CN113308937A - Cell culture paper and preparation method and application thereof - Google Patents
Cell culture paper and preparation method and application thereof Download PDFInfo
- Publication number
- CN113308937A CN113308937A CN202110588340.0A CN202110588340A CN113308937A CN 113308937 A CN113308937 A CN 113308937A CN 202110588340 A CN202110588340 A CN 202110588340A CN 113308937 A CN113308937 A CN 113308937A
- Authority
- CN
- China
- Prior art keywords
- cell culture
- paper
- solution
- culture paper
- filter paper
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 229920002678 cellulose Polymers 0.000 claims abstract description 36
- 239000001913 cellulose Substances 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229920001046 Nanocellulose Polymers 0.000 claims abstract description 14
- 239000006285 cell suspension Substances 0.000 claims abstract description 12
- 238000002791 soaking Methods 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 49
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 229920000742 Cotton Polymers 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
- LGZDNJBUAAXEMN-UHFFFAOYSA-N 1,2,2,3-tetramethyl-1-oxidopiperidin-1-ium Chemical compound CC1CCC[N+](C)([O-])C1(C)C LGZDNJBUAAXEMN-UHFFFAOYSA-N 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 239000002002 slurry Substances 0.000 claims description 2
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 239000011148 porous material Substances 0.000 abstract description 22
- 239000000463 material Substances 0.000 abstract description 15
- 230000010261 cell growth Effects 0.000 abstract description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 3
- 229920001222 biopolymer Polymers 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 41
- 239000002131 composite material Substances 0.000 description 9
- 206010006187 Breast cancer Diseases 0.000 description 5
- 208000026310 Breast neoplasm Diseases 0.000 description 5
- 206010008342 Cervix carcinoma Diseases 0.000 description 5
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 201000010881 cervical cancer Diseases 0.000 description 5
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical group O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 108010087230 Sincalide Proteins 0.000 description 4
- 238000007605 air drying Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- -1 2-methoxy-4-nitrophenyl Chemical group 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 235000012431 wafers Nutrition 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010041 electrostatic spinning Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002959 polymer blend Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000004627 regenerated cellulose Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 239000012744 reinforcing agent Substances 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H27/00—Special paper not otherwise provided for, e.g. made by multi-step processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H23/00—Processes or apparatus for adding material to the pulp or to the paper
- D21H23/02—Processes or apparatus for adding material to the pulp or to the paper characterised by the manner in which substances are added
- D21H23/22—Addition to the formed paper
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H25/00—After-treatment of paper not provided for in groups D21H17/00 - D21H23/00
- D21H25/04—Physical treatment, e.g. heating, irradiating
- D21H25/06—Physical treatment, e.g. heating, irradiating of impregnated or coated paper
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H27/00—Special paper not otherwise provided for, e.g. made by multi-step processes
- D21H27/08—Filter paper
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/78—Cellulose
Abstract
The invention relates to a cell culture paper and a preparation method and application thereof, relating to the field of biopolymer materials, wherein the method comprises the following steps: soaking the filter paper soaked in the alcohol solvent in 0.2-1.0 wt% of carboxylated nano cellulose solution, and naturally drying to obtain the cell culture paper. The method for culturing cells by adopting the cell culture paper is characterized by comprising the following steps: sterilizing the cell culture paper at 120-140 ℃ for 20-30 min, then inoculating cell suspension of cells to be cultured on the sterilized cell culture paper, and finally putting the sterilized cell culture paper into a culture medium for culturing. The carboxylated nano-cellulose has good water dispersibility due to the existence of a large amount of carboxyl on the surface, has small pore diameter, can uniformly enter large pores of filter paper, can adjust the pore diameter to a range suitable for cell growth, has good mechanical property and can be used for cell culture.
Description
Technical Field
The invention relates to the field of biological polymer materials, in particular to cell culture paper and a preparation method and application thereof.
