CN1289842A - Magnetosensitive microcarrier for culturing animal cells and its preparing process - Google Patents
Magnetosensitive microcarrier for culturing animal cells and its preparing process Download PDFInfo
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- CN1289842A CN1289842A CN99119643A CN99119643A CN1289842A CN 1289842 A CN1289842 A CN 1289842A CN 99119643 A CN99119643 A CN 99119643A CN 99119643 A CN99119643 A CN 99119643A CN 1289842 A CN1289842 A CN 1289842A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/06—Magnetic means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
Abstract
A magnetosensitive microcarrier for culturing animal cells is composed of the magnetosensitive kernal made of magnetic powder and high-molecular polymer for wrapping the magnetic powder partides and the shell which is the matrix suitable for adherence and growth of cells, and is prepared through silico alkanisation of magnetic powder, preparing magnetic kernel, surface treatmenting, coating matrix, and surface modifying of microcarrier. It has rigid compact structure.
Description
The present invention relates to a kind of animal cell culture technology, particularly relate to a kind of Magnetosensitive microcarrier that is used for animal cell culture and preparation method thereof.
The large scale culturing zooblast can be used for producing many biological products, as vaccine, pharmaceutical protein, cell metabolite etc.Cultivating anchorage-dependent cell is to adopt roller bottle system at first, and labour intensity is big, and the cell growth area that unit volume provides is little, and it is low to calculate culture efficiency by volume of culture.Van Wezel had developed microcarrier cultural system in 1967, cultivated anchorage-dependent cell.Microcarrier is the microballon of diameter tens to the hundreds of micron.When cultivating anchorage-dependent cell with microcarrier, cell attachment is on microcarrier, and microcarrier suspension is in substratum.Because microcarrier has very big specific surface area, thereby improved culture efficiency greatly.
Animal cell culture microcarrier commonly used is at present divided by matrix following a few class: (1) sephadex; (2) carbohydrate is as DEAE-Mierocrystalline cellulose microcarrier; (3) protein is as the gelatin microcarrier; (4) polyose is as chitin; (5) rubber; (6) glass is as hollow glass bead; (7) plastics are as polystyrene, polyacrylamide.Trade names have Biocarrier, Superbeads, and Biosilon, Cytosphere, Cytodex1, Cytodex2, Cytodex3 etc., these trades mark all do not have magnetic response.
The bio-reactor that is used for the microcarrier cultivation has types such as airlift bioreactor, aeration-agitation cage type bioreactor, column reactor, mechanical stir-reactor, sail formula stirred reactor, fluidized bed bio reactor.Because microcarrier suspension in liquid, is collided mutually, the growth of pair cell is unfavorable, so the amount of microcarrier is generally the 1-5 grams per liter, is up to the 12-15 grams per liter, and the cell density that obtains is generally 10
6-10
7/ ml.If give in the column reactor and add longitudinal magnetic field, microcarrier particle wherein has magnetosensitive again simultaneously, then can form stable particle layers under certain condition, particle is static relatively in the space, it is very high that packing density can reach, and liquid can freely pass through, and pressure drop is very low, therefore can realize high-density culture.
About the animal cell culture Magnetosensitive microcarrier, document 1:U.S.S.R.SU1567623 has reported the preparation method of a kind of eukaryotic cell cultivation with Magnetosensitive microcarrier, be with the oxide compound of Fe, Co, Ni and gelatin mixing granulation, grinding, use protease treatment up to smooth surface then, used oxide compound is the powder of 0.005-0.05 μ m.This carrier is owing to directly mix magnetic with matrix, is difficult to guarantee that magnetic do not reveal and injure cell.
The present invention provides a kind of Magnetosensitive microcarrier that is used for animal cell culture for the density that improves animal cell large-scale and cultivate and the efficient of reactor, and it is used in the animal cell culture reactor of magnetic field and cultivates zooblast.This microcarrier should have response to magnetic field, is suitable for adherent, the growth of cell again, and the leakage of magnetic can't be arranged, in order to avoid pollute nutrient solution and injury cell.The present invention also provides the above-mentioned preparation method who is used for the Magnetosensitive microcarrier of animal cell culture.
Technical scheme of the present invention is as follows:
The Magnetosensitive microcarrier that is used for animal cell culture is the core-shell type structure, kernel is a magnetosensitive nuclear, magnetosensitive nuclear is made of the high molecular polymer of magnetic and parcel magnetic, shell is the matrix that is suitable for cell attachment, growth, the median size of used magnetic is the 0.1-5 micron, the ratio of magnetosensitive nuclear diameter and microcarrier diameter is 1: 1.2-1: 5, and the median size of microcarrier is 120 microns～300 microns.
