CN109055294A - A kind of cell culture artificial cell membrane's coating microcarrier and preparation method thereof - Google Patents
A kind of cell culture artificial cell membrane's coating microcarrier and preparation method thereof Download PDFInfo
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- CN109055294A CN109055294A CN201811077979.7A CN201811077979A CN109055294A CN 109055294 A CN109055294 A CN 109055294A CN 201811077979 A CN201811077979 A CN 201811077979A CN 109055294 A CN109055294 A CN 109055294A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
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- C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
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- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
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Abstract
The present invention provides a kind of cell culture artificial cell membrane's coating microcarriers and preparation method thereof.Microcarrier of the invention is using Konjac Glucomannan Granules as matrix, with bionic new material artificial cell membrane's coating microballoon.Microcarrier of the invention the preparation method is as follows: matrix microspheres are washed with petroleum ether and dehydrated alcohol respectively after, be dried for standby；2- methylacryoyloxyethyl phosphocholine (MPC) and n-BMA are dissolved in ethyl alcohol, aggregated under the action of initiator azodiisobutyronitrile (AIBN) as artificial cell membrane material, is diluted to spare after 2% concentration；The Konjac Glucomannan Granules washed are added to after being impregnated in artificial cell membrane's ethanol solution, up to microcarrier product after being dried under vacuum with Rotary Evaporators.Micro-carrier surface artificial cell membrane material covering uniformly, is connected firmly, and has many advantages, such as that non-toxic, adherent good, the degradable, cell of cell is easy elution adhesion, and manufactured microcarrier is low in cost, production process is simple, and the period is shorter, is conducive to industrialized production.
Description
Technical field
The invention belongs to the tissue engineering technique fields of biomaterial more particularly to a kind of cell culture to use artificial cell membrane
Coating microcarrier and preparation method thereof.
Background technique
Microcarrier Culture Techniques are generally acknowledged at present most promising a kind of for zooblast large-scale culture
Technology.Since microcarrier is used for animal cell culture for the first time by Van Wezel within 1967, Microcarrier Culture Techniques are had evolved into
For the main method of current anchorage-dependent cell pilot scale culture.Microcarrier has large specific surface area, culture medium utilization rate high
The advantages that process is simple is collected with cell, is widely used in the life of the biological products such as culture and vaccine, antibody, the nucleic acid of various kinds of cell
It produces, and there is certain application prospect in organizational project and regenerative medicine field.So far the microcarrier quotient sold on international market
Category type is several up to ten, wherein commonly commercialization microcarrier there are three types of: Cytodex1,2,3, Cytopore and
Cytoline, currently most used is Cytodexl, 2,3 type microcarriers.
Konjaku glucomannan (KGM) is a kind of natural macromolecule amylose, has good biocompatibility and nontoxicity
Deng KGM microballoon transparency is high, good mechanical property and appropriate density is a kind of potential microcarrier matrix, at present studies in China
Report is relatively more, and numerous documents and patent are described synthetic method, such as: CN201210523380.8, but KGM is micro-
Ball carries out surface modification and is rarely reported for animal cell culture.
Twentieth century end, biomimetic material series of products of the NOF Corp (NOF CORPORATION.) in exploitation
(LIPIDURE) it marches at the forefront of the world on direction, this kind of medical bionics material that they develop is ventilative due to its high moisturizing
Performance and ingredient extremely similar with human cell membrane and characteristic, are known as artificial cell membrane.
This high-end medical bionics material has the biocompatibility of height, research shows that using LIPIDURE coated product
Cell line can be prevented to be adhered to substrate surface, while protein can be prevented to be adsorbed to substrate surface, the culture that makes to swim becomes can
Energy.Artificial cell membrane has been widely used for biomedicine field and medical instruments field, and has been commercialized: for manufacturing
Hemodialysis membrane treats uremic patient；Biological support (scaffold) material is applied to for expanding blood vessel；It is made into imitative cell membrane
Polymer micelle slow releasing pharmaceutical improves curative effect；Chip surface applied to biomedicine field is reduced for protein, DNA, thin
The non-specific adsorption of born of the same parents etc.；Its interface friction problem between soft tissue is reduced coated on artificial joint material surface, is increased
Service life；The aggregation of blood platelet is reduced coated on artificial blood vessel inner wall, reduces thrombotic risk；As soft contact lens material；It is high
The moisturizing raw material of shelves cosmetics.
