CN102329728A - Chitosan/arginine-glycine-aspartic acid (RGD) three-dimensional porous microcarrier and preparation method and application thereof - Google Patents
Chitosan/arginine-glycine-aspartic acid (RGD) three-dimensional porous microcarrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a chitosan/arginine-glycine-aspartic acid (RGD) three-dimensional porous microcarrier, a preparation method thereof and application of the chitosan/RGD three-dimensional porous microcarrier to the culture of liver cells. The chitosan/RGD three-dimensional porous microcarrier is prepared from chitosan and RGD. The preparation method comprises the following steps of: allowing a mixed solution of the chitosan and the RGD to pass through a high-voltage pulse microcapsule forming instrument, dripping into a sodium polyphosphate solution in which the RGD is dissolved under the action of high pressure so as to form microspheres, preparing holes through freeze-drying, and finally forming the chitosan/RGD porous microcarrier with a three-dimensional structure. The aperture of the chitosan/RGD three-dimensional porous microcarrier is 20 to 50mm, the particle size is 400 to 900mm, and the porosity is 91 percent; meanwhile, by the adding of the RGD, the biocompatibility of the chitosan is effectively improved, and the liver cells can grow on the microcarrier in a high-density and high-activity way. The process is simple and easy to operate.
Description
Technical field
The invention belongs to the porous material field of material preparation, relate to three-dimensional porous microcarrier of a kind of chitosan/RGD and preparation method thereof and the application aspect liver cell culture.
Background technology
At present, no matter be on laboratory or the industrial production, employed cell cultures microcarrier mainly is the carrier of solid Cytodex series, and belongs to individual layer monolayer culture mode.And in vivo; The growth and development process of tissue and cell is under the interior envrionment conditions of 3 D stereo, to carry out; Mutual signal transmission between cell and cell matrix and the cell has material impact for the propagation of reconciling cell, differentiation; Conventional cultured in monolayer in vitro method can not provide organizes normal growth to grow required three-dimensional environment condition, thereby prepares three-dimensional microcarrier and just have crucial meaning.
Chitosan is a kind of natural polymer inertia positive charge polysaccharide, especially is with a wide range of applications as the organizational project embedded material at biomedical aspect.And the tensile strength of sodium polyphosphate/chitosan composite package is significantly higher than pure chistosan film.Liang Kai [1] etc. places certain density Trisodium trimetaphosphate solution to carry out crosslinking reaction abundant swollen chitosan film and prepares Trisodium trimetaphosphate/chitosan complex film; When the concentration of Trisodium trimetaphosphate is 0.11mol/L; The dry state tensile strength of cross linking membrane reaches 83.74MPa, compares with pure chistosan film and has improved 44.81%.
What used more present microcarrier liver cell culture aspect is solid microcarrier; Can only carry out monolayer culture by pair cell, the cultured cells amount is little, and the shape of the microcarrier that has is not spherical; Then the specific surface area of microcarrier is little, and the amount of corresponding cultured cells also can reduce.
Reference:
[1] Liang Kai, Du Yumin, Li Yan opens graceful. the preparation of Trisodium trimetaphosphate crosslinked chitosan film and performance study thereof [J]. Journal of Analytical Science, 2008,24 (2): 136-140.
Summary of the invention
The objective of the invention is to provide in order to solve above-mentioned technical problem three-dimensional porous microcarrier of a kind of chitosan/RGD and preparation method thereof with its in the application aspect the liver cell culture.
Technical scheme of the present invention
The three-dimensional porous microcarrier of a kind of chitosan/RGD; Raw material comprises chitosan, it is characterized in that described raw material also comprises RGD, after soon chitosan and RGD form microballoon through high-voltage pulse microcapsule forming instrument; Pass through the lyophilize drilling again; The water-setting that is about in the microballoon is formed tiny ice crystal, makes the ice crystal distillation form small hole, the final three-dimensional porous microcarrier of chitosan/RGD with three-dimensional structure that forms;
The molecular weight of wherein said chitosan is 5.63 * 10
6, deacetylation is 91%; RGD is l-arginine, glycocoll, the formed tripeptides of aspartic acid.
