CN106754672A - A kind of cultural method of attached cell - Google Patents
A kind of cultural method of attached cell Download PDFInfo
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- CN106754672A CN106754672A CN201611087967.3A CN201611087967A CN106754672A CN 106754672 A CN106754672 A CN 106754672A CN 201611087967 A CN201611087967 A CN 201611087967A CN 106754672 A CN106754672 A CN 106754672A
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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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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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
- C12N2511/00—Cells for large scale production
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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
Abstract
The present invention relates to cell field, more particularly to a kind of cultural method of attached cell.The cultural method is taken and cultivated after the attached cell mixes with microcarrier, and attached cell is peeled off from the microcarrier, is collected.The invention provides a kind of technique for obtaining a large amount of amnion mesenchymal stem cells in a short time, using microcarrier for attached cell provides substantial amounts of growth area in limited space, amnion mesenchymal stem cell is expanded on a large scale.
Description
Technical field
The present invention relates to cell field, more particularly to a kind of cultural method of attached cell.
Background technology
1999,《Science》HESC's achievement in research is chosen as first of the big Progress ＆ New Products in the world ten then,
2000,《Time》Weekly be classified as 20 the end of the century world ten first of big technological achievement, and think embryonic stem cell and the mankind
Genome will most develop the field with application prospect as the new century simultaneously.So far stem-cell research turns into biology in recent years
And one of most noticeable focus in medical domain.Stem cell is that " trunk " work is played in the growth and development of bion
Initial cell, is a kind of cell colony with self-renewing, hyperproliferation and multi-lineage potential, and it is dry including embryo
Cell and adult stem cell.Although embryonic stem cell can be divided into various cell types, this differentiation is " non-locating ",
And there is a problem of the aspects such as ethics.Adult stem cell does not exist ethics, law, immunological rejection etc. then and asks
Topic, while have the advantages that convenient material drawing, source are wide, thus, adult stem cell is controlled in organizational project, gene therapy and individuation
There is good potential applicability in clinical practice in the research for the treatment of.
The raising of mass cell culture productivity can be by 2-3 × 106It is amplified under cell/mL cell densities big
Volume, or increase cell density come hot housing process by smaller volume., it is necessary to more frequency during raising cell density
Numerous replacing nutrient solution, finally also needs to apply perfusion cultures.As the amnion mesenchymal stem cell of attached cell because culture
The limitation in space can not on a large scale be cultivated as suspension cell, great culture surface product/body that micro-carrier system is provided
Product ratio, the equipment that improve cell yield and Large Copacity need not be relied on.For other kinds of monolayer cultivation, micro- load
Body culture needs relatively little of space, so that it may substantial amounts of cell is produced, so as to reach the purpose of large-scale culture.Now, micro- load
The main commercial Application of body is production vaccine, the carrier of gene therapy, natural and recombinant protein and growing number of Dan Ke
Grand antibody, may be influenceed using the application numbers of microcarrier by the selection of production cell and its type of glycosylation.Such as
Really more natural attached cell systems are chosen, then the application of microcarrier will be greatly increased.Generally speaking, with results
A large amount of attached cells are deficient comparings for final purpose Microcarrier Culture Techniques.
Amnion mesenchymal stem cell is used for clinical, organizational project and is required for substantial amounts of cell.Clinical practice is up to 108-1010
The order of magnitude.Because mescenchymal stem cell needs the cell quantity of adherent growth, this scale to be expanded using blake bottle in laboratory,
Consumptive material consumption is big, and artificial paying is also surprising.Bioreactor is generally used for extensive amplifying cells, but generally raw
Thing reactor is used for the extensive amplification of suspension cell, and is open system, it is necessary to many holding equipments, not only operate multiple
It is miscellaneous, the problems such as causing pollution is also easy to, there is sizable risk in clinical practice.
The content of the invention
In view of this, the present invention has found that the key for improving attached cell culture density is that have sufficiently large table by studying
The supplement of area and culture medium, the present invention realizes attached cell High Density Cultivation using microcarrier and bioreactor.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of cultural method of attached cell, take and cultivated after the attached cell mixes with microcarrier,
Attached cell is peeled off from the microcarrier, is collected.
