CN101624472A - Macroporous microcarrier for cell cultivation, preparation method and usage thereof - Google Patents
Macroporous microcarrier for cell cultivation, preparation method and usage thereof Download PDFInfo
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- CN101624472A CN101624472A CN200910041768A CN200910041768A CN101624472A CN 101624472 A CN101624472 A CN 101624472A CN 200910041768 A CN200910041768 A CN 200910041768A CN 200910041768 A CN200910041768 A CN 200910041768A CN 101624472 A CN101624472 A CN 101624472A
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Abstract
The invention provides fibroin/chitosan macroporous microcarrier prepared by taking silk fibroin as main raw material, a preparation method and the usage thereof. The macroporous microcarrier takes on sphere, has the diameter of 200-500mu m and the aperture of 40-80mu m, and made of the silk fibroin and the chitosan under the action of crosslinking agent. The fibroin/chitosan macroporous microcarrier takes the fibroin as main ingredient, and the silk fibroin has good biocompatibility, slow degradation and excellent mechanical property in a wet state, so that the structure of the microcarrier can be maintained by the silk fibroin for a longer time; therefore, the silk fibroin not only can support the growth of a large number of cells, but also can maintain the activity and the functions of the cells for a long time, thus having unique function in the high density cultivation of animal cells for a long time.
Description
Technical field
The present invention relates to a kind of macroporous microcarrier and preparation method and purposes that is used for the large scale culturing zooblast, being specifically related to a kind of is fibroin/chitosan macroporous microcarrier and preparation method and the purposes that main raw material is prepared from the silk fibroin.
Background technology
It is the most rising a kind of animal cell large-scale culture technique of generally acknowledging at present that microcarrier is cultivated, and it has the advantage of suspension culture and adherent culture concurrently, amplifies easily.Macroporous microcarrier is grown cell fixation in the hole, can enlarge specific surface area to greatest extent, compares with additive method, and its specific surface area is bigger, is several times even tens times of solid microcarrier; Cell is grown in the hole, is protected, and the shearing damage is little; The cell three-dimensional growth, cell density will further improve, and cell density is more than 10 times of solid microcarrier, and what have reaches 10
8Individual/ml; Macroporous microcarrier also has remarkable advantages aspect the long-term high expression level of foreign protein except being beneficial to cell high-density amplification culture, be suitable for the secretion of protein production and product; In addition, macroporous microcarrier and seed cell are cultivated altogether, and the complex body of formation can be regarded many little tissues as, and this provides new thinking for vitro tissue makes up.
It is one of hepatocellular Perfected process of high-density suspension culture that macroporous microcarrier is cultivated, and many research institutions are all at the higher microcarrier of the research ratio of performance to price.Yet existing microcarrier can not the long term maintenance cell growth, cell easily is mechanically damaged, cell comes off and adherent inhomogeneous easily during amplification culture, is necessary to provide a kind of novel microcarrier to overcome the defective that exists in the prior art for this reason.
Summary of the invention
One of purpose of the present invention is to provide a kind of macroporous microcarrier, it is a raw material with the good silk fibroin of biocompatibility, chitosan, thereby prepare a kind of cellular affinity that both had, help cell adhesion, support a large amount of cell growths, again can long term maintenance cytoactive and cell function and the macroporous microcarrier that can be used in the large scale culturing cell with low cost.
Another object of the present invention is to provide the preparation method of above-mentioned macroporous microcarrier, this method is simple to operate, and raw materials cost is low, can be widely used.
Another purpose of the present invention is to provide the application of above-mentioned macroporous microcarrier in extensive animal cell culture.
For achieving the above object, the invention provides a kind of macroporous microcarrier, this macroporous microcarrier is spherical in shape, and diameter is 200-500 μ m, and the aperture is 40-80 μ m, and it is to be made under the effect of linking agent by silk fibroin and chitosan.
