CN103525801B - A kind of cell stationary phase based on microcarrier cell and preparation method thereof - Google Patents
A kind of cell stationary phase based on microcarrier cell and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of cell stationary phase based on microcarrier cell and preparation method thereof, by cell cultures in micro-carrier surface, after paraformaldehyde is fixing, using microcarrier cultured cells together with supporting the microcarrier of its growth as stationary phase, wet method dress Column preparation cell chromatographic column.Application the method prepares stationary phase, simple to operate, reaction conditions is gentle, and be easy to amplify, this stationary phase has the characteristic of cell, and maintains the original fundamental characteristics of cell, and the acceptor of medicine also keeps basic space structure, the cell stationary phase applying this legal system standby can be applicable to drug screening, improves the specificity of drug screening.
Description
Technical field
The invention belongs to membrane flexibility technical field, relate to a kind of cell stationary phase based on microcarrier and preparation method thereof.
Background technology
Cellular membrane chromatography (CMC) is a kind of biocompatible chromatography that He Lang punching professor and seminar thereof propose in 1996, the screening being applicable to activeconstituents in traditional Chinese medicine complex system be separated, the method take silica gel as carrier, packing after cytolemma is mixed with silica gel, prepare membrane flexibility post, in conjunction with the activeconstituents in high performance liquid chromatography screening Chinese medicinal materials, the target spot of drug screening is the acceptor on cytolemma, membrane flexibility can directly identify from the complicated ingredient of traditional Chinese medicine extraction can with the activeconstituents of drug target effect, it is a kind of screening method rapidly and efficiently.But, also there are some defects in membrane flexibility, CMC method be natural biology film (histocyte), the density of membrane receptor is less, and post effect is lower, and the chromatographic column life-span is usually shorter, its circulation ratio be separated and precision can not be guaranteed, natural biology film has various active albumen, and medicine may be combined with multiple acceptor or channel protein, still can not completely in analogue body in complex environment and body other system medicine is played to the impact of drug action.
Microcarrier refers to that diameter is 60-250 μm, the microballoon of adherent type Growth of Cells can be applicable to, cell cultures uses ion-exchange gel as carrier the earliest, start from the latter stage sixties 19th century, mild agitation can suspend in the medium, this microcarrier, with electric charge, is beneficial to cell attachment and carries out growth and breeding on carrier.When carrier is in suspended state, so both can greatly increase cell attachment area, and make again cell fully can contact with substratum, and then be conducive to the growth of cell, therefore can reach the object of mass propgation cell.Microcarrier is selected to be beneficial to cell attachment growth, nontoxicity is principle again, but there is many shortcomings as carrier in ion-exchange gel, as toxic to cell to a certain extent, also have certain influence to the pH of substratum, thus improve carrier in recent years, the carrier used at present mainly contains three types, they are all the macromole be cross-linked with not isoplastic dextran, are respectively I, II, III type Cytopore (Cytodex1,2,3).Due to microcarrier with group different, just determine the difference between them, I type carrier is whole belt carrier positive charge, and only surface is with positive charge for II type, and III type is without electric charge, and its outside surface is wrapped up by collagen layer, is suitable for cell attachment and growth equally.The macroporous structure of Cytopore microcarrier promotes that cell grows in microballoon, and aperture provides nutritive ingredient as much as possible.This feature can help the Growth of Cells needing high cycling rate and high nutrition.Microcarrier has obvious advantage than ion-exchange gel: 1. size and surface properties Growth of Cells preferably; 2. there is certain transparency, be convenient to microscopic examination; 3. nontoxicity, is suitable for various types of cells apposition growth.
Microcarrier Culture Techniques is a kind of emerging mass cell culture technique, there is specific surface area large, have the advantage such as suspension culture and adherent culture concurrently, amplify easily, a large amount of cells can be obtained within a short period of time at microsphere surface culturing cell, and passage only needs to add new microcarrier, substantially can avoid the damage that cell is subject in trysinization process, therefore microcarrier culturing cell technology is very convenient and significant.
Summary of the invention
The problem that the present invention solves is to provide a kind of cell stationary phase based on microcarrier cell and preparation method thereof, and this stationary phase has the characteristic of single cell, and maintains the original fundamental characteristics of cell, and the acceptor of medicine also keeps basic space structure.
The present invention is achieved through the following technical solutions:
Based on a cell stationary phase for microcarrier cell, it is characterized in that, comprise chromatographic column, in chromatographic column, be filled with the microcarrier cell as chromatographic stationary phases; Described microcarrier cell is by the cell of adherent growth in micro-carrier surface, is fixed on micro-carrier surface obtains by cross linked chain.