Background
Paper has attracted considerable attention as an emerging cell culture platform. Paper is a cheaper and more abundant material than other materials, and has some special advantages, such as: (1) the raw material is cellulose or a cellulose-polymer mixture, and has good biocompatibility; (2) the device has a large number of pores, can simulate the structure of extracellular matrix, allows substances to pass through, can provide nutrients for cells and timely discharge metabolic waste; (3) the chemical modification is convenient, the immobilization of biomolecules is realized, and various substances are convenient to deposit so as to change the physical and chemical properties of the paper; (4) the structure and the thickness of the paper can be flexibly adjusted; (5) the plurality of paper-based units can realize 3D structural organization/organ construction by simple stacking, and can also realize structural transformation from 3D to 2D simply and conveniently by disassembling without damaging the cell structure, thereby facilitating the observation of the internal cell morphology and functions.
However, paper as a new cell culture platform also has some drawbacks. An ideal cell culture support material should have sufficient porosity to allow nutrient transport and metabolite excretion, and mechanical strength to support the scaffold structure, and the cells must adhere properly to the material for migration, differentiation and proliferation. Therefore, certain porosity and pore size are needed, and the pore size must be ensured to be slightly larger than the normal cell size (10-100 μm) so that the cell culture system can show a good cell growth environment and promote the adhesion, migration and proliferation of cells.
At present, filter paper is directly used as cell culture paper, but the qualitative filter paper has large pores (80-120 microns) and is not suitable for efficient cell adhesion, and the qualitative filter paper has poor wet strength and cannot play a good support role in the cell growth process. Also, a regenerated cellulose membrane prepared by a dissolution regeneration method has a pore diameter of about 10 to 30 nm. The average pore diameter of the cellulose porous membrane prepared by the electrostatic spinning method is about 80-1100 nm, but the pore diameters of the cellulose membranes prepared by the two methods are small, so that the space required by cell proliferation is difficult to meet. Later, cellulose membranes are prepared by combining a dissolving and regenerating cellulose with a template method, a pore-forming agent is added in the regeneration process, and cellulose membranes with different pore diameters and porosities are prepared by controlling the size of the pore-forming agent, but the operation is complicated, and the addition of an organic solvent is unfavorable for cell growth.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the cell culture paper and the preparation method and the application thereof, the filling performance and the hydrophilic characteristic of the carboxyl nano-cellulose are utilized to regulate and control the pores of the filter paper with larger pores, and the porosity of the obtained material is suitable for the growth of cells, has good mechanical property and can be used for cell culture.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of cell culture paper comprises the following steps:
soaking the filter paper soaked in the alcohol solvent in 0.2-1.0 wt% of carboxylated nano cellulose solution, and naturally drying to obtain the cell culture paper.
Preferably, the alcohol solvent is ethanol water solution.
Preferably, the filter paper is qualitative filter paper.
Preferably, the filter paper is soaked in the alcohol solvent for 5-10 min and then is soaked in the carboxylated nano-cellulose solution.
Preferably, the filter paper is soaked in the carboxylated nano-cellulose solution for 3-8 min, then drained and naturally dried.
Preferably, the carboxylated nano-cellulose solution is prepared by the following steps:
uniformly dispersing 1-3% by mass of cotton fiber slurry, then adding 2-3% by mass of tetramethylpiperidine oxide solution and 6-10% by mass of NaBr solution, uniformly mixing, then adding NaClO solution to enable the pH of the formed suspension to be 10-12, washing the suspension to be neutral by deionized water after the reaction is finished, and then adding deionized water for dilution to obtain the carboxylated nano-cellulose solution.
Further, the reaction is carried out for 2.5-3.5 h when the carboxylated nano-cellulose solution is prepared.
A cell culture paper obtained by the method for producing a cell culture paper described in any one of the above.
The method for culturing the cells by adopting the cell culture paper is characterized by comprising the following steps:
sterilizing the cell culture paper at 120-140 ℃ for 20-30 min, then inoculating cell suspension of cells to be cultured on the sterilized cell culture paper, and finally putting the sterilized cell culture paper into a culture medium for culturing.
Further, the cell culture paper is sterilized under the conditions, added with a gelatin aqueous solution and air-dried, and then the cell suspension of the cells to be cultured is inoculated onto the obtained cell culture paper.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention relates to a preparation method of cell culture paper, which takes filter paper as a raw material, and the filter paper is soaked in an alcohol solvent to displace the air in the filter paper, so that the filter paper is soaked in a carboxylated nano-cellulose solution, a certain amount of carboxylated nano-cellulose can be filled in gaps of the filter paper, and simultaneously the filter paper plays a role in adjusting aperture and reinforcing agent, and the cell culture paper with excellent performance can be obtained at low cost and high efficiency after being naturally dried. The carboxylated nano-cellulose has good water dispersibility due to the existence of a large amount of carboxyl on the surface, has small pore size, can uniformly enter large pores of filter paper, and can adjust the pores to a range suitable for cell growth.