In embodiments of the present invention, can prepare described macroporous microcarrier by following method, this method comprises:
(a) silk fibroin protein solution and chitosan-acetic acid solution are mixed in proportion, make that producing final concentration is fibroin-chitosan mixing solutions of 4-10w/v%;
(b) described fibroin-chitosan mixing solutions adding is contained in the oil phase of emulsifying agent, obtain white emulsion; And in this white emulsion, slowly add linking agent, fully stir and make the water crosslinking curing;
(c) pH with the adding of the white emulsion in the step (b) whipped state is in the polar solvent of 9-10, and continues to stir 30-60 minute, filters and obtains mutual NA microballoon;
(d) solidify described microballoon and remove the oil phase on surface, sieving obtains the microballoon of 200-500 μ m;
(e) remove residual linking agent in the microballoon, and lyophilize, described macroporous microcarrier obtained.
In preferred embodiment, the amount of the linking agent that adds in the step (b) is 2.5% glutaraldehyde solution that the microcarrier of every gram dry weight adds 1-2ml.
Preferably, described emulsifying agent is class of department 80, and oil phase is a whiteruss; Wherein the volume ratio of class of department 80, whiteruss is 3: 100 to 5: 100.Described linking agent is preferably glutaraldehyde.Described polar solvent is selected from Virahol, acetone and ethanol etc.
In preferred embodiment, the constant speed that stirs described in the step (c) to 250-400 rev/min stirs.
Preferably, in step (d), by removing described oil phase with described polar solvent, sherwood oil and water washing.
Preferably, in step (e),, microballoon removed residual linking agent in 2-4 hour in glycine solution by being soaked.
In yet another embodiment of the present invention, also comprise sterilisation step afterwards in described step (e).Described sterilisation step is with cobalt 60-gamma-ray irradiation, or with macroporous microcarrier with distilled water or PBS solution swelling after high-temperature sterilization.
Fibroin provided by the invention/chitosan macroporous microcarrier with fibroin as main component, because silk fibroin possesses excellent biological compatibility and slow degradation characteristic, and under hygrometric state, excellent mechanical property is arranged, so can keep the structure of microcarrier the long period, a large amount of cell growths both can have been supported, again can long term maintenance cytoactive and function, unique effect is arranged in the long-time high-density culture of zooblast.
Description of drawings
Fig. 1-Fig. 2 has shown the scanning electron microscope picture of fibroin of the present invention/chitosan macroporous microcarrier;
Fig. 3, Fig. 4 show the 3rd day and the cell observed liver cell situation of sticking on material under inverted microscope of sampling in the 7th day that fibroin of the present invention/chitosan macroporous microcarrier and liver cell CL-1 cultivate altogether respectively in microgravity rotating and culturing system;
Fig. 5-Fig. 6 shows that the MTT staining of the cell of the sampling in the 4th day that fibroin of the present invention/chitosan macroporous microcarrier and liver cell CL-1 cultivate altogether detects the picture of cell viability in microgravity rotating and culturing system.
Embodiment
For methods of this invention will be better understood, will further set forth the present invention and advantage thereof by embodiment and experimental evidence below.Before describing following examples and experiment in detail, at first need definition and clarify some terms.
Silk fibroin is a kind of natural structure albumen that does not have physiologically active, and mainly by three kinds of simple amino acid: glycine, L-Ala and Serine are formed, and they account for general 85% of Tot Prot.Silk fibroin has excellent biological compatibility, and is nontoxic, nonirritant; Silk fibroin has crystallizing field and noncrystalline domain mixed structure to exist, and electronegative on the whole, the fibroin noncrystalline domain has many basic aminoacidss, and pair cell has adsorption to a certain degree, so attached thereto of energy absorptive cell.Just because of having above-mentioned character, silk fibroin has obtained increasingly extensive application in the bio-medical field.And use it for the matrix of cell cultures, or some biopolymers are modified, improve their biological property, make it be applicable to organizational project, especially the focus of silk fibroin research in recent years.The biological characteristics of above-mentioned excellence is that solid basis has been set up in the application that silk fibroin becomes cell cultures and tissue engineering bracket material.
