CN101603007B - Method for preparing cell culture vector - Google Patents
Method for preparing cell culture vector Download PDFInfo
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- CN101603007B CN101603007B CN2009101043978A CN200910104397A CN101603007B CN 101603007 B CN101603007 B CN 101603007B CN 2009101043978 A CN2009101043978 A CN 2009101043978A CN 200910104397 A CN200910104397 A CN 200910104397A CN 101603007 B CN101603007 B CN 101603007B
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- cell culture
- silk fibroin
- culture vector
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- polyvinyl alcohol
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Abstract
The invention relates to a method for preparing a cell culture vector, which comprises the following steps: firstly, preparing nanoscale silk fibroin powder; secondly, dispersing or dissolving the nanoscale silk fibroin powder in an aqueous solution to blend with an aqueous solution of polyvinyl alcohol, and forming the blending solution into a fiber or a membrane; and finally, making the fiber or the membrane into a required three-dimensional shape to obtain the cell culture vector. The preparation method has the advantage of easy industrial application; and cells cultured by the obtained cell culture vector have the characteristics of high density, high yield and low cost. The cell culture vector prepared by the method is used for culturing target cells, and the cultured target cells can be used for gene expression and preparation of genomic medicaments. In addition, the cell culture vector also can be used for a tissue engineering bracket.
Description
Technical field
The present invention relates to a kind of method for preparing cell culture vector, specifically refer to utilize nano level silk fibroin particle (or segment) and polyvinyl alcohol to prepare the method for cell culture vector, the cell culture vector of present method preparation is used to cultivate target cell, its target cell of cultivating out can be used for genetic expression, the preparation genomic medicine, this cell culture vector also can be used for tissue engineering bracket, belongs to biomaterial, biochemical engineering and cell engineering field.
Background technology
Silk fibroin can be in a variety of forms (as film, film, reticulated structure, spongy etc.) sticking, breeding and breaking up at external sustenticular cell, promote the reparation of tissue in the body, especially three-dimensional silk fibroin support has enlarged the application of fibroin in cellular system engineering, becomes in the organizational project one of the most promising biomaterial.
The manufacture method of genomic medicine is that the method for goal gene with the DNA reorganization is connected on the carrier, then carrier is imported target cell, make goal gene in target cell, obtain expressing, at last the target protein matter of expressing is purified and make preparation, thereby become protein medicine or vaccine.In this production process, target cell is absolutely necessary and is the main determining factor that genomic medicine is produced the Chinese traditional medicine cost, and demand is very big.Target cell can be microorganism, mammalian cell or tissue target cell.Chinese hamster ovary cell (Chinese hamster ovary celI) as mammalian cell is prepared by the more genomic medicine that is applied in.The type of existing cultured cell in vitro has attaching type cell and suspension type cell, its cultural method mainly contains former foster, the cultivation of going down to posterity, the suspension culture and other are as microcarrier, micro-capsule, tubular fibre method etc. of being commissioned to train, and the cell matrix materials that adopts mostly is synthesized polymer material, its biocompatibility is relatively poor, and synthesized polymer material also may residual monomer occur and have certain toxicity.It has problems such as surface hydrophobicity or surface irregularity simultaneously, and its cultured cells density is also lower, the receipts amount is few, if adopt once more surface modification to improve surface hydrophilic performance, the more high again shortcoming of its cost.Based on cell sterility culture requirement, the realization of cheap disposable cultivation carrier also will be cultivated carrier and make contributions for the cost, the optimization that reduce genomic medicine.
Summary of the invention
At the prior art above shortcomings, the purpose of this invention is to provide a kind of method for preparing cell culture vector, the easy industrial application of this preparation method, the cell that the cell culture vector that obtains is turned out has that density is big, the receipts amount greatly, characteristics cheaply.
