CN103966097A - Preparation method and application of carboxymethyl cellulose-coated RGD short peptide-coupled cell culture plate - Google Patents
Preparation method and application of carboxymethyl cellulose-coated RGD short peptide-coupled cell culture plate Download PDFInfo
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- CN103966097A CN103966097A CN201410226749.8A CN201410226749A CN103966097A CN 103966097 A CN103966097 A CN 103966097A CN 201410226749 A CN201410226749 A CN 201410226749A CN 103966097 A CN103966097 A CN 103966097A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
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- 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
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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Abstract
The invention discloses a preparation method and application of a carboxymethyl cellulose-coated polypeptide-coupled cell culture plate. According to the invention, carboxymethyl cellulose forms a layer of polymer film on the surface of the cell culture plate, thus achieving the purpose of performing carbonation to couple an RGD short peptide, changing the hydrophilicity of the surface of the cell culture plate, and finally enhancing the wall adhesion capability of cells. Compared with the traditional cell plate, the cell plate disclosed by the invention has the advantages of low cost and simple operation process; and the culture plate is applicable to culture of primary cells and caducous cells growing slowly, can promote growth of the cells, and ensures that the cells can adhere to the wall more firmly and obtain a more stable growing state.
Description
Technical field
The present invention relates to a kind of Tissue Culture Plate that applies carboxymethyl cellulose coupling polypeptide and preparation method thereof and the application in cell cultures, belong to technical field of biological materials.
Background technology
Polystyrene has that transparency is good, nontoxic, easy cleaning, the good characteristic such as can reuse, and is widely used in cell culture material.But because its surface free energy is low, wetting ability is poor, p-poly-phenyl ethene carry out modification improving its surface biological consistency, strengthen cellular affinity and become an important research topic.
Through plasma body and electric arc processing, can improve polystyrene surface wetting ability, and there is biocompatibility relatively preferably, but the wetting ability time is short, generally at latter 3 days internal surface active groups of processing, disappear.The coated matrix that promotes cell attachment to use is at present Matrigel, is a kind of extracellular matrix from the secretion of EHS Mouse Bone sarcoma.Use the extracellular matrix extracting in animal may contain growth and the function that some morbid substances can damage cell.Encrusting substance research for culture surface over the past two years slightly got along with, and have report can use the multiple ECM of restructuring to mix, or with certain ECM albumen, reach merely the effect of similar Matrigel, but recombinant protein costs dearly, and be unfavorable for long-term preservation.In recent years, people recognize gradually except biochemical factors, and cell Microphysical environment (as elasticity, surface micronano pattern etc.) has material impact to the maintaining of the migration of cell, propagation, phenotype and function, apoptosis, differentiation.At present existing bibliographical information synthetic surfaces can be the impact that Growth of Cells provides the environment of a relative homogeneous and reduces detrimental impurity, therefore on the basis of investigation of materials, if can develop sustenticular cell adherent growth and maintain the controllable cell Microphysical environment of versatility, for the practical application of cell cultures, there is very large meaning.Patent CN1986778A has invented a kind of method that increases number of adherent mononuclear cells, has shortened the needed time of adherent mononuclear cells, obtains and can be divided into the monocyte of dendritic cell in a large number, has improved quantity prepared by dendritic cell.Patent CN103060264A discloses a kind of stem cell media and application and stem cell culture method, and in described stem cell media, not containing serum, described stem cell media comprises amino acid, VITAMIN, salt, lipid, cytokine and protein polypeptide.These prior aries exist preparation cost high, particularly CN103060264 is used the extracellular matrix Matrigel extracting in animal, Matrigel is a kind of extracellular matrix from the secretion of EHS Mouse Bone sarcoma, use the extracellular matrix extracting in animal may contain growth and the differentiation that some morbid substances can damage cell, different sources cytokine can affect Growth of Cells stability, thereby can have problems in actual use.Encrusting substance research for culture surface over the past two years slightly got along with, and have report can use the multiple ECM of restructuring to mix, or with certain ECM albumen, reach merely the effect of similar Matrigel, but recombinant protein costs dearly, and be unfavorable for long-term preservation.
