CN109306338B - Method for inducing differentiation of adipose-derived stem cells in vitro by using microporous coating - Google Patents
Method for inducing differentiation of adipose-derived stem cells in vitro by using microporous coating Download PDFInfo
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Abstract
The invention discloses a method for inducing differentiation of adipose-derived stem cells in vitro by using a microporous coating, belonging to the field of biomolecules. The method comprises the following steps: (1) preparing a polyelectrolyte coating by a layer-by-layer self-assembly method; (2) carrying out acid treatment on the coating, and then carrying out freeze drying to form a communicated microporous structure; (3) loading a plurality of bioactive molecules into the coating of the microporous structure; (4) the coating loaded with bioactive molecules realizes the healing of micropores in a saturated humidity environment; (5) the coating loaded with bioactive molecules can regulate and control the release of various bioactive molecules, and is maintained in a proper concentration range for more than 16 days; (6) the coating was placed in a culture plate for cell culture. The controlled-release bioactive molecule can keep good bioactivity under physiological conditions, reduce the replacement frequency of cell culture solution and reduce the cost of cell culture.
Description
Technical Field
The invention belongs to the field of biomolecules, and relates to a method for inducing differentiation of adipose-derived stem cells in vitro by using a microporous coating.
Background
Tissue engineering has been developed rapidly in recent years as an important subject for treating certain diseases instead of organ transplantation, and recently, adipose-derived stem cells (hASCs) isolated from human adipose tissues have been proven to be cells with self-renewal capacity and multi-directional differentiation potential, which can differentiate into osteoblasts, chondroblasts, adipoblasts, nerve cells, muscle cells, vascular cells and other cells from different germ layers, and have the advantages of easy self-material collection, wide source, less damage, easy mass acquisition, strong in vitro amplification capacity, difficult aging, low antigenicity, no ethical problems and the like, and thus the tissue engineering seed cells have a broad application prospect in aspects of organ repair, tissue engineering, gene therapy and the like.
Chinese patent CN104383611A discloses a method for preparing a bioactive molecule-loaded coating by polyelectrolyte assembly, which mainly comprises the following steps of (1) preparing a coating by a polyelectrolyte layered assembly method; (2) performing acid treatment on the coating to generate a microporous structure; (3) freeze-drying the coating to fix the microporous structure; (4) immersing the coating with the microporous structure into a drug solution for drug loading; (5) and (3) carrying out water vapor treatment on the coating loaded with the bioactive molecules to close micropores in the coating, so as to realize the embedding load of the medicine. The polyelectrolyte coating prepared by the method has a microporous structure, can be used for simultaneously loading various types of medicines, and has the advantages of simple and convenient loading process and controllable loading capacity.
Although the polyelectrolyte coating obtained by the preparation method has a microporous structure and can be used for loading drugs, the polycation electrolyte aqueous solution is required to be limited to be alkaline, and the polyanion electrolyte aqueous solution is acidic, so that the requirements on conditions are strict, the differentiation culture of the adipose-derived stem cell is not involved, and the specific operation and conditions for the differentiation culture of the adipose-derived stem cell are not disclosed, so that the application of the preparation method to the differentiation culture of the adipose-derived stem cell belongs to a blank stage. The drug-loaded coating in chinese patent CN104383611A is only used for releasing drugs. The acidity and alkalinity of the polyelectrolyte solution are not limited in the invention, compared with the drug released by Chinese patent CN104383611A alone, the surface of the coating of the invention can grow cells, and the time for releasing various bioactive molecules in proportion can be as long as 16 days, which is not possessed by Chinese patent CN 104383611A.
The traditional differentiation culture method of the adipose-derived stem cells at present is to culture the cells on a polystyrene cell culture plate (TCPS) in an adherent manner, and replace a cell culture medium containing cell bioactive molecules every 1-2 days. Unlike some cell cultures with short periods, the differentiation period of the adipose-derived stem cells exceeds 16 days, the frequent liquid change can cause adverse effects on the cells, increase the risk of bacterial infection, and consume excessive cell culture medium, thereby causing waste.
