CN106754669A - The preparation method and preparation system of the multi-cellular structure based on Reaction-Diffusion Models - Google Patents

The preparation method and preparation system of the multi-cellular structure based on Reaction-Diffusion Models Download PDF

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CN106754669A
CN106754669A CN201611035302.8A CN201611035302A CN106754669A CN 106754669 A CN106754669 A CN 106754669A CN 201611035302 A CN201611035302 A CN 201611035302A CN 106754669 A CN106754669 A CN 106754669A
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cellular structure
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朱晓璐
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Changzhou Campus of Hohai University
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Abstract

The present invention relates to the preparation method and preparation system of a kind of multi-cellular structure based on Reaction-Diffusion Models, this preparation method comprises the following steps：Step S1, in vitro culture is carried out by the three dimensional gel system for building to stem cell；And step S2, regulate and control multi-cellular structure form；The method of the multi-cellular structure pattern that regulation and control mescenchymal stem cell self-organizing proposed by the invention is formed fully relies on the self assembly of cell, the processing technology thereof of necessary external structure material and complexity in completely left out current existing tissue vitro construction method, whole preparation method more fully make use of the mutual ability to communicate between a large amount of cells, and process is naturally occurred closer to in-vivo tissue, for the preparation of artificial organ provides a kind of method high closer to internal natural tissues, reliability.

Description

The preparation method and preparation system of the multi-cellular structure based on diffusion-reaction model
Technical field
The present invention is a kind of how thin to regulate and control by adding suitable cell factor under the guiding of diffusion reaction Mathematical Modeling The method of born of the same parents' structure and tissue growth in vitro process, is related to the enabling tool of biological manufacture and organizational project, particularly bone tissue The technical field repaired with cartilaginous tissue.
Background technology
The reparation and regeneration for realizing biological tissue are the main targets of organizational project.To realize the function of tissue or even organ Regeneration, increasing researcher designs and manufactures various artificial substituting structures and be widely used in biological tissue's work Journey, and it is such manufacture gradually develop towards increasingly complex and fine direction.Biological tissue with various geometry features (such as tracheae, blood vessel, lymphatic vessel and intestines etc.) are widely present in higher organism body.The artificial replacement of such tissue constructs Thing generally needs to build corresponding three-dimensional structure in vitro using specific material, then carries out relevant art treatment, and then Obtain the substitute close with internal tissue profile.Such local organization substitute is required for first producing by macromolecule or gold The structure for possessing particular geometric feature that category material etc. exterior material is constituted, be then implanted into again in vivo and with corresponding human body Good connection and fusion are organized the formation of, and then completes to realize the function of artificial tissue.Although however, such tubular body structure is manufactured Finely, but by the material of itself it is still some synthetic materials (tissue of inorganic matter itself), thus still have very much can The problems such as rejection of human body and unpredictable complication can be caused.Although some foreign studies person's utilizationization in recent years The biomolecule that conjunction macromolecular material combination can preferably resist rejection has been produced some special " printing inks ", and Artificial organ substitute is produced using the three-dimensional printing technology based on " printing ink ", but the implementation process of this method is answered It is miscellaneous, and cost is very high.
Therefore, at present some research worker by using organism own cells self (self- Organizing) characteristic, building specific microenvironment carries out the external dimensional culture of stem cell, makes from the dry of human body itself Cell is self-assembled into a kind of multi-cellular structure for simulating natural biological tissue, and without by outside synthetic material preparation Artificial substituent with particular geometric feature, this makes it possible to the rejection for fundamentally mitigating human body significantly, and significantly Degree reduces the manufacturing cost of artificial organ or even organ.But the self-organizing behavior of pure cell is filled with more uncontrollable Property, some stem cells form specific multi-cellular structure, but these many cells through dimensional culture self-organizing after a while The appearance of structure is generally difficult to prediction in advance, and with certain randomness, this is just organizational project and the tool of regenerative medicine field The limitation of body application band.
If under the guiding of certain theory or rule, structure that just can be controllable by simple biochemical operations The multi-cellular structure of cell self-organizing formation is built and adjusts, then to solving the above problems with positive propulsion meaning.
The content of the invention
It is an object of the invention to provide a kind of preparation method, adjusted with the preparation and structural form that solve three-dimensional multi-cellular structure The technical problem of control.