When the cell culture paper is applied to cell culture, the introduction of the carboxylated nanocellulose can enhance the cell adhesion on the surface of the cell culture paper due to good hydrophilicity, and meanwhile, the introduction of the carboxylated nanocellulose with high length-diameter ratio can enhance the mechanical property of the filter paper, so that cell suspension of cells to be cultured can be inoculated after sterilization, and finally, the cells to be cultured can be cultured by placing the cells into a culture medium.
Drawings
FIG. 1 is an SEM image of the cell culture paper obtained in example 2 of the present invention.
FIG. 2 is a graph comparing the wet strength performance of the cell culture paper and the raw material qualitative filter paper obtained in examples 1 to 3 of the present invention.
FIG. 3 is a diagram showing the cell activity test using the CCK-8 kit in example 2 of the present invention.
FIG. 4 shows the results of cell growth staining on a cell culture paper obtained in example 2 of the present invention, in which FIGS. 4a and 4b are control groups in which a cell suspension containing cervical cancer cells was inoculated and not inoculated on the paper, respectively, and FIGS. 4c and 4d are control groups in which a cell suspension containing breast cancer cells was inoculated and not inoculated on the paper, respectively.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
The invention relates to a preparation method of cell culture paper with adjustable pores, which specifically comprises the following steps:
step 1, preparing carboxylated nanocellulose by a Tempo oxidation method:
defibering the pulped cotton fibers in a standard defibering device according to the concentration of 1% by mass fraction, uniformly dispersing the cotton fibers, respectively adding a tetramethylpiperidine oxide (TEMPO) solution with the mass fraction of 1.6% and a NaBr solution with the mass fraction of 10% which are dissolved in deionized water, mixing and stirring for 10min, dropwise adding a NaClO solution to enable the pH of a suspension to be 10-12, and continuing the whole process for 2.5-3.5 h. After the reaction is finished, washing the suspension to be neutral by using deionized water to form a carboxylated nano-cellulose solution, and adding deionized water to adjust the mass fraction of the carboxylated nano-cellulose solution to 0.2-1.0%.
And 2, soaking the qualitative filter paper in an ethanol water solution for 5-10 min, pretreating the qualitative filter paper, displacing air in the qualitative filter paper, and improving the soaking efficiency of the next step of the nano cellulose solution.
And 3, dipping the filter paper obtained in the step 2 into the carboxylated nano-cellulose solution obtained in the step 1, carrying out the dipping out after 3-8 minutes, draining, and hanging and naturally airing to obtain the nano-cellulose/filter paper composite paper base material, namely the cell culture paper.
And 4, when the cell culture paper is used, sterilizing the prepared nano-cellulose/filter paper composite paper base material in an autoclave at the temperature of 120-140 ℃ for 20-30 min, cutting the nano-cellulose/filter paper composite paper base material into wafers with the diameter of 15mm, dropwise adding 1-2 mL of gelatin aqueous solution with the mass fraction of 0.1%, and air-drying to improve the adhesion between cells and the culture paper to obtain the cell culture paper.
And 5, inoculating the cell suspension containing the cervical cancer cells and the breast cancer cells on culture paper, and putting the culture paper into a culture medium for culture. Subculturing every 1-2 days, and directly observing the cell growth condition under an inverted microscope.
Example 1
The invention relates to a preparation method of cell culture paper with adjustable pores, which specifically comprises the following steps:
step 1) preparing carboxylated nanocellulose by a Tempo oxidation method:
defibering the pulped cotton fibers in a standard defibering device according to the concentration of 1% by mass fraction to uniformly disperse the pulped cotton fibers, respectively adding a TEMPO solution with the mass fraction of 1.6% and a NaBr solution with the mass fraction of 10% which are dissolved in deionized water, mixing and stirring for 10min, dropwise adding a NaClO solution, controlling the pH of the suspension to be 11, and continuing the whole process for 3 h.