Chitosan is a kind of natural biological polymer of being used widely in biological medicine, field of tissue engineering technology in recent years, and it has biological functionality, biocompatibility, hypotoxicity and does not almost have characteristic such as supersensitivity.
With fibroin and the compound porous microcarrier of making of chitosan, so just further increased the surface-area of cell attachment.Method provided by the invention is main raw material with the silk fibroin, prepare fibroin, chitosan compound microsphere by emulsification-chemical crosslink technique, handle with polar solvent then, induce the silk fibroin occurred conformation to change, make it no longer water-soluble, it is stable to reach water, has obtained mutual NA glue pearl, the macroporous microcarrier with vesicular structure has been prepared in lyophilize then.It is cheap, and cell adheres to thereon and grows better, is a kind of comparatively ideal solid support material.
Below further describe details of the present invention by specific embodiment.Fibroin/chitosan the macroporous microcarrier that can be used for the large scale culturing cell provided by the invention can obtain by the following method.
Embodiment one
1) preparation of silk fibroin protein solution: 75g is given birth to silk in the sodium carbonate solution of the 5g/L of 4L, boiled 1 hour, then with a large amount of washed with de-ionized water to remove sericin, dry down, obtain silk fibroin for 60-70 ℃.An amount of silk fibroin is dissolved under 80 ± 2 ℃ in calcium chloride/water/ethanol (mol ratio 1: 8: 2) ternary solution, deionized water dialysis 3d at ambient temperature, remove salt and ethanol in the solution, filter and remove insoluble impurity, prepare the aqueous solution of silk fibroin.Silk fibroin water solution is concentrated 50 ± 2 ℃, 60 rev/mins stirrings, obtain the silk fibroin protein solution of the about 7-10w/v% of concentration;
2) preparation of fibroin-chitosan mixing solutions: the chitosan aqueous acetic acid of preparation 4w/v%, preparation fibroin-chitosan mixing solutions, silk fibroin, chitosan mass ratio are 7: 3, final concentration is 5w/v%;
3) whiteruss of getting a certain amount of 160ml is added in the 500ml beaker, add class of 6.4ml department 80 then, mixing the back stirs with 250 rev/mins of constant speed, slowly drip 40ml fibroin-chitosan mixing solutions, at room temperature continue constant speed and stirred 45 minutes, obtain white emulsion, in above-mentioned emulsion, slowly add 2.5ml 2.5% glutaraldehyde solution, continue to stir 3 hours, make the water crosslinking curing;
4) get the aqueous isopropanol of 100ml, the deionized water toward wherein adding equal volume drips the NaOH dilute solution again, regulates PH to 9-10;
5) with 4) in solution with 250 rev/mins of stirrings, with 3) in white emulsion slowly add wherein, continue to stir 45 minutes.Filter then, obtain mutual NA microballoon, microballoon was placed 4 ℃ of refrigerators 6 hours, microballoon is fully solidified;
6) with microballoon with 4) in aqueous isopropanol, sherwood oil, deionized water wash, remove the oil phase on surface; Sieve out the microballoon of 200-500 μ m then;
7) microballoon after will sieving soaked 2 hours with the glycine solution of 5g/L, removed unreacted glutaraldehyde; Use a large amount of distilled water washs again;
8) thorough washing is clean microballoon places-20 ℃ of refrigerators, and freezing 48 hours, lyophilize then obtained macroporous microcarrier; After the macroporous microcarrier packing, the sterilization of cobalt 60-gamma-ray irradiation, standby.