Disclosure of Invention
The invention provides a method for inducing differentiation of adipose-derived stem cells in vitro by using a microporous coating, which is characterized in that a plurality of bioactive molecules are loaded into the coating at the same time and put into a cell culture solution for cell culture, and the bioactive molecules in the coating can be regularly released, so that the aim of culturing the cells is fulfilled. Compared with the traditional culture method, the method can reduce the frequency of replacing the cell culture solution, reduce the influence of the solution replacement on cell culture and reduce the cell culture cost; because the coating structure has adjustability, the release concentration can be freely adjusted. In vitro release experiments show that the actual load capacity of the porous coating can be regulated and controlled by changing the number of the coating layers, the concentration of the load liquid and other factors, and various bioactive molecules can be proportionally released according to the actual load proportion, and the release time exceeds 16 days, so that the method is particularly suitable for cell differentiation of the adipose-derived stem cells; the cell active molecules are easy to denature under normal physiological conditions to cause the loss of the activity of the cell active molecules, and the controlled release of the bioactive molecules by the method can keep good bioactivity under the physiological conditions; for cells growing in an adherent manner, the collagen layer is coated on the surface of the coating, so that the cells can grow by being attached to the surface of the coating, the cells are easy to transfer, and a new idea is provided for cell culture.
The invention discloses a method for inducing differentiation of adipose-derived stem cells in vitro by using a microporous coating, which comprises the following steps:
(1) fully dissolving polycation electrolyte in a solvent A to prepare a polycation electrolyte solution; fully dissolving polyanionic electrolyte in the solution B to prepare polyanionic electrolyte solution;
(2) completely soaking the substrate material in a polycation electrolyte solution for 8-60 minutes, taking out, and washing with a solution C;
(3) completely soaking the substrate material obtained in the step (2) in a polyanion electrolyte solution for 8-60 minutes, taking out, and washing with a solution D;
(4) repeating the steps (2) to (3) for a plurality of times, taking out the substrate material, putting the substrate material into a solvent E, freezing the substrate material at the temperature of minus 80 ℃ for one night, and freeze-drying the substrate material the next day to obtain a coating with a communicated microporous structure;
(5) determining the concentration of the loading liquid, loading various bioactive molecules in the negative loading liquid into the coating through capillary action, and drying the coating through vacuum;
in the invention, the concentration of various bioactive molecules released by the coating in a cell culture solution is regulated and controlled by regulating the thickness of the coating and the concentration of a loading solution. The bioactive molecules released from the coating can always meet the requirement of cell culture, namely the concentration of the bioactive molecules released from the coating in the culture medium after each liquid change is greater than or equal to the concentration required by cell culture.
(6) Treating the coating loaded with bioactive molecules by a physical method F to close micropores into a compact structure;
(7) coating a layer of collagen on the surface of the coating, adding 3 rd generation adipose-derived stem cells and a complete culture medium, carrying out cell differentiation culture, and periodically replacing a solvent H.
In the invention, the polycation electrolyte is one or a mixture of chitosan, polyethyleneimine and polylysine.
In the invention, the polyanionic electrolyte is one or a mixture of polyacrylic acid and sodium alginate.
In the invention, the solvent A is 0.5-2% of glacial acetic acid aqueous solution with volume fraction.
In the invention, the solution B, the solution C and the solution D are the same solution and are deionized water.
In the invention, the solvent E is hydrochloric acid aqueous solution with the pH value of 2.2-2.9, and the soaking time is 30-200 minutes.
In the invention, the physical method F is to place the microporous structure coating under 100% relative humidity for 2-48 hours.
In the invention, the solvent H is a complete cell culture solution, and the replacement interval is 2-5 days.
In the invention, the substrate material is one of a PET film or a glass sheet.
In the invention, the steps (2) to (3) are repeated for 10 to 60 times.
In the present invention, the carrier liquid refers to a solution of bioactive molecules dissolved in a specific solvent, and is configured such that the carrier liquid serves to dissolve the bioactive molecules and can enter the membrane by capillary action, and then the solvent is removed by vacuum.
In the present invention, the specific solvent is absolute ethanol.
Maintaining the concentration of the bioactive molecule at an appropriate level in the present invention means that the concentration of the bioactive molecule released in the culture solution after each change is equal to or greater than the concentration of the bioactive molecule required for culturing cells by conventional methods.
The invention has the following effects and advantages:
(1) the invention adopts chitosan/polyacrylic acid with an exponential growth mode or polyethyleneimine/polyacrylic acid as a component for preparing the coating, so that the obtained coating material has certain thickness and viscoelasticity similar to a matrix tissue.
