion mode, and finally the microporous filter membrane is dissolved to obtain the aminated graphene culture medium.
(4) Preferably, in the preparation of the aminated graphene dispersion liquid, the content of the aminated graphene is 0.5-2mg/ml, and the aminated graphene can be well and uniformly dispersed.
(5) In the present invention, the microporous filter membrane is preferably a cellulose water-based filter membrane, which can be dissolved by an organic solvent without affecting the transfer of the aminated graphene film to the support layer.
(6) According to the invention, preferably, the gravity of gravity extrusion is 10N-50N, the extrusion time is 8h-12h, the transfer of the aminated graphene can be realized, and the aminated graphene is not easy to fall off from the support layer.
Drawings
Fig. 1 is an infrared spectrum of aminated graphene.
FIG. 2 is a schematic diagram of an aminated graphene culture substrate.
Fig. 3 is a scanning electron microscope image of an aminated graphene culture substrate.
Fig. 4 is a light mirror image of induced pluripotent stem cell-derived cardiomyocytes on an aminated graphene culture substrate.
Fig. 5 is a contractile force measurement of derived cardiomyocytes on a normal coverslip and aminated graphene culture substrate, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
10mg of aminated graphene was dissolved in 10ml of ultrapure water, and uniformly dispersed using ultrasonic treatment to obtain a 1mg/ml aminated graphene dispersion solution. 30. mu.l of the dispersion were aspirated and suction filtered onto a microfiltration membrane and then dried at room temperature. And (3) extruding one side of the filter membrane with the aminated graphene film on a cover glass, extruding for 10 hours by 20N gravity to transfer the aminated graphene film to the cover glass, dissolving the microporous filter membrane for 2 hours by acetone, and washing for 10 minutes by methanol to obtain the aminated graphene culture medium.
Example 2
5mg of aminated graphene was dissolved in 10ml of ultrapure water, and uniformly dispersed using ultrasonic treatment to obtain an aminated graphene dispersion liquid of 0.5 mg/ml. 30. mu.l of the dispersion were aspirated and suction filtered onto a microfiltration membrane and then dried at room temperature. And (3) extruding one side of the filter membrane with the aminated graphene film on a cover glass, extruding for 8 hours by the gravity of 10N to transfer the aminated graphene film to the cover glass, dissolving the microporous filter membrane for 2 hours by acetone, and washing for 10 minutes by methanol to obtain the aminated graphene culture medium.
Example 3
20mg of aminated graphene was dissolved in 10ml of ultrapure water, and uniformly dispersed using ultrasonic treatment to obtain a 2mg/ml aminated graphene dispersion. 30. mu.l of the dispersion were aspirated and suction filtered onto a microfiltration membrane and then dried at room temperature. And (3) extruding one side of the filter membrane with the aminated graphene film on a cover glass sheet, extruding for 12 hours by the aid of 50N gravity, transferring the aminated graphene film to a gelatin membrane, dissolving the microporous filter membrane for 2 hours by using ethyl acetate, and washing for 10 minutes by using methanol to obtain the aminated graphene culture medium.
Example 4
The culture medium prepared in example 1 was subjected to ultraviolet irradiation and alcohol sterilization, a layer of matrigel was applied, a WTC human induced pluripotent stem cell line was inoculated, the cell density was cultured to 90%, the cardiomyocyte differentiation medium was changed for cardiomyocyte differentiation, and after 14 days of differentiation, the cardiomyocyte maintenance medium was changed for continuous culture to obtain mature cardiomyocytes.
Fig. 1 is an infrared spectrum of aminated graphene. The infrared spectrogram is 1593cm-1The characteristic absorption peak of (A) may be the bending vibration of N-H, and is 1327cm-1The absorption peak at (A) may be a tensile oscillation of C-N, indicating the presence of an amino group.
FIG. 2 is a diagram of an aminated graphene culture substrate, wherein a light black thin film is arranged on a support layer cover glass, namely, an aminated graphene nano-film.
Fig. 3 is a scanning electron microscope image of an aminated graphene culture substrate, showing the wrinkle structure characteristic of graphene.
Fig. 4 is a light mirror image of induced pluripotent stem cell-derived cardiomyocytes on an aminated graphene culture substrate, wherein the dashed area is the differentiated cardiomyocytes.
Fig. 5 is a measurement of contractile force of the derived cardiomyocytes on a common coverslip and an aminated graphene culture substrate, respectively, with the control group being the common coverslip and the experimental group being the aminated graphene culture substrate. Compared with the myocardial cells cultured on a common cover glass, the synchronization of the contraction of the myocardial cells cultured by the aminated graphene culture medium is more stable and uniform, and the mark is a mark for measuring the maturation of the myocardial cells.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A preparation method of an aminated graphene culture medium is characterized by comprising the following steps:
(1) dissolving aminated graphene in water, and uniformly dispersing to obtain an aminated graphene dispersion liquid; in the aminated graphene dispersion liquid, the content of aminated graphene is 0.5-2 mg/ml;
(2) carrying out vacuum filtration on the aminated graphene dispersion liquid obtained in the step (1) to the surface of the microporous filter membrane, and drying to obtain the aminated graphene dispersion liquid, wherein an aminated graphene membrane is formed and loaded on the surface of the microporous filter membrane;
(3) performing gravity extrusion on the microporous filter membrane in the step (2) to transfer the aminated graphene membrane to a support layer, namely attaching the aminated graphene membrane to the surface of the support layer;
(4) and (4) soaking the three-layer superposed structure of the microporous filter membrane, the aminated graphene membrane and the support layer obtained in the step (3) in a solvent to dissolve the microporous filter membrane, washing away the residual solvent, and drying to obtain the aminated graphene culture medium.
2. The method for preparing an aminated graphene culture medium according to claim 1, wherein the microporous filtration membrane in step (2) is a cellulose water filtration membrane.
3. The method for preparing the aminated graphene culture medium according to claim 1, wherein the gravity extrusion in step (3) has a gravity value of 10N-50N and an extrusion time of 8h-12 h.
4. The method for preparing the aminated graphene culture substrate according to claim 1, wherein the support layer in step (3) is a cover glass, a silica gel sheet or a gelatin film.
5. The method for preparing an aminated graphene culture substrate according to claim 1, wherein the solvent in step (4) is acetone or ethyl acetate.
6. An aminated graphene culture substrate prepared according to any one of claims 1-5.
7. The use of the aminated graphene culture medium according to claim 6 for promoting the maturation of induced pluripotent stem cell-derived cardiomyocytes, wherein the amino group of the aminated graphene culture medium provides a positive charge microenvironment, so that the PDGFRB gene expression is up-regulated, thereby regulating and inducing the oriented cardiac differentiation of pluripotent stem cells, and effectively stimulating the maturation of cardiomyocyte precursor cells.
8. The use of claim 7, wherein a layer of matrigel is laid on the aminated graphene culture substrate, the matrigel is used for promoting cell adhesion, then induced pluripotent stem cells are inoculated on the matrigel for cell proliferation culture, when the cell density of the induced pluripotent stem cells reaches 85% -95%, the proliferation culture medium is replaced by a myocardial differentiation culture medium for myocardial cell differentiation, and the aminated graphene culture substrate can provide a positive-charge microenvironment to promote the maturation of the derived myocardial cells; and after the cardiac muscle cells are differentiated, replacing a cardiac muscle maintaining culture medium for continuous culture to obtain mature cardiac muscle cells.
9. The use of claim 8, wherein the cardiomyocytes are differentiated between 8 days and 14 days.
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