Said magnetic is γ-Fe (OH)
2, γ-Fe
2O
3Or Fe
3O
4Said high molecular polymer can be by monomer, initiator and the linking agent preparation that can form spherical particle through suspension polymerization commonly used, wherein monomer comprises the monomer that can form spherical particle through suspension polymerization, as phenylethylene, as vinylbenzene, to Vinyl toluene, a Vinyl toluene or alpha-methyl styrene, and methyl methacrylate.Initiator comprises the initiator that suspension polymerization is commonly used, as benzoyl peroxide (BPO) or Diisopropyl azodicarboxylate (AIBN).Linking agent comprises the linking agent that suspension polymerization is commonly used, as Vinylstyrene and triallyl isocyanurate.Shell matrix comprises the matrix that is fit to different types of cell attachment and growth, as gelatin, dextran etc.
The microcarrier surface can also be carried out chemically modified as required, to change its charge distribution, wetting ability etc.
The preparation of above-mentioned microcarrier is to adopt high molecular polymer parcel magnetic to form magnetosensitive nuclear, and bread through aftertreatment, is promptly obtained Magnetosensitive microcarrier by one deck matrix outside again.
Concrete preparation comprises following two steps: 1. magnetic nuclear preparation
With polymerization single polymerization monomer and linking agent is 100 by volume: 2-40 mixes, the initiator that adds the 0.5-1 gram by every 100ml monomer, keep stirring in 60 ℃-70 ℃, add 25-75 gram magnetic again, be warming up to 80-90 ℃ with 1.2-2 ℃/minute speed by every 100ml reaction solution.Improve mixing speed, adding is that monomer volume 6-15 weight percent doubly is the gelatin solution of 3-8%, polyase 13-4 hour.90-100 ℃ of slaking 2 hours.Filter collecting granules, embathe 3-5 time,,, spend the night,, get required magnetic nuclear in oven dry below 100 ℃ with soaked in absolute ethyl alcohol with the flushing of dehydrated alcohol room temperature with cold water flush with hot water more than 60 ℃.2. bag is by matrix
With the pH value is that the phosphoric acid buffer preparation weight percent of 5.0-7.2 is the gelatin solution of 5%-20%, needs the ratio of 10-100ml gelatin solution that magnetic is examined by every gram magnetic nuclear (wet) and joins in the gelatin solution, and stirring is more than 5 minutes.This liquid is joined 4-8 in the stirring doubly in the toluene liquid of gelatin solution volume, the ratio that is dissolved with Sp80 in the toluene liquid is that 0.5-4mlSp80/ restrains in gelatin, more than 5 minutes, the magnetic nuclear that is wrapped with gelatin is fully disperseed in 40-50 ℃ of stirring, be cooled to 0-10 ℃ afterwards, stir more than 5 minutes.Ratio dropping weight percent according to 0.2-3ml glutaraldehyde solution/gram dry gelatin is the glutaraldehyde solution of 10-50%, is warmed up to 20-30 ℃, crosslinked 1-3 hour.Filter collecting microballon, is that the phosphoric acid buffer of 7.0-7.2 is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution adjust pH to 9-10, add NaBH
4NaBH to the solution
4Weight percent concentration be 0.5-2%, reduced 10-24 hour.Wash microballoon three times with phosphoric acid buffer,,, get microcarrier of the present invention in oven dry below 70 ℃ with gradient alcohol flushing (weight percent is 60%, 70%, 80%, 95%).
In order to obtain better effect, also can adopt following five steps preparation.
I.e. " preparation of magnetic nuclear " in the 1st step adds a magnetic silanization step before: it is that the surface of magnetic is handled, and makes it easier and merges with high molecular polymer, to improve the particle content of magnetic nuclear.Add the solution of silane of 0.2-10 parts by volume in the weight percent of 100 parts by volume is the aqueous acetic acid of 0.1-1%, stirs, add a certain amount of magnetic as required, stirring at low speed was filtered more than 10 minutes, in oven dry below 100 ℃, porphyrize.
Add magnetic nuclear surface treatment step between " bag is by the matrix " of " magnetic nuclear preparation " of the 1st step and the 2nd step: it is that the surface that magnetic is examined is handled, and makes it the easier matrix that hangs up different types of cell attachment and growth.Magnetic nuclear (doing) joined magnetic nuclear is submerged and suspends, slightly stirring, soaking at room temperature is more than 3 minutes.Filtering, is neutrality with a large amount of deionized water rinsing particles to washing water.
