CN110093312A - A kind of cell large-scaled culture method, purification process and cell messenger - Google Patents
A kind of cell large-scaled culture method, purification process and cell messenger Download PDFInfo
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- CN110093312A CN110093312A CN201910405915.3A CN201910405915A CN110093312A CN 110093312 A CN110093312 A CN 110093312A CN 201910405915 A CN201910405915 A CN 201910405915A CN 110093312 A CN110093312 A CN 110093312A
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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
- C12N5/0653—Adipocytes; Adipose tissue
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- 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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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2531/00—Microcarriers
Abstract
The present invention provides a kind of large-scaled culture methods of cell; it include: that the GE microcarrier after cultivating cell and sterilizing is inoculated in the culture medium containing serum; high density is cultivated in containers for culturing organisms; then serum free medium is changed into; continue to cultivate 4-7d in serum free medium, collects supernatant.The cultural method is easy to operate, and operating condition is milder, and cell survival rate is high, and the culture for subsequent cell scale provides reference method, and should be widely promoted application.
Description
Technical field
The present invention relates to field of cell culture, in particular to a kind of cell large-scaled culture method, cell messenger and
Purification process.
Background technique
In each adult body, about contain 30,000,000,000 white adiposes, function is to store energy in the form of fat cell
Get up.In each fat cell, all contain triglyceride, is commonly called as fat globule.Fat globule amount becomes larger, and fat cell volume just expands
Increase, causes obesity；Otherwise burning triglyceride, cellular atrophy stature get off with regard to thin.Under normal circumstances, fat cell number
It is not just further added by after mesh to puberty.The distribution of body fat, depending in part on heredity and hormone etc. influences, such as women
Subcutaneous fat accumulate in underbelly, buttocks and thigh more, and male then hoards in upper abdomen and waist.
Umbilical chord mesenchymal cells (Mesenchymal Stem Cells, MSCs), which refer to, to be present in neonatal umbilical cord tissue
A kind of versatile stem cell, it can be divided into many kinds of histocytes, have wide potential applicability in clinical practice.Using inactivation navel
Band serum free culture system system can Successful amplification human umbilical cord mesenchymal stem cells, the cell of culture has the substantially special of mescenchymal stem cell
Property, theoretical foundation is provided to establish mesenchyma stem cell and clinical application.
Microcarrier refers to that diameter at 60-250 μm, can be suitably used for the microballon of adherent cell growth.Usually by natural glucan
Or the polymer composition of various synthesis.
The principle of microcarrier application is that the particle-microcarrier harmless to cell is added in the culture solution of culture vessel,
Cell is set so that microcarrier is remained suspended state in micro-carrier surface apposition growth, while by prolonged agitation as carrier.
Proliferation of the anchorage-dependent cell on micro-carrier surface, Yao Jingli stick it is adherent, grow and be extended to single layer three phases.Carefully
Born of the same parents are only attached to solid substrate and could be proliferated, therefore cell is further spread out and grows in the attaching of micro-carrier surface
It is crucial.Stick mainly by electrostatic attraction and Van der Waals force.Cells containing sequences stick in micro-carrier surface, depend primarily on cell with
The contact probability and blending of microcarrier.
The method and process of microcarrier culture cell in the prior art is complicated, and operating condition is harsh, and cell survival rate is not high,
Inoculum density is low, and the requirement to microcarrier itself is high, needs just to can guarantee cell culture using the microcarrier of tailored version
It is normally carried out, improves operating cost indirectly, limit the application surface of microcarrier.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of cultural method of cell scale, above-mentioned cultural method operation letter
Single, operating condition is milder, and cell survival rate is high, provides method for reference for the culture of subsequent cell scale,
Should be widely promoted application.
The second object of the present invention is to provide a kind of specific purification process of cell, is after purification natural non-recombinant structure
Characteristic, purity is high, and it simulates intracorporal optimal proportions concentration synergistic effect, regulating cell proliferation, differentiation maintain tissue and thin
The development of born of the same parents' ordering growth, the protein such as collagen, fibronectin, superoxide dismutase are the key that maintain skin youth state
Ingredient, it is very widely used that purifying obtains product.
The third object of the present invention is to provide the cell messenger that above-mentioned cultural method obtains, by using training of the invention
The cell messenger that the method for supporting obtains is not only active good, and each type of cell messenger functionality for cultivating acquisition is stronger, and
And this method itself is easy to operate, operating condition is milder, and cell survival rate is high, and the culture for subsequent cell scale provides
Method for reference, should be widely promoted application.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention provides a kind of cultural method of cell scale, including the following steps: will cell be cultivated and sterilizing
GE microcarrier afterwards is inoculated in the culture medium containing serum, and high density is cultivated in containers for culturing organisms, then changes serum-free training into
Base is supported, continues to cultivate 4-7d in serum free medium, collects supernatant.