In some specific embodiments of the invention, microcarrier described in the cultural method is Cytodex-3.
In some specific embodiments of the invention, microcarrier described in the cultural method is when attached cell is cultivated
Concentration be 0.5~5.0mg/mL final volumes.
In some specific embodiments of the invention, the inoculum density of attached cell is described in the cultural method
0.5~2 × 105cells/mL。
In some specific embodiments of the invention, the temperature cultivated described in the cultural method is 35.5 DEG C.
In some specific embodiments of the invention, the pH value cultivated described in the cultural method for 7.00~
7.15。
In some specific embodiments of the invention, incubation time described in the cultural method is 2~3d.
Peeled off in some specific embodiments of the invention, described in the cultural method include washing, enzymic digestion
Step.
In some specific embodiments of the invention, enzymic digestion described in the cultural method be specially addition with it is described
0.25% isometric (w/w) trypsase/EDTA of microcarrier [the working solution concentration of EDTA is 0.05% (w/w)], 10RPM is stirred
Mix 15 seconds, stand 1min, be repeated 1 times, 2.5 times of volumes are added in the complete medium of trypsase with the mixing speed of 10RPM
Terminate digestion.Under the method for combining piping and druming present invention also offers pancreatin/EDTA digestion peels off attached cell from microcarrier
Come.Within protection scope of the present invention, the present invention is not limited the digestion method of every this area herein.
In some specific embodiments of the invention, attached cell described in the cultural method is dry for amnion mesenchymal
Cell.
In some specific embodiments of the invention, 1 × 107Cell-500mL nutrient solution -1g microcarriers are cultivated for -3 days
Amplifiable about 16 times of pattern.The amplification limit depends on the average adherent area of amount and individual cells of microcarrier.
Cell and microcarrier combination：
Before inoculating cell, all culture parameters especially temperature and pH are balanced and stablize, this shows and decides whether to tie
Close complete key factor.Culture vessel containing culture medium is in humidity 95%, 5%CO2Incubator in 35.5 DEG C incubation
Balance, 30 minutes afterwards, and nutrient solution will just be suitable for inoculation.During inoculation, using the cell that exponential phase of growth vitality is vigorous, keep away
Exempt to use the cell of resting stage.The PBS in aseptic microcarrier is suctioned out, with the culture medium rinse for warming once.Adjustment culture medium
Temperature is to 35 DEG C, it is ensured that nutrient solution pH takes 50mL2 × 10 7.055Cell/mL cells are slowly added into 500mL and contain 2.0g/L
The said temperature of Cytodex3 and the nutrient solution of pH, are stirred 2 hours, afterwards 1 with the mixing speed of 20RPM under dark condition
Temperature is ramped up 37 DEG C, mixing speed is promoted to 25RPM, and pH is 7.00~7.15 for adjustment, into conventional training within hour
The stage of supporting.Microcarrier is incubated in evening before that day, is combined at once after morning next day preparation seed cell, afternoon 17：00 can first
Sub-sampling observation can accurately know the situation of combination.More than 90% cell is preferred with reference to upper microcarrier.
Cultivation stage：
Daily timing sampling (09：00 and 17：00), do not stop stirring, sampled from bioreactor with screw syringe
Module extracts about 2mL sample liquids, first determines pH, records pH modules measured value and pH meter measured value, is determined by pH meter and is defined, if pH
Value is outside 7.00~7.15, it is necessary to open pH adjustment modules, pH is within effective range for control, during batch cultur
, there are not the off-limits situations of pH in (2~3 days), and continuous culture needs always on pH adjustment modules and fluid infusion module.Survey
The uniform sample liquids of 1mL are taken in EP pipes after having determined pH, 2min is stood, and supernatant is abandoned in suction, adds the 0.1mol/L citric acids-crystallization of 1mL
Purple dye liquor (citric acid can be by microcarrier dissolving and broken cell membrane, and crystal violet dye liquor can be by nuclei dyeing into dark color), piping and druming is mixed
It is even, it is incubated 3 hours in 37 degrees Celsius of environment, 10uL is taken in blood cell counting plate, nucleus is counted under 200 times, to count knot
Fruit judges cell proliferative conditions.Other 1mL is placed in 12 well culture plates, in observation microcarrier situation under inverted microscope.