Add microcarrier finishing step afterwards in the 2nd step " bag is by matrix "; The microcarrier particle (doing) that wraps quilt is joined in the solution of 1～2M DEAEClHCl that can flood carrier and can make it to suspend, stir more than 10 minutes in 40-50 ℃.Slowly adding to system is 1～3M NaOH solution of 0.5～3 times of DEAEClHCl liquor capacity, stirs more than 1 hour.Filter, be the phosphoric acid buffer flush vehicle particle of 7.0-7.2 successively with deionized water, 0.05～0.5M HCl, 0.05～0.5mM HCl, deionized water, pH value, use the gradient alcohol flushing again,, or place 70% ethanol to preserve in oven dry below 70 ℃.
In preparation magnetic when nuclear, magnetic is joined in the mixture of monomer, linking agent and initiator after, can add the gelatin solution that disperses said mixture again through 10-20 minute prepolymerization.Prepared microcarrier can comprise further that DEAE modifies or the finishing of derivatize.
Magnetic of the present invention nuclear can be made through suspension polymerization through monomer, initiator and linking agent and the magnetic that suspension polymerization forms spherical particle by commonly used, and the median size that the magnetic of preparing is examined is 60 μ m-100 μ m, and particle content is 30%-50% (wt).
The median size of microcarrier is 120 μ m-300 μ m, wet proportion 1.1-1.5g/cm
3, microcarrier has the rigid dense structure.The microcarrier of shell nuclear formula structure, its shell is the matrix that helps cell attachment, growth, this structure had not only made microcarrier to magnetic field response be arranged. but also completely cut off magnetic and contact with the direct of cell, also prevented the leakage of magnetic in substratum, the injury of polluting nutrient solution and pair cell, cultivation results shows that being suitable for the Vero cell attachment grows.
The present invention is described in detail below in conjunction with embodiment.
Embodiment 1:1. magnetic silanization
To weight percent is to add the 0.2ml solution of silane among 0.1% the aqueous acetic acid 100ml, stirs, and adds 10 gram ferrite powder (Fe
3O
4), stirring at low speed 3 hours is filtered, with magnetic in 70 ℃ of oven dry, porphyrize, the ferrite powder (Fe of silanization
3O
4).2. magnetic nuclear preparation
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 0.4ml, mix the back and add benzoyl peroxide (BPO) 0.1g, stir down, add the ferrite powder (Fe of silanization in 65 ℃, 450rpm rotating speed
3O
4) 5g, be warming up to 80 ℃ with 1.2 ℃/minute speed.Improve mixing speed to 650rpm, adding weight percent is 8% gelatin solution 120ml, polymerization 4 hours.100 ℃ of slakings 2 hours.Filter collecting granules, embathe 5 times,,, spend the night,, must do magnetic nuclear in 70 ℃ of oven dry with soaked in absolute ethyl alcohol with the flushing of dehydrated alcohol room temperature with cold water flush with 60 ℃ of hot water.3. magnetic nuclear surface treatment
Get the 4ml chromic acid lotion, add 0.5g magnetic nuclear (doing) and slightly stir soaking at room temperature 3 minutes.Filtering, is neutrality with a large amount of deionized water rinsing particles to washing water.4. bag is by matrix
With the pH value is that 5.5 phosphoric acid buffer preparation 20ml weight percent is 15% gelatin solution, add 0.5g magnetic nuclear (wetting) then, stirred 0.5 hour, this liquid is joined in the 150ml toluene liquid that is dissolved with 1.5ml Sp80 in the stirring, 50 ℃ were stirred 5 minutes, and stirring velocity 300rpm changes ice-water bath afterwards over to, be cooled to 0 ℃, stirred 10 minutes.Dropping 9ml weight percent is 10% glutaraldehyde solution, is warmed up to 20 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.0 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution adjust pH to 9, add NaBH
4To 2% concentration, reduced 10 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 60 ℃ of oven dry.5. microcarrier finishing
Take by weighing and wrap, add 15ml 2M DEAEClHCl solution, stirred 10 minutes in 50 ℃ by microcarrier particle (doing) 0.5 gram.Slowly add 10ml 3M NaOH solution to system, stirred 1 hour.Filtering, is 7 phosphoric acid buffer flush vehicle particle successively with deionized water, 0.05MHCl, 0.05mM HCl, deionized water, pH value, and more successively with 60%, 70%, 80%, 95% alcohol flushing is in 60 ℃ of oven dry.Gained microcarrier particle diameter is the 60-300 micron, 200 microns of median sizes, wet proportion 1.37g/cm
3
Embodiment 2:
1. magnetic silanization
