CN106497867A - A kind of cell microcarrier based on egg white and preparation method thereof - Google Patents
A kind of cell microcarrier based on egg white and preparation method thereof Download PDFInfo
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- CN106497867A CN106497867A CN201610978810.3A CN201610978810A CN106497867A CN 106497867 A CN106497867 A CN 106497867A CN 201610978810 A CN201610978810 A CN 201610978810A CN 106497867 A CN106497867 A CN 106497867A
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- egg white
- microcarrier
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- egg
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention discloses a kind of cell microcarrier based on egg white and preparation method thereof, it is characterised in that the egg white microcarrier emulsifies egg white solution by micro fluidic device first and obtains egg white drop template, chemically or physically method is recycled to obtain template solidification.Egg white microcarrier can give its loose structure by subsequent treatments such as freeze-dryings.The unique boiomacromolecule composition of egg white microcarrier and loose structure become excellent cell culture microcarrier.The preparation method that the present invention is provided, operate simple, with low cost, be easy to mass produce, the microcarrier of preparation is nontoxic and with excellent biocompatibility, Biofunctional and biodegradability.
Description
Technical field
The invention belongs to Three-dimensional cell culture microcarrier investigation of materials technical field, and in particular to a kind of micro- load of egg white cell
Body and preparation method thereof.
Background technology
Microcarrier has greatly in the numerous areas of organizational engineering as the general utility tool for realizing Three-dimensional cell culture
Application potential.Microcarrier is a kind of supported matrix for allowing adherent cell to grow in bioreactor, and its yardstick is in micro-
Meter level, the suitable density of microcarrier make which keep suspended state under soft stirring.At present, multiple material can be used to make
Standby microcarrier, such as glucan, glass, polystyrene plastics, acrylamide copolymer, collagen and alginate etc..These
Microcarrier material combines different surface chemical compositions (for example：Extracellular matrix protein, recombinant protein, polypeptide and positively charged
Or the molecule of negative electricity) cell behaviors such as cellular morphology, cell amplification can be affected.Cell amplification is most important in organizational project
Part because cell is the basic of the reconstruction of tissue topography and function, and microcarrier can then make cell in bioreactor
Fast breeding.Microcarrier is that cell provides one in vitro to biological three-dimensional environment similar in vivo so that cell research is more
Convenient.In addition, the cell that cultivates on microcarrier easily can implant, and microcarrier reparation disappearance or impaired
The ability of tissue is confirmed by experiment and clinical research.At present, microcarrier can be used for the culture of various kinds of cell, including
Gegenbaur's cell, cartilage cell, epithelial cell, endothelial cell, liver cell and stem cell etc..But great majority are used for preparing micro- load
The material cost of body is high, and preparation method complexity is loaded down with trivial details, and be presented with terms of biocompatibility and degradability to be hoisted, therefore
And limit application of the microcarrier in clinic.
Bird egg white just receives food industry and medical field a long time ago as a kind of natural protein source
Extensive concern, for example, carry out wound repair and vaccine manufacture etc. using egg white.By taking egg as an example, contained albumen in egg
Matter can belong to different a few proteinoid families, including silk enzyme level albumen, transferrins, Kazal type protease inhibitors,
Glycosyl hydrolase, lipocalin protein, bacterium anatonosis albumen, clusterin, cystatin, VMO-1 albumen
And uPAR/CD59/Ly6/ snake venom neurotoxin folic acid receivers.In egg, main protein includes：Ovalbumin, ovum iron
Transferrin/conalbumin, ovomucoid and ovomucin；Other protein include：Lysozyme, ovoinhibitor, ovum are huge
Globulin/ovum solid albumen, cystatin and Avidin.Though bird egg white can not be degraded automatically, as egg white can be regarded as
A kind of protein of broad sense, can be finally absorbed by the body by related protease hydrolytic after which enters human body, so can also claim
For " degradable " material.Egg white has excellent biocompatibility, Biofunctional and its nontoxic, degradable characteristic, therefore
And egg white is a kind of ideal material for preparing cell microcarrier, meanwhile, which also has wide answering in biomedical materials field
Use prospect.