Carboxymethyl cellulose is that cellulosic carboxymethyl is rolled into a ball substitution product,, there is good biocompatibility, on biomaterial, obtained research widely.Carboxymethyl cellulose is a kind of anionic linear polymeric polysaccharose substance, appearance white or micro-yellow, tasteless, odorless, nontoxic, the vicidity solution that becomes soluble in water, there is unique physicochemical property, and the function such as tool thickening, suspension, stable emulsifying, rheological characteristics.Carboxymethyl cellulose exists with Mierocrystalline cellulose carboxylate form conventionally, the carboxymethyl cellulose water soluble occurring with the form of an alkali metal salt and ammonium salt, and its water-soluble rear pH value approaches neutrality.Carboxymethyl cellulose has good film-forming properties, after the carboxymethyl cellulose of high viscosity, relative high molecule mass is made film, has high strength and high flexibility.In recent years, carboxymethyl cellulose is used for to pharmaceutical carrier, Film with Preventing Adhesion, plastic surgery, surface of a wound auxiliary material etc. as a kind of biocompatible materials at biomedical aspect.RGD peptide is the small peptide that a class contains arginine-glycine-aspartic acid (Arg-Gly-Asp), as the recognition site of integrin and its ligand interaction, regulates the interaction between cell and extracellular matrix and cell.RGD small peptide is as the recognition reaction between common integrin bonding site mediated cell and iuntercellular and cell and extracellular matrix, regulate adhesion, migration and differentiation between cell and cell, cell and substrate material, can effectively promote adherent growth and the propagation of cell.
Summary of the invention
The object of the present invention is to provide a kind of for cell cultures be coated with Tissue Culture Plate of carboxymethyl cellulose and preparation method thereof, and make with the method the small peptide that Tissue Culture Plate surface-coated has carboxymethyl cellulose that biocompatibility is good and coupling to promote cell attachment growth.
The present invention is realized by following technical proposals, and a kind of Tissue Culture Plate of the coating carboxymethyl cellulose for cell cultures, has one deck carboxymethyl cellulose film in Tissue Culture Plate surface-coated, and on described film, coupling has rgd peptide
The invention also discloses the preparation method of described Tissue Culture Plate, use carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) and MES hydrate (MHS) coupling method to introduce polypeptide at culture plate surface grafting.
Concrete preparation process is as follows:
(1) with electronic balance, take carboxymethyl cellulose 0.25～1g, with 10～30% ammoniacal liquor of 20ml, mix, ultrasonic 200～600 seconds, add 10～30ml distilled water diluting, standby;
(2) by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
(3) solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 30 minutes;
(4) with electronic balance, take 5～10g carbodiimide, 4～8g N-Hydroxysuccinimide) and 12.1875g2-morpholino b acid hydrate, with 50ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby;
(5) solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 1～2 hour;
(6) mixing liquid in the culture plate in (5) is abandoned to it, and the polypeptide solution of 0.5～2g/L is added in the Tissue Culture Plate in (5), every hole 1～2mL, and be placed on and shake bed reaction 12～48 hours;
After (7) 24 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the phosphate buffer solution of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
The present invention also provides the application of described Tissue Culture Plate in cultivator scleroblast.
By human osteoblast cell's cell strain MG-63 cell with 5 * 10
3the density of cell is inoculated in 96 well culture plates and cultivates 7 days and test cell vigor (MTT test), carrys out the upgrowth situation of showed cell by cell number and cytoactive.
The present invention is by the way of spin-coating, after being dissolved, carboxymethyl cellulose (CMC) spreads upon uniformly Tissue Culture Plate surface, by carboxymethyl cellulose, on Tissue Culture Plate surface, form one deck polymeric membrane, thereby reach, introduce the hydrophilic ability that carboxyl closes the object of coupling rgd peptide (RGDKKK) by valence bond and changed Tissue Culture Plate surface, finally increased the adherent ability of cell.It is low that the more traditional cell plate of these cell plate not only possess cost, advantage simple to operate, and this culture plate is applicable to primary cell and easily comes off, the cultivation of poky cell, can Promote cell's growth, make cell attachment more firmly and obtain more stable growth state.
