CN111254108B - Silk protein hydrogel and preparation method and application thereof - Google Patents

Silk protein hydrogel and preparation method and application thereof Download PDF

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CN111254108B
CN111254108B CN201911380244.6A CN201911380244A CN111254108B CN 111254108 B CN111254108 B CN 111254108B CN 201911380244 A CN201911380244 A CN 201911380244A CN 111254108 B CN111254108 B CN 111254108B
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silk protein
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CN111254108A (en
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吕强
杭颖婕
陈红
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Suzhou University
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Abstract

The invention relates to the biomedical field, in particular to a silk protein hydrogel and a preparation method and application thereof. The silk protein hydrogel is prepared by coating silk protein oriented gel with coating material with concentration of 0.001% -10% (w/v). The invention utilizes different biological materials to coat the silk protein oriented gel, regulates and controls the interaction strength of the material and stem cells, obtains ideal cell adhesion effect, combines the structure bionic design, realizes the in vitro control of embryonic stem cell fate, and realizes the long-term maintenance and in vitro expansion of the stem of the embryonic stem cell by adopting the conventional cell culture means on the premise of no trophoblast cells and basically avoiding the use of exogenous growth factors. The technology solves the problems of strict technical requirements, high cost and the like of the existing embryonic stem cells, and provides a simple, economical and more stable culture medium and a technical method for the in-vitro amplification of the embryonic stem cells.

Description

Silk protein hydrogel and preparation method and application thereof
Technical Field
The invention relates to the biomedical field, in particular to a silk protein hydrogel and a preparation method and application thereof.
Background
Embryonic stem cells (embryonic stem cell, ESCs, ES or EK cells for short) are a class of cells isolated from early embryo (prior to gastrulation) or primordial gonads and have the properties of in vitro culture immortalization, self-renewal and multipotent differentiation. Embryonic stem cells can be induced to differentiate into almost all cell types of the body, both in an in vitro and in vivo environment.
Embryonic stem cells have multipotency (Pluripotency) and are characterized by the ability to differentiate (Cellular differentiation) into a variety of tissues (all tissues, including germ line cells), but cannot be independently grown into an individual (individuals that develop entirely from the ES cells used can be obtained using tetraploid fusion techniques). It can develop into cell tissue of ectodermal, mesodermal and endodermal germ layers.
Embryonic stem cells with multidirectional differentiation potential have great application prospects in tissue repair and regenerative medicine, and become the important development field of medical fronts in recent years. One key technology in stem cell therapy is efficient in vitro expansion, and maintaining stem cell stem property during expansion and preventing differentiation are key to in vitro expansion. The traditional culture method is to add leukemia inhibitory factor into the culture medium to maintain the cell stem property, but the leukemia inhibitory factor is taken as an exogenous factor, and the problems of high price, inherent tumorigenicity, easy inactivation and the like seriously obstruct the application of the leukemia inhibitory factor, so that the safe clinical transformation of embryonic stem cells is difficult to realize.
In recent years, various culture substrates for promoting embryonic stem cell proliferation have been developed, but in the use process, growth factors are still required to be added to assist in the maintenance of the stem property, and the maintenance time is short, so that the culture method of embryonic stem cells still needs to be further optimized.
Disclosure of Invention
In view of the above, the invention provides a silk protein hydrogel and a preparation method and application thereof. The silk protein hydrogel can be used for carrying out in-vitro high-efficiency amplification on mouse embryonic stem cells under the culture condition without leukemia inhibitory factor, and the cell stem expression level is high.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a silk protein hydrogel, which is prepared by coating silk protein oriented gel with a coating material with the concentration of 0.001% -10% (w/v);
the coating material comprises one or more than two of gelatin, hyaluronic acid and chitosan;
the preparation method of the silk protein oriented gel comprises the following steps: mixing silk and salt solution, boiling, cleaning, drying, mixing with LiBr solution, dialyzing, centrifuging, and collecting supernatant to obtain silk protein solution;
concentrating the silk protein solution, diluting to the concentration of 0.5-4wt%, and standing to obtain nanofiber hydrogel;
and stirring the nanofiber hydrogel, applying voltage, and collecting the silk fibroin oriented gel at the positive electrode.