Content of the invention
Present invention aim at a kind of cell microcarrier based on egg white and preparation method thereof is provided, its preparation process letter
Single, size is controllable, and homogeneity and repeatability are good, while with good biocompatibility, feature and degradability.
In order to solve these problems of the prior art, the technical scheme that the present invention is provided is：
It is characterized in that the egg white microcarrier emulsifies egg white solution by micro fluidic device first obtains egg white drop mould
Plate, recycles chemically or physically method to obtain template solidification.Egg white microcarrier can pass through the subsequent treatments such as freeze-drying and give
Its loose structure.The unique boiomacromolecule composition of egg white microcarrier and loose structure become the micro- load of excellent cell culture
Body.Microballoon can be by protease hydrolytic, so as to meet for the requirement of microcarrier degradability after entering human body.
Another object of the present invention is to providing a kind of cell microcarrier based on egg white and preparation method thereof, its feature exists
In the method comprising the steps of：
(1) extraction of egg white：
Thoroughly cleaning is carried out to selected eggs surface, egg white is separated with yolk with egg device, to isolated egg white
Filtered, taken the egg white weak solution for leaching standby；
(2) preparation of egg white emulsion：
Size tunable, uniform egg white emulsion are prepared using micro fluidic device and egg white weak solution；
(3) solidification of egg white emulsion：
Egg white emulsion is solidified by chemically or physically method, egg white microcarrier after cleaning, is obtained；
(4) acquisition of egg white microcarrier loose structure：
Freeze-drying is carried out by the egg white microcarrier to solidifying, you can give egg white microcarrier distinctive porous knot
Structure.
(5) cell culture is carried out using the egg white microcarrier for preparing：
The egg white micro-carrier surface for preparing is carried out microcarrier is soaked in after thoroughly cleaning PBS, cell successively
Each 2h in nutrient solution, then cell is inoculated on microcarrier carries out Three-dimensional cell culture, can be observed after 3 days cell cover with whole
Individual microcarrier.
Preferably, the egg white described in step (1) is retrieved from the egg ovum of the various birds such as chicken, duck, goose, quail or birds.
Preferably, described in step (2) obtained in microflow control technique egg white emulsion size between 20 μm of -5mm.
Preferably, the curing of the egg white emulsion described in step (3) can be heating, ultraviolet irradiation, strong acid-base, alcohol
Protein denaturation is made either physically or chemically etc. various.
Preferably, egg white microcarrier loose structure can be given by freeze-drying described in step (4), its hole size can
By factor controllings such as the concentration of egg white, the species of egg white, cryodesiccated times.
Preferably, be inoculated in described in step (5) on microcarrier carry out Three-dimensional cell culture cell can be HepG2 thin
Born of the same parents, 3T3 cells.
A kind of extraction of the cell microcarrier based on egg white of the present invention and preparation method thereof including early stage egg white, using extraction
Egg white weak solution carry out the preparation of egg white emulsion, emulsion is solidified after collection and is cleaned, you can obtained egg white microcarrier,
And pass through freeze-drying and can give egg white microcarrier loose structure, the microcarrier can meet the requirement of cell microcarrier, can carry out
Three-dimensional cell culture.The method comprising the steps of：
(1) extraction of egg white：
Thoroughly cleaning is carried out to selected eggs surface, egg white is separated with yolk with egg device, to isolated egg white
Filtered, taken the egg white weak solution for leaching standby；
(2) preparation of egg white emulsion：
Size tunable, uniform egg white emulsion are prepared using micro fluidic device and egg white weak solution；
(3) solidification of egg white emulsion：
Egg white emulsion is solidified by chemically or physically method, egg white microcarrier after cleaning, is obtained；
(4) acquisition of egg white microcarrier loose structure：
Freeze-drying is carried out by the egg white microcarrier to solidifying, you can give egg white microcarrier distinctive porous knot
Structure.