Embodiment two
1) preparation of silk fibroin protein solution: 75g is given birth to silk in the sodium carbonate solution of the 5g/L of 4L, boiled 1 hour, then with a large amount of washed with de-ionized water to remove sericin, dry down, obtain silk fibroin for 60-70 ℃.An amount of silk fibroin is dissolved under 80 ± 2 ℃ in calcium chloride/water/ethanol (mol ratio 1: 8: 2) ternary solution, deionized water dialysis 3d at ambient temperature, remove salt and ethanol in the solution, filter and remove insoluble impurity, prepare the aqueous solution of silk fibroin.Silk fibroin water solution rev/min stirred at 50 ± 2 ℃, 50-60 concentrate, obtain the silk fibroin protein solution of the about 7-10w/v% of concentration;
2) preparation of fibroin-chitosan mixing solutions: the chitosan aqueous acetic acid of preparation 4w/v%, preparation fibroin-chitosan mixing solutions, silk fibroin, chitosan mass ratio are 6: 4, final concentration is 4w/v%;
3) whiteruss of getting a certain amount of 160ml is added in the 500ml beaker, add class of 6.4ml department 80 then, mixing the back stirs with 300 rev/mins of constant speed, slowly drip 40ml fibroin-chitosan mixing solutions, at room temperature continue constant speed and stirred 45 minutes, obtain white emulsion, in above-mentioned emulsion, slowly add 3ml 2.5% glutaraldehyde solution, continue to stir 3 hours, make the water crosslinking curing;
4) get the aqueous isopropanol of 100ml, the deionized water toward wherein adding equal volume drips the NaOH dilute solution again, regulates PH to 9-10;
5) with 4) in solution with 300 rev/mins of stirrings, with 3) in white emulsion slowly add wherein, continue to stir 45 minutes.Filter then, obtain mutual NA microballoon, microballoon was placed 4 ℃ of refrigerators 8 hours, microballoon is fully solidified;
6) microballoon is used aqueous isopropanol, sherwood oil, deionized water wash, removed the oil phase on surface as having diluted as described in the step 4); Sieve out the microballoon of 200-400 μ m then;
7) microballoon after will sieving soaked 4 hours with the glycine solution of 5g/L, removed unreacted glutaraldehyde; Use a large amount of distilled water washs again;
8) thorough washing is clean microballoon places-20 ℃ of refrigerators, and freezing 48 hours, lyophilize then obtained macroporous microcarrier; After the macroporous microcarrier packing,, standby with high-temperature sterilization after distilled water or the PBS solution swelling.
Embodiment three
1) preparation of silk fibroin protein solution: 75g is given birth to silk in the sodium carbonate solution of the 5g/L of 4L, boiled 1 hour, then with a large amount of washed with de-ionized water to remove sericin, dry down, obtain silk fibroin for 60-70 ℃.An amount of silk fibroin is dissolved under 80 ± 2 ℃ in calcium chloride/water/ethanol (mol ratio 1: 8: 2) ternary solution, deionized water dialysis 3d at ambient temperature, remove salt and ethanol in the solution, filter and remove insoluble impurity, prepare the aqueous solution of silk fibroin.Silk fibroin water solution is concentrated 50 ± 2 ℃, 60 rev/mins stirrings, obtain the silk fibroin protein solution of the about 7-10w/v% of concentration;
2) preparation of fibroin-chitosan mixing solutions: the chitosan aqueous acetic acid of preparation 4w/v%, preparation fibroin-chitosan mixing solutions, silk fibroin, chitosan mass ratio are 5: 5, final concentration is 4w/v%;
3) whiteruss of getting a certain amount of 150ml is added in the 500ml beaker, add class of 6ml department 80 then, mixing the back stirs with 300 rev/mins of constant speed, slowly drip 30ml fibroin-chitosan mixing solutions, at room temperature continue constant speed and stirred 45 minutes, obtain white emulsion, in above-mentioned emulsion, slowly add the 3ml2.5% glutaraldehyde solution, continue to stir 3 hours, make the water crosslinking curing;
4) get the aqueous isopropanol of 100ml, the deionized water toward wherein adding 100ml drips the NaOH dilute solution again, regulates PH to 9-10;
5) with 4) in solution with 300 rev/mins of stirrings, with 3) in white emulsion slowly add wherein, continue to stir 45 minutes.Filter then, obtain mutual NA microballoon, microballoon was placed 4 ℃ of refrigerators 6 hours, microballoon is fully solidified;
6) microballoon is used as the aqueous isopropanol that diluted as described in the step 4) and sherwood oil, deionized water wash, removed the oil phase on surface; Sieve out the microballoon of 200-500 μ m then;
7) microballoon after will sieving soaked 3 hours with the glycine solution of 5g/L, removed unreacted glutaraldehyde; Use a large amount of distilled water washs again;
8) thorough washing is clean microballoon places-20 ℃ of refrigerators, and freezing 72 hours, lyophilize then obtained macroporous microcarrier; After the macroporous microcarrier packing, the sterilization of cobalt 60-gamma-ray irradiation, standby.