The mode that cell with microcarrier separate after culture maturation：
Whole processing procedure will ensure in 37 DEG C of environment.
Stop stirring, stand 2min, it is seen that microcarrier is settled completely, supernatant is abandoned in suction, with the injection isometric with microcarrier
Cleaned 2 times with physiological saline (being preheated to 37 DEG C in advance), stirred 15 seconds using 10RPM in cleaning process, be subsequently adding isometric
0.25% trypsase/EDTA, 10RPM stir 15 seconds, then stand 1min, be repeated 1 times, added with the mixing speed of 10RPM
The complete medium for entering 2.5 times of volumes in trypsase terminates digestion, and standing 2min makes microcarrier settle completely, draws supernatant mistake
70um filter membranes 2 times (can draw the microcarrier of a small amount of critical layer), gained supernatant is centrifuged 5min in 1500RPM, that is, obtain pure thin
Born of the same parents.
3rd day afternoon can harvesting, 1 × 107Cell-500mL nutrient solution -1g -3 days training modes of microcarrier can expand
Increase about 16 times.The amplification limit depends on the average adherent area of amount and individual cells of microcarrier.
In some specific embodiments of the invention, the washing is with the injection physiology salt isometric with microcarrier
Water (being preheated to 37 DEG C in advance) is cleaned 2 times, is stirred 15 seconds using 10RPM in cleaning process.
In some specific embodiments of the invention, with reference to microcarrier, filled using between bioreactor prepare with scale
Matter stem cell.Bioreactor use glass jar and have multiple functional modules available, including import/exhaust, fluid infusion system,
Sampler, all kinds agitating paddle, heat exchanger etc., can need swap modules according to experiment, its closed culture systems, have
Effect avoids pollution equivalent risk, is expanded on a large scale into clinical practice for attached cell and provides good technical foundation.
The invention provides a kind of cultural method of attached cell, take and cultivated after the attached cell mixes with microcarrier,
Attached cell is peeled off from the microcarrier, is collected.The use of microcarrier is in a short time adherent thin the invention provides one kind
Born of the same parents provide the technique that substantial amounts of growth area obtains a large amount of amnion mesenchymal stem cells in limited space.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 technical solution of the present invention schematic diagrames.
Specific embodiment
The invention discloses a kind of cultural method of attached cell, those skilled in the art can use for reference present disclosure, fit
When modified technique parameter is realized.In particular, all similar replacements and change for a person skilled in the art
It is it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are entered by preferred embodiment
Description is gone, related personnel can be not substantially being departed from present invention, spirit and scope to method described herein and application
It is modified or suitably change is realized and apply the technology of the present invention with combining.
The present invention is using bioreactor combination microcarrier culture attached cell and enzymic digestion and low-shearing force agitating paddle thing
What reason stirring was combined shells cellifugal method from microcarrier.
(1) separation and Extraction of amnion mesenchymal stem cell：Take people's amnion of fresh collection, trypsase and type i collagen enzyme
After treatment, filtered with 100 μm of cell screen clothes, with (1~1.5) × 105Cell/mL is in the training containing serum substitute cell culture fluid
In foster vessel, in 37 DEG C, 5%CO2Under the conditions of quiescent culture, liquid is changed to primary cell after 48h, obtain primary amnion mesenchymal do
Cell；
(2) the culture amplification of amnion mesenchymal stem cell：By the primary cell obtained by step (1) culture with 6 × 104-8×
104cell/cm2Density be inoculated in culture dish, add fresh cell medium, in 37 DEG C, 5%CO2Under the conditions of cultivate；
(3) detection of amnion mesenchymal stem cell：Carry out cytoactive detection, aseptic detection and FCM analysis；
(4) large-scale culture of amnion mesenchymal stem cell：Using bioreactor culture, by amnion mesenchymal stem cell
After being seeded in bioreactor, after mixing with microcarrier, adjustment dissolved oxygen concentration, pH value, speed of agitator and throughput, according to
The result of sampling analysis cell count, survival rate, nutritional ingredient and metabolin changes culture medium, so as to reach the high density of cell
Large-scale culture；
(5) freezen protective of amnion mesenchymal stem cell：To detect that qualified cell cryopreservation is interior in pipe, the detailed letter of cell
Breath is scanned into data management system.