The preparation method of the above-mentioned three-dimensional porous microcarrier of a kind of chitosan/RGD comprises the steps:
(1), the preparation of RGD/ chitosan-acetic acid solution and RGD/ polyphosphoric acid sodium solution
The preparation of RGD/ chitosan-acetic acid solution
Is RGD with RGD and described chitosan-acetic acid solution by the mass ratio of the chitosan in RGD and the chitosan-acetic acid solution: the chitosan in the chitosan-acetic acid solution is that 2.0g:2.5g mixes and promptly gets the RGD/ chitosan-acetic acid solution;
Wherein chitosan and concentration be that the aqueous acetic acid of 1% (V/V) calculates by mass volume ratio are chitosan in the chitosan-acetic acid solution: aqueous acetic acid is 2.5g:100ml;
The preparation of RGD/ polyphosphoric acid sodium water solution
Is RGD with RGD and polyphosphoric acid sodium water solution by the mass ratio of the sodium polyphosphate in RGD and the polyphosphoric acid sodium solution: sodium polyphosphate is that 2.0:4.5 mixes and promptly gets RGD/ polyphosphoric acid sodium water solution;
In the polyphosphoric acid sodium water solution wherein, press mass volume ratio and calculate, be i.e. sodium polyphosphate: water is 4.5g:100ml;
(2), high pressure electrostatic pulse balling-up
The RGD/ chitosan-acetic acid solution of the 3. gained in the step (1) and RGD/ polyphosphoric acid sodium water solution are carried out the high pressure electrostatic pulse prepare microballoon in high pressure electrostatic microcapsule forming instrument; The microballoon of gained soaks 4h in RGD/ polyphosphoric acid sodium water solution after; Use the washed with de-ionized water microballoon to be neutrality, obtain white solid microsphere until solution;
High pressure electrostatic pulse process control voltage is 39-50kv, and fltting speed is 90mm/h, and pulsewidth is 7ms, and frequency is 90Hz, and the liquid level distance is 20mm;
(3), lyophilize prepares the three-dimensional porous microcarrier of chitosan/RGD
The white solid microsphere of step (2) gained is put into Freeze Drying Equipment carry out lyophilize and come drilling, freezing dry process control pre-freeze temperature is-80 ℃, and the pre-freeze time is 1h; The temperature of primary drying is-30 ℃, and the time is 9-10h; The temperature of redrying is 10 ℃, and the time is 1.5-2h, the final three-dimensional porous microcarrier of chitosan/RGD that gets.
The three-dimensional porous microcarrier of the chitosan/RGD of above-mentioned gained is used for the liver cell adherent culture, and its cultural method comprises the steps:
(1), the pre-treatment of the three-dimensional porous microcarrier of chitosan/RGD
It is in 70% the aqueous ethanolic solution that the three-dimensional porous microcarrier of chitosan/RGD is immersed concentration, in 4 ℃ temperature environment, keeps 6h, leaches the chitosan microcarrier then; And with washed with de-ionized water 3 times; Again with the PBS damping fluid drip washing of the pH=7 for preparing 1 time, again the chitosan microcarrier is immersed in the PBS damping fluid afterwards, keep 12h in 4 ℃ the temperature environment; Leach the three-dimensional porous microcarrier microcarrier of chitosan/RGD once more, and clean repeatedly 3 times with the PBS damping fluid;
Described PBS damping fluid is promptly got potassium primary phosphate 0.68 g and is added 0.1mol/L sodium hydroxide solution 29.1ml, is diluted with water to 100 ml, promptly gets;
(2), hepatocellular inoculation and cultivation
On 48 orifice plates, press 5 * 10
6The cell inoculation density in every hole is inoculated in step (1) in the three-dimensional porous microcarrier of pretreated chitosan/RGD with human liver cell; 48 orifice plates are jiggled to be transferred to temperature behind the 10min be 37 ℃; Gas concentration lwevel is 5%, humidity be stick in 100% the CO2gas incubator cultivate 4h after, on the three-dimensional porous microcarrier of chitosan/RGD, do not stick liver cell closely to remove with the three-dimensional porous microcarrier of RPMI1640 nutrient solution flushing chitosan/RGD that contains serum; To stick the three-dimensional porous microcarrier of hepatocellular chitosan/RGD again and be transferred to 24 well culture plates; The RPMI1640 nutrient solution that every hole adding 1ml contains serum is cultivated, and cultivates after 4 hours, promptly gets the nutrient solution that contains three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body; Changed in every afterwards 1-2 days contain serum the RPMI1640 nutrient solution once; Cultivate the nutrient solution that will contain three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body after 4 days again and carry out spinning, the control centrifugal rotational speed is 1000r/min, time 5min; Get upper solution, can obtain high-density, highly active liver cell;
The described preparation that contains the RPMI1640 nutrient solution of serum; Being about to serum is serum with RPMI1640 by mass ratio: RPMI1640 is after 1:9 mixes; Add microbiotic penicillium mould and Streptomycin sulphate, the ultimate density of the two is 100U/ml, regulates pH between 7.0-7.2 with the PBS buffered soln described in the step (1); Filtration sterilization then, 4 ℃ of preservations are subsequent use.