Described microcarrier is microcarrier Cytodex prepared by dextrane gel, described cell is cytolemma is expressed the cell having the acceptor that can be combined with medicine, described cross linked chain utilizes between the protein terminal group in paraformaldehyde and cell to form cross linked chain, and be fixed on micro-carrier surface.
Described microcarrier cell is loaded in chromatographic column by wet method.
Based on a preparation method for the cell stationary phase of microcarrier cell, comprise following operation:
1) microcarrier is placed in the culture vessel of silication, sterilizing after activation, then adds DMEM substratum and spends the night, and inhales before use and abandons former substratum, change 37 DEG C of warm DMEM substratum, make microcarrier suspension;
2) microcarrier suspension is inserted in culture vessel, inoculating cell suspension, then add DMEM nutrient solution; Culture vessel is stood on CO
2cultivate 2 ~ 3h in incubator, remove and rotate shake 1 ~ 2min; Within every 1 ~ 2 hour afterwards, shake once, every 12 ~ 24h changes a subculture;
3) be grown on after microcarrier until cell attachment, get and be in the cell suspension of logarithmic phase adherent growth on microcarrier, add the paraformaldehyde solution of mass concentration 4%, after room temperature fixes 15 ~ 30min, cell is fixed on microcarrier by cross linked chain show, obtains microcarrier cell;
4) using microcarrier cell as chromatographic stationary phases, wet method loads in chromatographic column, obtains the cell chromatographic column for drug screening.
Described microcarrier is microcarrier Cytodex prepared by dextrane gel, and diameter is 100 ~ 250 μm, and its activation is: container microcarrier being placed in silication, after rushing liquid washing, changes fresh PBS soaked overnight with the PBS of PH=7.4;
Sterilizing after activation is autoclaving, renews fresh substratum soaked overnight after sterilizing, inhales before use and abandons former substratum, change 37 DEG C of warm DMEM substratum, and the content adjusting microcarrier is 10 ~ 20mg/ml.
The G418 of 0.3mg/l is also added with in described DMEM substratum, and the FBS of volumetric concentration 10%.
The silication of described container is: cleaned by container and dry, to be soaked in the dimethyl silicone oil-ethyl acetate solution of mass concentration 5% after uniform silicidation 4h, picks up empty dry, with ultrapure washing 3 times and soaked overnight, and dry for standby.
Described cell inoculation is 1 × 10 by cell density
5~ 1 × 10
7the cell suspension of individual/ml, draw 2 ~ 5ml and be inoculated in the culturing bottle adding microcarrier suspension, then add the DMEM substratum of 10 ~ 15ml, final volume is 15 ~ 20ml.
Described paraformaldehyde solution PBS prepares, and after adding paraformaldehyde solution, forms cross linked chain, the cell of adherent growth on microcarrier is fixed on micro-carrier surface between the protein terminal group in paraformaldehyde and cell.
The described cell stationary phase based on microcarrier cell can retain the application in medicine at screening vector cell.
Compared with prior art, the present invention has following useful technique effect:
Cell stationary phase based on microcarrier cell provided by the invention and preparation method thereof, the cytolemma in CMC method is replaced with the cell of adherent growth, using microcarrier cultured cells together with supporting the microcarrier of its growth as stationary phase, eliminate the process originally preparing cytolemma and silica gel combination, this stationary phase has the characteristic of single cell, and maintaining the original fundamental characteristics of cell, the acceptor of medicine also keeps basic space structure.Prepared cell stationary phase can improve the specificity of drug screening, has great importance to illustrating drug mechanism; Can be applicable to the medicine that screening vector cell can retain.
Cell stationary phase based on microcarrier cell provided by the invention and preparation method thereof, by cell cultures in micro-carrier surface, utilizes microcarrier culturing cell, easily realize the immobilization of cell, specific surface area is comparatively large, for cell provides suitable growing space, amplifies easily; And microcarrier is suitable for the growth of attached cell on its surface, and be transparently convenient to the state examining under a microscope cell.
Cell stationary phase based on microcarrier cell provided by the invention and preparation method thereof, after paraformaldehyde is fixing, using microcarrier cultured cells together with supporting the microcarrier of its growth as stationary phase, wet method dress Column preparation cell chromatographic column.Cell after fixing maintains Normocellular fundamental characteristics and protein structure, more can environment effectively in analogue body.Cell after fixing is loaded preparative chromatography post as stationary phase can the interaction of more deep drugs and acceptor.And it is simple to operate, reaction conditions is gentle, and is easy to amplify.
