CN111499899B - Mass production method and application of in-vitro cell culture substrate material polyacrylamide gel film with different hardness - Google Patents
Mass production method and application of in-vitro cell culture substrate material polyacrylamide gel film with different hardness Download PDFInfo
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Abstract
The invention provides a method for manufacturing polyacrylamide gel films of in vitro cell culture substrate materials with different hardness in large scale, which is characterized in that a gel solution is dripped between a hydrophobic glass slide and a hydrophilic surface of a flexible plastic film support to be covalently bonded with PA gel during deposition. Once the PA gel sheet is deposited and permanently fixed on the flexible plastic support, it is possible to handle gels of any thickness or hardness, cut them into any desired shape and size, and widely used in vitro culture for stiffness-dependent cellular reactions. The method not only can produce PA gel films with various sizes in a large scale, but also eliminates the necessity of using PA adhesive solution to pre-treat the glass surface (glass cover glass or holes of expensive glass bottom porous plates), greatly simplifies the research steps and the research time, and simultaneously, the uniform PA gel sheets can be dehydrated and stored for months.
Description
Technical Field
The invention belongs to the technical field of bioengineering hydrogel, and particularly relates to a mass production method and application of in-vitro cell culture substrate material polyacrylamide gel films with different hardness.
Background
At present, most in vitro cell research is carried out on polystyrene rigid Tissue Culture Plates (TCP), the Young modulus of the hydrogel is 1GPa, most tissues in vivo are soft viscoelastic materials, the modulus of the hydrogel ranges from 0.1kPa of brain to 100kPa of osteochondral, more and more researches show that the hardness of a substrate influences the reaction mode of cells to environment, and even changes the fate of the cells, so that the development of hydrogel materials with wide-range rigidity as the substrate for in vitro cell culture is a research hotspot in the technical field of biological engineering. Various hydrogels have been developed, including Polyacrylamide (PA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), and alginate, to help understand the study of stiffness-dependent cells. Although evidence of a significant influence of matrix hardness on cell fate is growing, most of the current hydrogel preparation processes are long and tedious, and are only suitable for small-scale production, so that research samples are limited to a small scale. Even though several high-throughput hydrogel technologies have been developed, including polyethylene glycol PEG-based microarrays, microfluidic devices for producing agarose hydrogel microbeads, or micro-and nanorods with stiffness adjusted by diameter and height, the technology for preparing such substrates is complicated and requires specialized equipment, and is only available in a limited number of laboratories.
Polyacrylamide gel (PA gel) is not only cheap and easy to implement, but also shows a physiologically relevant Young's modulus range, namely 0.3-300kPa, which can cover the hardness range of all soft tissues in vivo, and is the hydrogel of choice in the vast bioengineering research. However, the existing method for preparing the cell culture PA gel is labor-intensive, needs to be independently prepared one by one according to requirements, and is only limited to small-batch preparation. Several groups have been internationally attempting to produce PA gels in large quantities for cell culture. There have been studies attempting to prepare thick sheets of PA gel, "cut" with a punch and placed into 96-well plates, however this method is limited to harder gels, i.e., young's modulus >1kPa, because softer gels are "sticky" and difficult to cut directly and are extremely fragile. There has also been a study to develop a more sophisticated technique that allows the gels to polymerize directly in glass-bottom multi-well plates by pouring the gel solutions into functionalized glass-bottom plates and by "clipping" them with custom-made cover slips, which even though it is currently very promising, still allows a slight edge effect to be observed, i.e. gel formation polymerizes in glass-bottom multi-well plates, which can lead to wrinkles forming when the hydrogel is used with the glass-bottom plate removed, affecting the use. Furthermore, this technique requires custom designed arrays, cannot be directly employed by many laboratories, and requires expensive glass-based multi-well plates, which are still not suitable for large sample studies.
In order to solve the above problems, the present invention provides a method for preparing a polyacrylamide gel film by using a permanent flexible plastic film as a gel structure support, which can prepare and customize various forms of polyacrylamide gel films including multi-well plates into in vitro cell culture substrates with variable hardness. Compared with the existing method, the preparation method is quicker, more effective and lower in cost, allows mass preparation of gel films with required customized sizes, is easy to store and transport after drying, can meet adherent cell growth after reaching hydration balance in use, and can be easily used for preparing in-vitro cell culture substrates in any research laboratory due to no need of any special equipment, so that the preparation method is widely applied to series researches on rigidity-dependent cell reactions.