Add 2ml solution of silane (ratio) in 0.5% aqueous acetic acid 100ml, stir, add 15 gram magnetics, stirring at low speed was soaked 1 hour, filter, with magnetic in 85 ℃ of oven dry, porphyrize, the ferrite powder (Fe of silanization
3O
4).2. magnetic nuclear preparation
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 4ml, mix the back and add benzoyl peroxide (BPO) 0.2g, stir down, add the ferrite powder (Fe of silanization in 60 ℃, 450rpm rotating speed
3O
4) 10g, be warming up to 85 ℃ with 1.6 ℃/minute speed.Improve mixing speed to 650rpm, add 5% gelatin solution 200ml, polyase 13 .5 hour.95 ℃ of slakings 2 hours.Filter collecting granules, embathe 4 times,,, spend the night with soaked in absolute ethyl alcohol, in 85 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 80 ℃ of hot water.3. magnetic nuclear surface treatment
Get the 2ml sulfonated liquid, add 0.5g magnetic nuclear (doing) and slightly stir soaking at room temperature 10 minutes.Filtering, is neutrality with a large amount of deionized water rinsing particles to washing water.4. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 5ml 20% for preparing with the phosphoric acid buffer of pH5.0, stirred 1 hour, this liquid is joined in the 40ml toluene liquid that is dissolved with 2ml Sp80 in the stirring, 40 ℃ were stirred 20 minutes, stirring velocity 400rpm, change ice-water bath afterwards over to, be cooled to 5 ℃, stirred 20 minutes.Drip the glutaraldehyde solution of 1ml 20%, be warmed up to 25 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.1 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 9.5, add NaBH
4To 1% concentration, reduced 18 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 50 ℃ of oven dry.5. microcarrier finishing
Take by weighing and wrap, add 10ml 1M DEAEClHCl solution, stirred 0.5 hour in 50 ℃ by microcarrier particle (doing) 0.5 gram.Slowly add 5ml 2M NaOH solution to system, stirred 1 hour.Filter, use deionized water, 0.2MHCl, 0.2mM HCl, deionized water, phosphoric acid buffer liquid flush vehicle particle successively, more successively with 60%, 70%, 80%, 95% alcohol flushing is in 50 ℃ of oven dry.Gained microcarrier particle diameter is the 60-400 micron, 240 microns of median sizes, wet proportion 1.45g/cm
3
Embodiment 3:1. magnetic silanization
Add the 10ml solution of silane in 1% aqueous acetic acid 100ml, stir, add 20 gram magnetics, stirring at low speed 10 minutes is filtered, with magnetic in 100 ℃ of oven dry, porphyrize.2. magnetic nuclear preparation
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 8ml, mix the back and add Diisopropyl azodicarboxylate (AIBN) 0.15g, stir down, add the ferrite powder (Fe of silanization in 70 ℃, 450rpm rotating speed
3O
4) 15g, be warming up to 90 ℃ with 2 ℃/minute speed.Improve mixing speed to 650rpm, add 3% gelatin solution 300ml, polyase 13 hour.90 ℃ of slakings 2 hours.Filter collecting granules, embathe 3 times,,, spend the night with soaked in absolute ethyl alcohol, in 100 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 90 ℃ of hot water.3. magnetic nuclear surface treatment
Get the 1ml chromic acid lotion, add 0.5g magnetic nuclear (doing) and slightly stir soaking at room temperature 20 minutes.Filtering, is neutrality with a large amount of deionized water rinsing particles to washing water.4. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 50ml 5% for preparing with the phosphoric acid buffer of pH7.2, stirred 5 minutes, this liquid is joined in the 200ml toluene liquid that is dissolved with 10ml Sp80 in the stirring, 45 ℃ of constant temperature stirred 10 minutes, stirring velocity 200rpm, change ice-water bath afterwards over to, be cooled to 10 ℃, stirred 5 minutes.Drip the glutaraldehyde solution of 0.5ml 50%, be warmed up to 30 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.2 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 10, add NaBH
4To 0.5% concentration, reductase 12 4 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 70 ℃ of oven dry.5. microcarrier finishing
Take by weighing and wrap, add 5ml 1.5M DEAEClHCl solution, stirred 1 hour in 50 ℃ by microcarrier particle (doing) 0.5 gram.Slowly add 15ml 1M NaOH solution to system, stirred 1 hour.Filter, use deionized water, 0.5M HCl, 0.5mM HCl, deionized water, phosphoric acid buffer liquid flush vehicle particle successively, more successively with 60%, 70%, 80%, 95% alcohol flushing places 70% ethanol to preserve.Gained microcarrier particle diameter is the 60-280 micron, 180 microns of median sizes, wet proportion 1.50g/cm
3
The preparation of embodiment 4:1. magnetic nuclear