Embodiment four
With the foregoing description gained macroporous microcarrier, after the sterilization, with PBS washing 3 times, basic medium soaked overnight then is so that the abundant swelling of material; Scanning electron microscope (SEM) picture is presented among Fig. 1 and Fig. 2, and visible microcarrier surface is vesicular structure, aperture about 40-80 μ m, and opening is outside, is horn-like, the distribution uniform in hole.
With the macroporous microcarrier after the abundant swelling, add in the microgravity rotating and culturing system (RCCS) with the CL-1 cell; Rotating and culturing is observed the stick situation of liver cell on material in the different time sections sampling under inverted microscope.Fig. 3 takes a sample for cultivating the 7th day for cultivation the 3rd day, Fig. 4, as seen all sticked cell on all microcarriers, liver cell is many cells accumulative agglomerate, because the vesicular structure on fibroin/chitosan macroporous microcarrier surface, make cell attachment more firm, and liver cell can enter in the hole and grow, the pattern that shows when cell is dimensional culture---spherical; Fig. 5, Fig. 6 picture (inverted microscope is observed X 40 down) for cultivating the 4th day MTT staining detection cell viability, as seen MTT is reduced into the bluish voilet crystallisate of insoluble by the succinodehydrogenase on the intracellular plastochondria, be deposited in the cell or cell peripheral, make viable cell show black-and-blue, dead cell is then not painted; Find that by dyeing the whole uniformity of cell-composite body is good, cytoactive is high.
Though the present invention is described with reference to concrete embodiment, those skilled in the art can make conspicuous modification and modification to the present invention by after reading foregoing description, and without prejudice to the intent of the present invention and essence.The present invention comprises these modifications and modification within the scope of the claims intentionally.
Claims (12)
1. macroporous microcarrier, this macroporous microcarrier is spherical in shape, and diameter is 200-500 μ m, and the aperture is 40-80 μ m, is made under the effect of linking agent by silk fibroin and chitosan.
2. method for preparing the described macroporous microcarrier of claim 1, this method comprises:
(a) silk fibroin protein solution and chitosan-acetic acid solution are mixed in proportion, make that producing final concentration is fibroin-chitosan mixing solutions of 4-10w/v%;
(b) described fibroin-chitosan mixing solutions is disperseed to enter in the oil phase that contains emulsifying agent, obtain white emulsion; And in this white emulsion, slowly add linking agent, fully stir and make the water crosslinking curing;
(c) pH with the adding of the white emulsion in the step (b) whipped state is in the polar solvent of 9-10, and continues to stir 30-60 minute, filters and obtains mutual NA microballoon;
(d) and remove the oil phase of described microsphere surface, sieving obtains the microballoon of 200-500 μ m;
(e) remove linking agent residual in the microballoon, and lyophilize, described macroporous microcarrier obtained.
3. method as claimed in claim 2 is characterized in that, described emulsifying agent is a class of department 80 etc., and wherein the volume ratio of class of department 80, whiteruss is 3: 100 to 5: 100.
4. method as claimed in claim 2 is characterized in that described linking agent is preferably glutaraldehyde.
5. method as claimed in claim 2 is characterized in that described polar solvent is selected from Virahol, acetone and ethanol.
6. method as claimed in claim 2 is characterized in that, the amount of the linking agent that adds in the step (b) is 2.5% glutaraldehyde solution that the microcarrier of every gram dry weight adds about 2ml.
7. method as claimed in claim 2 is characterized in that, the constant speed that stirs described in the step (c) to 250-400 rev/min stirs.