In step (1), amnion tissue is aseptically sheared, with after wash buffer separate needed for tissue block,
Contain sugar, amino acid, somatomedin, inorganic salts, trace element for supporting stem cell growth etc. in the cell culture fluid.
In step (2), primary cell is digested with trypsin solution when culture vessel bottom 80%-90% is covered with and blown and beaten thin
Born of the same parents, completely disengage from cell and be inoculated in after original cuiture vessel culture vessel, the culture vessel may be selected from cell culture orifice plate,
Culture dish and tissue cultures square vase.
Term is explained：
Mescenchymal stem cell：It is the important member of stem cell line, from the mesoderm and ectoderm of mesoderm growing early stage, category
In multipotential stem cell, MSC initially has found in marrow, because it has multi-lineage potential, hematopoiesis support and promotes stem cell to plant
Enter, immunoregulation and be increasingly subject to the concern of people the features such as self-replacation.As mescenchymal stem cell is special in vivo or in vitro
Under fixed inductive condition, various groups of amnion, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium etc. can be divided into
Cell is knitted, still there is multi-lineage potential after continuous passage culture and freezen protective, can be used to decline as preferable seed cell
The injuries of tissues and organs reparation that old and lesion causes.
Microcarrier：Refer to diameter at 60-250 μm, can be suitably used for the microballon of adherent cell growth.Usually gathered by natural Portugal
The polymer composition of sugared or various synthesis.
Bioreactor：Refer to manufacture or the engineering equipment of any offer bioactivity environment.In one case, it is biological
Reactor is one to carry out being related to chemical process of the biological or CBAC material by specific biological production out
Container.This process both can with it is aerobic carry out can also anaerobic carry out.These bioreactors are generally cylindrical, and its volume is from several
Several cubic metres are raised to, are often made of stainless steel.
The invention provides a kind of cultural method of attached cell, take and cultivated after the attached cell mixes with microcarrier,
Attached cell is peeled off from the microcarrier, is collected.Compared with prior art, the amnion mesenchymal stem cell that the present invention is provided is big
Scale evaluation method, due to using with powerful control function, also bioreactor simple to operate and safe, enzymic digestion
Cellifugal method is shelled from microcarrier with what low-shearing force agitating paddle physical agitation was combined, thus can be concluded with following
Effect：
1st, micro-carrier surface product/volume ratio is big, and the yield of unit volume cultured cells is high, more traditional monolayer cell culture
Area is greatly increased, and amnion mesenchymal stem cell can be largely obtained in a short time；
2nd, sampling repeatability is good, and cell finally harvests that process is uncomplicated, and labour intensity is small, occupies little space, easy to operate,
Required personnel are few, and technique easily amplifies production, it is ensured that cell quality；
3rd, cell culture system meets newest statutory standard, and security is more preferable.
4th, the amplification times of the cultural method that the present invention is provided are about 8~20 times, and prior art three-dimensional amplification cultivation
About 3~4 times of amplification times, compared with prior art with significant difference (P ＜ 0.05).
Raw materials used and reagent can be bought by market in the cultural method of the attached cell that the present invention is provided.
Operation in embodiment 1,2,3,4,5,6,7 is carried out under rigorous aseptic environment.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1,
Table 1
Note：Compared with control group, * shows that P ＜ 0.05, * * show P ＜ 0.01.
As can be seen from Table 1：Step of the cellar culture ware incubation required for a plurality magnitude cell concentration is reached
It is rapid very cumbersome, can so cause cell to be greatly increased exposed to the time being unfavorable in the environment of cell growth, finally obtain
Cell state can also have a greatly reduced quality, and microcarrier culture intermediate link is few, and an operation for flow just can be completed, and be reduced carefully
The open-assembly time of born of the same parents, the cell state for finally obtaining must be excellent.In the consumption of material, microcarrier is except slightly many consumption
Some culture mediums, other main materials all can be recycling.