It is a kind of excellent microcarrier matrix by summarizing visible KGM microballoon above, artificial cell membrane LIPIDURE is fine
Microcarrier coating material, the advantage of the two is combined together, this artificial cell membrane's coating microcarrier is in cell culture
Have potential huge advantage, give we this field explore provide broad space.
Summary of the invention
In view of this, the present invention is directed to propose a kind of cell culture artificial cell membrane's coating microcarrier and its preparation side
Method, micro-carrier surface artificial cell membrane material covering uniformly, are connected firmly, have non-toxic, cell it is adherent it is good, degradable,
Cell is easy the advantages that elution adhesion, and manufactured microcarrier is low in cost, and production process is simple, and the period is shorter, is conducive to
Industrialized production.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of cell culture preparation method of artificial cell membrane's coating microcarrier, includes the following steps,
1) Konjac Glucomannan Granules are added in petroleum ether, agitator treating, are then added in ethyl alcohol after filtering, stirring is washed
It washs, it is spare after filtering drying；
2) 2- methylacryoyloxyethyl phosphocholine (MPC) and the n-BMA of 2-5 molar equivalent are dissolved in
It in ethyl alcohol, is added initiator azodiisobutyronitrile (AIBN), weight is the 0.5% of both the above raw material weight sum, and rear reflux 24 is small
When, it pours into the acetone being vigorously stirred and precipitates after cooling, filtering removes unreacted raw material and small molecular weight impurity, after vacuum drying
The concentration for being diluted to 2% with ethyl alcohol is spare；
3) konjaku for washing step 1) according to the ratio for the 3-5% that artificial cell membrane's weight is Konjac Glucomannan Granules weight
Glucomannan microballoon is added to step 2 and has been diluted in artificial cell membrane's ethanol solution of 2% concentration, is existed with Rotary Evaporators
Good fluidity is arrived in vacuum drying at 35-40 DEG C, under rotation, opens ultraviolet light irradiation and packs after 24 hours.
Preferably, the mole of n-BMA described in step 2 is 2- methylacryoyloxyethyl phosphoric acid gallbladder
3 times of alkali (MPC).
Preferably, Konjac Glucomannan Granules described in step 2 and artificial cell membrane's weight ratio are 4%.
By adopting the above-described technical solution, the beneficial effects of the present invention are:
1, there is excellent mechanical property under hygrometric state using artificial cell membrane's high molecular material LIPIDURE, by konjak portuguese gansu polyose
Sugared microcarrier is wrapped up, and the shortcomings that simple konjaku glucomannan microcarrier is easy expansion in aqueous solution is overcome.
2, artificial cell membrane LIPIDURE has good biocompatibility, water imbibition and the slow characteristic of degradation, and
LIPIDURE coated product can prevent cell line adherency and protein to be adsorbed to substrate surface, be especially beneficial cell and swim training
It supports, can not only support a large amount of cells to grow, but also cell activity and function can be maintained for a long time, in zooblast long-time High Density Cultivation
In have unique effect.
3, micro-carrier surface artificial cell membrane material covering uniformly, is connected firmly, have non-toxic, cell it is adherent it is good,
Degradable, cell is easy the advantages that elution adhesion.
4, raw material of the present invention is easy to get, cheap.Solvent can reuse, and realize the purpose of green syt.
5, present invention post-processing is simple and convenient, can obtain the product of high-quality, and the method can be enlarged, and realizes work
Industry.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the electron-microscope scanning figure of cell culture artificial cell membrane's coating microcarrier described in the embodiment of the present invention two；
Fig. 2 is that cell culture described in the embodiment of the present invention two is inoculated with artificial cell membrane's coating microcarrier Cultivation of Vero
Electron-microscope scanning figure after for 24 hours.