(2) According to the invention, the properties such as the thickness and the quality of the polyelectrolyte coating can be regulated and controlled by regulating the cycle times of layer-by-layer self-assembly, and then the release amount of each bioactive molecule can be regulated and controlled by the actual load of the coating.
(3) The invention uses the coating with the microporous structure to release bioactive molecules, so that the frequency of changing the cell culture solution can be reduced, the influence of the solution change on cells is reduced, and the cell culture cost is reduced.
(4) The cell active molecule is easy to denature under normal physiological conditions, so that the activity of the cell active molecule is lost, and the controlled release of the bioactive molecule by the method can keep good bioactivity under physiological conditions.
(5) In vitro bioactive molecule loading and releasing research shows that the actual loading of the porous coating can be regulated and controlled by changing the number of coating layers, the concentration of the loading solution and other factors, and various bioactive molecules can be proportionally released according to the actual loading proportion, namely the release amount of each bioactive molecule can be regulated and controlled through the actual loading.
(6) The time for releasing cell active molecules exceeds 16 days, so the method is suitable for differentiation of the adipose-derived stem cells and has potential application value in the fields of cell culture and the like.
(7) For cells growing in an adherent manner, the collagen layer is coated on the surface of the coating, so that the cells can grow by being attached to the surface of the coating, the cells are easy to transfer, and a new idea is provided for cell culture.
Drawings
FIG. 1 is a graph showing staining with oil red O after two weeks of adipocyte culture, comparing the method (A) of the present invention with the conventional culture method (B);
FIG. 2 is a graph showing staining using Hoechst 33342 after two weeks of culture of adipocytes, which is compared with the method (A) of the present invention and the conventional culture method (B).
Detailed Description
Example 1:
(1) cutting a PET film into a rectangular shape of 10mm multiplied by 30mm, sequentially cleaning the PET film with ethanol and acetone, flushing residual liquid on the surface with deionized water, and drying the residual liquid with nitrogen for later use;
(2) preparing a Chitosan (CS) solution with the concentration of 1mg/mL and a polyacrylic acid (PAA) solution with the concentration of 3mg/mL, and stirring until the CS and the PAA are fully dissolved;
(3) firstly, soaking a PET film in a CS solution for 10 minutes, and then washing the PET film for 3 times by using deionized water; then soaking in PAA solution for 10 minutes, and then washing with deionized water for 3 times (step (3) constitutes preparation of a double layer);
(4) repeating the process (3) 15 times to obtain (CS/PAA)15Coating;
(5) will (CS/PAA)15Taking out the coating, putting the coating into a hydrochloric acid solution with the pH value of 2.5 for 1 hour, taking out the coating, freezing the coating overnight at the temperature of minus 80 ℃, and freeze-drying the coating the next day to obtain the coating with a communicated microporous structure;
(6) preparing load solution of IBMX 5mmol/L, dexamethasone 10 mu mol/L and indometacin 2mmol/L, wherein the solvent is ethanol;
(7) one end of the microporous coating is lightly touched with the negative carrier liquid, so that the whole membrane is filled with the load liquid through capillary action, and the solvent ethanol is removed through a vacuum drier;
(8) when the coating loaded with the bioactive molecules is placed under 100 percent relative humidity for 5 hours, the micropore structures in the coating can be spontaneously healed, and the existing release result shows (CS/PAA)15The coating releases the bioactive molecule over a period of more than 16 days, so (CS/PAA)15Can carry out adipogenic differentiation of the adipose-derived stem cells;
(9) the coating loaded with bioactive molecules is enclosed around the culture plate according to the proportion of 5 multiplied by 103Adding 3 rd generation adipose-derived stem cells and complete culture medium into the culture plate per square centimeter;
(10) changing complete culture solution every 4 days, and continuously performing induction culture for 14 days;
(11) the existence of cell nucleus is observed under fluorescence after the Hoechst 33342 is stained, and fat is red after the oil red O staining, which indicates that the adipose-derived stem cells are successfully differentiated into the fat cells.
The 3 rd generation adipose-derived stem cells are obtained by extracting and separating and culturing by adopting a conventional process.
The preparation method of the complete culture solution comprises the following steps:
adding 5g DMEM powder, 1.85g sodium bicarbonate, 1.75g glucose, 5ml penicillin-streptomycin double antibody solution, 50ml Fetal Bovine Serum (FBS) and 100. mu.L basic fibroblast active molecule (bGFG) into a bottle, fixing the volume to 500ml, filtering with a sterilized filter, sealing, and storing in a refrigerator at 4 ℃.