(5) cell culture is carried out using the egg white microcarrier for preparing：
The egg white micro-carrier surface for preparing is carried out microcarrier is soaked in after thoroughly cleaning PBS, cell successively
Each 2h in nutrient solution, then cell is inoculated on microcarrier carries out Three-dimensional cell culture, can be observed after 3 days cell cover with whole
Individual microcarrier.
Relative to scheme of the prior art, it is an advantage of the invention that：
A kind of cell microcarrier based on egg white of the present invention, microballoon main component are bird or birds egg white, and egg white is nontoxic,
There is good biocompatibility and feature, Three-dimensional cell culture can be carried out, simultaneously because egg white can be considered a kind of broad sense egg
White matter, can be finally absorbed by the body by protease hydrolytic after entering human body, so can be regarded as degradation material, therefore, egg
It is clearly a kind of ideal material for preparing cell microcarrier, while egg white also has broad prospect of application in biomedical materials field.
Egg white microcarrier of the present invention as a kind of single emulsion microballoon, with preparation method simple, material is easy to get, low cost
Honest and clean, the features such as be easy to large-scale production, be conducive to application of the egg white cell microcarrier in clinic.
Description of the drawings
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Fig. 1 is the schematic diagram for preparing egg white emulsion using micro fluidic device；
Fig. 2 is the flow chart that the egg white emulsion that collects is solidified and given the microsphere porous structure of egg white；
Fig. 3 is the instance graph that HepG2 cells are grown in egg white cell microcarrier, and engineer's scale is 100 μm.
Specific embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are for illustrating
The present invention and be not limited to limit the scope of the present invention.The implementation condition adopted in embodiment can be done according to the condition of concrete producer
Further adjust, not marked implementation condition is usually the condition in normal experiment.
A kind of cell microcarrier preparation method based on egg of embodiment 1
1. the extraction of egg：Thoroughly cleaning is carried out to Fresh Egg surface with 70% alcohol, afterwards with egg device by chicken
Egg white is separated with egg yolk, and the egg that isolates is filtered with gauze, takes the egg clear weak solution for leaching standby.
2. the preparation of egg emulsion：The micro fluidic device material for preparing egg microballoon is borosilicate, inside mutually i.e. non-
Continuous state material is the egg clear weak solution for leaching, and it is leaching type soybean oil that foreign minister is continuous state material.Using stable liquid
Flow velocity (interior phase flow velocity 0.5mL/h) and shearing force (foreign minister flow velocity 5mL/h), continuous phase soybean oil (oil phase) is by discontinuous phase chicken
Egg white (water phase) is mutually cut into the Water-In-Oil single emulsion structure of size uniformity, and then emulsion fills soybean oil by collecting pipe injection
In collecting pit.
3. the solidification of egg emulsion：Collecting pit is placed on magnetic agitation heater, 90 DEG C is heated to, egg emulsion
Generation denaturation of being heated is solidified into egg microballoon.After collecting pit cooling, egg microballoon is suctioned out, and use acetone, nothing successively
Water-alcohol carries out thoroughly cleaning to which, to remove the soybean oil of remained on surface, that is, obtains egg cell microcarrier.Determine particle diameter
For 200 μm, pdi is 1.5%.
4. the acquisition of egg microcarrier loose structure：The microcarrier for not carrying out cell culture after cleaning is placed in sterilized water
Middle immersion, blots sterilized water after 2h, microcarrier is placed in freezing 30min in -80 DEG C of refrigerators, takes out microcarrier afterwards and is placed in freezing
Carry out freeze-drying in drying machine, overnight after can obtain have loose structure egg microcarrier.