In round-bottomed flask, add Vinyl toluene 20ml, triallyl isocyanurate 0.4ml, mix the back and add Diisopropyl azodicarboxylate (AIBN) 0.1g, stir down, add ferrite powder (Fe in 65 ℃, 500rpm rotating speed
3O
4) 5g, be warming up to 80 ℃ with 1.2 ℃/minute speed.Improve mixing speed to 750rpm, adding weight percent is 8% gelatin solution 120ml, polymerization 4 hours.100 ℃ of slakings 2 hours.Filter collecting granules, embathe 5 times,,, spend the night,, must do magnetic nuclear in 70 ℃ of oven dry with soaked in absolute ethyl alcohol with the flushing of dehydrated alcohol room temperature with cold water flush with 60 ℃ of hot water.2. bag is by matrix
With the pH value is that 6.0 phosphoric acid buffer preparation 20ml weight percent is 15% gelatin solution, add 0.5g magnetic nuclear (wetting) then, stirred 0.5 hour, this liquid is joined in the 150ml toluene liquid that is dissolved with 1.5ml Sp80 in the stirring, 50 ℃ were stirred 5 minutes, and stirring velocity 300rpm changes ice-water bath afterwards over to, be cooled to 0 ℃, stirred 10 minutes.Dropping 9ml weight percent is 10% glutaraldehyde solution, is warmed up to 20 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.0 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution adjust pH to 9, add NaBH
4To 2% concentration, reduced 10 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 60 ℃ of oven dry.Gained microcarrier particle diameter is the 45-300 micron, 190 microns of median sizes, wet proportion 1.28g/cm
3
The preparation of embodiment 5:1. magnetic nuclear
Vinyl toluene 20ml, Vinylstyrene (DVB) 4ml between adding in round-bottomed flask mix the back and add benzoyl peroxide (BPO) 0.2g, stir down in 60 ℃, 500rpm rotating speed, add ferrite powder (Fe
3O
4) 10g, be warming up to 85 ℃ with 1.6 ℃/minute speed.Improve mixing speed to 750rpm, add 5% gelatin solution 200ml, polyase 13 .5 hour.95 ℃ of slakings 2 hours.Filter collecting granules, embathe 4 times,,, spend the night with soaked in absolute ethyl alcohol, in 85 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 80 ℃ of hot water.2. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 5ml 20% for preparing with the phosphoric acid buffer of pH6.5, stirred 1 hour, this liquid is joined in the 40ml toluene liquid that is dissolved with 2ml Sp80 in the stirring, 40 ℃ were stirred 20 minutes, stirring velocity 400rpm, change ice-water bath afterwards over to, be cooled to 5 ℃, stirred 20 minutes.Drip the glutaraldehyde solution of 1ml 20%, be warmed up to 25 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.1 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 9.5, add NaBH
4To 1% concentration, reduced 18 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 50 ℃ of oven dry.Gained microcarrier particle diameter is the 50-350 micron, 230 microns of median sizes, wet proportion 1.36g/cm
3
The preparation of embodiment 6:1. magnetic nuclear
In round-bottomed flask, add alpha-methyl styrene 20ml, triallyl isocyanurate 8ml, mix the back and add Diisopropyl azodicarboxylate (AIBN) 0.15g, stir down, add ferrite powder (Fe in 70 ℃, 500rpm rotating speed
3O
4) 15g, be warming up to 90 ℃ with 2 ℃/minute speed.Improve mixing speed to 750rpm, add 3% gelatin solution 300ml, polyase 13 hour.90 ℃ of slakings 2 hours.Filter collecting granules, embathe 3 times,,, spend the night with soaked in absolute ethyl alcohol, in 100 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 90 ℃ of hot water.2. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 50ml 5% for preparing with the phosphoric acid buffer of pH7.0, stirred 5 minutes, this liquid is joined in the 200ml toluene liquid that is dissolved with 10ml Sp80 in the stirring, 45 ℃ of constant temperature stirred 10 minutes, stirring velocity 200rpm, change ice-water bath afterwards over to, be cooled to 10 ℃, stirred 5 minutes.Drip the glutaraldehyde solution of 0.5ml 50%, be warmed up to 30 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.2 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 10, add NaBH
4To 0.5% concentration, reductase 12 4 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 70 ℃ of oven dry.Gained microcarrier particle diameter is the 50-280 micron, 165 microns of median sizes, wet proportion 1.39g/cm
3
The preparation of embodiment 7:1. magnetic nuclear
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 0.4ml, mix the back and add Diisopropyl azodicarboxylate (AIBN) 0.1g, stir down, add γ-Fe (OH) in 65 ℃, 500rpm rotating speed