Silk proteins are ideal candidate materials for studying cell behaviors such as stem cell differentiation, proliferation and stem maintenance due to excellent biocompatibility, excellent mechanical properties and controllable nanostructure, conformation and surface characteristics.
The bionic design of the in-vivo microenvironment is a reliable means for regulating and controlling the cell behaviors, and the structure of the cystodendron can be simulated to a certain extent on the basis of preparing the silk protein hydrogel with an orientation interval structure, however, the silk protein has high negative charge density and has strong repulsive force with cells, so that the cells are difficult to adhere and expand on the gel. In consideration of the fact that the too strong adhesion of materials to cells can induce stem cells to differentiate, the invention utilizes different biological materials to coat oriented gel, regulates and controls the interaction strength of the materials and the stem cells, obtains ideal cell adhesion effect, combines with the structure bionic design, realizes the in vitro control of embryonic stem cell fate, and realizes the long-term maintenance and in vitro expansion of the stem of the embryonic stem cell by adopting the conventional cell culture means on the premise of no trophoblast cells and basically avoiding the use of exogenous growth factors. The technology solves the problems of strict technical requirements, high cost and the like of the existing embryonic stem cells, and provides a simple, economical and more stable culture medium and a technical method for the in-vitro amplification of the embryonic stem cells.
In some embodiments of the invention, the silk protein oriented gel has an orientation interval of 50 to 500 μm.
In some embodiments of the invention, the nanofibers in the nanofiber hydrogel have a diameter of 10 to 20nm and a length of 1 to 2 μm.
Based on the above research, the invention also provides the application of the silk protein hydrogel in preparing stem cell culture substrates.
In addition, the invention also provides a stem cell culture substrate which is characterized by comprising the silk protein hydrogel.
The invention also provides a method for culturing stem cells, which comprises the step of inoculating the stem cells into the silk fibroin hydrogel or the stem cell culture substrate and culturing.
In some embodiments of the invention, the seeding is by culturing embryonic stem cells (2X 10) in complete medium (0.5-2 ml) 4 /cm 2 ～10×10 4 /cm 2 ) Inoculating the mixture into silk protein hydrogel, and adhering for 0.5-8 h;
the stem cell complete medium comprises the following components: leukemia inhibitory factor at a final concentration of 1000U/mL, high-sugar DMEM basal medium, fetal bovine serum at a final concentration of 10%, and green chain mycin at a final concentration of 100U/mL and 0.1mg/mL, respectively, beta-mercaptoethanol at a final concentration of 0.1mM, MEM nonessential amino acid solution at a final concentration of 1X.
In some embodiments of the invention, the culture medium used for the culture does not include leukemia inhibitory factor.
In some embodiments of the invention, the culturing is culturing the stem cells using leukemia inhibitory factor-free medium (0.5-2 ml);
the leukemia inhibitory factor-free medium comprises the following components: high sugar DMEM basal medium, fetal bovine serum at a final concentration of 10% and penicillin streptomycin at a final concentration of 100U/mL and 0.1mg/mL, respectively, beta-mercaptoethanol at a final concentration of 0.1mM, MEM nonessential amino acid solution at a final concentration of 1X.
In some embodiments of the invention, the seeded cell density is 2X 10 4 /cm 2 ～10×10 4 /cm 2 The dosage of the stem cell complete culture medium adopted by the inoculation or the leukemia inhibitory factor-free culture medium adopted by the culture is 0.5-2 mL.
In some embodiments of the invention the stem cells are embryonic stem cells.