Embodiment 2, amnion mesenchymal stem cell separation and Extraction
Take people's amnion of fresh collection, after PBS 3 times, cut to (20-50) cm2After size, Trypsin Induced is first used
Into small tissue block, then with PBS, it is wholly absent with type i collagen enzymic digestion to tissue block again after shredding, 100 μm of cells
With (1-1.5) × 10 after screen filtration5Cell/mL in the culture vessel containing serum substitute cell culture fluid, in 37 DEG C,
5%CO2Under the conditions of quiescent culture, change liquid after 2 days, when observe ware inner cell degree of converging reach more than 80%, obtain primary sheep
Intermembranous mesenchymal stem cells.
The culture vessel culture amplification of embodiment 3, amnion mesenchymal stem cell
When cell covers with the 80% of plate, original fluid is discarded, washed with PBS 2 times, add containing for covering ware bottom amount
The trypsase of EDTA, digests 1-2min, and period is uninterruptedly observed with inverted microscope, sees that kytoplasm bounces back, space between cells increase,
Cell rounding, adds the cell culture medium of respective volume to terminate digestion immediately, attached cell is blown and beaten repeatedly with pipette, by cell
Piping and druming is got off, low-speed centrifugal 5min, then uses cell culture fluid re-suspended cell, is 6-8 × 10 according to density4Individual cell/ml, inoculation
To in plate, fresh cell medium is added in 37 DEG C, 5%CO2Under the conditions of cultivate, change liquid within 3 days.About 2 × 10 can be obtained5It is individual
Cell/ml cells, cell quantity expands about 3 times.
Embodiment 4, amnion mesenchymal stem cell cultivates amplification in bioreactor containing microcarrier
The glass blake bottle culture amplifying cells containing microcarrier, the work of glass blake bottle are used in the present embodiment
Volume is 500ml, and microcarrier is Cytodex-3.
1. glass blake bottle pretreatment：500ml glass blake bottles are cleaned up, and drying adds silication liquid to being completely dried
In bottle, slow rotating and culturing bottle makes silication immersion moisten bottle wall, after sucking silication liquid, rolling bottle is placed in into ventilation and air-dries 12h, steams
Distilled water is rinsed standby；
2. microcarrier pretreatment：Microcarrier density is set to 2g/L in the present embodiment, because volume of culture is 500mL, therefore
Weigh in 1gCytodex-3 rolling bottles, add 200ml PBS immersion 3h, the new PBS of 200ml are added after sucking, 121 DEG C of high pressures are steamed
Vapour sterilizing 20min, sucks PBS and adds 200ml containing cell culture fluid, 37 DEG C of soaked overnights；
3. the inoculation of amnion mesenchymal stem cell：It is thin to what is converged rather than length by the cell for being inoculated with exponential phase of growth
Born of the same parents, the cell yield of microcarrier culture can improve 2-3 times, and microcarrier culture should be inoculated with as much as possible has silk in active
Division stage, about grow to 80% cell for converging.Microcarrier inoculum density is 0.5-2 × 105Cells/mL, at 1/3-1/2 ends
Inoculating cell in volume.Then the homogeneous suspension of microcarrier is kept with minimum speed stirring at once.After cell attachment, culture is added
Base is to final volume.The cell yield of microcarrier culture is directly related thus also direct with microcarrier concentration with the surface area of growth
Correlation, it is 0.5-5.0mg/mL final volumes that Cytodex3 microcarriers are used in the concentration used in stir culture.
4. the supply of cell culture fluid：Carefully supply culture is for maintaining microcarrier culture in cultivation cycle
Critically important one side, the program for amnion mesenchymal stem cell is to start to change within every 3 days 50% culture medium, works as sampling
When, the volume of culture of 10-20% can be changed with fresh culture medium, in order to utilize equivocation, in first 2 days of culture not
Culture medium should be changed；
5. the acquisition of amnion mesenchymal stem cell：Stop stirring, allow microcarrier to precipitate, pour out supernatant culture medium, use pH7.6
Containing EDTA without Ca2+、Mg2+The PBS washings of ion.The total amount of EDTA-PBS solution should be in 50-100mL/g
Cytodex3.EDTA-PBS solution is discarded, is then stirred every now and then in 37 DEG C of incubations with pancreas enzyme -EDTA solution, after 15min, plus
Enter the effect that the nutrient solution containing 10% (v/v) serum substitute terminates pancreatin.Harvesting, can obtain (0.4~1.6) × 106
The cell of individual cell/ml density, cell quantity expands about 8 times.