The object of the present invention is achieved like this: a kind of method for preparing cell culture vector, and preparation process is:
(1) at first prepares nano level silk fibroin powder;
(2) nano level silk fibroin powder is dispersed or dissolved in the aqueous solution, realize blend with polyvinyl alcohol water solution, blend nano level silk fibroin and polyvinyl alcohol mass ratio are 1: 1～9, evenly concentrate after the blend, the concentration that makes nano level silk fibroin and polyvinyl alcohol total mass is shaped to blended liquid fiber or diaphragm again between 5% to 10%;
(3) fiber or the diaphragm that at last (2) step was obtained made required three-dimensional shape, promptly becomes cell culture vector.
This preparation method adopts nano level silk fibroin particle (or segment) to be dispersed or dissolved in the pure water, again with the polyvinyl alcohol water solution blend, ultra tiny being embedded in the PVA (PVOH) FILM of nano level silk fibroin particle (or segment) is made.Method is simple, and preparation cost is low, but suitability for industrialized production.Obtain the cell culture vector product with this preparation method, possess high-tech, low cost, high quality, field of medicaments has competitive power at home.Product that this preparation method obtains, constructed a biomimetic culture system of cell cultures, analog cell outer room matter, be tridimensional network, the duct is many and be evenly distributed, and links up mutually between the duct, there be not " blind area ", contact with intercellular " physiological " to strengthen cell, cell fully is exposed in the nutrient solution in whole culturing process, thus and one way or another growth increase cell stand density.Because it is network structure, can prevent that also iuntercellular is piled into piece and influences its merisis.These goods (1), can be used for the production of genetically engineered monoclonal antibody medicine; (2), can cultivate at engineered eukaryotic cell; (3), in conjunction with its degradation property, can be used for tissue engineering bracket.Because the requirement of cell sterile culture, this cell culture vector can be a kind of disposable articles for use that make, the market requirement that comprises all bio-pharmaceuticals enterprises, medical institutions, research institute is bigger, as a kind of high-tech product, will bring very big economic benefit.
Embodiment
Below the present invention is further illustrated.
Method for preparing cell culture vector of the present invention, its preparation process be,
(1) at first from silk, cocoon shell (available waste material), makes pure fibroin fiber through dewaxing matter, decarburization hydrate, sericin removal, and by 50～60%NaSCN solution dissolving dialysis, re-use Chymetin, trypsinase, neutral protease and carry out enzymolysis and get the nano level silk fibroin solution, lyophilize gets nano level silk fibroin powder; The prior art that is prepared as owing to nano level silk fibroin powder, do not give unnecessary details at this, detailed method can be called " a kind of preparation method of nano natural silk protein powder " referring to the name that the applicant applies for before this, application number is the patent of invention of " 200710092969.6 ".
(2) nano level silk fibroin powder is dissolved (or dispersion) in the aqueous solution, with the polyvinyl alcohol water solution blend, blend nano level silk fibroin/polyvinyl alcohol mass ratio is 1: 1～9 (once to adopt 10/90 among the embodiment, 20/80,30/70,40/60,50/50 equal proportion), evenly concentrate after the blend, the concentration that makes nano level silk fibroin and polyvinyl alcohol total mass is between 5% to 10%, again with blended liquid moulding (fiber or diaphragm); The present invention does not require the concentration of silk fibroin water solution and polyvinyl alcohol water solution, and concentration is big or little without any influence; And follow-up concentrating do not have any influence to solution itself yet, and be just influential to the thickness of last film forming speed, film.If do not concentrate, solution is too rare, add a lot of amounts and just can make film have certain thickness, and film formation time is oversize.Require when therefore the present invention is in the end film forming to be concentrated between 5% to 10%, film forming is very fast like this.
(3) nano level silk fibroin/polyvinyl alcohol fiber or diaphragm are processed into various three-dimensional shapes (as " worker " font etc.) as required, promptly become cell culture vector.