The invention provides a silk protein hydrogel with adjustable and controllable material and stem cell mutual adhesion strength, and a method for realizing in-vitro expansion of stem cells without trophoblasts and exogenous growth factors by taking the silk protein hydrogel as a matrix, which comprises the following steps: s1: preparing silk protein oriented gel, wherein the orientation interval is 50-500 μm; s2: coating the silk protein oriented gel obtained in the step S1 by using gelatin, hyaluronic acid, chitosan and the like, wherein the concentration of a coating material is 0.001% -10%; s3: taking the silk fibroin oriented gel coated by different materials as a culture substrate; s4: inoculating the mouse embryo stem cells on the culture substrate by adopting a complete embryo stem cell culture medium, placing the culture substrate in a cell culture box, and continuously culturing the mouse embryo stem cells by changing the culture medium into a leukemia inhibitory factor-free culture medium after the cells adhere for 0.5-8 hours; s5: continuously culturing stem cells by using a leukemia inhibitory factor-free culture medium for 24-48 hours, and changing the liquid until the required cell density is reached. The invention utilizes different biological materials to coat the silk protein oriented gel, regulates and controls the interaction strength of the material and stem cells, obtains ideal cell adhesion effect, combines the structure bionic design, realizes the in vitro control of embryonic stem cell fate, and realizes the long-term maintenance and in vitro expansion of the stem of the embryonic stem cell by adopting the conventional cell culture means on the premise of no trophoblast cells and basically avoiding the use of exogenous growth factors. The technology solves the problems of strict technical requirements, high cost and the like of the existing embryonic stem cells, and provides a simple, economical and more stable culture medium and a technical method for the in-vitro amplification of the embryonic stem cells.
The beneficial effects of the invention include, but are not limited to:
(1) Realizes the efficient in-vitro expansion of the mouse embryonic stem cells under the culture condition without leukemia inhibitory factor, and has high cell stem expression level;
(2) Avoiding the risks of tumorigenicity and the like caused by using growth factors in normal culture and providing a safer and more reliable technical scheme for realizing stem cell treatment;
(3) The method reduces the usage amount of leukemia inhibitory factor, and greatly reduces cell culture cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows an electron microscopy image of an uncoated silk protein oriented gel, wherein FIG. 1 (a) shows a macroscopic view of the silk protein oriented gel of the invention; FIG. 1 (b) is a scanning electron microscope image showing the alignment structure of the silk fibroin alignment gel of the present invention; FIG. 1 (c) is a scanning electron microscope image of nanofibers of the silk fibroin oriented gel of the present invention;
FIG. 2 shows a graph of adhesion of mouse embryonic stem cells to a material surface after 4h of silk protein oriented gel coated with gelatin; wherein line 1 shows the results for gelatin concentrations of 0.001%; line 2 shows the results for a gelatin concentration of 0.01%; line 3 shows the results for a gelatin concentration of 0.1%; line 4 shows the results for gelatin concentration of 1%;
FIG. 3 shows the results of the silk fibroin hydrogels of the present invention (prepared using gelatin coated silk fibroin oriented gels according to the preparation method of example 2) after 7 days of culturing mouse embryonic stem cells in the absence of leukemia inhibitory factor (SA); wherein, the cell morphology of FIG. 3 (a), the cell proliferation condition of FIG. 3 (b) and the cell stem expression result of FIG. 3 (c) are shown, wherein, a classical culture Method (MEF) with a mouse embryo fibroblast cell as a feeder layer and a leukemia inhibitory factor added is used as a positive control, and a culture method (Gelatin) without oriented gel and leukemia inhibitory factor is used as a negative control;
FIG. 4 shows the cell adhesion amount of embryonic stem cells cultured with silk protein hydrogels without gelatin coating in comparative example;
FIG. 5 shows the three-way differentiation of mouse embryonic stem cells in vitro by culturing the mouse embryonic stem cells in the absence of leukemia inhibitory factor (SA) in silk protein hydrogel of the invention (prepared by using gelatin-coated silk protein oriented gel according to the preparation method of example 2); wherein, the result of three-way differentiation of the mouse embryonic stem cells cultured by a classical culture Method (MEF) with the mouse embryonic fibroblasts as a feeder layer and added with leukemia inhibitory factor is used as a control;
FIG. 6 shows the results of a silk fibroin hydrogel of the present invention (prepared by using gelatin-coated silk fibroin oriented gel according to the preparation method of example 2) for culturing mouse embryonic stem cells under leukemia inhibitory factor-free conditions (SA) to form chimeric mice in vivo, wherein the results of a classical culture Method (MEF) in which mouse embryonic stem cells were cultured with mouse embryonic fibroblasts as a feeder layer and leukemia inhibitory factor added to the cultured mouse embryonic stem cells were used as a control; wherein, FIG. 6 (a) shows a photograph of a chimeric mouse; FIG. 6 (b) shows the comparison of the number of surviving chimeric mice and the number of chimeric mice; FIG. 6 (c) shows the comparison of the chimeric rate of the obtained chimeric mice with sex statistics.