Disclosure of Invention
The invention aims to provide a method for manufacturing polyacrylamide gel films of in vitro cell culture substrate materials with different hardness in a large scale, which has the advantages of simple, convenient, quick and effective operation, suitability for preparing the required customized size in a large scale, low economic cost, easy storage and transportation of the prepared polyacrylamide gel films after drying, capability of meeting the adherent growth of cells after reaching hydration balance during use, reduction of the complexity of researching cell mechanics and contribution to the relevant research on cell mechanics properties.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mass production method of in vitro cell culture substrate material polyacrylamide gel films with different hardness comprises the following steps:
(1) Preparing an acrylamide solution, a bisacrylamide solution and an ammonium persulfate aqueous solution for later use;
(2) Preparing a hydrophobic glass slide and a hydrophobic flexible plastic film support for later use;
(3) Degassing the acrylamide solution and the bisacrylamide solution, adding an ammonium persulfate aqueous solution and N, N, N ', N' -Tetramethylethylenediamine (TEMED), and mixing to obtain a gel solution;
(4) Taking the hydrophobic glass slide as the bottom, supporting the periphery of the hydrophobic glass slide by a silicon resin spacer, and dripping gel solution; then the hydrophilic side of the flexible plastic film support is laid on the gel solution, a hydrophobic glass slide is covered on the flexible plastic film support, and the flexible plastic film support is dried and polymerized.
Said acrylamide solution 40% w/v; 2% w/v of the bisacrylamide solution; the aqueous ammonium persulfate solution 10% w/v.
The dry polymerization can be room-temperature standing dry polymerization, convection dry polymerization or heating dry polymerization.
After the dry polymerization, the flexible plastic film support is peeled off.
The polyacrylamide gel film is used as an in vitro cell culture substrate material with different hardness.
The application method of the polyacrylamide gel film in-vitro cell culture substrate materials with different hardness comprises the following steps:
(1) Preparing a polyacrylamide gel film into a size suitable for a cell culture dish for later use;
(2) Placing PDMS in a culture dish, then placing a prepared polyacrylamide gel film with the gel surface facing upwards, and standing at room temperature until the polyacrylamide gel film is adhered to the bottom of the culture dish;
(3) The polyacrylamide gel surface is activated, so that cells can grow better in an adhesion manner.
(1) Uniformly coating the surface of a gel film with an aqueous solution of N-sulfosuccinimidyl-6- (4 '-azido-2' -nitrophenylamino) hexanoate Sulfo-SANPAH;
(2) UV lamp (intensity =37mW, λ =302-365 nm) treatment 5-10min, pbs rinsing excess Sulfo-SANPAH;
(3) soaking the gel in the type I collagen solution, and standing for 2 hours at room temperature or 10-15 hours at 4 ℃;
(4) PBS rinsing excess type I collagen solution, UV (λ =200 nm) sterilization, ready for use;
(4) Adding cell culture solution immersion gel into culture dish for hydration equilibration, 10-15h at 4 deg.C, and immediately inoculating cell, or storing at 4 deg.C, and optimally using within 48 h.
The sulfol-SANPAH aqueous solution is 0.35mg/ml.
The type I collagen solution is 0.2mg/ml.
After the gel is immersed by adding the cell culture solution, the gel is immediately used for cell inoculation after being immersed for 10-15h at 4 ℃, or the gel is stored at 4 ℃, and the using effect is optimal when the gel is not more than 48 h.
