CN115992050A - Controllable rigidity modulus culture dish and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of culture dish preparation, and provides a controllable rigidity modulus culture dish, and a preparation method and application thereof. The preparation method of the culture dish with the controllable rigidity modulus comprises the following steps: (1) Mixing PDMS monomer with a cross-linking agent, and vacuumizing and defoaming to obtain a PDMS solution; (2) And (3) coating the culture dish with the PDMS solution obtained in the step (1), sterilizing and standing to obtain the culture dish with the controllable rigidity modulus. The controllable rigidity modulus culture dish obtained by the preparation method can be used for 3D organoid culture, cell culture and tissue culture. The method integrates the microfluid technology into the cell culture, replaces the expensive photoetching process, has extremely strong plasticity, can customize different rigidity moduli and culture modes, meets the 2D/3D conversion and culture requirements of cells and tissues, promotes the application and progress of stem cell conversion medicine, and has extremely great application value and popularization significance.
Description
Technical Field
The invention relates to the technical field of culture dish preparation, in particular to a controllable rigidity modulus culture dish, and a preparation method and application thereof.
Background
Cells are the basic units constituting higher organisms, and constitute tissues and further organs from various cells having different functions such as cardiomyocytes, nerve cells, blood cells, and the like. These cells grow in different tissues and form different microenvironments, one special feature being that these microenvironments have different tissue moduli of rigidity, e.g. the brain tissue has a low tissue modulus of rigidity, is the most soft and fragile, but the skull has the highest tissue modulus of rigidity, which protects the soft brain tissue. The rigidity modulus of the tissue microenvironment plays an important role in the dynamic growth and functional maintenance of cells, and various functional cells adhere to the tissue microenvironment, perform cell differentiation and function, and perform self-renewal and tissue metabolism.
Cell culture is the most important fundamental technology in life development research, biomedical development and medical tissue engineering. Typically we will culture cells in dishes, which may be glass, plastic, even textile materials and cellulose membranes. Typically, cell culture dishes made of these materials have only a single modulus of rigidity, so different materials are required for different cells requiring different moduli of rigidity. For example, mesenchymal stem cells grow on a surface with a high modulus of rigidity to facilitate differentiation into functional tissues, whereas mesenchymal stem cells tend to remain in a dry and undifferentiated state on the surface of a material with a low modulus of rigidity. Also, embryonic pluripotent stem cells tend to spontaneously differentiate into fibroblasts at surfaces with high modulus of rigidity, but grow at surfaces with low modulus of rigidity in favor of maintaining an undifferentiated state and high proliferation. Therefore, scientists have developed dishes made of materials with different moduli of rigidity to meet the growth and function execution needs of different cells. However, both cellulose membranes and dishes made of textile materials have the disadvantage that the modulus of rigidity is fixed and cannot be custom-adjusted, and replacement of the material increases costs and is not suitable for cell culture observation and identification. Therefore, for scientific research and industrial transformation, a simple and general material is needed as a material for cell and tissue culture, and an essential important feature of the material is that dynamic adjustment of rigidity modulus can be realized so as to meet the culture requirement of different tissue cells.
The chemical monomer of polydimethylsiloxane PDMS is dimethylsiloxane, which presents oily liquid at normal temperature, has extremely high viscosity, is not easy to flow, but can be horizontally spread on any vessel surface. The glass fiber reinforced plastic composite material is nontoxic and odorless, has high transparency, is heat-resistant and cold-resistant, can be used for a long time at the temperature of between 50 ℃ below zero and 200 ℃, has small viscosity change along with the temperature, has thermal conductivity, and has the thermal conductivity coefficient of between 0.134 and 0.159W/(m.K) and the light transmittance of 100 percent. In addition, PDMS surface is hydrophobic, has excellent waterproof property, physiological inertia and good chemical stability. In addition, the micro-fluidic chip has high shearing resistance and can maintain the shape unchanged for a long time, so that the micro-fluidic chip is used as an important material for manufacturing micro-fluidic chips, and the manufactured micro-fluidic chips are further used for cell culture and property analysis. The preparation of microfluidic chips generally requires the addition of a crosslinking reagent to polymerize the PDMS chemical monomer into polydimethylsiloxane, and in the preparation of microfluidic chips, the ratio of PDMS chemical monomer to crosslinking reagent is generally set to 10:1 in order to ensure a certain stability against deformation of the prepared chips. In addition, the PDMS chip fabrication process also requires the use of hundred-level dust-free workshops and photolithography equipment, which are difficult to burden and operate in general biological laboratories and hospital units, so that the preparation of PDMS-based microfluidic chips is an expensive and complex procedure, and also limits its wide application.