About 3~4 times of the amplification times of prior art three-dimensional amplification cultivation, compared with prior art with significant difference (P ＜
0.05)。
Embodiment 5, stirring type bioreactor culture amplification amnion mesenchymal stem cell
Form health is selected, well-grown amnion mesenchymal stem cell trypsase/EDTA digestion is given birth to as cell
The seed cell of thing reactor, with 2 × 105The density of individual cell/ml is inoculated on microcarrier, is expanded with cell culture fluid
Culture, bioreactor condition of culture is pH7.2, and 37 DEG C of temperature, oxyty is the 50% of the saturation of the air.
The parameters such as glucose and lactic acid content are detected by Biochemical Analyzer daily, is cultivated after inoculation about 7 days, emptying culture
Liquid, after PBS rinses 2-3 times, addition pancreatin/EDTA, digestion 15min, the isometric nutrient solution termination digestion of addition, harvesting,
1.6 × 10 can be obtained6The cell of individual cell/ml density, cell quantity expands 8 times.
About 3~4 times of the amplification times of prior art three-dimensional amplification cultivation, compared with prior art with significant difference (P ＜
0.05)。
Embodiment 6, amnion mesenchymal stem cell cultivates amplification in bioreactor containing microcarrier
Cell and microcarrier combination：
Before inoculating cell, all culture parameters especially temperature and pH are balanced and stablized.Culture containing culture medium
Container is in humidity 95%, 5%CO2Incubator in 35.5 DEG C incubate balance, 30 minutes are afterwards, and nutrient solution will just be suitable for connecing
Kind.During inoculation, the cell that exponential phase of growth vitality is vigorous is used, it is to avoid use the cell of resting stage.Suction out aseptic microcarrier
In PBS, with warm culture medium rinse once.The temperature of culture medium is adjusted to 35 DEG C, it is ensured that nutrient solution pH takes 7.05
50mL2×105Cell/mL cells are slowly added into 500mL and contain the said temperature of 2.0g/L Cytodex3 and the nutrient solution of pH,
Stirred 2 hours under dark condition (illumination condition can or can not have an impact not known) with the mixing speed of 20RPM, it is small 1 afterwards
When within temperature is ramped up 37 DEG C, mixing speed is promoted to 25RPM, and pH is 7.00~7.15 for adjustment, into cellar culture
Stage.
Microcarrier is incubated in evening before that day, is combined at once after morning next day preparation seed cell, afternoon 17：00 can first
Sub-sampling observation can accurately know the situation of combination.More than 90% cell is preferred with reference to upper microcarrier.
Cultivation stage：
Daily timing sampling (09：00 and 17：00), do not stop stirring, sampled from bioreactor with screw syringe
Module extracts about 2mL sample liquids, first determines pH, records pH modules measured value and pH meter measured value, is determined by pH meter and is defined, if pH
Value is outside 7.00~7.15, it is necessary to open pH adjustment modules, pH is within effective range for control, during batch cultur
, there are not the off-limits situations of pH in (2~3 days), and continuous culture needs always on pH adjustment modules and fluid infusion module.Survey
The uniform sample liquids of 1mL are taken in EP pipes after having determined pH, 2min is stood, and supernatant is abandoned in suction, adds the 0.1mol/L citric acids-crystallization of 1mL
Purple dye liquor (citric acid can be by microcarrier dissolving and broken cell membrane, and crystal violet dye liquor can be by nuclei dyeing into dark color), piping and druming is mixed
It is even, it is incubated 3 hours in 37 degrees Celsius of environment, 10uL is taken in blood cell counting plate, nucleus is counted under 200 times, to count knot
Fruit judges cell proliferative conditions.Other 1mL is placed in 12 well culture plates, in observation microcarrier situation under inverted microscope.