The cell to be cultivated is one or both of umbilical chord mesenchymal cells, fat cell；
Preferably, the inoculum density of the cell to be cultivated is (8-9) * 103cell/ml；
It is highly preferred that the inoculum density of the cell to be cultivated is 8.6*103cell/ml。
Microcarrier refers to that diameter at 60-250 μm, can be suitably used for the microballon of adherent cell growth.Usually by natural glucan
Or the polymer composition of various synthesis.
The principle of microcarrier application is that the particle-microcarrier harmless to cell is added in the culture solution of culture vessel,
Cell is set so that microcarrier is remained suspended state in micro-carrier surface apposition growth, while by prolonged agitation as carrier.
Proliferation of the anchorage-dependent cell on micro-carrier surface, Yao Jingli stick it is adherent, grow and be extended to single layer three phases.Carefully
Born of the same parents are only attached to solid substrate and could be proliferated, therefore cell is further spread out and grows in the attaching of micro-carrier surface
It is crucial.Stick mainly by electrostatic attraction and Van der Waals force.Cells containing sequences stick in micro-carrier surface, depend primarily on cell with
The contact probability and blending of microcarrier.
The method and process of microcarrier culture cell in the prior art is complicated, and operating condition is harsh, and cell survival rate is not high,
Inoculum density is low, and the requirement to microcarrier itself is high, needs just to can guarantee cell culture using the microcarrier of tailored version
It is normally carried out, improves operating cost indirectly, limit the application surface of microcarrier.
In order to solve the above technical problems, The present invention provides a kind of cultural method of cell scale, the cultural methods
Easy to operate, operating condition is mild, finds out and establish optimal proportion condition by constantly exploring, and microcarrier is combined wait train
The mode that cell carries out pilot scale culture is supported, cell survival rate is guaranteed, and inoculum density is high, and the requirement to microcarrier itself is low,
It does not need to be equipped with dedicated microcarrier, reduces operating cost, simplify training method, should be widely promoted application.
Preferably, the total concentration for the culture solution being formed by after inoculation is between 0.1-1wt%.
It is highly preferred that being surveyed after cell inoculation to microcarrier and the total concentration of cell to be cultivated in the medium
Fixed, optimum concentration needs to control between 0.2-0.8wt%, can also for 0.3wt%, 0.4wt%, 0.6wt%,
0.7wt% etc..
As cell to be cultivated after the attached cell of growth can be taken to have carried out digestion operation.
Above-mentioned GE microcarrier needs sterilization treatment, specific Biocidal treatment method before with cell combination are as follows:
PBS 0.01M pH7.4 prepares 121 DEG C of 15min of 4L high pressure sterilization；
Microcarrier is impregnated with above-mentioned PBS to stay overnight, is washed 3 times, and beads precipitates bottom, discards the sterilizing rear chamber being crushed in supernatant
It is balanced overnight under the conditions of temperature with culture medium, a culture solution is changed in centre.10ml/ pipe, 4 DEG C of preservations are dispensed, the used time takes 1ml to add
100ml culture medium.
Preferably as further enforceable scheme, 10-15rpm stir culture 24-26h, 18-30rpm are stirred after inoculation
Mix culture 24-72h.It is general first at a slow speed for 24 hours more than, be then quickly stirred overnight again, and observe the upgrowth situation of cell.
Preferably, in the basic culture solution incubation without serum, the concentration of oxygen is 4-7wt%, preferably oxygen
The concentration of gas is 5wt%.
If also needing the step of additionally increasing removal using the culture solution containing serum, during later-period purification, increase
Add operation complexity, and the culture solution containing serum will affect the product after subsequent purification and further use situation, because
This preferably selects the basic culture solution without serum, by adjusting the content of oxygen in atmosphere, to guarantee that cell messenger is a large amount of
Secretion and guarantee its activity.
Preferably as further enforceable scheme, the GE microcarrier is Cytodex 1GE.
Preferably as further enforceable scheme, after stir culture 72h or more, inhales and abandon culture supernatant, using life
Salt water washing is managed the culture medium containing serum is added, after microcarrier falls off, then adds new GE microcarrier after addition pancreatin digestion,
?.
Preferably as further enforceable scheme, addition pancreatin digests 40-60s.
The method specifically passed on carries out in accordance with the following steps:
Culture is passed on after 72 hours, draws culture supernatant, and physiology salt is added and washes one time, discards, adds pancreatin
About 40-60sec is digested, the culture medium containing serum is added and shakes rolling bottle, observation carrier falls off, and is disappeared several times with Dispette piping and druming
The carrier changed, makes it be uniformly dispersed, and is added in new rolling bottle, then rejoins new microcarrier 1ml, and sealing is laid flat
And rotate rolling bottle and tile by carrier to rolling bottle surface, it places on quick Rotary Machine, continues to observe.