Disclosure of Invention
The invention aims to provide a controllable rigidity modulus culture dish, a preparation method and application thereof, and the rigidity modulus and the surface hydrophobicity of polydimethylsiloxane are controlled by adjusting the proportion of PDMS monomers and cross-linking agents, so that the culture dish can be used for 3D organoid culture, cell culture, tissue culture and cell proliferation and migration observation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a controllable rigidity modulus culture dish, which comprises the following steps:
(1) Mixing PDMS monomer with a cross-linking agent, and vacuumizing and defoaming to obtain a PDMS solution;
(2) And (3) coating the culture dish with the PDMS solution obtained in the step (1), sterilizing and standing to obtain the culture dish with the controllable rigidity modulus.
Preferably, the ratio of the PDMS monomer to the crosslinker in step (1) is determined according to the cell culture mode:
when 3D cell culture is carried out, the ratio of PDMS monomer to cross-linking agent is 5:1-10:1;
when 2D cell culture is performed, the ratio of PDMS monomer to cross-linking agent is 10:1-30:1.
Preferably, the vacuumizing time in the step (1) is 10-15 min, and the vacuumizing pressure is 0.5-1 MPa.
Preferably, the temperature of the coating in the step (2) is 0-5 ℃, and the coating time is 20-40 min.
Preferably, the temperature of the sterilization in the step (2) is 65-75 ℃, and the time of the sterilization is 2-4 hours.
Preferably, the time of the standing in the step (2) is 12 to 24 hours.
Preferably, when the controllable rigidity modulus culture dish is used for 2D cell culture, the method further comprises the step of modifying the obtained controllable rigidity modulus culture dish, wherein the modification method is to coat the PDMS surface with a modifying substance;
the modifying substance comprises one or more of gelatin, collagen or serum.
Preferably, when the culture chamber is customized for culturing different cells by using the controllable rigidity modulus culture dish, the thickness of the PDMS colloid coated in the culture dish is more than 5mm, and the PDMS colloid is perforated, so that the perforated bulk PDMS colloid is used as the culture chamber for culturing different cells.
The invention also provides the culture dish with controllable rigidity modulus, which is prepared by the preparation method.
The invention also provides application of the controllable rigidity modulus culture dish in cell culture.
Compared with the prior art, the invention has the beneficial effects that:
the invention integrates PDMS for manufacturing the micro-fluid chip into the field of cell and tissue culture, and meets different requirements of preparing 3D organoids, cell and tissue adherent culture through simple formula adjustment, aseptic treatment, coating and surface modification. In addition, the method of the invention does not need any photoetching equipment and ionization treatment in the whole cell and tissue culture process, has simple procedure and easy operation, can customize different culture chambers in the same culture dish, realizes the co-growth of a plurality of cells in the same culture dish, can form clones with controllable size and clear edges, is convenient for measuring the proliferation performance and the cell interaction migration of the cell clones, and has great scientific research and application value.
Drawings
FIG. 1 is a mesenchymal stem cell pellet (organoid, scale 200 μm) formed by culturing rat bone marrow mesenchymal stem cells using the petri dish of the present invention;
FIG. 2 shows the adherent growth of MCF-7 on PDMS surface (scale 100 μm) of human breast cancer cells cultured in the petri dishes according to the present invention;
FIG. 3 is a clone of rat bone marrow mesenchymal stem cells revealed after removal of PDMS gel at the bottom of the culture dish.
Detailed Description
The invention provides a preparation method of a controllable rigidity modulus culture dish, which comprises the following steps:
(1) Mixing PDMS monomer with a cross-linking agent, and vacuumizing and defoaming to obtain a PDMS solution;
(2) And (3) coating the culture dish with the PDMS solution obtained in the step (1), sterilizing and standing to obtain the culture dish with the controllable rigidity modulus.