8. method as claimed in claim 2 is characterized in that, in step (d), by removing described oil phase with described polar solvent, sherwood oil and water washing.
9. method as claimed in claim 2 is characterized in that, in step (e), removes residual linking agent in 2-4 hour by microballoon is soaked in glycine solution.
10. method as claimed in claim 2 is characterized in that, also comprises sterilisation step afterwards in described step (e).
11. method as claimed in claim 2 is characterized in that, described sterilisation step is with cobalt 60-gamma-ray irradiation, perhaps with macroporous microcarrier with distilled water or PBS solution swelling after high-temperature sterilization.
12. the application of the described macroporous microcarrier of claim 1 in extensive animal cell culture.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011017930A1 (en) * | 2009-08-11 | 2011-02-17 | 南方医科大学珠江医院 | Macroporous microcarrier specific to liver cell, preparation mathod and use thereof |
| CN102492648A (en) * | 2012-01-04 | 2012-06-13 | 上海理工大学 | Preparation method of porous chitosan scaffold |
| CN103146672A (en) * | 2013-01-29 | 2013-06-12 | 广西壮族自治区水产研究所 | Method for immobilizing effective microorganisms (EM) during solid fermentation |
| CN103417497A (en) * | 2013-09-03 | 2013-12-04 | 重庆理工大学 | Oily drug controlled-release particles and preparation method of oily drug controlled-release particles |
| CN103898041A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Culture method of hybridomas |
| CN104099372A (en) * | 2014-07-23 | 2014-10-15 | 苏州大学 | Cationic silk fibroin/gene compound, and preparation method and application thereof |
| CN106754626A (en) * | 2016-12-30 | 2017-05-31 | 潍坊医学院 | A kind of cell culture microcarrier and preparation method thereof |
| CN106834204A (en) * | 2017-01-16 | 2017-06-13 | 西北民族大学 | A kind of cell culture SFL microcarriers and its preparation method and application |
| CN111269444A (en) * | 2020-01-22 | 2020-06-12 | 苏州新丝原生物科技有限公司 | Crosslinked microsphere and preparation method and application thereof |
| CN112354011A (en) * | 2020-10-12 | 2021-02-12 | 华南师范大学 | Liver tissue engineering scaffold and preparation method thereof |
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2009
- 2009-08-11 CN CN2009100417682A patent/CN101624472B/en active Active
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011017930A1 (en) * | 2009-08-11 | 2011-02-17 | 南方医科大学珠江医院 | Macroporous microcarrier specific to liver cell, preparation mathod and use thereof |
| CN102492648A (en) * | 2012-01-04 | 2012-06-13 | 上海理工大学 | Preparation method of porous chitosan scaffold |
| CN103898041A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Culture method of hybridomas |
| CN103898041B (en) * | 2012-12-25 | 2017-09-29 | 深圳先进技术研究院 | The cultural method of hybridoma |
| CN103146672A (en) * | 2013-01-29 | 2013-06-12 | 广西壮族自治区水产研究所 | Method for immobilizing effective microorganisms (EM) during solid fermentation |
| CN103417497A (en) * | 2013-09-03 | 2013-12-04 | 重庆理工大学 | Oily drug controlled-release particles and preparation method of oily drug controlled-release particles |
| CN104099372A (en) * | 2014-07-23 | 2014-10-15 | 苏州大学 | Cationic silk fibroin/gene compound, and preparation method and application thereof |
| CN106754626A (en) * | 2016-12-30 | 2017-05-31 | 潍坊医学院 | A kind of cell culture microcarrier and preparation method thereof |
| CN106834204A (en) * | 2017-01-16 | 2017-06-13 | 西北民族大学 | A kind of cell culture SFL microcarriers and its preparation method and application |
| CN111269444A (en) * | 2020-01-22 | 2020-06-12 | 苏州新丝原生物科技有限公司 | Crosslinked microsphere and preparation method and application thereof |
| CN112354011A (en) * | 2020-10-12 | 2021-02-12 | 华南师范大学 | Liver tissue engineering scaffold and preparation method thereof |
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