By observation, carrier will can be observed in discovery under mirror after microcarrier and cell while secondary culture rounded,
It is even, it is adherent, it is laid on culture dish and rolling bottle surface, carrier surface, which can be observed, a large amount of combination cells, equal adherent growth.One
Section time culture solution turns yellow, and when renewing culture solution, by bottle squint, gentle aspiration is fallen, and pours into the new culture medium of 10 or 100ml, it is seen that
Beads is laid on surface.
The present invention also provides the cell messenger obtained using above method culture, the cell messenger cultivated is not only active
Good, each type of cell messenger functionality for cultivating acquisition is stronger, and this method itself is easy to operate, operating condition ratio
Relatively mild, cell survival rate is high, provides method for reference for the culture of subsequent cell scale, should be widely promoted application.
Further, the present invention also provides the sides that the cell obtained using above-mentioned cultural method carries out a step culture and purifying
Method specifically comprises the following steps:
Cell messenger is extracted to obtain 100000MW component below using hollow fiber column, carries out thickening filtration, irradiation
Sterilizing, freeze-drying.
Preferably, it can be extracted 1-2 times repeatedly being concentrated in hollow fiber column.
Preferably, the temperature of freeze-drying is between -40~-50 DEG C.
The cell stoste obtained after purification by the above method is added to rhamnolipid to further increase its activity.
Preferably, the additive amount of the rhamnolipid is the 0.001-1wt% of the cell stock solution quality.
Rhamnolipid has cleaning, antibacterial functions from biology, as Glycolipids Biosurfactantss, despite one
Kind anionic surfactant, still there is lower critical micelle concentration, it can keep the stable system of mixture, controls product
Viscosity, have good cell permeability, can be dissipated by dispersion paving and wettability make small-molecule substance infiltration table
Skin can emulsify the grease in pore, reduce the generation of acne to help to retain moisture, lubrication, while as a kind of cleaning agent.
Rhamnolipid can form biomembrane on surface when in use, prevent fungi and bacterial growth, can be used for controlling
Skin burn is treated, cell messenger is had an impact based on wound healing, rhamnolipid can prevent or reduce the production of cell messenger
It is raw, reduce adverse effect therein as active constituent, in the healing of general wound, rhamnolipid is to skin epidermis
It is effectively for re-forming, because it provides one kind for wound healing and reduces fibrosis, adds the mouse of 0.001%-1%
Lee's glycolipid, can inhibit the Apoptosis in newborn horn cell, and it is horizontal to reduce Caspase Activity, promote cell growth and
Vigor, is particularly suitable for wound healing and reduces fibrosis, also treats gum disease especially gingivitis and periodontal regenerative.
It is lasting to be found by experiment that the action time of cell is used alone compared with rhamnolipid for cell messenger and rhamnolipid.
The addition of rhamnolipid has inhibiting effect to cell growth, directly related with its amphipathic molecule structure, utilizes biological surface
Activating agent dissolves heterologous cells film, reduces the generation of cell messenger, to inhibit cellular overgrowth, improves the activity of cell.
The effect of rhamnolipid is tested using fibroblast as experimental subjects, concrete outcome see the table below shown in 1-2.
Table 1 individually dilutes the rhamnolipid of various concentration to fibroblastic effect
| 10-1 | 10-2 | 10-3 | 10-4 | 10-5 | 10-6 | Positive control | Negative control | |
| 24 hours | - | - | + | + | + | + | + | - |
| 48 hours | - | - | + | + | + | + | + | - |
| 72 hours | - | - | - | + | + | - | + | -- |
| 96 hours | - | - | - | - | - | - | + | |
| 120 hours | - | - | - | - | - | - | + | - |
To fibroblastic effect after 2 cell stoste of table and rhamnolipid cooperation dilution various concentration
It is illustrated below for cell culture apparatus of the invention:
Cell is cultivated using big rolling bottle, small rolling bottle when above-mentioned carry out laboratory small-scale experiment, is had a try when in progress
It tests and also needs specially culture apparatus to be used to be cultivated, therefore the present invention provides a kind of dedicated for cell pilot scale culture
Culture apparatus, the culture apparatus is at low cost, is inventor's designed, designed, than bioreactor low cost on the market, and
The quality of cell culture is guaranteed.
In the prior art, bioreactor is made of disposable Tissue Culture Flask and control system platform, each culture
Bottle includes the special carrier of certain mass, these installation costs are expensive, and consumptive material relies on import producer.
Although bioreactor equipment can accomplish to automate, scale, it is expensive, and common laboratory is unwilling
Investment consumes big experimental facilities, needs high concentration cell and carrier, this experimental size is at high cost, and influence factor is more, needs
Producer carries out equipment and uses professional training and maintenance, and those skilled in the art is needed to complete, and preliminary experiment early period is established and gropes to train
Feeding condition is very arduous.Medical disposable material relies on high.
Cell factory includes that 5 layers and 10 layers of structure expands cell culture area, but the confined space of plane is come in fact
Existing cell adhere-wall culture, it has not been convenient to observe cell growth status under simple microscope, it is difficult to realize maximum space scale evaluation.