After the reaction is finished, the suspension is washed to be neutral by deionized water, and the mass fraction of the carboxylated nano-cellulose solution is adjusted to 0.2% by adding the deionized water.
And 2) soaking the qualitative filter paper for 5min by using ethanol water solution with the volume fraction of 1:1, and pretreating the filter paper.
And 3) soaking the filter paper obtained in the step 2 in 0.2% of carboxylated nano-cellulose solution, carrying out the filter paper out after 5 minutes, draining the water, and hanging and naturally airing the filter paper to obtain the nano-cellulose/filter paper composite paper base material.
And 4) sterilizing the prepared nano-cellulose/filter paper composite paper base material in an autoclave at 120 ℃ for 20min, cutting into a wafer with the diameter of 15mm, dropwise adding 1mL of 0.1% gelatin aqueous solution in mass concentration, and air-drying to improve the adhesion between cells and culture paper and obtain a cell culture paper base.
And 5) inoculating the cell suspension containing the cervical cancer cells and the breast cancer cells on culture paper, and putting the culture paper into a culture medium for culture. Subculturing every 1-2 days, and directly observing the cell growth condition under an inverted microscope.
Example 2
The invention relates to a preparation method of cell culture paper with adjustable pores, which specifically comprises the following steps:
step 1) preparing carboxylated nanocellulose by a Tempo oxidation method:
defibering the pulped cotton fibers in a standard defibering device according to the concentration of 1% by mass fraction to uniformly disperse the pulped cotton fibers, respectively adding a TEMPO solution with the mass fraction of 1.6% and a NaBr solution with the mass fraction of 10% which are dissolved in deionized water, mixing and stirring for 10min, dropwise adding a NaClO solution, controlling the pH of the suspension to be 11, and continuing the whole process for 3 h.
After the reaction is finished, the suspension is washed to be neutral by deionized water, and the mass fraction of the carboxylated nano-cellulose solution is adjusted to 0.6% by adding the deionized water.
And 2) soaking the qualitative filter paper for 5min by using ethanol water solution with the volume fraction of 1:1, and pretreating the filter paper.
And 3) soaking the filter paper obtained in the step 2 in 0.6% of carboxylated nano-cellulose solution, carrying out the filter paper out after 5 minutes, draining the water, and hanging and naturally airing the filter paper to obtain the nano-cellulose/filter paper composite paper base material.
And 4) sterilizing the prepared nano-cellulose/filter paper composite paper base material in an autoclave at 120 ℃ for 20min, cutting into a wafer with the diameter of 15mm, dropwise adding 2mL of 0.1% gelatin aqueous solution in mass concentration, and air-drying to improve the adhesion between cells and culture paper and obtain a cell culture paper base.
And 5) inoculating the cell suspension containing the cervical cancer cells and the breast cancer cells on culture paper, and putting the culture paper into a culture medium for culture. Subculturing every 1-2 days, and directly observing the cell growth condition under an inverted microscope.
As shown in FIG. 1, the pores on the surface of the cell culture paper are uniform and about 20-50 μm in size, which is suitable for the cell growth environment.
The CCK-8 Kit is short for Cell Counting Kit-8, and the principle is that WST-8 tetrazolium salt (the name of England is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-sulfophenyl) -2H-tetrazolium) can be reduced into orange formazan dye by dehydrogenase in living cells, then the light absorption value at 450nm is measured, and the amount of the generated formazan is in direct proportion to the number of the living cells. In this example, the CCK-8 kit is used to verify the effect of cell culture paper, and for adherent cells, the method includes the following steps:
step 1, adding pancreatin to the adherent cells in the logarithmic phase for digestion to enable the cells to fall off, collecting the cells in the logarithmic phase, and adjusting the concentration.
And 2, planting the cells in a 96-well plate, and adding a reagent (WST-8 tetrazolium salt) after the cells adhere to the wall.
And 4, adding 10 mu L of CCK-8 solution into each hole, and culturing for 1-4 h.
And 5, taking out the 96-well plate, placing the 96-well plate on an enzyme-linked immunosorbent assay instrument, oscillating for 15s, and measuring light absorption at 450 nm.
And 6, carrying out statistical analysis on results.