In order to solve the above-mentioned technical problem, the invention provides a kind of preparation method of three-dimensional multi-cellular structure, including such as Lower step：
Step S1, in vitro culture is carried out by the three dimensional gel system for building to stem cell；And
Step S2, regulates and controls multi-cellular structure form.
Further, the three dimensional gel system in the step S1 by building carries out the method bag of in vitro culture to stem cell Include following steps：
Step S11, exogenous cytokines, i.e. activator, retardance, matrix are mixed according to a certain percentage, and uniformly It is scattered in cell culture medium；
Step S12, adds a number of stem cell and gel compositions formulated to be mixed in cell culture medium, matches somebody with somebody Make gel precursors solution；
Step S13, the gel premise solution is mixed in certain proportion with corsslinking molecular solution, by certain hour Incubation, realize gelation；
Step S14, appropriate culture medium is added in the top of three dimensional gel body, is cultivated, and constitutes a multi-cellular structure.
Further, in the step S1 by build three dimensional gel system in vitro culture is carried out to stem cell method also Including：
Repeat the above steps S11 to step S14, configures the cell culture medium of the exogenous cytokines containing different ratio, To constitute a series of multi-cellular structures with different geometry features.
Further, the method that multi-cellular structure form is regulated and controled in the step S2 comprises the following steps：
Step S21, builds diffusion-reaction model；
Step S22, is guided by diffusion-reaction model, and morphology control is carried out to the multi-cellular structure in three dimensional gel body, With the multi-cellular structure needed for formation.
Further, the mathematic(al) representation of the diffusion-reaction model includes：
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output system that retardance is produced by activator Number；And in formula (3)To be suitable to represent cell to chemoattractant concentration regional movement higher Chemotaxis；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
Further, guided by diffusion-reaction model in the step S22, to the multi-cellular structure in three dimensional gel body Morphology control is carried out, is comprised the following steps in the method for the multi-cellular structure needed for being formed：
A series of multi-cellular structures are taken pictures by step S221 by Laser Scanning Confocal Microscope,
Step S222, is simulated by diffusion-reaction model and is similarly configuring Parameter Conditions with a series of multi-cellular structures Under simulation multi-cellular structure series；
Step S223, will cultivate the multi-cellular structure for obtaining with simulation multi-cellular structure respectively according to similarly configuring parameter bar Tendency information is extracted in part contrast, to adjust the related exogenous cytokines parameter of addition.
Another aspect, present invention also offers a kind of diffusion-reaction model, to realize adjusting multi-cellular structure form Control.
In order to solve the above-mentioned technical problem, the mathematic(al) representation of this diffusion-reaction model includes：
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output system that retardance is produced by activator Number；And
In formula (3)To be suitable to represent cell to chemoattractant concentration regional movement higher Chemotaxis；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
The third aspect, present invention also offers a kind of three-dimensional multi-cellular structure preparation system, to solve three-dimensional many cells knot The preparation of structure and the technical problem of structural form regulation and control.
The three-dimensional multi-cellular structure preparation system includes：
Preliminary culture unit, in vitro culture is carried out to stem cell by the three dimensional gel system for building, and forms a series of tools There is the multi-cellular structure of different geometry features；
Experiment material unit, regulates and controls multi-cellular structure form.
Further, the experiment material unit is suitable to build diffusion-reaction model, and is guided by diffusion-reaction model, Morphology control is carried out to the multi-cellular structure in three dimensional gel body, to form required multi-cellular structure.
Further, the mathematic(al) representation of the diffusion-reaction model includes：
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output system that retardance is produced by activator Number；And
In formula (3)To be suitable to represent cell to chemoattractant concentration regional movement higher Chemotaxis；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
The beneficial effects of the invention are as follows many cells knot that regulation and control mescenchymal stem cell self-organizing proposed by the invention is formed The method of configuration looks fully relies on the self assembly of cell, eliminates necessary outer in existing tissue vitro construction method at present The processing technology thereof of portion's structural material and complexity, whole preparation method more fully make use of the phase intercommunication between a large amount of cells News ability, and process is naturally occurred closer to in-vivo tissue, for the preparation of artificial organ provides one kind closer in vivo Natural tissues, reliability method high.Multi-cellular structure in the present invention is complete by cellularity, without other synthetic materials, The substitute by material manufactures such as polymer needed for current artificial tissue can significantly be mitigated brings the problem of rejection, And cost can be greatly lowered.Exogenous cytokines mixture in the present invention can make to the regulating and controlling effect of multi-cellular structure Predicted with the reaction-Diffusion Simulation model of cell three-dimensional process of self-organization, and the various concentrations of every kind of cell factor can take There must be the multi-cellular structure body of different structure feature, this reduces the species of required cell factor to a certain extent, i.e., Complete to regulate and control out a variety of many cells microstructures with different characteristic by only needing a small amount of several cell factors.