It is simple in order to provide a kind of structure, facilitate observation cell specifically to cultivate the culture apparatus of situation, it is provided by the invention
Culture apparatus specifically includes: for being equipped with the containers for culturing organisms of GE microcarrier and cell to be cultivated, sterile culture bag；
Sterile culture bag is connect with the import of the containers for culturing organisms by connector, and wherein joint is to can according to need
What connection disconnected, therefore multiple connectors can be set as needed in the culture apparatus, in this way when a certain in entire culture apparatus
When equipment needs replacing, it can disconnect and be replaced from joint.
Preferably as further enforceable scheme, it is provided with and is used between sterile culture bag and containers for culturing organisms
By the speed-variable pump of culture medium circulation discharge.
Since microcarrier is different from combination cell volume size and shape, the rate of settling is different, can be by adjusting speed change
The revolving speed of pump simultaneously extends the hematocrit time to reach the combination of maximum cell and microcarrier.
Preferably as further enforceable scheme, sterile culture bag be it is multiple, containers for culturing organisms is multiple, speed change
It is single for pumping, and pump head can be multiple, and each sterile culture bag passes through the speed-variable pump with corresponding containers for culturing organisms and connects；
Preferably, the sterile culture bag is two, mutually symmetrical with setting；
Preferably, the containers for culturing organisms is two, mutually symmetrical with setting.
Sterile culture bag, containers for culturing organisms in above-mentioned apparatus can be multiple simultaneously, so that more set culture systems are formed,
But these culture systems can share the same speed-variable pump, optimal mode is two groups of culture symmetry system having symmetry settings, and speed-variable pump is put
Set middle position.
Using above-mentioned culture apparatus, microcarrier can 360 degree of surface area combination cells being total to by foundation under identical environment
It cultivates cell combination microcarrier method and applies this Cyclic culture microcarrier cell method, realize the scale of space environment to greatest extent
To change culture, cell is suitble to co-culture to reach large-scale production benefit, visualized operation is flexibly simple, and it is economical and practical, greatly
It saves space and saves operating process.Extremely facilitate control production high concentration product preparation amount and be easy to separate from culture medium and carries
Body cell.
Cell is used for the newly-designed apparatus system of the present invention and has realized prepare with scale, this system is based on biological respinse
Device principle combination actual conditions carry out designed, designed.
The carrying out practically technique of the device are as follows: containers for culturing organisms is sterilized in advance, then with an adjustable change
Speed pump provides culture medium circulation to nutrient, and culture medium, which is pre-installed in sterile sterilizing bag, utilizes CO2Adjust Medium's PH Value, and
The system for forming circulation supply culture medium with containers for culturing organisms.
In fact, can also containers for culturing organisms pass in and out pipeline on installation and adjustment valve, can sample at any time in this way observation cell knot
The case where closing microcarrier then needs replacing culture solution, passes through the channel of connection when the culture solution color jaundice in sterile culture bag
New and old culture fluid exchange is carried out, is taken off from joint by sterile culture bag.
The surface area combination cell of certain density microcarrier can amplify culture area, realize scale High Density Cultivation
Cell, high efficiency prepare Related product.One layer of web plate is provided in containers for culturing organisms, the web plate is to stop microcarrier, training
Liquid band will not be cultured when culture solution circulates and walk by supporting cell, when microcarrier, culture cell need further replacement or
When moving to other places storage, containers for culturing organisms directly can entirely be removed and be handled, it is very convenient, it is reusable after processing.
In addition, to usually require that its color clarification, contained substance are formed after merging into each other uniform for general cell culture fluid
Solution, therefore in order to promote the degrees of fusion in culture solution between each substance, increase the time that each substance contacts with each other, keeps
The stable mobility of culture solution promotes its effect to cell culture, and improves the service life cycle of culture solution, improves culture
The quality of liquid, the present invention provides a kind of culture solutions to promote mixed device, which promotees mixed device setting in sterile culture bag and biology training
It supports between device, is made of porous sample introduction extra heavy pipe and porous sample introduction tubule interworking,
Preferably, porous sample introduction extra heavy pipe is disposed side by side on close to the position of import, and porous sample introduction tubule, which is disposed side by side on, to be leaned on
The position closely exported.
Preferably, the diameter control of porous sample introduction extra heavy pipe is in 5-10cm, and the diameter control of porous sample introduction tubule is between 2-4cm.
Preferably, the number control of porous sample introduction extra heavy pipe is at 3-4, and the number control of porous sample introduction tubule is at 3-4.