Detailed Description
The invention discloses a silk protein hydrogel, a preparation method and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the silk protein hydrogel. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention provides a silk protein hydrogel capable of maintaining the stem property of embryonic stem cells, which comprises the following steps:
(1) Obtaining a silk protein oriented gel;
(2) The silk protein oriented gel is coated by gelatin, hyaluronic acid, chitosan and the like, and the concentration of the coating material is 0.001% -10%.
Preferably, the orientation interval of the oriented gel formed in the step 1) is 50-500 μm.
Preferably, the step 2) is coated with 0.001-10% gelatin for 0.5-8 hours.
Preferably, the step 2) uses 0.001-10% hyaluronic acid for coating for 0.5-8 hours.
Preferably, the step 2) uses 0.001-10% chitosan to coat for 0.5-8 hours.
The invention also provides a method for realizing in-vitro expansion of stem cells by utilizing the silk protein hydrogel, which comprises the following steps:
(1) Silk protein hydrogel (silk protein oriented gel coated by different materials) is used as a culture substrate;
(2) Inoculating the mouse embryo stem cells on the culture substrate by adopting a complete embryo stem cell culture medium, placing the culture substrate in a cell culture box, and continuously culturing the mouse embryo stem cells by changing the culture medium into a leukemia inhibitory factor-free culture medium after the cells adhere for 0.5-8 h;
(3) Continuously culturing stem cells by using a leukemia inhibitory factor-free culture medium for 24-48 hours, and changing the liquid until the required cell density is reached.
Preferably, the step 2) has a cell seeding density of 2×10 4 /cm 2 ～10×10 4 /cm 2 。
Preferably, the leukemia inhibitory factor-free medium of step 2) comprises the following components: high-sugar DMEM basal medium, 10% fetal bovine serum, 100U/mL and 0.1mg/mL of green streptomycin, 0.1mM of beta-mercaptoethanol, 1x of MEM nonessential amino acid solution; the embryonic stem cell complete medium comprises the following components: leukemia inhibitory factor (leukemia inhibitory factor) at final concentration of 1000U/mL, without leukemia inhibitory factor medium.
Preferably, the cell culture conditions in the step 3) are normal culture conditions of 37 ℃ and 5% CO 2 。
Preferably, the step 3) is normal subculture, normal liquid exchange and culture time of 1-21d.
The invention has the advantages that:
(1) Realizes the efficient in-vitro expansion of the mouse embryonic stem cells under the culture condition without leukemia inhibitory factor, and has high cell stem expression level;
(2) Avoiding the risks of tumorigenicity and the like caused by using growth factors in normal culture and providing a safer and more reliable technical scheme for realizing stem cell treatment;
(3) The method reduces the usage amount of leukemia inhibitory factor, and greatly reduces cell culture cost.
The silk protein hydrogel, the preparation method and the application thereof provided by the invention can be obtained from the market by using raw materials and reagents.
The invention is further illustrated by the following examples:
example 1
(1) Preparation of silk fibroin nanofiber hydrogels with high crystalline structure oriented at 50-100 μm intervals
(2) Slicing the oriented hydrogel, sterilizing, and coating with 10% gelatin for 0.5h to obtain a culture substrate;
(3) Mouse embryonic stem cells were grown in 2X 10 cells using embryonic stem cell complete medium (0.5 ml) 4 /cm 2 Is inoculated onto the culture substrate at 37℃with 5% CO 2 In a cell culture box, after cell adhesion, changing the culture medium into a leukemia inhibitory factor-free culture medium (0.5 ml) to continuously culture the mouse embryonic stem cells for 1, 3 and 7 days; classical culture Method (MEF) with mouse embryo fibroblast as feeder layer and leukemia inhibitory factor (Gelatin) without oriented gelCells after 7 days are used as a control group;
(4) Discarding the original culture solution, fixing cells and breaking membranes by sequentially using a 4% paraformaldehyde solution and a 0.1% Triton X-100/PBS solution, then performing fluorescent staining treatment on cytoskeletal proteins by using FITC-phalloidin, and observing morphological changes of the cells after 1, 3 and 7 days under a confocal fluorescent microscope after the nuclei are stained by using DAPI;
(5) Collecting cells after culturing for 1, 3 and 7 days respectively, and analyzing the proliferation condition of the cells by detecting the DNA content of each group of cells;
(6) And (3) respectively extracting all RNA in the cells after 7 days of culture by using an RNA total extraction kit, performing reverse transcription to obtain cDNA, and quantitatively analyzing the dry related genes expressed in the cells by using a real-time PCR system.