Technique effect of the present invention
The three-dimensional porous microcarrier of a kind of chitosan/RGD of the present invention; Owing to covering the RGD tripeptide sequence of biologically active in its preparation process on the chitosan microball surface through high-voltage pulse, form the functional composite bed of material type extracellular matrix, reach the purpose of the affine improved performance of pair cell; Improve the biocompatibility of chitosan; And adopt the freeze-drying drilling in the preparation process, do pore-creating agent with water, pair cell does not have toxicity; Make when this carrier is used for liver cell culture that liver cell can high-density, highly active growth on microcarrier.And the preparation method of this carrier is simple to operate, and controllability is strong, and its aperture is 20-50mm, and particle diameter is 400-900mm, and porosity is 91%.
In addition, the three-dimensional porous microcarrier of of the present invention a kind of chitosan/RGD prepares in the process that to adopt sodium polyphosphate be that linking agent prepares microballoon, thereby has strengthened the mechanical property of microballoon.Simultaneously,, strengthen the electronegativity of solution, be easy to the deposition of microballoon, and can strengthen the mechanical property of microballoon owing in chitosan and polyphosphoric acid sodium solution, all add RGD.
Description of drawings
Fig. 1 is the ESEM picture of the three-dimensional porous microcarrier of chitosan/RGD of the present invention;
Fig. 2 be among the present invention human liver cell at the fluorescence labelling photo on microcarrier surface.
Embodiment
Below through embodiment and combine accompanying drawing that the present invention is further specified, but do not limit the present invention.
The human liver cell that the present invention is used is that the normal liver cell is L-02, Shanghai Inst. of Cytobiology, Chinese Academy of Sciences;
The RPMI1640 nutrient solution that the present invention is used is available from Beijing Suo Laibao Science and Technology Ltd.;
Used serum is foetal calf serum, available from Hangzhou SIJIQING ltd;
Other various raw materials or reagent are analytical pure, from Shanghai traditional Chinese medicines group.
The Freeze Drying Equipment that the present invention is used, model are ES, and the instrument name is an AdVantage PLUS frozen type drying machine, and manufacturer is a U.S. Virtis company;
The high pressure electrostatic microcapsule forming instrument that the present invention is used, fltting speed is 1-99mm/h, Shanghai University of Science and Technology;
Used whizzer is that the TDL-60B type is desk-top, Eppendorf company.
Embodiment 1
The three-dimensional porous microcarrier of a kind of chitosan/RGD; Its raw material comprises chitosan; It is characterized in that described raw material also comprises RGD; After soon chitosan and RGD mixing solutions will form microballoon through high-voltage pulse microcapsule forming instrument, pass through the lyophilize drilling again, form the three-dimensional porous microcarrier of chitosan/RGD with three-dimensional structure.