Preferably, each outlet of porous sample introduction extra heavy pipe and porous sample introduction tubule is cone-shaped, the inclined-plane of cone-shaped with
Parallel surface compares the angle to concave in 30-50 degree, is more preferably in 40 degree.Why to control in such angle in order that can
To allow the culture solution come out from each pipeline to eject, to accelerate the flowing of culture solution, in this way by by pipe design at this
The structure of kind outlet pointed cone type, effectively controls the flow velocity of culture solution to reach, due to culture solution in the duct
Rate is slow, and the mixed effect of rush is more excellent, and the culture solution speed ratio at nozzle is very fast, and accelerates flowing, promotes its fortune
It is defeated, because if rate is relatively slower always, to influence whether the service life of culture solution itself into containers for culturing organisms, because
This achievees the effect that promote culture solution quality preferably by the mode of transformation rate.
The structure that culture solution of the invention promotees mixed device is designed according to the self-characteristic of culture solution, is had single-minded
Property, it improves the quality of culture solution itself conscientiously especially by the conversion of culture solution rate speed, also improves the thin of culture
The survival rate of born of the same parents.
Compared with prior art, the invention has the benefit that
(1) cell culture of the invention can turn culture in glassware, at any time in different cell envelope microcarriers under the microscope
Situation can also cultivate in the closed filtration cycle culture systems of design, and this Cyclic culture method can be adjusted by speed-variable pump and be trained
Base flow speed is supported, to the shearing force of cell when reducing Cell infusion stirring and filtering.Continuing mild cycle rate is in liquid
State is circulated, to the nutrient environment that microcarrier cell provides, nutriment between hole inner cell is can promote and exchanges, it is micro-
Carrier combination cell can use free serum culture under high concentration environment, while cell is such as metabolized from the influence of chemical factor
Toxicity of the Product bulk to cell；
(2) microcarrier co-culture system of the invention can realize pilot scale culture cell, body in an incubator
Funds, waste treatment process in saving space, time and production process have greatly been saved in existing efficient preparation.Simultaneously
High-density culturing cell can reduce additional extra-nutrition, without adding serum.Because of cell concentration and cell messenger gene expression
At positivity dependence, more promotion cell secretion activity ingredient, while easily reaching and being separated and collected in cell and culture supernatant；
(3) culture apparatus of the invention sampling is simple, sample tap can be installed on containers for culturing organisms and sampled at any time, then set
In the cell growth status of microscopically observation carrier surface；
(4) processing is carried out after the carrier recovery after dissociating may be reused, and greatly reduce production cost.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram of the culture apparatus of the cell scale of the embodiment of the present invention three；
Fig. 2 is the structural schematic diagram of the culture apparatus of the cell scale of the embodiment of the present invention six；
Fig. 3 is that the specific structure of the mixed device of culture solution rush in the culture apparatus of the cell scale of the embodiment of the present invention six shows
It is intended to.
Appended drawing reference:
10- sterile culture bag；20- containers for culturing organisms；
30- speed-variable pump；40- culture solution promotees mixed device；
The porous sample introduction extra heavy pipe of 401-；The porous sample introduction tubule of 402-；
50- connector.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The cultural method and subsequent purification processes of cell scale carry out in accordance with the following steps:
1) it after taking adherent fat cell and mesenchymal cell to be digested, is inoculated in micro- with GE in containers for culturing organisms 20
Carrier co-cultures, and the inoculum density of cell is 8*103Cell/ml, the final concentration of 0.2wt% of culture solution after inoculation；
2) sterilizing culture bag 10 be provided with culture solution, sterilizing culture bag side wall be equipped with connector 50, by the connector with
The import of containers for culturing organisms 20 connects, to convey nutrition, the outlet setting of containers for culturing organisms 20 by adjustable speed-variable pump 30
There is connector 50, and the culture solution in containers for culturing organisms 20 is discharged by speed-variable pump 30；
3) at a slow speed 10-15rpm stir culture for 24 hours after, then quick 18-30rpm stir culture 72h, and biology training can be passed through
It supports and takes carrier cell at the connector 50 of the outlet of device 20, observe the growth conditions of cell；
4) it is passed on after cultivating, draws culture supernatant, physiology salt is added and washes one time, discards, add pancreatin digestion
About 40-60sec is added the culture medium containing serum and shakes containers for culturing organisms 20, and observation carrier falls off, and is blown and beaten with Dispette several
The secondary carrier digested, makes it be uniformly dispersed, and is added in new containers for culturing organisms 20, then rejoin new microcarrier,
Sealing, lays flat and rotates carrier is allowed to tile to 20 surface of containers for culturing organisms, continues to observe；
5) containers for culturing organisms 20 is irradiated into 10min in the UV lamp, the basic nutrient solution without serum is changed and continues in hypoxemia
Cyclic culture 4 days under 5wt%'s and nitrogen atmosphere, the cell messenger for collecting secretion supernatant extracts purifying；
6) cell messenger hollow fiber column is tentatively extracted, removes the component of macromolecular, collected efflux and mentioned again
It takes, retains the component of 10000MW or less molecular weight, while carrying out thickening filtration, it is dry to carry out -50 DEG C of vacuum refrigerations for irradiation sterilization
Dry preservation, the rhamnolipid that cell messenger can be added to 0.01wt% mix.