The N-sulfosuccinimidyl-6- (4 '-azido-2' -nitrophenylamino) hexanoate (Sulfo-SANPAH, M) r 492.40 g/mol), comprising the following steps: dissolving Sulfo-SANPAH in dimethyl sulfoxide (DMSO) at 50mg/ml, dividing equally as required, and reserving at-80 deg.C for later use;
the ammonium persulfate (M) r 228.18 g/mol) comprising the following steps: dissolving ammonium persulfate in deionized water at a concentration of 10% w/v, subpackaging as required, and storing at-20 deg.C for use;
the preparation method of the type I collagen solution comprises the following steps: the collagen stock solution was diluted in PBS to 0.2mg/ml and kept on ice or used immediately.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for manufacturing polyacrylamide gel films of in vitro cell culture substrate materials with different hardness on a large scale, which breaks through the current situations that the polyacrylamide gel films can only be prepared in a small scale in a laboratory and the preparation process is long and complicated; the preparation method is simple and convenient to operate, quick and effective, suitable for mass preparation of the required customized size, and low in economic cost, the prepared polyacrylamide gel film is easy to store and transport after being dried, and the polyacrylamide gel which reaches hydration balance during use can also meet the requirement of cell adherent growth, so that the complexity of researching cell mechanics is reduced, and the relevant research on cell mechanics properties is facilitated.
Drawings
FIG. 1 is a flow chart of polyacrylamide gel films of in vitro cell culture substrate materials of different hardness prepared by the mass production method of the present invention;
FIG. 2 is a diagram and schematic diagram of polyacrylamide gel films of different hardness of in vitro cell culture substrate materials prepared by the mass preparation method of the invention, wherein: a is a schematic diagram of polyacrylamide gel of in vitro cell culture substrate materials with different hardness prepared by the mass preparation method, and B is a diagram of polyacrylamide gel films of in vitro cell culture substrate materials with different hardness prepared by the mass preparation method;
wherein: 1-PDMS PA gel film was fixed to the bottom of the culture dish, 2-collagen layer.
Detailed Description
A mass production method of in vitro cell culture substrate materials with different hardnesses specifically comprises the following steps:
and (I) preparing materials.
1. Preparation of Polyacrylamide gel precursor solution and aliquots
(1) Preparation of a polyacrylamide hydrogel precursor solution: by sequential addition of acrylamide (A) (40% w/v, M) r 71.08 g/mol), bisacrylamide (B) (2%; w/v, M) r 154.17 g/mol) and deionized water, the percentages of the components are exemplified in table 1. These solutions can be prepared in bulk and stored at 4 ℃ for up to several months.
(2) Preparation and use of N-Sulfosuccinimidyl-6- (4 '-azido-2' -nitrophenylamino) hexanoate (Sulfo-SANPAH, M) r 492.40 g/mol) aliquots: sulfo-SANPAH was dissolved at 50mg/ml in dimethyl sulfoxide (DMSO), and the stock solution was divided into 50 microliter (. Mu.l) centrifuge tubes, 20. Mu.l each, snap frozen in dry ice or liquid nitrogen and stored at-80 ℃ for several months.
(3) Preparation of ammonium persulfate (M) r 228.18 g/mol) aliquot of solution: dissolving ammonium persulfate in deionized water to a final ammonium persulfate concentration of 10% w/v, subpackaging and storing at-20 deg.C, and thawing immediately before use.
(4) Preparation of type I collagen solution: a0.2 mg/ml collagen solution was prepared by diluting the collagen stock solution in 1 Xphosphate buffered saline (PBS) at pH 7.4, either temporarily on ice or immediately after dilution.
2. Preparation of hydrophobic glass slides
3-5 drops of hydrophobic solution were dropped onto the glass plate, then wiped with a tissue paper to homogenize the hydrophobic coating, left to air dry.
3. Preparation of flexible plastic film support
(1) 3-5 drops of hydrophobic solution were dropped on one side of a flexible plastic film support, then wiped with a thin paper to homogenize the hydrophobic coating, left to air dry. The flexible plastic film support was then cut to match the dimensions of the previously hydrophobic glass slide.
(2) The surface of the flexible plastic film support is lightly scratched with a sharp instrument (e.g., scalpel) to mark the hydrophobic side, and once completely clear and dry, the polyacrylamide gel permanently adheres to the hydrophilic side of the plastic film support, and the scratch will help to distinguish which flexible plastic film support contains the gel. (II) preparation method of polyacrylamide gel film (brief flow chart shown in figure 1).
Taking a volume of 5ml of the gel precursor solution as an example, 40 polyacrylamide gel films with a thickness of 0.5mm can be prepared.
1. 4972.5. Mu.l of the desired final concentration of polyacrylamide precursor solution (as in Table 1) was placed in a 50ml conical tube, the lid opened, and placed in a degassing chamber to degas for 30 minutes.