Embodiment
For more complete understanding is a little of the present invention and feature, below in conjunction with concrete case study on implementation, further introduce the present invention.Embodiment mono-: apply the preparation of the Tissue Culture Plate of carboxymethyl cellulose
1. with electronic balance, take carboxymethyl cellulose 0.25g, with 25% ammoniacal liquor of 20ml, mix, ultrasonic 600 seconds, add 20ml distilled water diluting, standby.
2. by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
3. the solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 30 minutes.
4. with electronic balance, take 7.17g carbodiimide, 4.604g N-Hydroxysuccinimide and 12.1875g2-morpholino b acid hydrate, with 50ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby.
5. the solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 2 hours.
6. the mixing liquid in the culture plate in (5) is abandoned to it, and the protein solution of 1g/L is added in the Tissue Culture Plate in (5), every hole 2mL, and be placed on and shake bed reaction 24 hours.
After 7.24 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the PBS (phosphate buffer solution) of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
8. cell experiment MTT meta-bolites result shows, the Tissue Culture Plate cell proliferation mean value of the above-mentioned processing of process is higher than untreated culture plate 5.9%.
Embodiment bis-: apply the preparation of the Tissue Culture Plate of carboxymethyl cellulose
1. with electronic balance, take CMC (carboxymethyl cellulose) 0.1g, with 25% ammoniacal liquor of 20ml, mix, ultrasonic 600 seconds, add 20ml distilled water diluting, standby.
2. by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
3. the solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 20 minutes.
4. with electronic balance, take 2.39g carbodiimide, 1.53g N-Hydroxysuccinimide) and 4.06g2-morpholino b acid hydrate, with 50ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby.
5. the solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 3 hours.
6. the mixing liquid in the culture plate in (5) is abandoned to it, and the polypeptide solution of 0.5g/L is added in the Tissue Culture Plate in (5), every hole 3mL, and be placed on and shake bed reaction 36 hours.
After 7.36 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the PBS (phosphate buffer solution) of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
8. cell experiment MTT meta-bolites result shows, the Tissue Culture Plate cell proliferation mean value of the above-mentioned processing of process is higher than untreated culture plate 4.3%.
Case three: apply the preparation of the Tissue Culture Plate of carboxymethyl cellulose
1. with electronic balance, take CMC (carboxymethyl cellulose) 0.5g, with 25% ammoniacal liquor of 20ml, mix, ultrasonic 800 seconds, add 30ml distilled water diluting, standby.
2. by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
3. the solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 60 minutes.
4. with electronic balance, take 4.78g carbodiimide, 6.906g N-Hydroxysuccinimide and 18.28g2-morpholino b acid hydrate, with 100ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby.
5. the solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 5 hours.
6. the mixing liquid in the culture plate in (5) is abandoned to it, and the polypeptide solution of 2g/L is added in the Tissue Culture Plate in (5), every hole 3mL, and be placed on and shake bed reaction 36 hours.
After 7.36 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the PBS (phosphate buffer solution) of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
The demonstration of cell experiment MTT meta-bolites result, the Tissue Culture Plate cell proliferation mean value of the above-mentioned processing of process is higher than untreated culture plate 7.2%.
Claims (6)
1. a Tissue Culture Plate that scribbles carboxymethyl cellulose, is characterized in that, at Tissue Culture Plate surface-coated one deck carboxymethyl cellulose film, on described film, coupling has rgd peptide.
2. Tissue Culture Plate as claimed in claim 1, is characterized in that preparing in accordance with the following methods:
(1) with electronic balance, take carboxymethyl cellulose 0.25～1g, with 10～30% ammoniacal liquor of 20ml, mix, ultrasonic 200～600 seconds, add 10～30ml distilled water diluting, standby;
(2) by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
(3) solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 30 minutes;
(4) with electronic balance, take 5～10g carbodiimide, 4～8g N-Hydroxysuccinimide) and 12.1875g2-morpholino b acid hydrate, with 50ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby;
(5) solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 1～2 hour;
(6) mixing liquid in the culture plate in (5) is abandoned to it, and the polypeptide solution of 0.5～2g/L is added in the Tissue Culture Plate in (5), every hole 1～2mL, and be placed on and shake bed reaction 12～48 hours;
After (7) 24 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the phosphate buffer solution of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
3. prepare a method that is coated with the Tissue Culture Plate of carboxymethyl cellulose claimed in claim 1, it is characterized in that using carbodiimide and N-Hydroxysuccinimide and MES hydrate coupling method to introduce polypeptide at culture plate surface grafting.