Specific embodiment
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
Embodiment one
A kind of cell culture preparation method of artificial cell membrane's coating microcarrier, includes the following steps:
1) 100 grams of Konjac Glucomannan Granules are added in 500 milliliters of petroleum ethers, agitator treating, after filtered on buchner funnel
It is then added in 500 ethyl alcohol, agitator treating, is dried for standby after filtering；
2) 29.5 grams of 2- methylacryoyloxyethyl phosphocholines (MPC) are dissolved in 42.6 grams of n-BMAs
In 350 milliliters of ethyl alcohol, 0.4 gram of azodiisobutyronitrile (AIBN) is added, then vacuumizes reaction system then with vacuum pump
It is passed through high pure nitrogen, is high pure nitrogen by gas displacement in reaction system repeatedly three times, after being protected from light with aluminium foil package, stirs
Mix back flow reaction 24 hours.Efficient liquid phase detects raw material MPC, and consumption is net substantially, stops reaction, pours into and be vigorously stirred after cooling
Acetone in precipitate, acetone washing three times, removes unreacted raw material and small molecular weight impurity, uses ethyl alcohol after vacuum drying after filtering
It is spare to be diluted to 2% concentration；
3) Konjac Glucomannan Granules washed in 100 grams of step 1) are added in 200 milliliters of step 2 and are diluted to 2% concentration
Artificial cell membrane's ethanol solution in, be dried in vacuo at 35-40 DEG C with Rotary Evaporators to good fluidity, under rotation, beaten
Ultraviolet light irradiation is opened to pack after 24 hours.
Embodiment two
A kind of cell culture preparation method of artificial cell membrane's coating microcarrier, includes the following steps:
1) 100 grams of Konjac Glucomannan Granules are added in 500 milliliters of petroleum ethers, agitator treating, after filtered on buchner funnel
It is then added in 500 ethyl alcohol, agitator treating, is dried for standby after filtering；
2) 29.5 grams of 2- methylacryoyloxyethyl phosphocholines (MPC) are dissolved in 28.4 grams of n-BMAs
In 260 milliliters of ethyl alcohol, 0.3 gram of azodiisobutyronitrile (AIBN) is added, then vacuumizes reaction system then with vacuum pump
It is passed through high pure nitrogen, is high pure nitrogen by gas displacement in reaction system repeatedly, after being protected from light with aluminium foil package, stirs back
Stream reaction 24 hours, efficient liquid phase detects raw material MPC, and consumption is net substantially, stops reaction, pours into third be vigorously stirred after cooling
It is precipitated in ketone, acetone washing three times, removes unreacted raw material and small molecular weight impurity, diluted after vacuum drying with ethyl alcohol after filtering
It is spare to 2% concentration；
3) Konjac Glucomannan Granules washed in 100 grams of step 1) are added in 150 milliliters of step 2 and are diluted to 2% concentration
Artificial cell membrane's ethanol solution in, be dried in vacuo at 35-40 DEG C with Rotary Evaporators to good fluidity, under rotation, beaten
Ultraviolet light irradiation is opened to pack after 24 hours.
Embodiment three
A kind of cell culture preparation method of artificial cell membrane's coating microcarrier, includes the following steps:
1) 100 grams of Konjac Glucomannan Granules are added in 500 milliliters of petroleum ethers, agitator treating, after filtered on buchner funnel
It is then added in 500 ethyl alcohol, agitator treating, is dried for standby after filtering；
2) 29.5 grams of 2- methylacryoyloxyethyl phosphocholines (MPC) are dissolved in 42.6 grams of n-BMAs
In 350 milliliters of ethyl alcohol, 0.4 gram of azodiisobutyronitrile (AIBN) is added, then vacuumizes reaction system then with vacuum pump
It is passed through high pure nitrogen, is high pure nitrogen by gas displacement in reaction system repeatedly, after being protected from light with aluminium foil package, stirs back
Stream reaction 24 hours, efficient liquid phase detects raw material MPC, and consumption is net substantially, stops reaction, pours into third be vigorously stirred after cooling
It is precipitated in ketone, acetone washing three times, removes unreacted raw material and small molecular weight impurity, diluted after vacuum drying with ethyl alcohol after filtering
It is spare to 2% concentration；
3) Konjac Glucomannan Granules washed in 100 grams of step 1) are added in 250 milliliters of step 2 and are diluted to 2% concentration
Artificial cell membrane's ethanol solution in, be dried in vacuo at 35-40 DEG C with Rotary Evaporators to good fluidity, under rotation, beaten
Ultraviolet light irradiation is opened to pack after 24 hours.