Embodiment 2
The cultural method and subsequent purification processes of cell scale carry out in accordance with the following steps:
1) handle microcarrier: PBS 0.01M pH7.4 prepares 121 DEG C of 15min of 4L high pressure sterilization, with above-mentioned PBS
It impregnates Cytodex 1GE microcarrier to stay overnight, wash 3 times, beads precipitates bottom, discards room temperature condition after the sterilizing being crushed in supernatant
Lower to be balanced overnight with culture medium, a culture solution is changed in centre.10ml/ pipe, 4 DEG C of preservations are dispensed, the used time takes 1ml to add 100ml culture medium；
2) it after taking adherent fat cell, mesenchymal cell to be digested, is inoculated in containers for culturing organisms 20 and microcarrier
It co-cultures, the inoculum density of cell is 9.0*103Cell/ml, the final concentration of 0.8wt% of culture solution after inoculation；
3) sterilizing culture bag 10 be provided with culture solution, sterilizing culture bag side wall be equipped with connector 50, by the connector with
The import of containers for culturing organisms 20 connects, to convey nutrition, the outlet setting of containers for culturing organisms 20 by adjustable speed-variable pump 30
There is connector 50, and the culture solution in containers for culturing organisms 20 is discharged by speed-variable pump 30；
4) at a slow speed after 10-15rpm stir culture 26h, then quick 18-30rpm stir culture is for 24 hours, and can be trained by biology
It supports and takes carrier cell at the connector 50 of the outlet of device 20, observe the growth conditions of cell；
5) it is passed on after cultivating, draws culture supernatant, physiology salt is added and washes one time, discards, add pancreatin digestion
About 40-60sec is added the culture medium containing serum and shakes containers for culturing organisms 20, and observation carrier falls off, and is blown and beaten with Dispette several
The secondary carrier digested, makes it be uniformly dispersed, and is added in new containers for culturing organisms 20, then rejoin new microcarrier,
Sealing, lays flat and rotates carrier is allowed to tile to 20 surface of containers for culturing organisms, continues to observe.
6) it by containers for culturing organisms 20, changes basic nutrient solution without serum and continues in hypoxemia 1.5wt%'s and nitrogen gas
Cyclic culture 5 days under atmosphere environment, the cell messenger for collecting secretion supernatant extract purifying；
7) cell messenger hollow fiber column is tentatively extracted, removes the component of macromolecular, collected efflux and mentioned again
It takes, retains the component of 100000MW or less molecular weight, while carrying out thickening filtration, it is dry to carry out -45 DEG C of vacuum refrigerations for irradiation sterilization
Dry preservation, the rhamnolipid that cell messenger can be added to 1wt% mix.
Embodiment 3
The cultural method and subsequent purification processes of fat cell scale carry out in accordance with the following steps:
1) handle microcarrier: PBS 0.01M pH7.4 prepares 121 DEG C of 15min of 4L high pressure sterilization, with above-mentioned PBS
It impregnates Cytodex 1GE microcarrier to stay overnight, wash 3 times, beads precipitates bottom, discards room temperature condition after the sterilizing being crushed in supernatant
Lower to be balanced overnight with culture medium, a culture solution is changed in centre.10ml/ pipe, 4 DEG C of preservations are dispensed, the used time takes 1ml to add 100ml culture medium；
2) it after taking adherent fat cell to be digested, is inoculated in containers for culturing organisms 20 and is co-cultured with microcarrier, fat
The inoculum density of cell is 8.6*103Cell/ml, the final concentration of 0.1wt% of culture solution after inoculation；
3) sterilizing culture bag 10 be provided with culture solution, sterilizing culture bag side wall be equipped with connector 50, by the connector with
The import of containers for culturing organisms 20 connects, to convey nutrition, the outlet setting of containers for culturing organisms 20 by adjustable speed-variable pump 30
There is connector 50, and the culture solution in containers for culturing organisms 20 is discharged by speed-variable pump 30, sterilizing culture bag 10 is two, biology training
Supporting device 20 is also two, is oppositely arranged the circulation that a shared speed-variable pump 30 realizes culture solution；
4) at a slow speed after 10-15rpm stir culture 25h, then quick 18-30rpm stir culture 48h, and can be trained by biology
It supports and takes carrier cell at the connector 50 of the outlet of device 20, observe the growth conditions of cell；
5) it is passed on after cultivating, draws culture supernatant, physiology salt is added and washes one time, discards, add pancreatin digestion
About 40-60sec is added the culture medium containing serum and shakes containers for culturing organisms 20, and observation carrier falls off, and is blown and beaten with Dispette several
The secondary carrier digested, makes it be uniformly dispersed, and is added in new containers for culturing organisms 20, then rejoin new microcarrier,
Sealing, lays flat and rotates carrier is allowed to tile to 20 surface of containers for culturing organisms, continues to observe, the structure of specific culture apparatus is shown in Fig. 1.