2. 25. Mu.l of 10% w/v ammonium persulfate was added (see 1 (3)) to give a final ammonium persulfate concentration of 0.05%.
3. 2.5. Mu.l of N, N, N ', N' -tetramethylethylenediamine (TEMED, M) were added r 116.24 g/mol) to a final TEMED concentration of 0.5%.
4. The pipette tip gently blows the mixed solution up and down for 3 to 5 times without vortex to mix evenly to avoid oxygen from diffusing into the gel solution.
5. A hydrophobic glass slide is used as a bottom support, a silicone resin partition with the required thickness (such as 0.5 mm) is used as a peripheral support at the outer edge of the slide, a proper amount of the prepared gel solution is dripped in the center of the hydrophobic glass slide, then the hydrophilic side of the flexible plastic film support is laid on the gel solution, and finally another hydrophobic glass slide is placed on the flexible plastic film support to ensure that the surface of the polymerized gel is flat and uniform. After standing at room temperature, polymerization of the gel mixture (about 100. Mu.l) remaining in a 50ml conical tube was observed, and the polymerization time was determined, and the polyacrylamide gel was generally completely polymerized for about 45 minutes.
6. After the gel was completely polymerized, the flexible plastic film support was peeled off and the gel polymer was left to dry standing with its upper side at room temperature. Convection or heat drying may also be used to accelerate this step. Once dried onto the flexible plastic film support, the polyacrylamide gel film is stored for long periods of time.
(III) use of polyacrylamide gel film.
Take a 60mm cell culture dish as an example.
1. The polyacrylamide gel film is prepared to 50-55mm by using scissors for standby.
2. Approximately 500. Mu.l of PDMS was placed in the center of each 60mm dish, a thin film of gel was placed in each dish using tweezers, the gel-containing surface was placed facing up, the PDMS was allowed to solidify by standing overnight at room temperature, and the polyacrylamide gel film was adhered to the bottom of the dish.
3. Activating the surface of polyacrylamide gel to ensure that cells grow better in an adhesion manner:
(1) Aliquots of Sulfo-SANPAH were removed and diluted to 0.35mg/ml in water, and approximately 600. Mu.l of each 60mm dish was placed using a pipette and the dishes were slowly shaken to coat the gel surface evenly. Because Sulfo-SANPAH is unstable in water, it should be diluted immediately.
(2) The dishes were placed under a high intensity UV lamp (intensity =37mW, λ =302-365 nm) for 5-10 minutes, and the gel was rinsed with PBS to remove excess Sulfo-SANPAH.
(3) About 3.5ml of 0.2mg/ml type I collagen solution was added to each dish, the gel was completely soaked, and left to stand at room temperature for at least 2 hours or overnight at 4 ℃.
(4) The excess collagen solution was removed by washing with PBS and sterilized in a tissue culture hood under UV (λ =200 nm) for 2 hours for use.
4. Approximately 5ml of culture medium for the desired cells was added per 60mm dish (ensuring that the added culture medium was sufficient to completely submerge the gel), hydrated equilibrated overnight at 4 ℃ and then used immediately for cell seeding or stored at 4 ℃ for optimal use within 48 hours.
5. In conventional cell seeding and collection procedures, special care was taken to avoid damaging the gel surface by gently tilting the dishes laterally and contacting the pipette tips to the side walls of each dish to aspirate or pipette media, rather than contacting the hydrogel.
Experiment for influence of concentration of acrylamide (A) and bisacrylamide (B) on gel stiffness of prepared PA
In the process of preparing the polyacrylamide gel film, the concentrations of acrylamide (a) and bisacrylamide (B) were changed, and the rigidity of the prepared polyacrylamide gel was changed, as shown in table 1.
The rigidity measured by rheology is represented by G' and E. G 'is the storage modulus at a frequency of 1hz, young's modulus E = G '2 (1 + υ), calculated as poisson's ratio of approximately 0.5 with hydrogel. The Standard Deviation (SD) calculation was based on three independent experiments, each measuring 3-5 samples.
TABLE 1 concentration of acrylamide (A) and bisacrylamide (B) and resulting PA gel stiffness
As can be seen from Table 1, by adjusting the proportion of solvents such as acrylamide, bisacrylamide and the like, polyacrylamide gels with different rigidities can be obtained, and the rigidity range can cover most of soft tissues in vivo, so that the polyacrylamide gels can be used as a substrate of in vitro cell culture for related research.