The preparation method of the above-mentioned three-dimensional porous microcarrier of a kind of chitosan/RGD comprises the steps:
(1), the preparation of RGD/ chitosan-acetic acid solution and RGD/ polyphosphoric acid sodium solution
1., preparation 2.5% (W/V) chitosan acid solution:
Measure 1ml acetic acid earlier and add constant volume in the volumetric flask of 100ml, make 1% (V/V) acetum, take by weighing the 2.5g chitosan again, add in 1% (V/V) acetum and get final product;
Used deacetylating degree of chitosan >=91%;
2., preparation 4.5% (W/V) polyphosphoric acid sodium water solution:
Taking by weighing the 4.5g sodium polyphosphate is dissolved in the deionized water of 100ml and gets final product;
3., the preparation of RGD/ chitosan-acetic acid solution and RGD/ polyphosphoric acid sodium water solution
The RGD that takes by weighing 2.0g respectively adds in 2.5% (W/V) chitosan acid solution and 4.5% (W/V) polyphosphoric acid sodium solution, has both obtained being dissolved with RGD/ chitosan-acetic acid solution that contains 2.0gRGD and the RGD/ polyphosphoric acid sodium water solution that contains 2.0gRGD respectively;
(2), high pressure electrostatic pulse balling-up
The RGD/ chitosan-acetic acid solution of the 3. gained in the step (1) and RGD/ polyphosphoric acid sodium water solution are carried out the high pressure electrostatic pulse prepare microballoon in high pressure electrostatic microcapsule forming instrument; The microballoon of gained soaks 4h in RGD/ polyphosphoric acid sodium water solution after; Use the washed with de-ionized water microballoon to be neutrality, obtain white solid microsphere until solution;
High pressure electrostatic pulse process control voltage is 39-50kv, and fltting speed is 90mm/h, and pulsewidth is 7ms, and frequency is 90Hz, and the liquid level distance is 20mm;
(3), lyophilize prepares the three-dimensional porous microcarrier of chitosan/RGD
The white solid microsphere of step (2) gained is put into Freeze Drying Equipment equipment carry out lyophilize and come drilling, concrete parameter setting is-80 ℃ for the pre-freeze temperature, and the pre-freeze time is 1h; Baffle temperature in the Freeze Drying Equipment equipment of primary drying is-30 ℃, and the time is 9-10h; Baffle temperature in the Freeze Drying Equipment equipment of redrying is 10 ℃, and the time is 1.5-2h, the final three-dimensional porous microcarrier of chitosan/RGD that gets.
Final gained gets the ESEM picture of the three-dimensional porous microcarrier of chitosan/RGD, and as shown in Figure 1, as can be seen from Figure 1 there are many apertures on the microcarrier surface, and its aperture is 20-50mm, and particle diameter is 600mm, and porosity is 91%.
Embodiment 2
The three-dimensional porous microcarrier of chitosan/RGD of embodiment 1 gained is used for the liver cell adherent culture, and its cultural method comprises the steps:
(1), the pre-treatment of the three-dimensional porous microcarrier of chitosan/RGD
It is in 70% the aqueous ethanolic solution that the three-dimensional porous microcarrier of the chitosan/RGD of embodiment 1 gained is immersed concentration, in 4 ℃ temperature environment, keeps 6h, leaches the three-dimensional porous microcarrier of chitosan/RGD then; And with washed with de-ionized water 3 times; Again with the PBS damping fluid drip washing of the pH=7 for preparing 1 time, again the chitosan microcarrier is immersed in the PBS damping fluid afterwards, keep 12h in 4 ℃ the temperature environment; Leach microcarrier once more, and clean repeatedly 3 times with the PBS damping fluid;
Described PBS damping fluid is promptly got potassium primary phosphate 0.68 g and is added 0.1mol/L sodium hydroxide solution 29.1ml, is diluted with water to 100 ml, promptly gets;
(2), hepatocellular inoculation and cultivation
With 5 * 10
6The cell inoculation density in every hole (48 orifice plate) is inoculated in the three-dimensional porous microcarrier of the pretreated chitosan/RGD of step (1) with human liver cell; 48 orifice plates are jiggled to be transferred to temperature behind the 10min be 37 ℃, gas concentration lwevel is 5%, humidity be stick in 100% the CO2gas incubator cultivate 4h after; The three-dimensional porous microcarrier of RPMI1640 nutrient solution flushing chitosan/RGD with containing serum does not stick liver cell closely to remove on the three-dimensional porous microcarrier of chitosan/RGD; Again the three-dimensional porous microcarrier of the chitosan/RGD of adherent cell is transferred to 24 well culture plates, the RPMI1640 that every hole adding 1ml contains serum cultivates, and cultivates 4 hours; Promptly get the nutrient solution that contains three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body; Changed nutrient solution liquid once in every afterwards 1-2 days, and every other day carry out the scanning electron microscopic observation result, cultivate the nutrient solution that will contain three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body after 4 days again and carry out spinning; The control rotating speed is 1000r/min; Time is 5min, gets upper solution, can obtain high-density, highly active liver cell.