Accompanying drawing explanation
The growth conditions of Fig. 1 basis of microscopic observation cell on Cytodex; Wherein A is empty carrier Cytodex, B is FGFR4 cell;
The cell stationary phase of Fig. 2 electricity Microscopic observation; A is empty carrier Cytodex, B is FGFR4 cell;
Fig. 3 Schuttgelb retains color atlas on immobilization ECV304 cell chromatographic stationary phases; A is Schuttgelb (blank microcarrier), and b is Schuttgelb (the long microcarrier having cell), and c is methyl alcohol (in contrast); Peak 1 is Schuttgelb peak.
Embodiment
Cell stationary phase based on microcarrier cell provided by the invention, comprises chromatographic column, is filled with the microcarrier cell as chromatographic stationary phases in chromatographic column; Described microcarrier cell is by the cell of adherent growth in micro-carrier surface, is fixed on micro-carrier surface obtains by cross linked chain.The cytolemma in CMC method is replaced like this with the cell of adherent growth, using microcarrier cultured cells together with supporting the microcarrier of its growth as stationary phase, eliminate the process originally preparing cytolemma and silica gel combination, this stationary phase has the characteristic of single cell, and maintaining the original fundamental characteristics of cell, the acceptor of medicine also keeps basic space structure.Applying the specificity that the standby cell stationary phase of this legal system can improve drug screening, having great importance to illustrating drug mechanism.
The preparation method of the cell stationary phase based on microcarrier cell provided, comprises following operation:
1) microcarrier is placed in the culture vessel of silication, sterilizing after activation, then adds DMEM substratum and spends the night, and inhales before use and abandons former substratum, change 37 DEG C of warm DMEM substratum, make microcarrier suspension;
2) microcarrier suspension is inserted in culture vessel, inoculating cell suspension, then add DMEM nutrient solution; By culture vessel in standing on CO
2cultivate 2 ~ 3h in incubator, remove and rotate shake 1 ~ 2min; Within every 1 ~ 2 hour afterwards, shake once, every 12 ~ 24h changes a subculture;
3) be grown on after microcarrier until cell attachment, get and be in the cell suspension of logarithmic phase adherent growth on microcarrier, add the paraformaldehyde solution of mass concentration 4%, after room temperature fixes 15 ~ 30min, cell is fixed on microcarrier by cross linked chain show, obtains microcarrier cell;
4) using microcarrier cell as chromatographic stationary phases, wet method loads in chromatographic column, obtains the cell chromatographic column for drug screening.
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Embodiment 1
Based on the preparation of the FGFR4 cell chromatographic column of microcarrier cell, comprise following operation:
1) process of microcarrier
The pre-treatment of glassware: all glasswares used for microcarrier pre-treatment are cleaned and dries, to be soaked in the dimethyl silicone oil-ethyl acetate solution of 5% after uniform silicidation 4h, to pick up empty dry, with ultrapure washing 3 times and soaked overnight, dry for standby.
The process of microcarrier Cytodex: take the beaker that 0.2g microcarrier Cytodex is placed in silication, with the PBS(PH=7.4 of 20mL without calcium magnesium) wash 2 times, renew fresh PBS soaked overnight, autoclaving, inhales and abandons PBS, renew fresh DMEM substratum (containing the G418 of 0.3mg/l, the FBS of 10%) soaked overnight, inhale before use and abandon former substratum, change 37 DEG C of warm substratum appropriate, and the content adjusting culture in glassware base is 20mg/ml.
The process on culturing bottle surface: by for subsequent use after culturing bottle autoclaving good for silication, use substratum rinse before use.
2) cell inoculation
1. the microcarrier suspension 5mL handled well is added in the culturing bottle handled well.
2. get the good fibroblast growth factor receptor of growth conditions (FGFR4) cell, become cell suspension with tryptic digestion, counting, adjustment cell density is 1 × 10
5individual/ml, absorption 5ml is inoculated into and adds in the culturing bottle of microcarrier, then adds the substratum of 15ml.
3. culturing bottle is stood on CO
2in incubator after 2h, take out and rotate shake 1min, within every 2 hours afterwards, shake once, every 24h changes a subculture.