2Powder 5g is warming up to 80 ℃ with 1.2 ℃/minute speed.Improve mixing speed to 750rpm, adding weight percent is 8% gelatin solution 120ml, polymerization 4 hours.100 ℃ of slakings 2 hours.Filter collecting granules, embathe 5 times,,, spend the night,, must do magnetic nuclear in 70 ℃ of oven dry with soaked in absolute ethyl alcohol with the flushing of dehydrated alcohol room temperature with cold water flush with 60 ℃ of hot water.2. bag is by matrix
With the pH value is that 6.0 phosphoric acid buffer preparation 20ml weight percent is 15% gelatin solution, add 0.5g magnetic nuclear (wetting) then, stirred 0.5 hour, this liquid is joined in the 150ml toluene liquid that is dissolved with 1.5ml Sp80 in the stirring, 50 ℃ were stirred 5 minutes, and stirring velocity 300rpm changes ice-water bath afterwards over to, be cooled to 0 ℃, stirred 10 minutes.Dropping 9ml weight percent is 10% glutaraldehyde solution, is warmed up to 20 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.0 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution adjust pH to 9, add NaBH
4To 2% concentration, reduced 10 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 60 ℃ of oven dry.Gained microcarrier particle diameter is the 40-270 micron, 165 microns of median sizes, wet proportion 1.10g/cm
3
The preparation of embodiment 8:1. magnetic nuclear
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 4ml, mix the back and add benzoyl peroxide (BPO) 0.2g, stir down, add γ-Fe in 60 ℃, 500rpm rotating speed
2O
3Powder 10g is warming up to 85 ℃ with 1.6 ℃/minute speed.Improve mixing speed to 750rpm, add 5% gelatin solution 200ml, polyase 13 .5 hour.95 ℃ of slakings 2 hours.Filter collecting granules, embathe 4 times,,, spend the night with soaked in absolute ethyl alcohol, in 85 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 80 ℃ of hot water.2. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 5ml 20% for preparing with the phosphoric acid buffer of pH6.5, stirred 1 hour, this liquid is joined in the 40ml toluene liquid that is dissolved with 2ml Sp80 in the stirring, 40 ℃ were stirred 20 minutes, stirring velocity 400rpm, change ice-water bath afterwards over to, be cooled to 5 ℃, stirred 20 minutes.Drip the glutaraldehyde solution of 1ml 20%, be warmed up to 25 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.1 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 9.5, add NaBH
4To 1% concentration, reduced 18 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 50 ℃ of oven dry.Gained microcarrier particle diameter is the 40-360 micron, 210 microns of median sizes, wet proportion 1.24g/cm
3
The preparation of embodiment 9:1. magnetic nuclear
In round-bottomed flask, add vinylbenzene (St) 20ml, Vinylstyrene (DVB) 8ml, mix the back and add Diisopropyl azodicarboxylate (AIBN) 0.15g, stir down, add γ-Fe (OH) in 70 ℃, 500rpm rotating speed
2Powder 15g is warming up to 90 ℃ with 2 ℃/minute speed.Improve mixing speed to 750rpm, add 3% gelatin solution 300ml, polyase 13 hour.90 ℃ of slakings 2 hours.Filter collecting granules, embathe 3 times,,, spend the night with soaked in absolute ethyl alcohol, in 100 ℃ of oven dry with the flushing of dehydrated alcohol room temperature with cold water flush with 90 ℃ of hot water.2. bag is by matrix
0.5g magnetic nuclear (wetting) is joined in the gelatin solution of the 50ml 5% for preparing with the phosphoric acid buffer of pH7.0, stirred 5 minutes, this liquid is joined in the 200ml toluene liquid that is dissolved with 10ml Sp80 in the stirring, 45 ℃ of constant temperature stirred 10 minutes, stirring velocity 200rpm, change ice-water bath afterwards over to, be cooled to 10 ℃, stirred 5 minutes.Drip the glutaraldehyde solution of 0.5ml 50%, be warmed up to 30 ℃, crosslinked 2 hours.Filter collecting microballon, is that 7.2 phosphoric acid buffer is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution and transfer pH to 10, add NaBH
4To 0.5% concentration, reductase 12 4 hours.Wash microballoon three times with phosphoric acid buffer, use 60%, 70%, 80%, 95% alcohol flushing successively, in 70 ℃ of oven dry.Gained microcarrier particle diameter is the 40-200 micron, 140 microns of median sizes, wet proportion 1.19g/cm
3
Claims (10)
1. Magnetosensitive microcarrier that is used for animal cell culture, it is characterized in that: this microcarrier is the core-shell type structure, kernel is a magnetosensitive nuclear, magnetosensitive nuclear is made of the high molecular polymer of magnetic and parcel magnetic, shell is the matrix that is suitable for cell attachment, growth, the median size of used magnetic is the 0.1-5 micron, and the ratio of magnetosensitive nuclear diameter and microcarrier diameter is 1: 1.2-1: 5, and the median size of microcarrier is 120 microns～300 microns.