The described preparation that contains the RPMI1640 nutrient solution of serum; Being about to serum is serum with RPMI1640 by mass ratio: RPMI1640 is after 1:9 mixes; Add microbiotic penicillium mould and Streptomycin sulphate, the ultimate density of the two is 100U/ml, regulates pH between 7.0-7.2 with PBS buffered soln; Filtration sterilization then, 4 ℃ of preservations are subsequent use.
The three-dimensional porous microcarrier of chitosan/RGD after cultivating 4 days of gained and the fluorescence labelling scanning electron microscopic observation result of liver cell complex body are as shown in Figure 2, as can be seen from Figure 2 liver cell well-grown on the three-dimensional porous microcarrier of chitosan/RGD.
Foregoing is merely the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (8)
1. three-dimensional porous microcarrier of chitosan/RGD; Its raw material comprises chitosan; It is characterized in that described raw material also comprises RGD; After soon chitosan and RGD mixing solutions will form microballoon through high-voltage pulse microcapsule forming instrument, pass through the lyophilize drilling again, the final three-dimensional porous microcarrier of chitosan/RGD that forms with three-dimensional structure.
2. the three-dimensional porous microcarrier of chitosan/RGD as claimed in claim 1, the molecular weight that it is characterized in that described chitosan is 5.63 * 10
6, deacetylation is 91%.
3. according to claim 1 or claim 2 the preparation method of the three-dimensional porous microcarrier of a kind of chitosan/RGD is characterized in that comprising the steps:
(1), the preparation of RGD/ chitosan-acetic acid solution and RGD/ polyphosphoric acid sodium solution
The preparation of RGD/ chitosan-acetic acid solution
Is RGD with RGD and described chitosan-acetic acid solution by the mass ratio of the chitosan in RGD and the chitosan-acetic acid solution: the chitosan in the chitosan-acetic acid solution is that 2.0g:2.5g mixes and promptly gets the RGD/ chitosan-acetic acid solution;
Wherein chitosan and concentration be that the aqueous acetic acid of 1% (V/V) calculates by mass volume ratio are chitosan in the chitosan-acetic acid solution: aqueous acetic acid is 2.5g:100ml;
The preparation of RGD/ polyphosphoric acid sodium water solution
Is RGD with RGD and polyphosphoric acid sodium water solution by the mass ratio of the sodium polyphosphate in RGD and the polyphosphoric acid sodium solution: sodium polyphosphate is that 2.0:4.5 mixes and promptly gets RGD/ polyphosphoric acid sodium water solution;
In the polyphosphoric acid sodium water solution wherein, press mass volume ratio and calculate, be i.e. sodium polyphosphate: water is 4.5g:100ml;
(2), high pressure electrostatic pulse balling-up
The RGD/ chitosan-acetic acid solution of the 3. gained in the step (1) and RGD/ polyphosphoric acid sodium water solution are carried out the high pressure electrostatic pulse prepare microballoon in high pressure electrostatic microcapsule forming instrument; The microballoon of gained soaks 4h in RGD/ polyphosphoric acid sodium water solution after; Use the washed with de-ionized water microballoon to be neutrality, obtain white solid microsphere until solution;
Described high pressure electrostatic pulse process control voltage is 39-50kv, and fltting speed is 90mm/h, and pulsewidth is 7ms, and frequency is 90Hz, and the liquid level distance is 20mm;
(3), lyophilize prepares the three-dimensional porous microcarrier of chitosan/RGD
The white solid microsphere of step (2) gained is carried out lyophilize come drilling, final the three-dimensional porous microcarrier of chitosan/RGD.