What adopt among the present invention is nano level silk fibroin particle (or segment), can be partly dissolved, part is scattered in the water, ultra tinyly during with the polyvinyl alcohol water solution blend is embedded in (or in fiber) in the film, film surfacing, smooth, do not influence its biocompatibility, be easy to the cell growth.And after micron order silk fibroin particle (or segment) and the polyvinyl alcohol film forming, membrane surface is coarse, and biocompatibility is poor, and cell growth condition is poor.
What the present invention adopted is the blend of nano level silk fibroin water solution and polyvinyl alcohol water solution, has avoided organic solvent or the small molecules organic monomer toxicity due to residual, is beneficial to the cell growth.
The diaphragm or the fiber of the present invention preparation be smooth, soft, do not break because of external force, can arbitrarily cut out the three-dimensional rack of producing prefabricated form, can further be applied to tissue engineering bracket.
Claims (1)
1. method for preparing cell culture vector is characterized in that: preparation process is,
(1) at first prepares nano level silk fibroin powder;
(2) nano level silk fibroin powder is dissolved or dispersed in the aqueous solution, realize blend with polyvinyl alcohol water solution, blend nano level silk fibroin and polyvinyl alcohol mass ratio are 1: 1～9, evenly concentrate after the blend, the concentration that makes nano level silk fibroin and polyvinyl alcohol total mass is shaped to blended liquid fiber or diaphragm again between 5% to 10%;
(3) fiber or the diaphragm that at last (2) step was obtained made required three-dimensional shape, promptly becomes cell culture vector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101043978A CN101603007B (en) | 2009-07-21 | 2009-07-21 | Method for preparing cell culture vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101043978A CN101603007B (en) | 2009-07-21 | 2009-07-21 | Method for preparing cell culture vector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101603007A CN101603007A (en) | 2009-12-16 |
| CN101603007B true CN101603007B (en) | 2011-08-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2009101043978A Expired - Fee Related CN101603007B (en) | 2009-07-21 | 2009-07-21 | Method for preparing cell culture vector |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304259B (en) * | 2011-07-18 | 2012-12-19 | 重庆理工大学 | Preparation method of fibroin blending material with gradually degrading performance |
| CN109266608B (en) * | 2018-08-30 | 2021-12-10 | 宁波大学 | Method for regulating and controlling growth of nerve cells by using polyvinyl alcohol/silk fibroin hydrogel |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168763A (en) * | 2007-11-09 | 2008-04-30 | 重庆工学院 | Method for preparing nano natural silk protein powder |
| CN101187089A (en) * | 2007-11-22 | 2008-05-28 | 苏州大学 | Silk fibroin and polyvinyl alcohol blending antibacterial nanometer fiber and its preparation method |
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2009
- 2009-07-21 CN CN2009101043978A patent/CN101603007B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168763A (en) * | 2007-11-09 | 2008-04-30 | 重庆工学院 | Method for preparing nano natural silk protein powder |
| CN101187089A (en) * | 2007-11-22 | 2008-05-28 | 苏州大学 | Silk fibroin and polyvinyl alcohol blending antibacterial nanometer fiber and its preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 刘芸等.再生丝素蛋白/聚乙烯醇共混纳米纤维的静电纺丝研究.《合成纤维》.2006,(第8期),13－19. * |
| 陈忠敏等.再生蚕丝丝素蛋白纳米颗粒的制备及抗菌性.《纺织学报》.2008,(第7期),17-20. * |
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| CN101603007A (en) | 2009-12-16 |
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Owner name: CHONGQING HUISEN CHIHENG MEDICAL EQUIPMENT CO., LT Free format text: FORMER OWNER: CHONGQING UNIVERSITY OF TECHNOLOGY Effective date: 20150716 |
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Effective date of registration: 20150716 Address after: 404000 Lin Yuan, village 192, cave village, Shapingba District, Chongqing Patentee after: Chongqing Huisen Chiheng Medical Equipment Co., Ltd. Address before: No. 69 lijiatuo Chongqing District of Banan City Road 400054 red Patentee before: Chongqing University of Technology |
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