2. according to the described Magnetosensitive microcarrier that is used for animal cell culture of claim 1, it is characterized in that: said magnetic is γ-Fe (OH)
2, γ-Fe
2O
3Or Fe
3O
4
3. according to the described Magnetosensitive microcarrier that is used for animal cell culture of claim 1, it is characterized in that: said high molecular polymer is by commonly used monomer, initiator and the linking agent preparation that can form spherical particle through suspension polymerization, wherein monomer be selected from vinylbenzene, to Vinyl toluene, a Vinyl toluene, alpha-methyl styrene or methyl methacrylate; Initiator comprises benzoyl peroxide or Diisopropyl azodicarboxylate: linking agent comprises Vinylstyrene and triallyl isocyanurate.
4. according to the described Magnetosensitive microcarrier that is used for animal cell culture of claim 1, it is characterized in that: shell matrix is gelatin, dextran.
5. one kind prepares the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 1～4, it is characterized in that: comprise following two steps: the preparation of (1) magnetic nuclear:
With polymerization single polymerization monomer and linking agent is 100 by volume: 2-40 mixes, the initiator that adds the 0.5-1 gram by every 100ml monomer, keep stirring in 60 ℃-70 ℃, add 25-75 gram magnetic again by every 100ml reaction solution, be warming up to 80-90 ℃ with 1.2-2 ℃/minute speed, improve mixing speed, adding is that monomer volume 6-15 weight percent doubly is the gelatin solution of 3-8%, polyase 13-4 hour, 90-100 ℃ of slaking 2 hours, filter collecting granules, embathe with hot water successively, cold water flush is with the flushing of dehydrated alcohol room temperature, spend the night with soaked in absolute ethyl alcohol, in oven dry below 100 ℃; (2) wrap by matrix:
With the pH value is that the phosphoric acid buffer preparation weight percent of 5.0-7.2 is the gelatin solution of 5%-20%, need the ratio of 10-100ml gelatin solution that magnetic nuclear is joined in the gelatin solution in the wet magnetic nuclear of every gram, stir more than 5 minutes, this liquid is joined 4-8 in the stirring doubly in the toluene liquid of gelatin solution volume, the ratio that is dissolved with Sp80 in the toluene liquid is a 0.5-4mlSp80/ gram dry gelatin, stir more than 5 minutes in 40-50 ℃, the magnetic nuclear that is wrapped with gelatin is fully disperseed, be cooled to 0-10 ℃ afterwards, stir more than 5 minutes, ratio dropping weight percent according to 0.2-3ml glutaraldehyde solution/gram dry gelatin is the glutaraldehyde solution of 10-50%, be warmed up to 20-30 ℃, crosslinked 1-3 hour, filter to collect microballon, be that the phosphoric acid buffer of 7.0-7.2 is given a baby a bath on the third day after its birth inferior with pH value.In carrier-phosphoric acid buffer liquid system, add saturated borax solution adjust pH to 9-10, add NaBH
4NaBH to the solution
4Weight percent concentration be 0.5-2%, reduced 10-24 hour, wash microballoon three times with phosphoric acid buffer, use the gradient alcohol flushing, in below 70 ℃ the oven dry.