6) it by containers for culturing organisms 20, changes basic nutrient solution without serum and continues in hypoxemia 4wt%'s and nitrogen atmosphere
Cyclic culture 7 days under environment, the cell messenger for collecting secretion supernatant extract purifying；
7) cell messenger hollow fiber column is tentatively extracted, removes the component of macromolecular, collected efflux and mentioned again
It takes, retains the component of 100000MW or less molecular weight, while carrying out thickening filtration, it is dry to carry out -40 DEG C of vacuum refrigerations for irradiation sterilization
Dry preservation, the rhamnolipid that cell messenger can be added to 0.001wt% mix.
Embodiment 4
Concrete operation step and embodiment 3 are consistent, only the final concentration of 1wt% of culture solution.
Embodiment 5
Concrete operation step and embodiment 3 are consistent, only the final concentration of 0.5wt% of culture solution.
Comparative example 1
Concrete operation step and embodiment 3 are consistent, only the final concentration of 1.5wt% of culture solution.
Comparative example 2
Concrete operation step and embodiment 3 are consistent, only the final concentration of 0.01wt% of culture solution.
Comparative example 3
Concrete operation step and embodiment 3 are consistent, do not use microcarrier culture only.
Experimental example 1
The quality and survival rate for the cell that the cultural method of 1-5 of the embodiment of the present invention and comparative example 1-3 are turned out into
Row statistics, concrete outcome see the table below shown in 1:
1 statistical result of table
From the data in upper table 1 can be seen that by using cultural method of the invention can significantly improve cell at
Motility rate adds beads sealing and CO by microscopical observation after cell adhere-wall culture2It is cultivated under environment, it is visible under mirror
The surface beads combination cell, cell are creeped in the surface beads, illustrate that beads combination cell is better than vessel surface, cell combination
Rate is high, tiling, adherent, finds the final concentration of 0.5wt% of optimal operation by constantly groping, and inventor is by a large amount of
Practice concrete operations parameter is advanced optimized, have certain reference value.
Embodiment 6
Concrete operation step and embodiment 5 are consistent, and only the culture solutions that are added to promote mixed device 40, culture more in culture apparatus
Liquid promotees mixed device 40 between sterile culture bag 10 and the import of containers for culturing organisms 20, and idiographic flow schematic diagram is shown in Fig. 2, the equipment
Specific structure it is as follows, be specifically shown in Fig. 3:
It is made of 3 porous porous sample introduction tubules 402 of sample introduction extra heavy pipe 401 and 4, the diameter of porous sample introduction extra heavy pipe 401 is
5cm, the import for promoting mixed device 40 close to culture solution are arranged side by side mutually, and the diameter of porous sample introduction tubule is 2cm, is promoted close to culture solution
The outlet of mixed device 40 is arranged side by side mutually, and each outlet of porous sample introduction extra heavy pipe 401 and porous sample introduction tubule 402 is cone
Shape, the angle that the inclined-plane of the cone-shaped concaves compared with parallel surface are 30 degree, and culture solution successively circulates porous sample introduction extra heavy pipe
401, porous sample introduction tubule 402, then flows into containers for culturing organisms 20.
Embodiment 7
Concrete operation step and embodiment 6 are consistent, and only culture solution promotees the structure of mixed device 40 are as follows: thick by 4 porous sample introductions
The porous sample introduction tubule 402 of pipe 401 and 3 forms, and the diameter of porous sample introduction extra heavy pipe 401 is 10cm, promotees mixed device 40 close to culture solution
Import be arranged side by side mutually, the diameter of porous sample introduction tubule is 4cm, is set side by side mutually close to the outlet that culture solution promotees mixed device 40
Set, and each outlet of porous sample introduction extra heavy pipe 401 and porous sample introduction tubule 402 is cone-shaped, the inclined-plane of the cone-shaped with
It is 50 degree that parallel surface, which compares the angle that concaves, and culture solution successively circulates porous sample introduction extra heavy pipe 401, porous sample introduction tubule 402, so
After flow into containers for culturing organisms 20.
Embodiment 8
Concrete operation step and embodiment 6 are consistent, and only culture solution promotees the structure of mixed device 40 are as follows: thick by 4 porous sample introductions
The porous sample introduction tubule 402 of pipe 401 and 4 forms, and the diameter of porous sample introduction extra heavy pipe 401 is 6cm, promotees mixed device 40 close to culture solution
Import is arranged side by side mutually, and the diameter of porous sample introduction tubule is 3cm, sets side by side mutually close to the outlet that culture solution promotees mixed device 40
Set, and each outlet of porous sample introduction extra heavy pipe 401 and porous sample introduction tubule 402 is cone-shaped, the inclined-plane of the cone-shaped with
It is 50 degree that parallel surface, which compares the angle that concaves, and culture solution successively circulates porous sample introduction extra heavy pipe 401, porous sample introduction tubule 402, so
After flow into containers for culturing organisms 20.