In the invention, PDMS monomer and cross-linking agent are mixed first, and then the PDMS solution is obtained after vacuumizing and defoaming.
In the invention, the ratio of the PDMS monomer to the cross-linking agent is determined according to the cell culture mode: when 3D cell culture is performed, the ratio of PDMS monomer to cross-linking agent is 5:1-10:1, preferably 6:1-9:1, and more preferably 7:1-8:1; when 2D cell culture is performed, the ratio of PDMS monomer to cross-linking agent is 10:1 to 30:1, preferably 15:1 to 25:1, and more preferably 18:1 to 23:1.
In the invention, the vacuumizing time is 10-15 min, preferably 11-14 min, and more preferably 12-13 min; the pressure at the time of vacuum pumping is 0.5 to 1MPa, preferably 0.66 to 0.9MPa, and more preferably 0.7 to 0.8MPa. In the invention, the vacuumizing function is to eliminate bubbles generated in the mixing process of the PDMS monomer and the cross-linking agent.
In the invention, the PDMS solution is used for coating the culture dish, and the culture dish is subjected to sterilization and then is placed still to obtain the culture dish with controllable rigidity modulus; the amount of the PDMS solution is determined according to the cell culture mode, and when 3D cell culture and 2D cell culture are performed, the amount of the PDMS solution is 0.3 to 0.8mL, preferably 0.4 to 0.7mL, and more preferably 0.5 to 0.6mL; when the custom culture chamber is used for culturing different cells, the PDMS solution is used in an amount of 1.3 to 1.8mL, preferably 1.4 to 1.7mL, and more preferably 1.5 to 1.6mL; the dish may also be replaced with a culture plate.
In the present invention, the temperature of the coating is 0 to 5 ℃, preferably 1 to 4 ℃, and more preferably 2 to 3 ℃; the coating time is 20 to 40 minutes, preferably 25 to 35 minutes, and more preferably 28 to 32 minutes. In the invention, the PDMS liquid after coating can naturally spread to cover the bottom of the culture dish under the action of gravity to form a liquid membranous structure.
In the present invention, the sterilization temperature is 65 to 75 ℃, preferably 67 to 73 ℃, and more preferably 69 to 71 ℃; the sterilization time is 2-4 hours, preferably 2.5-3.5 hours, and more preferably 3 hours; the sterilization is performed in an oven. According to the invention, the sterilization can consume the cross-linking agent and fully cross-link the PDMS monomer to form stable PDMS without deformation, and the dry heat sterilization can perform aseptic treatment on the culture dish, so that the safety of subsequent cell and tissue culture is ensured.
In the present invention, the time for the standing is 12 to 24 hours, preferably 15 to 20 hours, and more preferably 16 to 18 hours; the standing is performed in a bio-clean bench.
In the invention, when the controllable rigidity modulus culture dish is used for 2D cell culture, the method further comprises the step of modifying the obtained controllable rigidity modulus culture dish, wherein the modification method is to coat the PDMS surface with a modifying substance; the modifying substance preferably comprises one or more of gelatin, collagen or serum; the coating step is as follows: covering the surface of PDMS with the modified substance, standing in a 37 ℃ incubator for 30min, or standing in a 4 ℃ refrigerator for 12h, and removing the modified substance after standing.
In the present invention, when the culture chamber is customized for culturing different cells using the controllable modulus of rigidity culture dish, the thickness of the PDMS gel coated in the culture dish is more than 5mm, preferably 6 to 10mm, and more preferably 7 to 8mm; punching on the PDMS colloid, wherein the diameter of the hole is 2-5 mm, preferably 2.5-4.5 mm, and more preferably 3-4 mm; the block PDMS colloid formed by punching is used as a culture chamber for culturing different cells.
The invention also provides the culture dish with controllable rigidity modulus, which is prepared by the preparation method.