5. cell culture is carried out using microcarrier：Thoroughly cleaning is soaked successively with PBS phosphate buffers, cell culture fluid
Egg white microballoon 2h, is inoculated in HepG2 cells on egg microcarrier afterwards, observes cells on microcarriers growing state after 3 days.
A kind of cell microcarrier preparation method based on pale blue of example 2
1. the extraction of pale blue：Thoroughly cleaning is carried out to fresh duck egg surface with 70% alcohol, afterwards with egg device by duck
Egg white is separated with Ovum Anas domestica yolk, and the pale blue that isolates is filtered with gauze, takes the duck's egg clear weak solution for leaching standby.
2. the preparation of pale blue emulsion：The micro fluidic device material for preparing pale blue microballoon is borosilicate, inside mutually i.e. non-
Continuous state material is the duck's egg clear weak solution for leaching, and it is 100cs methyl-silicone oils that foreign minister is continuous state material.Using stable liquid
Flow velocity (interior phase flow velocity 0.2mL/h) and shearing force (foreign minister flow velocity 2mL/h), continuous phase methyl-silicone oil (oil phase) is by discontinuous phase
Pale blue (water phase) is mutually cut into the Water-In-Oil single emulsion structure of size uniformity, and then emulsion fills methyl silicon by collecting pipe injection
In the collecting pit of oil.
3. the solidification of pale blue emulsion：30min ultraviolet irradiations are carried out to collecting pit, and pale blue emulsion is under ultraviolet irradiation
Generation denaturation is solidified into pale blue microballoon.Afterwards pale blue microballoon is suctioned out, and which is carried out with n-hexane, absolute alcohol successively
Thoroughly cleaning, to remove the methyl-silicone oil of remained on surface, you can obtains pale blue cell microcarrier, and it is 500 μm to determine particle diameter,
Pdi is 1.9%.
4. the acquisition of pale blue microcarrier loose structure：Microcarrier first to having been used for cell culture cell fixer
Cell is fixed, microcarrier is cleaned with sterilized water afterwards, to remove the culture medium and PBS of micro-carrier surface residual
Buffer solution.Then the microcarrier that completes of cleaning is carried out with liquid nitrogen quick-frozen, quick-frozen after be immediately placed in freeze drier, overnight after
Take out, cell growth can be observed in the loose structure of pale blue microcarrier by Scanning Electron microscope.
5. cell culture is carried out using microcarrier：Thoroughly cleaning is soaked successively with PBS phosphate buffers, cell culture fluid
Egg white microballoon 2h, is inoculated in 3T3 cells on pale blue microcarrier afterwards, observes cells on microcarriers growing state after 3 days.
A kind of cell microcarrier preparation method based on quail egg of example 3
1. the extraction of quail egg white：Thoroughly cleaning is carried out to new fresh quail egg surface with 70% alcohol, egg device is used afterwards
Quail egg white is separated with Quail Egg, the quail egg white that isolates is filtered with gauze, take the quail egg for leaching thin
Solution for standby.
2. the preparation of quail egg white emulsion：The quail egg white weak solution leached with liquid nitrogen flash freezer, afterwards promptly will be quick-frozen
Quail egg white be placed in freeze drier, quail egg white powder can be overnight obtained.By obtained quail egg white powder 2M vinegar
Acid dissolving (6%, W/V) as interior mutually be discontinuous phase material.It is leaching type soybean oil that foreign minister is continuous state material.Using stable
Flow rate of liquid (interior phase flow velocity 0.3mL/h) and shearing force (foreign minister flow velocity 3mL/h), continuous phase soybean oil (oil phase) is by non-company
Continuous phase quail egg white powder solution (water phase) is mutually cut into the Water-In-Oil single emulsion structure of size uniformity, and then emulsion passes through collecting pipe
Injection is filled in the collecting pit of soybean oil.