Example 1
A method for mass production of in vitro cell culture substrate materials with different hardness is characterized in that polyacrylamide gel thin films which are used in 10 parts of 60mm culture dishes and have the hardness of Young modulus E =29.14 +/-1.93 are produced.
1. 4972.5. Mu.l of polyacrylamide precursor solution was prepared according to the PA3 ratio in Table 1, placed in a 50ml conical tube, the lid opened, and placed in a degassing chamber for 30 minutes.
2. 25. Mu.l of 10% w/v ammonium persulfate was added (see 1 (3)) to give a final ammonium persulfate concentration of 0.05%.
3. 2.5. Mu.l of N, N, N ', N' -tetramethylethylenediamine (TEMED, M) was added r 116.24 g/mol) to a final TEMED concentration of 0.5%.
4. The pipette tip gently blows the mixed solution up and down for 3 to 5 times without vortex to mix evenly to avoid oxygen from diffusing into the gel solution.
5. Respectively using 10 60mm-60mm hydrophobic glass slides as bottom supports, using 0.5mm silicone resin spacers as peripheral supports at the outer edges of the slides, dripping 450 mu l of the prepared gel solution into the centers of the hydrophobic glass slides, respectively paving hydrophilic sides of the flexible plastic film supports with the same size on the gel solution, and finally placing the other hydrophobic glass slide on the flexible plastic film supports to ensure that the surfaces of the polymerized hydrogel are flat and uniform. The mixture was allowed to stand at room temperature and polymerized for about 45 minutes.
6. After the gel was completely polymerized, the flexible plastic film support was peeled off and the hydrogel polymer was left to dry overnight, standing with the side up to room temperature.
7. The polyacrylamide gel film was prepared to a circular size of 50mm in diameter using scissors.
8. About 500. Mu.l of PDMS was placed in the center of each 60mm dish, one circular thin film of gel was placed in each dish using tweezers, the gel-containing face was placed upward, PDMS was cured by standing overnight at room temperature, and the thin film of polyacrylamide gel was adhered to the bottom of the dish.
9. About 600 μ l of Sulfo-SANPAH at a concentration of 0.35mg/ml was placed in each 60mm petri dish, followed by 10 minutes under a high intensity UV lamp (intensity =37mW, λ =302-365 nm), water gel was washed with PBS to remove excess Sulfo-SANPAH, about 3.5ml of collagen type I solution at a concentration of 0.2mg/ml was added to each petri dish, the gel was completely soaked, left to stand at room temperature for at least 2 hours (or overnight at 4 ℃), washed with PBS to remove excess collagen solution, and sterilized in a tissue culture hood under UV (λ =200 nm) for 2 hours.
10. 5ml of the desired cell culture broth (to ensure that the broth added is sufficient to completely submerge the gel) was added per 60mm dish, equilibrated by hydration overnight at 4 ℃, placed in an incubator for 30 minutes and used for cell culture (stored at 4 ℃ for up to 48 hours).
FIG. 2 is a diagram and a schematic diagram of polyacrylamide gel films of different hardness of in vitro cell culture substrate materials prepared by the mass production method of this embodiment. From fig. 2 (a), which is a comparison of MDA-MB-231 breast cancer cell line seeded on 100kPa PA gel (top right panel) and directly seeded on petri dish (bottom right panel TCP), the edge image clearly shows the extent of coverage of the bottom of the dish by PA gel-flexible material. As can be seen from FIG. 2, the polyacrylamide gel film prepared by the invention has no edge effect, can be cut into a shape and a size according to needs, can be produced in large scale and stored at normal temperature, and is convenient to use.
Example 2
A method for mass production of in vitro cell culture substrate materials with different hardness is used for manufacturing 10 parts of polyacrylamide gel thin films which are used in 60mm culture dishes and have the hardness of Young modulus E =1.85 +/-0.57.
1. 4972.5. Mu.l of polyacrylamide precursor solution was prepared according to the PA1 ratio in Table 1, placed in a 50ml conical tube, the lid opened, and placed in a degassing chamber for 30 minutes.
2. 25. Mu.l of 10% w/v ammonium persulfate was added (see 1 (3)) to give a final ammonium persulfate concentration of 0.05%.