4. the preparation method of the three-dimensional porous microcarrier of chitosan/RGD as claimed in claim 3 is characterized in that the lyophilize described in the step (3), and process control pre-freeze temperature is-80 ℃, and the pre-freeze time is 1h; The temperature of primary drying is-30 ℃, and the time is 9-10h; The temperature of redrying is 10 ℃, and the time is 1.5-2h.
5. according to claim 1 or claim 2 the three-dimensional porous microcarrier of chitosan/RGD is used for hepatocellular adherent culture.
6. the application of the three-dimensional porous microcarrier of chitosan/RGD as claimed in claim 5 aspect the liver cell adherent culture is characterized in that culturing process comprises the steps:
(1), the pre-treatment of the three-dimensional porous microcarrier of chitosan/RGD
It is in 70% the aqueous ethanolic solution that the three-dimensional porous microcarrier of chitosan/RGD is immersed concentration, in 4 ℃ temperature environment, keeps 6h, leaches the chitosan microcarrier then; And with washed with de-ionized water 3 times; Again with the PBS damping fluid drip washing of the pH=7 for preparing 1 time, again the chitosan microcarrier is immersed in the PBS damping fluid afterwards, keep 12h in 4 ℃ the temperature environment; Leach microcarrier once more, and clean repeatedly 3 times with the PBS damping fluid;
(2), hepatocellular inoculation and cultivation
On 48 orifice plates, press 5 * 10
6The cell inoculation density in every hole is inoculated in step (1) in the three-dimensional porous microcarrier microcarrier of pretreated chitosan/RGD with human liver cell; 48 orifice plates are jiggled to be transferred to temperature behind the 10min be 37 ℃; Gas concentration lwevel is 5%, humidity be stick in 100% the CO2gas incubator cultivate 4h after, on the three-dimensional porous microcarrier of chitosan/RGD, do not stick liver cell closely to remove with the three-dimensional porous microcarrier of RPMI1640 nutrient solution flushing chitosan/RGD that contains serum; To stick the three-dimensional porous microcarrier of hepatocellular chitosan/RGD again and be transferred to 24 well culture plates; The RPMI1640 nutrient solution that every hole adding 1ml contains serum is cultivated, and cultivates after 4 hours, promptly gets the nutrient solution that contains three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body; Changed in every afterwards 1-2 days contain serum the RPMI1640 nutrient solution once; Cultivate the nutrient solution that will contain three-dimensional porous microcarrier of chitosan/RGD and liver cell complex body after 4 days again and carry out spinning, the control centrifugal rotational speed is 1000r/min, and the time is 5min; Get upper solution, can obtain high-density, highly active liver cell.
7. the application of the three-dimensional porous microcarrier of chitosan/RGD as claimed in claim 6 aspect the liver cell adherent culture; It is characterized in that the preparation of the RPMI1640 nutrient solution that contains serum described in the step (2); Being about to serum is serum with RPMI1640 by mass ratio: RPMI1640 is after 1:9 mixes; Add microbiotic penicillium mould and Streptomycin sulphate, the ultimate density of the two is 100U/ml, regulates pH between 7.0-7.2 with the PBS buffered soln described in the step (1); Filtration sterilization then, 4 ℃ of preservations.
8. the application of the three-dimensional porous microcarrier of described chitosan/RGD as claimed in claim 7 aspect the liver cell adherent culture; It is characterized in that the PBS damping fluid described in the step (1); Promptly get potassium primary phosphate 0.68 g and add 0.1mol/L sodium hydroxide solution 29.1ml; Be diluted with water to 100 ml, promptly get the PBS damping fluid.
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| WO2023208212A1 (en) * | 2022-04-29 | 2023-11-02 | 北京华龛生物科技有限公司 | Method for producing virus vector on basis of three-dimensional porous microcarrier |
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| CN107198771A (en) * | 2017-05-08 | 2017-09-26 | 广东渔跃生物技术有限公司 | The method that microcarrier suspension culture cell produces pseudorabies gE gene delection viral vaccines |
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| WO2023208212A1 (en) * | 2022-04-29 | 2023-11-02 | 北京华龛生物科技有限公司 | Method for producing virus vector on basis of three-dimensional porous microcarrier |
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