6. according to the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 5, it is characterized in that: add a magnetic silanization step before above-mentioned (1) step: the solution of silane that in the weight percent of 100 parts by volume is the aqueous acetic acid of 0.1-1%, adds the 0.2-10 parts by volume, stir, add a certain amount of magnetic as required, stirring at low speed is more than 10 minutes, filter, in oven dry below 100 ℃, porphyrize.
7. according to the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 5, it is characterized in that: between above-mentioned (1) step and above-mentioned (2) step, add a magnetic nuclear surface treatment step: dried magnetic nuclear is joined magnetic nuclear is submerged and suspends, slightly stir, soaking at room temperature is more than 3 minutes, filtering, is neutrality with a large amount of deionized water rinsing particles to washing water.
8. according to the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 5, it is characterized in that: after (2) step, add a microcarrier finishing step: the dried microcarrier particle that will wrap quilt, join in 1～2M DEAEClHCl solution that can flood carrier and can make it to suspend, stir more than 10 minutes in 40-50 ℃, slowly adding to system is 1～3M NaOH solution of 0.5～3 times of DEAEClHCl liquor capacity, stir more than 1 hour, filter, use deionized water successively, 0.05～0.5M HCl, 0.05～0.5mM HCl, deionized water, the pH value is the phosphoric acid buffer flush vehicle particle of 7.0-7.2, use the gradient alcohol flushing again, in oven dry below 70 ℃, or place 70% ethanol to preserve.
9. according to the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 5, it is characterized in that: during preparation magnetic nuclear, after magnetic being joined in the mixture of monomer, linking agent and initiator, through 10-20 minute prepolymerization, add the gelatin solution that disperses said mixture again.
10. according to the described preparation method who is used for the Magnetosensitive microcarrier of animal cell culture of claim 5～9, it is characterized in that: prepared microcarrier can comprise further that DEAE modifies or the finishing of derivatize.
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| CN99119643A CN1108374C (en) | 1999-09-23 | 1999-09-23 | Magnetosensitive microcarrier for culturing animal cells and its preparing process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99119643A CN1108374C (en) | 1999-09-23 | 1999-09-23 | Magnetosensitive microcarrier for culturing animal cells and its preparing process |
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| Publication Number | Publication Date |
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| CN1289842A true CN1289842A (en) | 2001-04-04 |
| CN1108374C CN1108374C (en) | 2003-05-14 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005010139A3 (en) * | 2003-07-23 | 2005-07-07 | Univ South Florida | Ferromagnetic cell and tissue culture microcarriers |
| WO2005083057A1 (en) * | 2004-03-01 | 2005-09-09 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Magnetic manipulation of biological samples |
| CN103232891A (en) * | 2013-05-09 | 2013-08-07 | 湖北中烟工业有限责任公司 | Method for fermenting and preparing tobacco flavor by using magnetically-stabilized fluidized bed |
| US9752139B2 (en) | 2003-06-30 | 2017-09-05 | University Of South Florida | Magnetic three-dimensional cell culture apparatus and method |
| CN109943517A (en) * | 2017-12-21 | 2019-06-28 | 车医科学大学校产学协力团 | A kind of carrier for cell culture, preparation method and a kind of method of the cell culture using it |
-
1999
- 1999-09-23 CN CN99119643A patent/CN1108374C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9752139B2 (en) | 2003-06-30 | 2017-09-05 | University Of South Florida | Magnetic three-dimensional cell culture apparatus and method |
| WO2005010139A3 (en) * | 2003-07-23 | 2005-07-07 | Univ South Florida | Ferromagnetic cell and tissue culture microcarriers |
| US9476025B2 (en) | 2003-07-23 | 2016-10-25 | University Of South Florida | Ferromagnetic cell and tissue culture microcarriers |
| WO2005083057A1 (en) * | 2004-03-01 | 2005-09-09 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Magnetic manipulation of biological samples |
| KR100801518B1 (en) | 2004-03-01 | 2008-02-12 | 프라운호퍼-게젤샤프트 추르 푀르데룽 데어 안제반텐 포르슝 에 파우 | Magnetic manipulation of biological samples |
| US8241906B2 (en) | 2004-03-01 | 2012-08-14 | Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. | Magnetic manipulation of biological samples |
| CN103232891A (en) * | 2013-05-09 | 2013-08-07 | 湖北中烟工业有限责任公司 | Method for fermenting and preparing tobacco flavor by using magnetically-stabilized fluidized bed |
| CN109943517A (en) * | 2017-12-21 | 2019-06-28 | 车医科学大学校产学协力团 | A kind of carrier for cell culture, preparation method and a kind of method of the cell culture using it |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1108374C (en) | 2003-05-14 |
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