3. 2.5. Mu.l of N, N, N ', N' -tetramethylethylenediamine (TEMED, M) were added r 116.24 g/mol) to a final TEMED concentration of 0.5%.
4. The pipette tip gently blows the mixed solution up and down for 3 to 5 times without vortex to mix evenly to avoid oxygen from diffusing into the gel solution.
5. Respectively using 10 60mm-60mm hydrophobic glass slides as bottom supports, using 0.5mm silicone resin spacers as peripheral supports at the outer edges of the slides, dripping 450 mul of the prepared gel solution into the centers of the hydrophobic glass slides, respectively spreading hydrophilic sides of the flexible plastic film supports with the same size on the gel solution, and finally placing the other hydrophobic glass slide on the flexible plastic film supports to ensure that the surfaces of the polymerized hydrogel are flat and uniform. The mixture was allowed to stand at room temperature and polymerized for about 40 minutes.
6. After the gel was fully polymerized, the flexible plastic film support was peeled off and the hydrogel polymer side was left to dry overnight at room temperature.
7. The polyacrylamide gel film was prepared to a circular size of 50mm in diameter using scissors.
8. Approximately 500. Mu.l of PDMS was placed in the center of each 60mm dish, a circular thin film of gel was placed in each dish using tweezers, the gel-containing surface was placed facing up, the PDMS was allowed to solidify by standing overnight at room temperature, and the polyacrylamide gel film was adhered to the bottom of the dish.
9. About 600 μ l of Sulfo-SANPAH at a concentration of 0.35mg/ml was placed in each 60mm petri dish, followed by a high intensity UV lamp (intensity =37mW, λ =302-365 nm) for 10 minutes, the water gel was washed with PBS to remove excess Sulfo-SANPAH, about 3.5ml of collagen type I solution at a concentration of 0.2mg/ml was added to each petri dish, the gel was completely soaked, left to stand at room temperature for at least 2 hours (or overnight at 4 ℃), washed with PBS to remove excess collagen solution, and sterilized in a tissue culture hood under UV (λ =200 nm) for 2 hours.
10. 5ml of the desired cell culture broth was added per 60mm dish (ensuring that the broth added was sufficient to completely submerge the gel), equilibrated by hydration overnight at 4 ℃ and placed in an incubator for 30 minutes before being used for cell culture (4 ℃ for up to 48 hours).
Example 3
A mass production method of in vitro cell culture substrate materials with different hardness is characterized in that polyacrylamide gel films which are used in 10 parts of 60mm culture dishes and have the Young modulus of 112.25 +/-8.03 are manufactured.
1. 4972.5. Mu.l of polyacrylamide precursor solution was prepared according to the PA5 ratio in Table 1, placed in a 50ml conical tube, the lid opened, and placed in a degassing chamber for 30 minutes.
2. 25. Mu.l of 10% w/v ammonium persulfate was added (see 1 (3)) to give a final ammonium persulfate concentration of 0.05%.
3. 2.5. Mu.l of N, N, N ', N' -tetramethylethylenediamine (TEMED, mr 116.24 g/mol) were added to bring the final TEMED concentration to 0.5%.
4. The pipette tip gently blows the mixed solution up and down for 3 to 5 times without vortex to mix evenly to avoid oxygen from diffusing into the gel solution.
5. Respectively using 10 60mm-60mm hydrophobic glass slides as bottom supports, using 0.5mm silicone resin spacers as peripheral supports at the outer edges of the slides, dripping 450 mu l of the prepared gel solution into the centers of the hydrophobic glass slides, respectively paving hydrophilic sides of the flexible plastic film supports with the same size on the gel solution, and finally placing the other hydrophobic glass slide on the flexible plastic film supports to ensure that the surfaces of the polymerized hydrogel are flat and uniform. The mixture was allowed to stand at room temperature and polymerized for about 40 minutes.
6. After the gel was fully polymerized, the flexible plastic film support was peeled off and the hydrogel polymer side was left to dry overnight at room temperature.
7. The polyacrylamide gel film was prepared to a circular size of 50mm in diameter using scissors.
8. Approximately 500. Mu.l of PDMS was placed in the center of each 60mm dish, a circular thin film of gel was placed in each dish using tweezers, the gel-containing surface was placed facing up, the PDMS was allowed to solidify by standing overnight at room temperature, and the polyacrylamide gel film was adhered to the bottom of the dish.