(7) Cells (MEF group and SA group) after 7 days of culture were collected, respectively, and inoculated into gelatin-coated dishes, cultured in leukemia inhibitory factor-free medium for 3 weeks, respectively, tuj-1 (ectoderm), gata (endoderm), sma (mesoderm) immunostained on the cultured cells, and three-way differentiation behavior of the cells was observed under confocal fluorescence microscope after staining the nuclei with DAPI.
(8) The cells (MEF group and SA group) after 7 days of culture were collected and injected into blasts derived from C57BL/6J-Tyr mice by microinjection, the blasts after the injection of the cells were transplanted into pseudopregnant mice ICR, and whether ESCs were chimeric with the cell mass in the blasts cavity or not was detected to obtain chimeric mice, which demonstrated whether ESCs were dry and functional.
Example 2
(1) Preparation of silk fibroin nanofiber hydrogels with high crystalline structure oriented at 100-200 μm intervals
(2) Slicing the oriented hydrogel, sterilizing, and coating with 0.1% gelatin for 4 hours to obtain a culture substrate;
(3) Mouse embryonic stem cells were grown in 2X 10 cells using embryonic stem cell complete medium (0.5 ml) 4 /cm 2 Is inoculated onto the culture substrate at 37℃with 5% CO 2 In a cell culture box, the cells are adhered and then culturedThe medium is replaced by a leukemia inhibitory factor-free medium (0.5 ml) to continuously culture the embryonic stem cells of the mice for 1, 3 and 7 days; a classical culture Method (MEF) with mouse embryo fibroblast cells as a feeder layer and leukemia inhibitory factor (leukemia inhibitory factor) and a culture method (Gelatin) with unoriented gel and no leukemia inhibitory factor are used for respectively culturing the cells after 1day, 3 days and 7 days of mouse embryo stem as a control group;
(4) Discarding the original culture solution, fixing cells and breaking membranes by sequentially using a 4% paraformaldehyde solution and a 0.1% Triton X-100/PBS solution, then performing fluorescent staining treatment on cytoskeletal proteins by using FITC-phalloidin, and observing morphological changes of the cells after 1, 3 and 7 days under a confocal fluorescent microscope after the nuclei are stained by using DAPI;
(5) Collecting cells after culturing for 1, 3 and 7 days respectively, and analyzing the proliferation condition of the cells by detecting the DNA content of each group of cells;
(6) And (3) respectively extracting all RNA in the cells after 7 days of culture by using an RNA total extraction kit, performing reverse transcription to obtain cDNA, and quantitatively analyzing the dry related genes expressed in the cells by using a real-time PCR system.
(7) Cells (MEF group and SA group) after 7 days of culture were collected, respectively, and inoculated into gelatin-coated dishes, cultured in leukemia inhibitory factor-free medium for 3 weeks, respectively, tuj-1 (ectoderm), gata (endoderm), sma (mesoderm) immunostained on the cultured cells, and three-way differentiation behavior of the cells was observed under confocal fluorescence microscope after staining the nuclei with DAPI.
(8) The cells (MEF group and SA group) after 7 days of culture were collected and injected into blasts derived from C57BL/6J-Tyr mice by microinjection, the blasts after the injection of the cells were transplanted into pseudopregnant mice ICR, and whether ESCs were chimeric with the cell mass in the blasts cavity or not was detected to obtain chimeric mice, which demonstrated whether ESCs were dry and functional.