As can be seen from fig. 3, the cell viability was greater than 95% after the cells were cultured in the culture medium containing paper for 1, 3, 5 and 7 days, and good biocompatibility was exhibited.
As can be seen from fig. 4a, the visual field was green living cells, and the cells were uniformly distributed in a fusiform shape, and almost no dead cells were observed, and as can be seen from fig. 4b, the living cells were fusiform, and a very small amount of red dead cells were observed, which is consistent with the experimental result of fig. 3. The phenomena in fig. 4c are the same as in fig. 4a, and in fig. 4d, and in fig. 4 b.
The good biocompatibility of the transparency paper prepared in this study was verified by means of fig. 3 and 4.
Example 3
The invention relates to a preparation method of cell culture paper with adjustable pores, which specifically comprises the following steps:
step 1) preparing carboxylated nanocellulose by a Tempo oxidation method:
defibering the pulped cotton fibers in a standard defibering device according to the concentration of 1% by mass fraction to uniformly disperse the pulped cotton fibers, respectively adding a TEMPO solution with the mass fraction of 1.6% and a NaBr solution with the mass fraction of 10% which are dissolved in deionized water, mixing and stirring for 10min, dropwise adding a NaClO solution, controlling the pH of the suspension to be 11, and continuing the whole process for 3 h.
After the reaction is finished, the suspension is washed to be neutral by deionized water, and the mass fraction of the carboxylated nano-cellulose solution is adjusted to 1.0% by adding the deionized water.
And 2) soaking the qualitative filter paper for 5min by using ethanol water solution with the volume fraction of 1:1, and pretreating the filter paper.
And 3) soaking the filter paper obtained in the step 2 in 1.0% of carboxylated nano-cellulose solution, carrying out the filter paper out after 5 minutes, draining the water, and hanging and naturally airing the filter paper to obtain the nano-cellulose/filter paper composite paper base material.
And 4) sterilizing the prepared nano-cellulose/filter paper composite paper base material in an autoclave at 120 ℃ for 20min, cutting into round pieces with the diameter of 15mm, dropwise adding 1.5mL of 0.1% gelatin aqueous solution in mass concentration, and air-drying to improve the adhesion between cells and culture paper to obtain the cell culture paper.
And 5) inoculating the cell suspension containing the cervical cancer cells and the breast cancer cells on culture paper, and putting the culture paper into a culture medium for culture. Subculturing every 1-2 days, and directly observing the cell growth condition under an inverted microscope.
FIG. 2 is a graph comparing the wet strength performance of the cell culture paper and the raw material qualitative filter paper obtained in examples 1 to 3, and it can be seen that the pores of the filter paper are filled more effectively, the structure of the filter paper becomes dense, and the strength increases with the increase of the concentration of the nano-cellulose impregnation solution.
Claims (10)
1. A preparation method of cell culture paper is characterized by comprising the following steps:
soaking the filter paper soaked in the alcohol solvent in 0.2-1.0 wt% of carboxylated nano cellulose solution, and naturally drying to obtain the cell culture paper.
2. The method of producing a cell culture paper according to claim 1, wherein the alcohol solvent is an aqueous ethanol solution.
3. The method of preparing a cell culture paper according to claim 1, wherein the filter paper is a qualitative filter paper.
4. The method for preparing cell culture paper according to claim 1, wherein the filter paper is immersed in the alcoholic solvent for 5 to 10min and then immersed in the carboxylated nanocellulose solution.
5. The method for preparing cell culture paper according to claim 1, wherein the filter paper is immersed in the carboxylated nanocellulose solution for 3-8 min, drained, and then naturally dried.
6. The method for preparing cell culture paper according to claim 1, wherein the carboxylated nanocellulose solution is prepared by the following process:
uniformly dispersing 1-3% by mass of cotton fiber slurry, then adding 2-3% by mass of tetramethylpiperidine oxide solution and 6-10% by mass of NaBr solution, uniformly mixing, then adding NaClO solution to enable the pH of the formed suspension to be 10-12, washing the suspension to be neutral by deionized water after the reaction is finished, and then adding deionized water for dilution to obtain the carboxylated nano-cellulose solution.
7. The method for producing cell culture paper according to claim 6, wherein the reaction is carried out for 2.5 to 3.5 hours in the production of the carboxylated nanocellulose solution.