9. About 600 μ l of Sulfo-SANPAH at a concentration of 0.35mg/ml was placed in each 60mm petri dish, followed by 10 minutes under a high intensity UV lamp (intensity =37mW, λ =302-365 nm), water gel was washed with PBS to remove excess Sulfo-SANPAH, about 3.5ml of collagen type I solution at a concentration of 0.2mg/ml was added to each petri dish, the gel was completely soaked, left to stand at room temperature for at least 2 hours (overnight at 4 ℃), washed with PBS to remove excess collagen solution, and sterilized in a tissue culture hood under UV (λ =200 nm) for 2 hours.
10. 5ml of the desired cell culture broth was added per 60mm dish (ensuring that the added broth was sufficient to completely submerge the gel), equilibrated by hydration overnight at 4 ℃, placed in an incubator for 30 minutes and used for cell culture (4 ℃ for up to 2 days).
Claims (7)
1. A mass production method of in vitro cell culture substrate material polyacrylamide gel films with different hardnesses is characterized by comprising the following steps:
(1) Preparing an acrylamide solution, a bisacrylamide solution and an ammonium persulfate aqueous solution for later use;
(2) Preparing a hydrophobic glass slide and a hydrophobic flexible plastic film support for later use;
(3) Degassing the acrylamide solution and the bisacrylamide solution, adding an ammonium persulfate aqueous solution and N, N, N ', N' -tetramethyl ethylenediamine, and mixing to obtain a gel solution;
(4) Taking the hydrophobic glass slide as the bottom, supporting the periphery of the hydrophobic glass slide by a silicon resin spacer, and dripping a gel solution; then, the hydrophilic surface of the flexible plastic film support is paved on the gel solution, and the hydrophobic glass slide is covered on the flexible plastic film support, dried and polymerized.
2. The method for mass-producing polyacrylamide gel films as substrate materials for in vitro cell culture with different hardness according to claim 1, wherein the concentration of the acrylamide solution is 40% w/v; the concentration of the bisacrylamide solution is 2%; the concentration of the aqueous ammonium persulfate solution was 10% by weight w/v.
3. The method for mass production of in vitro cell culture substrate material polyacrylamide gel film with different hardness according to claim 1, wherein the dry polymerization is room temperature static dry polymerization, convection dry polymerization or heating dry polymerization.
4. The method for mass-producing polyacrylamide gel films of different hardness as claimed in claim 1, wherein the flexible plastic film support is peeled off after the drying polymerization.
5. The mass production method of the polyacrylamide gel films with different hardness as claimed in claim 1, wherein the method for using the polyacrylamide gel films in the in vitro cell culture substrate materials with different hardness comprises the following steps:
(1) Preparing a polyacrylamide gel film into a size suitable for a cell culture dish for later use;
(2) Placing PDMS in a culture dish, then placing a prepared polyacrylamide gel film with the gel surface facing upwards, and standing at room temperature until the polyacrylamide gel film is adhered to the bottom of the culture dish;
(3) Activating the surface of polyacrylamide gel to ensure that cells can better adhere and grow;
(1) uniformly coating the surface of the gel film with a Sulfo-SANPAH aqueous solution;
(2) treating with UV lamp with intensity of 37mW and lambda of 302-365nm for 5-10min, and washing excessive Sulfo-SANPAH with PBS;
(3) soaking the gel in the type I collagen solution, and standing for 2 hours at room temperature or 10-15 hours at 4 ℃;
(4) washing excessive type I collagen solution with PBS, and sterilizing under UV with lambda of 200nm for later use;
(4) Adding cell culture solution immersion gel into culture dish for hydration equilibration, 10-15h at 4 deg.C, and immediately inoculating cells, or storing at 4 deg.C for use within 48 h.
6. The method for mass-producing in vitro cell culture substrate material polyacrylamide gel films with different hardness according to claim 5, wherein the aqueous solution of Sulfo-SANPAH is 0.35mg/ml.
7. The method for mass production of in vitro cell culture substrate material polyacrylamide gel films with different hardness according to claim 5, wherein the type I collagen solution is 0.2mg/ml.
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