Example 2:
(1) soaking a circular glass sheet with the diameter of 20mm in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (the volume ratio is 7: 3) for 40min, taking out, washing residual liquid on the surface with deionized water, and drying with nitrogen for later use;
(2) step (2) is carried out in the same way as the example 1;
(3) firstly, soaking a circular glass slide in a CS solution for 10 minutes, and then washing the circular glass slide for 4 times by using deionized water; then soaking in PAA solution for 10 minutes, and then washing with deionized water for 4 times (step (3) constitutes preparation of a double layer);
(4) repeating the process (3) 20 times to obtain (CS/PAA)20Coating;
(5) will (CS/PAA)20Taking out the coating, putting the coating into a hydrochloric acid solution with the pH value of 2.5 for 1 hour, taking out the coating, freezing the coating overnight at the temperature of minus 80 ℃, and freeze-drying the coating the next day to obtain the coating with a communicated microporous structure;
(6) preparing load solution of IBMX 3.75mmol/L, dexamethasone 7.5 mu mol/L and indometacin 1.5mmol/L, wherein the solvent is ethanol;
(7) step (7) is carried out in the same way as in the example 1;
(8) when the coating loaded with the bioactive molecules is placed under 100 percent relative humidity for 8 hours, the micropore structures in the coating can be spontaneously healed, and the existing release result shows (CS/PAA)20The bioactive molecule of the coating is released for more than 16 days, so (CS/PAA)20Is suitable for adipogenic differentiation of the adipose-derived stem cells;
(9) placing the coating loaded with bioactive molecules at the bottom of a culture plate, coating a layer of collagen on the surface of the coating, and adding 3 rd generation of adipose-derived stem cells and a complete culture medium into the culture plate;
(10) changing complete culture solution every 5 days, and continuously performing induction culture for 17 days;
(11) the presence of cell nuclei was observed under fluorescence after Hoechst 33342 staining, and fat appeared red after oil red O staining, indicating successful differentiation of adipose-derived stem cells into adipocytes, which allowed the cells to grow on (CS/PAA)20The cell culture method of the coating proves (CS/PAA)20The coating has good biocompatibility and low cytotoxicity.
Example 3:
(1) step (1) is carried out in the same way as the example;
(2) preparing a Polyethyleneimine (PEI) solution with the concentration of 1mg/mL and a polyacrylic acid (PAA) solution with the concentration of 3mg/mL, and stirring until the PEI and the PAA are fully dissolved;
(3) firstly, soaking a PET film in a PEI solution for 10 minutes, and then washing the PET film for 3 times by using deionized water; then soaking in PAA solution for 10 minutes, and then washing with deionized water for 3 times (step (3) constitutes preparation of a double layer);
(4) repeating the process (3) 15 times to obtain (PEI/PAA)15Coating;
(5) will (PEI/PAA)15Taking out the coating, putting the coating into a hydrochloric acid solution with the pH value of 2.5 for 1 hour, taking out the coating, freezing the coating overnight at the temperature of minus 80 ℃, and freeze-drying the coating the next day to obtain the coating with a communicated microporous structure;
(6) step (6) is carried out in the same way as the example 1;
(7) preparing load solution of IBMX 5mmol/L, dexamethasone 10 mu mol/L and indometacin 2mmol/L, wherein the solvent is ethanol;
(8) placing the coating loaded with bioactive molecules under 100% relative humidity for 12h, wherein the microporous structure in the coating can heal spontaneously, and the release result shows that the release time of the bioactive molecules exceeds 16 days, so that the coating is suitable for adipogenic differentiation of the adipose-derived stem cells;
(9) step (9) is carried out in the same way as the example 1;
(10) changing complete culture solution every 5 days, and continuously performing induction culture for 14 days;
(11) the existence of cell nucleus is observed under fluorescence after Hoechst 33342 is stained, fat is red after oil red O staining, the fat-derived stem cells are successfully differentiated into the fat cells, the growth condition of the fat-derived stem cells is not greatly different from that of a control group, and the method can be used for long-time culture of the cells.