Example 3
(1) Preparation of silk fibroin nanofiber hydrogels with high crystalline structure with orientation spacing of 200-400 μm
(2) Slicing the oriented hydrogel, sterilizing, and coating with 0.001% hyaluronic acid for 8 hr to obtain culture substrate
(3) Mouse embryonic stem cells were grown at 10X 10 using embryonic stem cell complete medium (2 ml) 4 /cm 2 Is inoculated onto the culture substrate at 37℃with 5% CO 2 In a cell culture box, after cell adhesion, changing the culture medium into a leukemia inhibitory factor-free culture medium (2 ml) to continuously culture the mouse embryonic stem cells for 1, 3 and 7 days; a classical culture Method (MEF) with mouse embryo fibroblast cells as a feeder layer and leukemia inhibitory factor (leukemia inhibitory factor) and a culture method (Gelatin) with unoriented gel and no leukemia inhibitory factor are used for respectively culturing the cells after 1day, 3 days and 7 days of mouse embryo stem as a control group;
(4) Discarding the original culture solution, fixing cells and breaking membranes by sequentially using a 4% paraformaldehyde solution and a 0.1% Triton X-100/PBS solution, then performing fluorescent staining treatment on cytoskeletal proteins by using FITC-phalloidin, and observing morphological changes of the cells after 1, 3 and 7 days under a confocal fluorescent microscope after the nuclei are stained by using DAPI;
(5) Collecting cells after culturing for 1, 3 and 7 days respectively, and analyzing the proliferation condition of the cells by detecting the DNA content of each group of cells;
(6) And (3) respectively extracting all RNA in the cells after 7 days of culture by using an RNA total extraction kit, performing reverse transcription to obtain cDNA, and quantitatively analyzing the dry related genes expressed in the cells by using a real-time PCR system.
(7) Cells (MEF group and SA group) after 7 days of culture were collected, respectively, and inoculated into gelatin-coated dishes, cultured in leukemia inhibitory factor-free medium for 3 weeks, respectively, tuj-1 (ectoderm), gata (endoderm), sma (mesoderm) immunostained on the cultured cells, and three-way differentiation behavior of the cells was observed under confocal fluorescence microscope after staining the nuclei with DAPI.
(8) The cells (MEF group and SA group) after 7 days of culture were collected and injected into blasts derived from C57BL/6J-Tyr mice by microinjection, the blasts after the injection of the cells were transplanted into pseudopregnant mice ICR, and whether ESCs were chimeric with the cell mass in the blasts cavity or not was detected to obtain chimeric mice, which demonstrated whether ESCs were dry and functional.
Example 4
(1) Preparing silk fibroin nanofiber hydrogel with high crystal structure and orientation interval of 50-80 mu m;
(2) Slicing and sterilizing the oriented hydrogel, and coating the oriented hydrogel with 1% chitosan for 2 hours to obtain a culture substrate;
(3) Mouse embryonic stem cells were grown in 6X 10 cells using embryonic stem cell complete medium (1.5 ml) 4 /cm 2 Is inoculated onto the culture substrate at 37℃with 5% CO 2 In a cell culture box, after cell adhesion, changing a culture medium (1 ml) into a leukemia inhibitory factor-free culture medium to continuously culture the mouse embryonic stem cells for 1, 3 and 7 days; a classical culture Method (MEF) with mouse embryo fibroblast cells as a feeder layer and leukemia inhibitory factor (leukemia inhibitory factor) and a culture method (Gelatin) with unoriented gel and no leukemia inhibitory factor are used for respectively culturing the cells after 1day, 3 days and 7 days of mouse embryo stem as a control group;
(4) Discarding the original culture solution, fixing cells and breaking membranes by sequentially using a 4% paraformaldehyde solution and a 0.1% Triton X-100/PBS solution, then performing fluorescent staining treatment on cytoskeletal proteins by using FITC-phalloidin, and observing morphological changes of the cells after 1, 3 and 7 days under a confocal fluorescent microscope after the nuclei are stained by using DAPI;
(5) Collecting cells after culturing for 1, 3 and 7 days respectively, and analyzing the proliferation condition of the cells by detecting the DNA content of each group of cells;
(6) And (3) respectively extracting all RNA in the cells after 7 days of culture by using an RNA total extraction kit, performing reverse transcription to obtain cDNA, and quantitatively analyzing the dry related genes expressed in the cells by using a real-time PCR system.