Brief description of the drawings
The present invention is further described with reference to the accompanying drawings and examples.
Fig. 1 is the flow chart of the preparation method of three-dimensional multi-cellular structure of the invention；
Fig. 2 is signal the step of regulating and controlling multi-cellular structure pattern method by cytokine mixture in the embodiment of the present invention Figure；
Fig. 3 (a), Fig. 3 (b) and Fig. 3 (c) be respectively the embodiment of the present invention in combined type cell factor to mescenchymal stem cell Exercising result exemplary plot；
Method under the guiding of Fig. 4 diffusion-reaction models by applying cell factor and preparing and regulate and control three-dimensional multi-cellular structure Flow embodiment is illustrated.
Have in figure above：
It is activator 01, retardance son 02, stem cell 03, matrix (substrate) 04, thin and thin multi-cellular structure 11, thick and close Multi-cellular structure 12, distributed spherical multi-cellular structure 13, it includes single spherical structure 131, and between adjacent ball The strip multi-cellular structure 132,133 for exhaling；
The step of test operation program 21, addition cell factor or combined type cell factor 22, multi-cellular structure series 23rd, it is how thin that the step of desired multi-cellular structure 24, foundation Reaction-Diffusion Models carry out Computer Simulation 25, simulation draws Born of the same parents' structural series 26, knowledge base 27.
Specific embodiment
In conjunction with the accompanying drawings, the present invention is further explained in detail.These accompanying drawings are simplified schematic diagram, only with Illustration illustrates basic structure of the invention, therefore it only shows the composition relevant with the present invention.
Principle of the invention is to build three dimensional gel system by exogenous cytokines, and by ad hoc fashion to the gel System adds corresponding stem cell, and then progressively causes that the process of self-organization of its internal mescenchymal stem cell changes；It is another Aspect, by building a kind of reaction-diffusion Mathematical Modeling, carrys out approximate simulation just in the (letter below of mescenchymal stem cell 03 of calcification Claim cell) self-organizing behavior, and compare with test structure, and then tendency is provided guides to test next time.So repeatedly Repeatedly, until so that the pattern of the multi-cellular structure formed in experiment also occurs performance of expected change, and object construction shape is leveled off to Formula.
Embodiment 1
In the present embodiment, the extracellular matrix for three-dimensional cell cultivation is modified hyaluronic acid gel.By adjusting The concentration of macromolecular or connection peptide chain, can construct with different mechanical properties and different protein moleculars in water saving gel The three dimensional gel body of diffusivity.Using the mescenchymal stem cell 03 in preliminary calcification state in the present embodiment, because it has Stronger aggregation and structure formation ability, and it can be divided into cartilage, Gegenbaur's cell.Density of the cell in gel is uniform , various multi-cellular structures are also to be substantially uniformly distributed in three dimensional gel body.Uniformly added by by the solution of cell factor Into nutrient solution, you can cause that each local location of whole three-dimensional multi-cellular structure produces different changes in three dimensions.
As shown in figure 1, the present embodiment 1 provides a kind of preparation method of three-dimensional multi-cellular structure, comprise the following steps：
Step S1, in vitro culture is carried out by the three dimensional gel system for building to stem cell；And
Step S2, regulates and controls multi-cellular structure form.
Specifically, the three dimensional gel system in the step S1 by building carries out the method bag of in vitro culture to stem cell Include following steps：
Step S11, exogenous cytokines, i.e. activator, retardance, matrix are mixed according to a certain percentage, and uniformly It is scattered in cell culture medium；The activator can be BMP-2 and BMP-4, and retardance is MGP, and matrix is Noggin.This In activator be an Equivalent Physical concept, it is equivalent to the various comprehensive function effects of the molecule of activation.Similarly, Retardance and matrix (a kind of protein molecular) are also corresponding Equivalent Physical concept.Self-organizing of the cell in three dimensional gel Journey；
Specifically, by the Bone in Transforming growth factor (TGF)-beta superfamilies The albumen of morphogenetic proteins (BMP) -2 and BMP-4 albumen and Noggin albumen and Matrix gla Protein (MGP) albumen etc. mixes according to certain ratio, and is dispersed in cell culture medium.Change according to specific needs Become proportioning, can prepare multigroup.