3) cell cultivation process is observed
In the process that cell microcarrier is cultivated, carry out continuing, observe dynamically, and record the change occurred in cell growth process and comprise cellular form, the change of quantity and signaling situation.
The growth conditions of basis of microscopic observation cell on Cytodex as shown in Figure 1, sees cell well-grown on Cytodex as seen.
4) micro-carrier surface cell is fixing
Get and be in logarithmic phase adherent growth in the FGFR4 cell of micro-carrier surface, room temperature fixes 15min, obtains cell stationary phase.
The cell stationary phase of electricity Microscopic observation as shown in Figure 2, can see that cell is all fixed on the surface of microcarrier.
5) preparation of chromatographic column
Get the microcarrier cell that fixes as chromatographic stationary phases, wet method loads in previously prepared good chromatographic column, obtains FGFR4 cell chromatographic column.
Embodiment 2
Difference from Example 1 is, replaces fibroblastic growth acceptor (FGFR4) cell with human umbilical vein endothelial cell (ECV304), ECV304 cell DMEM culture medium culturing, and other operations prepare ECV304 cell chromatographic column according to embodiment 1.
Further, with the prepared reservation of ECV304 chromatogram column analysis Schuttgelb in ECV304 cell chromatogram, specific as follows:
The preparation of Schuttgelb reference substance solution: get Schuttgelb appropriate, add dissolve with methanol and be settled to 10ml, obtaining concentration is 0.5mg/ml.
Moving phase: 0.8%NaH
2pO
4-0.974%Na
2hPO
4(20:80), wherein every 100ml adds NaCl0.432g;
Chromatographic column: 10mm × 1mm;
Flow velocity: 0.2ml/min; Determined wavelength: 254nm.
As shown in Figure 3, wherein a is Schuttgelb (blank microcarrier) to detected result, and b is Schuttgelb (the long microcarrier having cell), and c is methyl alcohol (in contrast); Peak 1 is Schuttgelb peak.Can see that prepared ECV304 chromatographic column can have certain reservation to Schuttgelb, thus occur crest after its wash-out.
Above-mentioned detection shows the cell stationary phase based on microcarrier cell prepared by the present invention, has the characteristic of single cell, and maintains the original fundamental characteristics of cell, and the acceptor of medicine also keeps basic space structure.The medicine that screening vector cell can retain can be applied to.
Claims (2)
1. based on a cell stationary phase for microcarrier cell, it is characterized in that, comprise chromatographic column, in chromatographic column, be filled with the microcarrier cell as chromatographic stationary phases; Described microcarrier cell is by the cell of adherent growth in micro-carrier surface, is fixed on micro-carrier surface obtains by cross linked chain; Described microcarrier cell loads in chromatographic column by wet method;
Described microcarrier is microcarrier Cytodex prepared by dextrane gel, described cell is cytolemma is expressed the cell having the acceptor that can be combined with medicine, described cross linked chain utilizes between the protein terminal group in paraformaldehyde and cell to form cross linked chain, and be fixed on micro-carrier surface;
Described microcarrier cell is by following operation:
1) microcarrier is placed in the culture vessel of silication, sterilizing after activation, then adds DMEM substratum and spends the night, and inhales before use and abandons former substratum, change 37 DEG C of warm DMEM substratum, make microcarrier suspension;
2) microcarrier suspension is inserted in culture vessel, inoculating cell suspension, then add DMEM nutrient solution; Culture vessel is stood on CO
2cultivate 2 ~ 3h in incubator, remove and rotate shake 1 ~ 2min; Within every 1 ~ 2 hour afterwards, shake once, every 12 ~ 24h changes a subculture;
3) be grown on after microcarrier until cell attachment, get and be in the cell suspension of logarithmic phase adherent growth on microcarrier, add the paraformaldehyde solution of mass concentration 4%, after room temperature fixes 15 ~ 30min, cell is fixed on micro-carrier surface by cross linked chain, obtains microcarrier cell.
2. the cell stationary phase based on microcarrier cell according to claim 1 can retain the application in medicine at screening vector cell.
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| Vero 细胞在不同微载体固定化培养中的生长和代谢;刘红 等;《现代生物医学进展》;20121231;第12卷(第36期);7002页第 1.4节，第7003页左栏第一段，第7004页左栏第2-3段，图1E-G * |
| 用高表达EGFR 细胞膜色谱-HPLC/MS 联用快速筛选独活中抗EGFR 活性成分;王嗣岑 等;《中国科学: 化学》;20121231;第751页第2.3节，标题，摘要 * |
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