(7) Cells (MEF group and SA group) after 7 days of culture were collected, respectively, and inoculated into gelatin-coated dishes, cultured in leukemia inhibitory factor-free medium for 3 weeks, respectively, tuj-1 (ectoderm), gata (endoderm), sma (mesoderm) immunostained on the cultured cells, and three-way differentiation behavior of the cells was observed under confocal fluorescence microscope after staining the nuclei with DAPI.
(8) The cells (MEF group and SA group) after 7 days of culture were collected and injected into blasts derived from C57BL/6J-Tyr mice by microinjection, the blasts after the injection of the cells were transplanted into pseudopregnant mice ICR, and whether ESCs were chimeric with the cell mass in the blasts cavity or not was detected to obtain chimeric mice, which demonstrated whether ESCs were dry and functional.
Effect example 1
The results of the test for uncoated oriented hydrogels and oriented hydrogels coated with different concentrations of gelatin were as follows:
referring to fig. 1, fig. 1 (a) is a macroscopic view of an uncoated oriented hydrogel, fig. 1 (b) is a scanning electron microscope view of an oriented hydrogel structure, and fig. 1 (c) is a scanning electron microscope view of a silk fibroin nanofiber, wherein the oriented structure provides a rich water environment for cell growth, and the nanofiber with high negative charge inhibits spreading of cells on the surface of a material, and the structure of the material can simulate the structure of a cystlayer to a certain extent.
As a result of microscopic examination of the coated oriented hydrogel, see fig. 2, line 1 shows 0.001% of gelatin, line 2 shows 0.01% of gelatin, line 3 shows 0.1% of gelatin, and line 4 shows 1% of gelatin, which is coated with the hydrogel material for 2 hours, it was found that the interaction strength of the material and stem cells can be controlled by using the concentration of the coating, thereby controlling the adhesion behavior of the cells on the material.
Effect example 2
FIG. 3, table 1 shows the results of example 2, FIG. 3 (a) shows the cell morphology of the silk fibroin hydrogel after 7 days of culture of mouse embryonic stem cells in the absence of leukemia inhibitory factor (SA); FIG. 3 (b) shows the proliferation of cells; FIG. 3 (c) shows the results of cell stem gene expression, wherein a classical culture Method (MEF) in which leukemia inhibitory factor is added with mouse embryonic fibroblasts as a feeder layer was used as a positive control, and a culture method (Gelatin) in which leukemia inhibitory factor was not added with an unoriented gel was used as a negative control.
Compared with the dispersion of cells in the Gelatin group, the SA group can maintain the special clone-shaped cell morphology of the mouse embryonic stem cells under the condition of no leukemia inhibitory factor, and can reach the proliferation rate equivalent to that of the MEF method, and the expression quantity of the cell stem related genes oct4, nanog and sox2 after the amplification culture is highest, so that the amplification method provided by the invention can be proved to effectively realize the efficient amplification and the dryness maintenance of the mouse embryonic stem cells under the condition of no leukemia inhibitory factor.
TABLE 1
The expression of oct4, nanog, sox2 genes was analyzed by RT-qPCR after 7 days of culture of mESCs cells on MEF, gelatin and SA. By taking mESCs cultured on MEF as a control and setting the value as 1, experiments show that the oct4, nanog and sox2 gene expression amounts of cells on SA are all obviously higher than those of MEF groups (p <0.05, p <0.01 and p < 0.001), the values of the mESCs are respectively improved by 100%,100% and more than 50%, and the expression amounts of three dry related genes on Gelatin are respectively reduced by about 50%,40% and 40%. The above results demonstrate that SA can achieve high expression of mESCs in the absence of LIF, and that oct4, nanog, sox2 gene expression levels are higher than in classical MEF methods with LIF added for culture.
Effect example 3
As shown in fig. 5 and 6.
The stem ability of the different cells after in vitro culture was further verified by in vitro three-germ layer differentiation experiments, see figure 5. After mESCs were cultured on SA and MEF for 21day, the cells were further cultured on a common gelatin plate with mESCs medium for 14 days, and immunofluorescent staining experiments were performed to observe the signal expression of early three germ layers (ectoderm, endoderm and mesoderm) of mESCs, and the differentiation ability of the cells into three germ layers was analyzed by detecting the expression of the protein represented by Tuj-1 (ectoderm), GATA4 (endoderm) and SMA (mesoderm). Experiments show that after mESCs cultured on SA and MEF are continuously cultured on a common gelatin plate by using mESCs culture medium of leukemia inhibitory factor for 14 days, tuj-1 and SMA are both green in staining, and cells are red after GATA4 staining, so that cells Tuj-1, GATA4 and SMA are positive expression, and the cells can express three-germ layer signals and have the capacity of differentiating towards three germ layers.
