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Large-batch manufacturing method and application of polyacrylamide gel films of in-vitro cell culture substrate materials with different hardness

A polyacrylamide gel, in vitro cell culture technology, used in general culture methods, cell culture supports/coatings, tissue culture, etc., can solve problems such as being unsuitable for large sample research, difficult to directly cut, and edge wrinkles. , to achieve the effect of easy storage and transportation, simple operation and reduced complexity

Active Publication Date: 2020-08-07
辽宁省肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Attempts have been made to prepare thick PA gel sheets, "cut" with a hole punch and placed into 96-well plates, however this method is limited to harder gels, i.e. Young's modulus > Gels are "sticky", difficult to cut directly, and easily damaged
A more sophisticated technique has also been developed to allow gels to polymerize directly in glass-bottomed multiwell plates by pouring gel solutions into functionalized glass-bottomed plates and by "clamping" them with custom-made coverslips, Even though it is currently very promising, this technique can still observe a slight edge effect, that is, the gel formation aggregates in the glass bottom multiwell plate, and when the glass bottom plate is removed to use the hydrogel, the edge will form wrinkles, which affects the use.
In addition, the technique requires custom-designed arrays, which many laboratories cannot directly adopt, and requires expensive glass-bottom multiwell plates, which are still not suitable for large-sample studies

Method used

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  • Large-batch manufacturing method and application of polyacrylamide gel films of in-vitro cell culture substrate materials with different hardness
  • Large-batch manufacturing method and application of polyacrylamide gel films of in-vitro cell culture substrate materials with different hardness
  • Large-batch manufacturing method and application of polyacrylamide gel films of in-vitro cell culture substrate materials with different hardness

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Experimental program
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preparation example Construction

[0042] 2. Preparation of Hydrophobic Glass Slides

[0043] Put 3-5 drops of hydrophobic solution on the glass plate, then wipe with tissue paper to make the hydrophobic coating even, let it air dry.

[0044] 3. Preparation of Flexible Plastic Film Supports

[0045] (1) Drop 3-5 drops of hydrophobic solution on one side of the flexible plastic film support, then wipe it with tissue paper to make the hydrophobic coating uniform, and let it air dry. The flexible plastic film support was then cut to match the size of the previous hydrophobic glass slide.

[0046] (2) Gently scrape the surface of the flexible plastic film support with a sharp tool (such as a scalpel) to mark the hydrophobic side, and once completely transparent and dry, the polyacrylamide gel permanently adheres to the hydrophilic side of the plastic film support , the scratches will help distinguish which side of the flexible plastic film support contains the gel. (2) Preparation method of polyacrylamide gel fi...

Embodiment 1

[0072] A method for mass production of in vitro cell culture base materials with different hardnesses. Ten copies of polyacrylamide gel films with a hardness of Young's modulus E=29.14±1.93 for use in 60mm petri dishes are produced.

[0073] 1. Prepare 4972.5 μl of polyacrylamide precursor solution according to the ratio of PA3 in Table 1, put it into a 50ml conical tube, open the lid, and put it in a degassing chamber for degassing for 30 minutes.

[0074] 2. Add 25 µl of 10% w / v ammonium persulfate (see 1(3)) to make a final ammonium persulfate concentration of 0.05%.

[0075] 3. Add 2.5 μl N, N, N', N'-tetramethylethylenediamine (TEMED, M r 116.24 g / mol), so that the final TEMED concentration reached 0.5%.

[0076] 4. Gently pipette the mixed solution up and down for 3 to 5 times, do not vortex and shake well to avoid oxygen diffusion into the gel solution.

[0077] 5. Use 10 60mm*60mm hydrophobic glass slides as bottom supports, and use 0.5mm silicone resin spacers as s...

Embodiment 2

[0085] A method for mass production of in vitro cell culture base materials with different hardnesses. Ten copies of polyacrylamide gel films with a hardness of Young's modulus E=1.85±0.57 are prepared for 60mm petri dishes.

[0086]1. Prepare 4972.5 μl of polyacrylamide precursor solution according to the ratio of PA1 in Table 1, put it into a 50ml conical tube, open the lid, and put it in a degassing chamber for 30 minutes to degas.

[0087] 2. Add 25 µl of 10% w / v ammonium persulfate (see 1(3)) to make a final ammonium persulfate concentration of 0.05%.

[0088] 3. Add 2.5 μl N, N, N', N'-tetramethylethylenediamine (TEMED, M r 116.24 g / mol), so that the final TEMED concentration reached 0.5%.

[0089] 4. Gently pipette the mixed solution up and down for 3 to 5 times, do not vortex and shake well to avoid oxygen diffusion into the gel solution.

[0090] 5. Use 10 60mm*60mm hydrophobic glass slides as bottom supports, and use 0.5mm silicone resin spacers as support around ...

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Abstract

The invention provides a large-batch manufacturing method of polyacrylamide gel films of in-vitro cell culture substrate materials with different hardness, which is characterized in that a gel solution is dropwise added between a hydrophobic glass slide and a hydrophilic surface of a flexible plastic film support, so that the gel solution is covalently combined with PA gel during deposition. Oncethe PA gel sheet is deposited and permanently fixed on the flexible plastic support, gel with any thickness or hardness can be treated and cut into any required shape and size, and the invention is widely applied to in-vitro culture of rigidity-dependent cell reaction. According to the method, PA gel films of various sizes can be produced and customized on a large scale, the necessity of pretreating the glass surface (glass cover glass or holes of an expensive glass bottom porous plate) by using a PA adhesive solution is eliminated, the research steps and the research time are greatly simplified, and meanwhile, the uniform PA gel film can be dehydrated and stored for several months.

Description

technical field [0001] The invention belongs to the technical field of bioengineering hydrogels, and in particular relates to a mass production method and application of a polyacrylamide gel film, which is an in vitro cell culture base material with different hardness. Background technique [0002] Currently, most in vitro cell studies are performed on polystyrene rigid tissue culture plates (TCP), which have a Young's modulus of ~1 GPa, however, most tissues in vivo are soft viscoelastic materials with moduli derived from the brain From 0.1kPa to 100kPa of osteocartilage, more and more studies have found that the substrate stiffness affects the way cells respond to the environment, and even changes the fate of cells. Substrate is a research hotspot in the field of bioengineering technology. A variety of hydrogels including polyacrylamide (PA), polyethylene glycol (PEG), polydimethylsiloxane (PDMS), and alginate have been developed to help understand the study of stiffness-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J5/18C08L33/26C08F220/56C08F222/38C12N5/00
CPCC08J5/18C08F220/56C12N5/0075C12N2533/30C08J2335/00C08F222/38
Inventor 杨卓李玮珊
Owner 辽宁省肿瘤医院
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