The invention also provides application of the controllable rigidity modulus culture dish in cell culture; the cell culture includes 3D cell culture, 2D cell culture and culture of different cell custom culture chambers; such applications include cell cloning, cell proliferation and cell interaction migration.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a preparation method of a controllable rigidity modulus culture dish for 3D suspension culture, which comprises the following steps:
uniformly mixing PDMS monomer and cross-linking agent according to the weight ratio of 6:1, and then placing the mixture in a vacuum pumping device with the pressure of 0.66MPa for bubble removal for 15min to obtain PDMS solution; sucking 4mL of PDMS solution through a 5mL syringe, adding 0.5mL of the PDMS solution into each hole of a 6-hole culture plate, uniformly wiping the PDMS solution by using a cell scraper, placing the culture plate in a refrigerator at 4 ℃ for 30min, taking the culture plate out, placing the culture plate in an oven at 70 ℃ for crosslinking and dry heat sterilization for 3h, taking the culture plate out, and placing the culture plate in an ultra-clean workbench for standing for 12h for later use.
Example 2
The embodiment provides a preparation method of a controllable rigidity modulus culture dish for 2D adherence culture, which comprises the following steps:
uniformly mixing PDMS monomer and cross-linking agent according to the weight ratio of 25:1, placing the mixture in a vacuumizing device with the pressure of 0.66MPa for bubble removal for 15min, sucking 4mL of the bubble-removed PDMS liquid by a 5mL syringe, adding 0.5mL of the bubble-removed PDMS liquid into each hole of a 6-hole culture plate, uniformly wiping the mixture by using cell scraping, placing the mixture in a refrigerator with the temperature of 4 ℃ for 25min, taking the mixture out, placing the mixture in an oven with the temperature of 75 ℃ for cross-linking and dry heat sterilization for 2h, taking the mixture out, placing the mixture in an ultra-clean workbench, and standing the mixture for 15h.
Covering 0.1% gelatin on the surface of PDMS for modification, placing in a 37 ℃ incubator for standing for 30min, and removing 0.1% gelatin solution to obtain a modified culture dish suitable for cell inoculation culture.
Example 3
The present embodiment provides a method for preparing a controlled modulus of rigidity culture dish for customizing a culture chamber to culture different cells, comprising the steps of:
uniformly mixing PDMS monomer and cross-linking agent according to the weight ratio of 10:1, placing the mixture into a vacuumizing device with the pressure of 0.66MPa for removing bubbles for 10min, sucking 4mL of the bubble-removed PDMS liquid by a 10mL syringe, adding 1.5mL of the bubble-removed PDMS liquid into each hole of a 6-hole culture plate, uniformly wiping the PDMS monomer by using a cell scraper, placing the mixture into a refrigerator with the temperature of 4 ℃ for 30min, taking the mixture out, placing the mixture into a baking oven with the temperature of 65 ℃ for cross-linking and dry heat sterilization for 4h, taking the mixture out, placing the mixture into an ultra-clean workbench, and standing the mixture for 24h for later use.
And marking the position of a culture room needing to be perforated by using a marker pen at the bottom of the culture plate, perforating the PDMS glue by using a disposable sterile biological sampler of 5mm, taking out the PDMS glue block caused by perforation by using sterile fine tweezers, and then irradiating the PDMS glue block in an ultra-clean workbench for 10 minutes by using ultraviolet rays for later use.
Experimental example 1
The experimental example adopts the culture dish prepared in the embodiment 1 to carry out 3D suspension culture on the mesenchymal stem cells of the rat, and the steps are as follows:
1. the mesenchymal stem cells of rat bone marrow (supplied by Shenzhen second people hospital orthopedics laboratory) were resuscitated and cultured in the 6-well plate obtained in example 1 according to the conventional thawing method, 2mL of medium was added to each well, 1 Xdiab was added to the medium, and the cell density of each well was 5X 10 5 cells/mL;
2. placing a 6-hole plate loaded with rat bone marrow mesenchymal stem cells into an incubator with 37 ℃ and 5% carbon dioxide and 95% humidity for culturing for 36 hours;
3. after 36h of culture, the culture plate is taken out, so that mesenchymal stem cells of the rat bone marrow can be formed into mesenchymal stem cell spheres (organoids, see figure 1), the mesenchymal stem cell spheres are collected and centrifuged to be applied to directional induction differentiation experiments of adipogenesis, cartilage and bones.