Example 4:
(1) step (1) is carried out in the same way as the example 2;
(2) step (2) is carried out in the same way as the example 3;
(3) firstly, soaking a circular glass slide in a PEI solution for 10 minutes, and then washing the circular glass slide for 4 times by using deionized water; then soaking in PAA solution for 10 minutes, and then washing with deionized water for 4 times (step (3) constitutes preparation of a double layer);
(4) repeating the process (3) 25 times to obtain (PEI/PAA)25Coating;
(5) will (PEI/PAA)25Taking out the coating, putting the coating into a hydrochloric acid solution with the pH value of 2.5 for 1 hour, taking out the coating, freezing the coating overnight at the temperature of minus 80 ℃, and freeze-drying the coating the next day to obtain the coating with a communicated microporous structure;
(6) step (6) is carried out in the same way as the example 1;
(7) preparing load solution of IBMX 3mmol/L, dexamethasone 6 mu mol/L and indometacin 1.2mmol/L, wherein the solvent is ethanol;
(8) the coating loaded with the bioactive molecules is placed under 100% relative humidity for 24h, the microporous structure in the coating can be spontaneously healed, and the existing release result shows that the release time of the bioactive molecules exceeds 16 days, so that the coating is suitable for adipogenic differentiation of the adipose-derived stem cells;
(9) step (9) is carried out in the same way as the example 2;
(10) changing the culture solution every 4 days, and continuously performing induction culture for 17 days;
(11) the existence of cell nucleus is observed under fluorescence after Hoechst 33342 is stained, fat is red after oil red O staining, which indicates that the adipose-derived stem cells are successfully differentiated into fat cells.
The above-mentioned embodiments are only for the purpose of understanding the present invention and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (4)
1. A method for inducing differentiation of adipose-derived stem cells in vitro using a microporous coating, comprising the steps of:
(1) fully dissolving polycation electrolyte in glacial acetic acid water solution with volume fraction of 0.5% -2% to prepare polycation electrolyte solution; fully dissolving polyanionic electrolyte in deionized water to prepare polyanionic electrolyte solution; the polycation electrolyte is chitosan or polyethyleneimine; the polyanionic electrolyte is polyacrylic acid;
(2) completely soaking the PET film or glass sheet substrate material in a polycation electrolyte solution for 8-60 minutes, taking out, and washing with deionized water;
(3) completely soaking the substrate material obtained in the step (2) in a polyanion electrolyte solution for 8-60 minutes, taking out, and washing with deionized water;
(4) repeating the step (2) and the step (3) for 10-60 times to prepare a polymer coating substrate material, taking out the substrate material, soaking the substrate material in a hydrochloric acid aqueous solution, taking out the substrate material, freezing the substrate material at-80 ℃ overnight, and freeze-drying the substrate material the next day to obtain a coating with a communicated microporous structure; wherein the polymer coating substrate material is a PEI/PAA coating or a CS/PAA coating;
(5) determining the concentration of a load solution, wherein the load solution is prepared from IBMX, dexamethasone and indometacin in an ethanol solvent, a plurality of bioactive molecules in the negative carrier solution are loaded into the coating through capillary action, so that the concentration of the bioactive molecules released by the coating in a cell culture solution is always higher than the concentration required by cell culture, and then the coating is dried in vacuum;
(6) treating the coating loaded with bioactive molecules for 2-48 hours at 100% relative humidity to close micropores into a compact structure; and (3) coating a layer of collagen on the surface of the coating, adding 3 rd generation adipose-derived stem cells and a complete culture medium, carrying out cell differentiation culture, and periodically replacing a cell complete culture solution at intervals of 2-5 days.
2. The method for inducing differentiation of adipose-derived stem cells in vitro using microporous coating according to claim 1, wherein the concentration of the polycationic electrolyte solution is 0.5-5 mg/ml; the concentration of the polyanionic electrolyte solution is 0.5-5 mg/ml.
3. The method for inducing differentiation of adipose-derived stem cells in vitro using microporous coating according to claim 1, wherein the aqueous hydrochloric acid solution of step (4) has a pH = 2.2-2.9 and the soaking time is 30-200 minutes.
4. The method for inducing differentiation of adipose-derived stem cells in vitro using microporous coatings according to claim 1, wherein the negative carrier liquid is:
IBMX 5mmol/L, dexamethasone 10. mu. mol/L and indometacin 2mmol/L, and ethanol as solvent; or load liquid of IBMX 3.75mmol/L, dexamethasone 7.5 mu mol/L and indometacin 1.5mmol/L, wherein the solvent is ethanol or load liquid of IBMX 3mmol/L, dexamethasone 6 mu mol/L and indometacin 1.2mmol/L, and the solvent is ethanol.
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