8. A cell culture paper obtained by the method for producing a cell culture paper according to any one of claims 1 to 7.
9. A method of culturing cells using the cell culture paper according to claim 8, comprising the steps of:
sterilizing the cell culture paper of claim 8 at 120-140 ℃ for 20-30 min, then inoculating the cell suspension of the cells to be cultured on the sterilized cell culture paper, and finally placing the cell culture paper in a culture medium for culturing.
10. The method of claim 9, wherein the cell culture paper is sterilized under the above conditions, an aqueous solution of gelatin is added thereto, air-dried, and the cell suspension of the cells to be cultured is inoculated onto the obtained cell culture paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110588340.0A CN113308937A (en) | 2021-05-28 | 2021-05-28 | Cell culture paper and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110588340.0A CN113308937A (en) | 2021-05-28 | 2021-05-28 | Cell culture paper and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113308937A true CN113308937A (en) | 2021-08-27 |
Family
ID=77375730
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110588340.0A Pending CN113308937A (en) | 2021-05-28 | 2021-05-28 | Cell culture paper and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113308937A (en) |
Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02276593A (en) * | 1989-04-19 | 1990-11-13 | Kazuyuki Sugawara | Paper disk for examining cell culture and filter paper piece |
| US5558861A (en) * | 1986-04-22 | 1996-09-24 | Ajinomoto Co., Inc. | Modified microbially-produced cellulose gel with human epidermal cells adsorbed thereon for use as a skin graft or vulnerary cover |
| JP2004105043A (en) * | 2002-09-17 | 2004-04-08 | Sumitomo Bakelite Co Ltd | Cell culture substrate and method for producing the same |
| CN1887362A (en) * | 2006-07-13 | 2007-01-03 | 苏州大学 | Cell culturing rack material and its prepn |
| JP2007325543A (en) * | 2006-06-08 | 2007-12-20 | Toray Ind Inc | Cell scaffold material and method for producing the same |
| US20090186412A1 (en) * | 2008-01-21 | 2009-07-23 | Gc Corporation | Porous cell scaffold and production method thereof |
| CN104420400A (en) * | 2013-08-30 | 2015-03-18 | 珠海市红旌发展有限公司 | Preparation method of air filtering paper containing nanocellulose |
| CN104479368A (en) * | 2014-12-10 | 2015-04-01 | 国家纳米科学中心 | Nanocellulose enhanced full-biodegradable thin film and preparation method thereof |
| CN104531624A (en) * | 2014-12-23 | 2015-04-22 | 山东信得动物疫苗有限公司 | Method for culturing MDCK (medin-darby canine kidney) cell proliferation recombination H5N1 subtype avian influenza virus |
| US20160032272A1 (en) * | 2012-12-13 | 2016-02-04 | Nanjing University Of Technology | Preparation method of yeast cell immobilization medium and application thereof |
| CN105483178A (en) * | 2015-12-27 | 2016-04-13 | 常州亚环环保科技有限公司 | Method for preparing bacterial cellulose composite surgical dressing through waste paper pulp |
| CN105694110A (en) * | 2016-01-26 | 2016-06-22 | 中南林业科技大学 | Aperture-adjustable cellulose and montmorillonite nano-composite carrier material and preparation method thereof |
| CN106012667A (en) * | 2016-05-20 | 2016-10-12 | 广州纽楷美新材料科技有限公司 | Hollocellulose transparent paper and preparation method thereof |
| CN106638081A (en) * | 2016-10-28 | 2017-05-10 | 河南华禹环保科技有限公司 | Method for preparing filter paper fiber carrier for white-rot fungus culture |
| CN108118025A (en) * | 2016-11-28 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of foundation and its application of the three-dimensional liver model based on qualitative filter paper |
| CN108931565A (en) * | 2018-08-13 | 2018-12-04 | 山东农业大学 | The construction method of nano-cellulose paper base biosensor |
| CN109164149A (en) * | 2018-07-21 | 2019-01-08 | 福建医科大学 | A kind of electrochemica biological sensor based on paper chip combination screen printing electrode on-line checking intracellular hydrogen peroxide |