Experimental example 2
The experimental example adopts the culture dish prepared in the embodiment 2 to carry out 2D adherence culture on human breast cancer cells MCF-7, and the steps are as follows:
1. resuscitating human breast cancer cells MCF-7 (purchased from China center for type culture Collection) by conventional thawing method, culturing in 6-well plate obtained in example 2, 2mL of culture medium per well, adding 1 Xdiab to the culture medium, and cell density per well being 1×10 5 cells/mL;
2. placing a 6-hole plate loaded with human breast cancer cells MCF-7 into an incubator with 37 ℃ and 5% carbon dioxide and 95% humidity for culturing for 36 hours;
3. after 12h of incubation, the plates were removed and the human breast cancer cells MCF-7 were seen to grow adherent (see FIG. 2).
Experimental example 3
The experimental example adopts the culture chamber prepared in the embodiment 3 to culture the rat bone marrow mesenchymal stem cells, and the cell migration condition is observed, and the steps are as follows:
1. thawing rat bone marrow mesenchymal stem cells (supplied by Shenzhen second people hospital orthopedics laboratory) by conventional thawing method, and re-suspending in fresh culture medium, wherein 20% FBS,1% diabody and 1% NEAA are added to adjust cell density to 2×10 5 cells/mL, 2mL of cell suspension was injected into each well;
2. placing a culture plate loaded with rat bone marrow mesenchymal stem cells into an incubator with 37 ℃ temperature, 5% carbon dioxide and 95% humidity for culturing for 1h;
3. observing the adherence of the mesenchymal stem cells of the rat under a microscope, and then adhering the mesenchymal stem cells to form clones in a culture room, wherein other cells which are not adhered to the wall are removed together with cell sap;
4. rat bone marrow mesenchymal stem cells were washed 2 times using 1 x DPBS;
5. PDMS was removed using sterile, fine forceps, leaving uniform rat bone marrow mesenchymal stem cell clones (see fig. 3);
6. the migration of mesenchymal stem cells from the clones was observed by adding fresh medium.
From the above examples, the invention provides a controllable modulus of rigidity culture dish, a preparation method and application thereof, and the invention controls the modulus of rigidity and surface hydrophobicity of polydimethylsiloxane by adjusting the proportion of PDMS monomer and cross-linking agent, and can be used for 3D organoid culture, cell culture, tissue culture and cell proliferation and migration observation.
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 (10)
1. The preparation method of the culture dish with the controllable rigidity modulus is characterized by comprising the following steps of:
(1) Mixing PDMS monomer with a cross-linking agent, and vacuumizing and defoaming to obtain a PDMS solution;
(2) And (3) coating the culture dish with the PDMS solution obtained in the step (1), sterilizing and standing to obtain the culture dish with the controllable rigidity modulus.
2. The method of claim 1, wherein the weight ratio of PDMS monomer to crosslinker in step (1) is determined according to the cell culture method:
when 3D cell culture is carried out, the ratio of PDMS monomer to cross-linking agent is 5:1-10:1;
when 2D cell culture is performed, the ratio of PDMS monomer to cross-linking agent is 10:1-30:1.
3. The method according to claim 2, wherein the time of the vacuum in the step (1) is 10 to 15 minutes, and the pressure of the vacuum is 0.5 to 1MPa.
4. The method according to claim 1, wherein the coating temperature in the step (2) is 0 to 5 ℃ and the coating time is 20 to 40min.
5. The method according to claim 1, wherein the sterilization temperature in the step (2) is 65 to 75 ℃, and the sterilization time is 2 to 4 hours.
6. The method according to claim 1, wherein the time of the standing in the step (2) is 12 to 24 hours.
7. The method of claim 1, further comprising the step of modifying the resulting controlled modulus of rigidity culture dish when 2D cell culture is performed using the controlled modulus of rigidity culture dish, the modification being coating the PDMS surface with a modifying substance;
the modifying substance comprises one or more of gelatin, collagen and serum.
8. The preparation method according to claim 1, wherein when the culture chamber is customized for culturing different cells using the controllable modulus of rigidity culture dish, the thickness of the PDMS colloid coated in the culture dish is greater than 5mm, and the PDMS colloid is perforated, and the perforated block-shaped PDMS colloid is used as the culture chamber for culturing different cells.
9. A controllable modulus of rigidity culture dish prepared by the method of any one of claims 1 to 8.
10. Use of a controlled modulus of rigidity culture dish according to claim 9 in cell culture.
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Citations (12)
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