The mode that cell with microcarrier separate after culture maturation：
Whole processing procedure will ensure in 37 DEG C of environment.
Stop stirring, stand 2min, it is seen that microcarrier is settled completely, supernatant is abandoned in suction, with the injection isometric with microcarrier
Cleaned 2 times with physiological saline (being preheated to 37 DEG C in advance), stirred 15 seconds using 10RPM in cleaning process, be subsequently adding isometric
0.25% trypsase, 10RPM stirred 15 seconds, then stands 1min, is repeated 1 times, and 2.5 are added with the mixing speed of 10RPM
Times volume terminates digestion in the complete medium of trypsase, and standing 2min makes microcarrier settle completely, draws supernatant and crosses 70um
Filter membrane 2 times (can draw the microcarrier of a small amount of critical layer), gained supernatant is centrifuged 5min in 1500RPM, that is, obtain pure cell.
3rd day afternoon can harvesting, 1 × 107Cell-500mL nutrient solution -1g -3 days training modes of microcarrier can expand
Increase about 16 times.The amplification limit depends on the average adherent area of amount and individual cells of microcarrier.
About 3~4 times of the amplification times of prior art three-dimensional amplification cultivation, compared with prior art with significant difference (P ＜
0.05)。
The freezen protective of embodiment 7, amnion mesenchymal stem cell
The amnion mesenchymal stem cell to 80-90% will be covered with the plate of 4~embodiment of embodiment 6 and comparative example,
The old nutrient solution of removal, with PBS 2 times, is subsequently added into the 37 DEG C of digestion 1-2min of pancreatin containing EDTA, treats that cell is all de-
Fall behind, add cell culture fluid to terminate digestion, low-speed centrifugal 5min removes supernatant, then with stem cell cryopreserving liquid that cell is resuspended,
The amnion mesenchymal stem cell suspension of acquisition is added in cell cryopreservation tube, is placed in -80 in the program mode cooling box containing isopropanol
DEG C overnight, it is transferred within second day in -196 DEG C of liquid nitrogen and preserves for a long time.
10, the recovery Liquid nitrogen storage cell of 3 months, detects its Cell viability, as a result such as table 2：
The Cell viability of table 2
Note：Compared with control group, * shows that P ＜ 0.05, * * show P ＜ 0.01.
As can be seen from Table 2：It can be seen that still keeping cell quality very high after freezing by the cell of microcarrier culture.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of cultural method of attached cell, it is characterised in that take and cultivated after the attached cell mixes with microcarrier, from institute
State and attached cell is peeled off on microcarrier, collect.
2. cultural method according to claim 1, it is characterised in that the microcarrier is Cytodex-3.
3. cultural method according to claim 1 and 2, it is characterised in that the microcarrier is when cultivating attached cell
Concentration is 0.5~5.0mg/mL final volumes.
4. cultural method according to claim 3, it is characterised in that the inoculum density of the attached cell is 0.5~2 ×
105cells/mL。
5. the cultural method according to any one of Claims 1-4, it is characterised in that the temperature of the culture is 35.5 DEG C.
6. the cultural method according to any one of claim 1 to 5, it is characterised in that the pH value of the culture is 7.00~
7.15。
7. the cultural method according to any one of claim 1 to 6, it is characterised in that the incubation time is 2~3d.
8. the cultural method according to any one of claim 1 to 7, it is characterised in that the stripping includes washing, enzymic digestion
The step of.
9. cultural method according to claim 8, it is characterised in that the enzymic digestion is specially addition and the microcarrier
0.25% isometric (w/w) trypsase/EDTA, 10RPM are stirred 15 seconds, stand 1min, are repeated 1 times, with the stirring of 10RPM
Speed adds 2.5 times of volumes to terminate digestion in the complete medium of trypsase.
10. the cultural method according to any one of claim 1 to 9, it is characterised in that the attached cell is to fill between amnion
Matter stem cell.
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| CN109628372A (en) * | 2019-01-10 | 2019-04-16 | 武汉济源高科技有限公司 | Paramagnetic particle method culture attached cell |
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