| CN110387060A (en) * | 2019-07-15 | 2019-10-29 | 陕西科技大学 | A kind of underwater transparency porous cellulose paper base material and its preparation method and application for cell culture |
| CN110577672A (en) * | 2018-06-07 | 2019-12-17 | 国家纳米科学中心 | Controllable hydrophilic and hydrophobic nano cellulose film, and preparation method and application thereof |
| CN112480479A (en) * | 2020-11-30 | 2021-03-12 | 江南大学 | Preparation method of nanocellulose-based fluorescent film |
| US20210138454A1 (en) * | 2019-11-12 | 2021-05-13 | LYOPOR, s.r.o. | Carrier for cell culturing and a method of preparation thereof |
-
2021
- 2021-05-28 CN CN202110588340.0A patent/CN113308937A/en active Pending
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5558861A (en) * | 1986-04-22 | 1996-09-24 | Ajinomoto Co., Inc. | Modified microbially-produced cellulose gel with human epidermal cells adsorbed thereon for use as a skin graft or vulnerary cover |
| JPH02276593A (en) * | 1989-04-19 | 1990-11-13 | Kazuyuki Sugawara | Paper disk for examining cell culture and filter paper piece |
| JP2004105043A (en) * | 2002-09-17 | 2004-04-08 | Sumitomo Bakelite Co Ltd | Cell culture substrate and method for producing the same |
| JP2007325543A (en) * | 2006-06-08 | 2007-12-20 | Toray Ind Inc | Cell scaffold material and method for producing the same |
| CN1887362A (en) * | 2006-07-13 | 2007-01-03 | 苏州大学 | Cell culturing rack material and its prepn |
| US20090186412A1 (en) * | 2008-01-21 | 2009-07-23 | Gc Corporation | Porous cell scaffold and production method thereof |
| US20160032272A1 (en) * | 2012-12-13 | 2016-02-04 | Nanjing University Of Technology | Preparation method of yeast cell immobilization medium and application thereof |
| CN104420400A (en) * | 2013-08-30 | 2015-03-18 | 珠海市红旌发展有限公司 | Preparation method of air filtering paper containing nanocellulose |
| CN104479368A (en) * | 2014-12-10 | 2015-04-01 | 国家纳米科学中心 | Nanocellulose enhanced full-biodegradable thin film and preparation method thereof |
| CN104531624A (en) * | 2014-12-23 | 2015-04-22 | 山东信得动物疫苗有限公司 | Method for culturing MDCK (medin-darby canine kidney) cell proliferation recombination H5N1 subtype avian influenza virus |
| CN105483178A (en) * | 2015-12-27 | 2016-04-13 | 常州亚环环保科技有限公司 | Method for preparing bacterial cellulose composite surgical dressing through waste paper pulp |
| CN105694110A (en) * | 2016-01-26 | 2016-06-22 | 中南林业科技大学 | Aperture-adjustable cellulose and montmorillonite nano-composite carrier material and preparation method thereof |
| CN106012667A (en) * | 2016-05-20 | 2016-10-12 | 广州纽楷美新材料科技有限公司 | Hollocellulose transparent paper and preparation method thereof |
| CN106638081A (en) * | 2016-10-28 | 2017-05-10 | 河南华禹环保科技有限公司 | Method for preparing filter paper fiber carrier for white-rot fungus culture |
| CN108118025A (en) * | 2016-11-28 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of foundation and its application of the three-dimensional liver model based on qualitative filter paper |
| CN110577672A (en) * | 2018-06-07 | 2019-12-17 | 国家纳米科学中心 | Controllable hydrophilic and hydrophobic nano cellulose film, and preparation method and application thereof |
| CN109164149A (en) * | 2018-07-21 | 2019-01-08 | 福建医科大学 | A kind of electrochemica biological sensor based on paper chip combination screen printing electrode on-line checking intracellular hydrogen peroxide |
| CN108931565A (en) * | 2018-08-13 | 2018-12-04 | 山东农业大学 | The construction method of nano-cellulose paper base biosensor |
| CN110387060A (en) * | 2019-07-15 | 2019-10-29 | 陕西科技大学 | A kind of underwater transparency porous cellulose paper base material and its preparation method and application for cell culture |
| US20210138454A1 (en) * | 2019-11-12 | 2021-05-13 | LYOPOR, s.r.o. | Carrier for cell culturing and a method of preparation thereof |