3. the solidification of quail egg white emulsion：Collecting pit is placed in 24h in 4 DEG C of refrigerators, as acetic acid is for the change of protein
Property effect, you can solidification quail egg white emulsion.Quail egg white microballoon is suctioned out, and thoroughly cleaning is carried out to which with absolute alcohol,
To remove the soybean oil of remained on surface, you can obtain quail egg white cell microcarrier, it is 250 μm to determine particle diameter, and pdi is 2.0%.
4. the acquisition of quail egg white microcarrier loose structure：The microcarrier for not carrying out cell culture after cleaning is placed in aseptic
Soak in water, after 2h, blot sterilized water, microcarrier is placed in freezing 30min in -80 DEG C of refrigerators, taking-up microcarrier is placed in cold afterwards
Carry out freeze-drying in lyophilizer, overnight after can obtain have loose structure quail egg white microcarrier.
5. cell culture is carried out using microcarrier：Thoroughly cleaning is soaked successively with PBS phosphate buffers, cell culture fluid
Egg white microballoon 2h, is inoculated in HepG2 cells on quail egg white microcarrier afterwards, observation cells on microcarriers growth feelings after 3 days
Condition.
Claims (6)
1. a kind of cell microcarrier based on egg white, it is characterised in that the egg white microcarrier is emulsified by micro fluidic device first
Egg white solution obtains egg white drop template, recycles chemically or physically method to obtain template solidification, and concrete preparation process is as follows：
（1）The extraction of egg white：
Thoroughly cleaning is carried out to selected eggs surface, egg white is separated with yolk with egg device, isolated egg white is carried out
Filter, take the egg white weak solution for leaching standby；
（2）The preparation of egg white emulsion：
Size tunable, uniform egg white emulsion are prepared using micro fluidic device and egg white weak solution；
（3）The solidification of egg white emulsion：
Egg white emulsion is solidified by chemically or physically method, egg white microcarrier after cleaning, is obtained；
（4）The acquisition of egg white microcarrier loose structure：
Freeze-drying is carried out by the egg white microcarrier to solidifying, you can give egg white microcarrier distinctive loose structure；
（5）Cell culture is carried out using the egg white microcarrier for preparing：
The egg white micro-carrier surface for preparing is carried out microcarrier is soaked in after thoroughly cleaning PBS, cell culture successively
Each 2h in liquid, then cell is inoculated on microcarrier carries out Three-dimensional cell culture, can be observed after 3 days cell cover with entirely micro-
Carrier.
2. egg white cell microcarrier according to claim 1 and preparation method thereof, it is characterised in that step（1）Described in
Egg white is retrieved from the egg ovum of all kinds of birds of chicken, duck, goose, quail or birds.
3. egg white cell microcarrier according to claim 1 and preparation method thereof, it is characterised in that step（2）Described in by
Egg white emulsion size obtained in microflow control technique is 20 μm of -5mm.
4. egg white cell microcarrier according to claim 1 and preparation method thereof, it is characterised in that step（3）Described in
The curing of egg white emulsion is heating, ultraviolet irradiation, strong acid-base, alcohol denaturation.
5. egg white cell microcarrier according to claim 1 and preparation method thereof, it is characterised in that step（4）Described in lead to
Cross freeze-drying and can give egg white microcarrier loose structure, its hole size can be dry by the concentration of egg white, the species of egg white, freezing
All kinds of factor controllings of dry time.
6. egg white cell microcarrier according to claim 1 and preparation method thereof, it is characterised in that step（5）Described in connect
It can be HepG2 cells, 3T3 cells that kind carries out the cell of Three-dimensional cell culture on microcarrier.
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| CN201610978810.3A CN106497867A (en) | 2016-11-08 | 2016-11-08 | A kind of cell microcarrier based on egg white and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107601463A (en) * | 2017-08-18 | 2018-01-19 | 济南大学 | A kind of graphene aerosol method for preparing microsphere based on microflow control technique |
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Application publication date: 20170315 |