Whether ESCs can be chimeric with the cell mass in the blastula cavity to obtain chimeric mice is an effective method to demonstrate whether ESCs are dry and functional. As shown in fig. 6. The hair color of the mice from which mESCs are derived is black, the donor embryo used for injection is derived from a C57BL/6J-Tyr albino mouse, and if the cells are successfully embedded, the hair color of the chimeric mice born is black or black-white, so that whether the embedding is successful or not can be judged by detecting the hair color of the mice born, and whether the cells have dryness or not can be verified. The mESCs cultured on SA and MEF are microinjected into 88 donor embryos and transplanted into the uterus of an ICR female mouse, and the result shows that the survival rate of the obtained mice after the mESCs are injected into the embryo after SA culture is higher, cells can be embedded with the donor embryo, and the chimeric mice with black and white hair are obtained by differentiation expression in the embryo, the average chimeric rate of male mice is as high as 80%, and the SA can maintain the dryness of the mESCs under the condition of leukemia inhibitory factor and has functionality.
In conclusion, the silk fibroin oriented gel is coated by using different biological materials, and the ideal cell adhesion effect is expected to be obtained by regulating and controlling the interaction strength of the material and stem cells. The invention combines the structure bionic design to realize the in vitro control of embryonic stem cell fate, and adopts the conventional cell culture means to realize the long-term maintenance and in vitro expansion of the stem of the embryonic stem cell under the premise of no trophoblast cell and basically avoiding the use of exogenous growth factors.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (2)

1. The culture method of the mouse embryonic stem cells is characterized in that the mouse embryonic stem cells are inoculated into silk protein hydrogel for culture;
the silk protein hydrogel is prepared by coating silk protein oriented gel with a coating material;
the coating material is gelatin 0.1% -10%, hyaluronic acid 0.001% or chitosan 1%;
the preparation method of the silk protein oriented gel comprises the following steps: mixing silk and salt solution, boiling, cleaning, drying, mixing with LiBr solution, dialyzing, centrifuging, and collecting supernatant to obtain silk protein solution; concentrating the silk fibroin solution, diluting to a concentration of 0.5-4wt%, and standing to obtain nanofiber hydrogel; stirring the nanofiber hydrogel, applying voltage, and collecting the silk fibroin oriented gel at the positive electrode;
the orientation interval of the silk protein oriented gel is 50-500 mu m;
the diameter of the nanofiber in the nanofiber hydrogel is 10-20 nm, and the length of the nanofiber is 1-2 mu m;
the inoculation is to inoculate the mouse embryo stem cells into the silk protein hydrogel by adopting a stem cell complete culture medium, and adhere for 0.5-8 h;
the stem cell complete culture medium comprises the following components: leukemia inhibitory factor at a final concentration of 1000U/mL, high-sugar DMEM basal medium, fetal calf serum at a final concentration of 10%, and penicillin streptomycin at a final concentration of 100U/mL and 0.1mg/mL, respectively, beta-mercaptoethanol at a final concentration of 0.1mM, MEM nonessential amino acid solution at a final concentration of 1X;
after cell adhesion, the culture medium is changed into a leukemia inhibitory factor-free culture medium to continuously culture stem cells;
the leukemia inhibitory factor-free medium comprises the following components: high sugar DMEM basal medium, fetal bovine serum at a final concentration of 10%, penicillin streptomycin at a final concentration of 100U/mL and 0.1mg/mL, respectively, beta-mercaptoethanol at a final concentration of 0.1mM, MEM nonessential amino acid solution at a final concentration of 1X.
2. The culture method according to claim 1, wherein the inoculated cells have a density of 2X 10 4 /cm 2 ~10×10 4 /cm 2 The dosage of the stem cell complete culture medium or the leukemia inhibitory factor-free culture medium is 0.5 mL-2 mL.
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