| CN112480479A (en) * | 2020-11-30 | 2021-03-12 | 江南大学 | Preparation method of nanocellulose-based fluorescent film |
Non-Patent Citations (8)
| Title |
|---|
| SUFENG ZHANG: ""A polyethylenimine/salicylaldehyde modified cellulose Schiff base for selective and sensitive Fe3+ detection"", 《CARBOHYDRATE POLYMERS》, 24 September 2019 (2019-09-24), pages 1 - 8 * |
| 刘丽娜: ""3D细胞培养用透明纸的制备及应用研究"", 《中国优秀硕士论文 工程科技Ⅰ辑》, 15 February 2021 (2021-02-15), pages 10 - 12 * |
| 刘晓瀛 主编: "《医用化学实验实训》", 31 January 2013, 沈阳：东北大学出版社, pages: 24 * |
| 李鑫等: "纳米纤维素的制备工艺及其研究进展", 《纸和造纸》, no. 04, 15 July 2018 (2018-07-15) * |
| 赵永芳 编: "《生物化学技术原理及其应用》", 30 September 1988, 武汉：武汉大学出版社, pages: 79 * |
| 陈志南, 中国协和医科大学出版社 * |
| 陈志南, 中国协和医科大学出版社, pages: 58 - 59 * |
| 陈港等: "透明纸基材料的研究与应用", 《中国造纸》, no. 07, 15 July 2018 (2018-07-15) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109675104B (en) | Preparation method of mineralized hydrogel and biomimetic mineralized bone repair material | |
| CN105254917B (en) | A method of preparing cell patch using Sodium Alginate Hydrogel Films | |
| US8691973B2 (en) | Method of producing chitosan scaffold having high tensile strength and chitosan scaffold produced using the method | |
| Hirayama et al. | Cellular building unit integrated with microstrand-shaped bacterial cellulose | |
| CN104053459B (en) | Method and the device of three-dimensional cell microenvironment is built based on transparent sponge bracket | |
| US9090868B2 (en) | Alginate hydrogel fibers and related materials | |
| CN113101419B (en) | Hydrogel stent with polydopamine coating and preparation method thereof | |
| JP2005502351A (en) | Methods and structures for growing living organic tissue | |
| US20110294214A1 (en) | Seeding Cells on Porous Supports | |
| CN107551311A (en) | A kind of porous composite electrospun tunica fibrosa of orientation of controlled drug release and its preparation method and application | |
| CN109289075A (en) | A kind of preparation method and wound dressing of cellulose-collagen composite nano-fiber membrane | |
| Son et al. | Hydro-nanofibrous mesh deep cell penetration: a strategy based on peeling of electrospun coaxial nanofibers | |
| Wang et al. | Bacterial cellulose nanofiber reinforced poly (glycerol-sebacate) biomimetic matrix for 3D cell culture | |
| CN108676769B (en) | Non-woven fabric support material for promoting growth of liver cells and preparation method thereof | |
| CN114042191A (en) | Cell-printed osteogenic functional scaffold and preparation method and application thereof | |
| CN108273130B (en) | Three-dimensional micro-nano fiber composite support and preparation method thereof | |
| CN113308937A (en) | Cell culture paper and preparation method and application thereof | |
| Wu et al. | Bacterial cellulose nanofiber distribution on gelatin and silk fibroin scaffolds and the cell behavior | |
| CN110639058B (en) | Acicular HA/PBLG porous composite microcarrier material for bone tissue engineering and preparation method thereof | |
| CN113248778A (en) | Preparation method and application of surface modified cellulose-based porous membrane | |
| CN110387060B (en) | Underwater transparent porous cellulose paper base material for cell culture and preparation method and application thereof | |
| Zhang et al. | Recent advances of cellulose nanofiber-based materials in cell culture: From population to single-cell | |
| CN107684637B (en) | A kind of polylactic acid/hydroxy apatite/de- cell amnion compound rest and its construction method | |
| WO2021079931A1 (en) | Cell-culturing substrate and cell-provided cell-culturing substrate | |
| CN109081933A (en) | A kind of in-situ TEM modified bacteria cellulose aquagel and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210827 |
|
| RJ01 | Rejection of invention patent application after publication |