Example IV
A kind of cell culture preparation method of artificial cell membrane's coating microcarrier, includes the following steps:
1) 100 grams of Konjac Glucomannan Granules are added in 500 milliliters of petroleum ethers, agitator treating, after filtered on buchner funnel
It is then added in 500 ethyl alcohol, agitator treating, is dried for standby after filtering；
2) 29.5 grams of 2- methylacryoyloxyethyl phosphocholines (MPC) are dissolved in 56.8 grams of n-BMAs
In 450 milliliters of ethyl alcohol, 0.45 gram of azodiisobutyronitrile (AIBN) is added, then vacuumizes reaction system then with vacuum pump
It is passed through high pure nitrogen, is high pure nitrogen by gas displacement in reaction system repeatedly, after being protected from light with aluminium foil package, stirs back
Stream reaction 24 hours, efficient liquid phase detects raw material MPC, and consumption is net substantially, stops reaction, pours into third be vigorously stirred after cooling
It is precipitated in ketone, acetone washing three times, removes unreacted raw material and small molecular weight impurity, diluted after vacuum drying with ethyl alcohol after filtering
It is spare to 2% concentration；
3) Konjac Glucomannan Granules washed in 100 grams of step 1) are added in 200 milliliters of step 2 and are diluted to 2% concentration
Artificial cell membrane's ethanol solution in, be dried in vacuo at 35-40 DEG C with Rotary Evaporators to good fluidity, under rotation, beaten
Ultraviolet light irradiation is opened to pack after 24 hours.
By the microcarrier that above-described embodiment produces there is non-toxic, adherent good, the degradable, cell of cell to be easy elution etc.
Advantage.The microcarrier mobility as obtained in outer viewing embodiment two is best.
The characterization result for the product that two ~ example IV of embodiment obtains and embodiment one are almost the same.
The foregoing is merely the preferred embodiments of the invention, are not intended to limit the invention creation, all at this
Within the spirit and principle of innovation and creation, any modification, equivalent replacement, improvement and so on should be included in the invention
Protection scope within.
Claims (6)
1. a kind of cell culture artificial cell membrane's coating microcarrier, it is characterised in that: the artificial cell membrane material for coat
Material is the bionical new material of macromolecule that 2- methylacryoyloxyethyl phosphocholine (MPC) is synthesized with n-BMA.
2. a kind of cell culture artificial cell membrane's coating microcarrier, it is characterised in that: the matrix of the microcarrier is konjaku Portugal
Sweet glycan microballoon, artificial cell membrane's coating quality are the 3%-5% of matrix microspheres quality, and the artificial cell membrane of coating is dry by vacuum
Dry aging is coated on matrix microspheres surface.
3. a kind of cell culture preparation method of artificial cell membrane's coating microcarrier, it is characterised in that: include the following steps,
1) Konjac Glucomannan Granules are added in petroleum ether, agitator treating, are then added in ethyl alcohol after filtering, stirring is washed
It washs, it is spare after filtering drying；
2) 2- methylacryoyloxyethyl phosphocholine (MPC) and the n-BMA of 2-5 times of molar equivalent are dissolved
It in ethyl alcohol, is added initiator azodiisobutyronitrile (AIBN), weight is the 0.5% of both the above raw material weight sum, rear to flow back
It 24 hours, pours into the acetone being vigorously stirred and precipitates after cooling, filtering removes unreacted raw material and small molecular weight impurity, and vacuum is dry
It is spare that 2% concentration is diluted with ethanol to after dry；
3) konjaku for washing step 1) according to the ratio for the 3-5% that artificial cell membrane's weight is Konjac Glucomannan Granules weight
Glucomannan microballoon is added to step 2 and has been diluted in artificial cell membrane's ethanol solution of 2% concentration, is existed with Rotary Evaporators
Good fluidity is arrived in vacuum drying at 35-40 DEG C, under then rotating in Rotary Evaporators, opens ultraviolet light irradiation after 24 hours
Packaging.
4. a kind of preparation method of the cell culture according to claim 3 with artificial cell membrane's coating microcarrier, feature
Be: in step 2, the mole of the n-BMA is 2- methylacryoyloxyethyl phosphocholine (MPC)
2-5 times；Preferably, the mole of the n-BMA is the 3 of 2- methylacryoyloxyethyl phosphocholine (MPC)
Times.
5. a kind of preparation method of the cell culture according to claim 3 with artificial cell membrane's coating microcarrier, feature
Be: in step 2, artificial cell membrane's weight is the 3-5% of Konjac Glucomannan Granules weight；Preferably, described artificial
Cell membrane weight is the 4% of Konjac Glucomannan Granules weight.
6. a kind of preparation method of the cell culture according to claim 3 with artificial cell membrane's coating microcarrier, feature
Be: microcarrier is to be sterilized when rotating on Rotary Evaporators with ultraviolet light irradiation 24 hours in step 2.
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