4. preparation method as claimed in claim 3, the sequence that it is characterized in that described polypeptide is RGDKKK.
5. preparation method as claimed in claim 3, is characterized in that comprising the following steps:
(1) with electronic balance, take carboxymethyl cellulose 0.25～1g, with 10～30% ammoniacal liquor of 20ml, mix, ultrasonic 200～600 seconds, add 10～30ml distilled water diluting, standby;
(2) by polystyrene dehydrated alcohol for culture plate, distillation washing three times, vacuum-drying, standby.
(3) solution in step (1) is added drop-wise in the culture plate of step (2), liquid level will cover culture plate bottom (approximately 8) completely, then the polystyrene culture plate that dripped (1) solution is put into 60 ℃ of vacuum drying ovens 30 minutes;
(4) with electronic balance, take 5～10g carbodiimide, 4～8g N-Hydroxysuccinimide) and 12.1875g2-morpholino b acid hydrate, with 50ml distilled water, dissolved, put into 4 ℃ of refrigerators, standby;
(5) solution of joining in (4) is added drop-wise to liquid level in the culture plate of step (3) and will covers culture plate bottom (approximately 5) completely, put into 30 ℃ of baking ovens 1～2 hour;
(6) mixing liquid in the culture plate in (5) is abandoned to it, and the polypeptide solution of 0.5～2g/L is added in the Tissue Culture Plate in (5), every hole 1～2mL, and be placed on and shake bed reaction 12～48 hours;
After (7) 24 hours, the liquid in the Tissue Culture Plate in (6) is thrown aside, and rinsed with the phosphate buffer solution of a large amount of PH=7.35, rinse post-drying, seal up for safekeeping.
6. the application of Tissue Culture Plate in cultivator scleroblast described in claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635970A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Method for culturing cartilage tissues |
| CN115873712A (en) * | 2022-11-28 | 2023-03-31 | 杭州皓丰生物技术有限公司 | Deep-hole cell culture plate, preparation method and application thereof |
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| US20060051867A1 (en) * | 1999-01-21 | 2006-03-09 | Hamilton Douglas W | Cell growth |
| US20110251677A1 (en) * | 2007-10-23 | 2011-10-13 | Abbott Cardiovascular Systems Inc. | Coating Designs For The Tailored Release Of Dual Drugs From Polymeric Coatings |
| CN103087916A (en) * | 2013-01-17 | 2013-05-08 | 黑龙江省重生生物科技有限公司 | Cell culture plate coated with Matrigel as well as preparation method and application of cell culture plate |
-
2014
- 2014-05-26 CN CN201410226749.8A patent/CN103966097A/en active Pending
Patent Citations (3)
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| US20060051867A1 (en) * | 1999-01-21 | 2006-03-09 | Hamilton Douglas W | Cell growth |
| US20110251677A1 (en) * | 2007-10-23 | 2011-10-13 | Abbott Cardiovascular Systems Inc. | Coating Designs For The Tailored Release Of Dual Drugs From Polymeric Coatings |
| CN103087916A (en) * | 2013-01-17 | 2013-05-08 | 黑龙江省重生生物科技有限公司 | Cell culture plate coated with Matrigel as well as preparation method and application of cell culture plate |
Non-Patent Citations (2)
| Title |
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| 马亮: "RGD序列肽修饰的丝素蛋白仿生支架材料对骨髓间充质干细胞粘附、增值的影响", 《医药卫生科技辑》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635970A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Method for culturing cartilage tissues |
| CN115873712A (en) * | 2022-11-28 | 2023-03-31 | 杭州皓丰生物技术有限公司 | Deep-hole cell culture plate, preparation method and application thereof |
| CN115873712B (en) * | 2022-11-28 | 2023-05-12 | 杭州皓丰生物技术有限公司 | Deep hole cell culture plate, preparation method and application thereof |
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Application publication date: 20140806 |