Experimental example 2
Upgrowth situation of the culture apparatus of embodiment 5-8 to cell is tested, concrete outcome see the table below shown in 2:
2 statistical result of table
As can be seen that the embodiment of the present invention promotees mixed this structure of device 40, culture solution by increasing culture solution from upper table 2
Service life extended, the culture quality of cell is also improved.
Experimental example 3
Determination of activity is carried out to the cell messenger after purification of embodiment 3, is specifically shown in Table 1-2, the data in table 1 be using
The determination of activity situation of the stoste for the cell messenger that the method for the embodiment of the present invention 3 obtains, the data in table 2 are without ultraviolet
Irradiation and hypoxemia processing, the determination of activity situation for the cell messenger stoste that direct purification obtains, stoste be made into 8 parallel samples into
Row detection.
Method: using basal medium two-fold dilution sample, and addition is completed in tissue culture plate adherent overnight in advance, every hole
Add 1ml, positive control and negative control is arranged from 1:2-1:1280 in concentration.Each dilution adds multiple holes, sets carbon dioxide culture
Observation 10 days in case.Positive control is basic culture medium increase serum, and negative control is dilution.
Result judgement: positive hole: cell Proliferation is paved with hole, and cell is in fiber-like adherent growth, and space between cells is fine and close, side
Boundary is clear, and plus sige is shown in table.Negative hole cell Proliferation is not until grow, and circle cell gradually increases not adherent, and cell boundaries are not
Clear gap becomes larger, and minus sign is shown in table.The result of record observation daily.
1 determination of activity result of table
2 determination of activity result of table
Stability test: 37 degree of freeze-dried powder placement, room temperature, 4 degree, -20 degree varying environments, one week, two weeks, one month, two
A month, half a year, 1 year, 18 months, 2 years, ibid method measurement was active.4 degree can be reserved for 2 years with -20 degree, and 37 degree of activity can be tieed up
It holds 2 months or more, room temperature can be reserved for four months or more.
Toxicity test: blab/c mouse is injected intraperitoneally in freeze-dried powder after being diluted with pure water, only, observation has no different in 2 weeks to 0.5ml/
Often.
The cell messenger of preparation exists rich in hundreds of growth factor and various cell messengers relevant to cytothesis signal
Several hundred kinds of interior human body rabphilin Rabs are pure natural non-recombinant architectural characteristic, and it simulates intracorporal optimal proportions concentration collaboration
Effect, regulating cell proliferation, maintain tissue and the development of cell ordering growth at differentiation.Collagen, fibronectin, superoxides
The protein such as mutase are the key components for maintaining skin youth state, so the cell messenger of preparation has its relevant multiple function
Effect, is the incomparable effect of recombinant protein.If TYR enzyme is dermal melanin biosynthesis key enzyme, in cell messenger
TGF-β 1 reduces TYR enzyme and TRP1 activity, inhibits B16 cell；TGF-β is EMT early stage regulatory molecule, from transcription level
Upper TGF-β activates EMT process, the change for causing downstream passages and series of genes to be expressed, and is invaded and migratory activity.Cell
Courier is by promoting fibroblast proliferation liveness and mobility, thus the effect of playing it.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (9)
1. a kind of cultural method of cell scale, which is characterized in that the GE microcarrier after cultivating cell and sterilizing to be inoculated with
It in the culture medium containing serum, is cultivated in containers for culturing organisms, then changes serum free medium into, continued in serum free medium
4-7d is cultivated, supernatant is collected；
The cell to be cultivated is one or both of umbilical chord mesenchymal cells, fat cell；
Preferably, the inoculum density of the cell to be cultivated is (8-9) * 103cell/ml；
It is highly preferred that the inoculum density of the cell to be cultivated is 8.6*103cell/ml。
2. cultural method according to claim 1, which is characterized in that in serum free medium incubation, oxygen
Concentration is 4-7wt%, and preferably the concentration of oxygen is 5wt%.
3. cultural method according to claim 1, which is characterized in that 10-15rpm stir culture 24-26h after inoculation, then
With 18-30rpm stir culture 24-72h.
4. cultural method according to claim 3, which is characterized in that after culture 24-72h, inhale and abandon culture supernatant, use
Brine is added the culture medium containing serum, after microcarrier falls off, then adds the micro- load of new GE after addition pancreatin digestion
Body.
5. cultural method according to claim 4, which is characterized in that addition pancreatin digests 40-60s.
6. the purification process of cell obtained by the described in any item cultural methods of claim 1-5, which is characterized in that including walking as follows
It is rapid:
Cell is extracted to obtain 100000MW component below using hollow fiber column, carries out thickening filtration, irradiation sterilization, freezing
Drying.
7. purification process according to claim 6, which is characterized in that extracted 1-2 times repeatedly in hollow fiber column.
8. purification process according to claim 6, which is characterized in that the temperature of freeze-drying is between -40~-50 DEG C.
9. a kind of cell messenger is obtained using the described in any item cultural method cultures of claim 1-8.
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