Step S12, adds a number of stem cell and gel compositions formulated to be mixed in cell culture medium, matches somebody with somebody Make gel precursors solution；Specifically, by the mesenchyma of polymer backbone macromolecular, adhesion protein and the preliminary calcification of certain amount Stem cell is mixed according to a certain percentage, makes gel precursors solution.
Step S13, the gel premise solution is mixed in certain proportion with corsslinking molecular solution, by certain hour Incubation, realize gelation.
Step S14, appropriate culture medium is added in the top of three dimensional gel body, is then inserted and is trained in 37 degree of incubators Support, one multi-cellular structure of (a few houres to some days) composition after certain hour length.
The present invention utilizes the figure spirit unstability in the macromolecular reaction diffusion process in three dimensional gel system, by certain The three-dimensional cell cultivation of time so that uniform distribution of the concentration of activator and retardance inside gel is broken, and The activator high local concentrations region of many distributions is gradually constructed, the geometry in these high concentration topical regions can be line Shape, ellipsoid, or irregular shape etc. (these figures can be described as graphics spirit spot figure, the region of abbreviation cell aggregation here). After this, using the Chemotaxis characteristic (being intended to activator concentration region high) and diffusion mobility of cell so that big Amount cell gradually continues to cell aggregation region clustering, or away from the direction diffusive migration in current cell aggregation region. As cell spreads to all directions, this causes that cell has outside scattered trend in some positions；On the other hand, chemotaxis is same When cause that cell is migrated towards the intensive position of activator, so can also cause that cell has the trend of aggregation in local location.In This simultaneously, retardance generated by activator and cytosis also can in turn weaken the self-catalysis generating process of activator, And then cause that the localized clusters of cell produce the trend for weakening.Each mechanism is according to the dynamic of certain sequential rule more than Under state effect, the migration of cell gradually tends towards stability at each location point, and causes that cell process of self-organization is gradually intended to receive Hold back (i.e. the change of structure is more and more slower, and is gradually intended to the pattern for determining).
Three dimensional gel system in the step S1 by building also includes to the method that stem cell carries out in vitro culture：
Repeat the above steps S11 to step S14, configures the cell culture medium of the exogenous cytokines containing different ratio, To constitute a series of multi-cellular structures with different geometry features.
The method for regulating and controlling multi-cellular structure form in the step S2 comprises the following steps：
Step S21, builds diffusion-reaction model；
Step S22, is guided by diffusion-reaction model, and morphology control is carried out to the multi-cellular structure in three dimensional gel body, With the multi-cellular structure needed for formation.
The present invention is particularly caused by the utilization to scheming clever unstability principle in biosystem to the clever unstability of figure Activator, the reaction and diffusion process of retardance sub (the two is referred to as morphogen) guide and regulate and control, be in particular in addition Can as the exogenous cytokines of activator or retardance and/or other can influence the outer of morphogen reaction and diffusion process The source cell factor；And can be from side to the self-organizing pattern of cell multi-cellular structure by the model of reaction-diffusion system Qualitative forecasting is carried out, and for the multi-cellular structure in experiment builds the guiding that tendency is provided with regulation and control.
The mathematic(al) representation of the diffusion-reaction model includes：
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output system that retardance is produced by activator Number；And
In formula (3)To be suitable to represent cell to chemoattractant concentration regional movement higher Chemotaxis；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time (dimensionless) of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rn(rn=L2RN/Dh) it is cell largest value-added rate (RN) Dimensionless Form, block son diffusivity DhApproximately set It is 1 × 10-7cm2/s；
ExU, ExV are respectively activator, the outer source item of retardance.
Guided by diffusion-reaction model in the step S22, form is carried out to the multi-cellular structure in three dimensional gel body Regulation and control, comprise the following steps in the method for the multi-cellular structure needed for being formed：
A series of multi-cellular structures are taken pictures by step S221 by Laser Scanning Confocal Microscope,
Step S222, is simulated by diffusion-reaction model and is similarly configuring Parameter Conditions with a series of multi-cellular structures Under simulation multi-cellular structure series；Parameter is wherein prepared except the proportioning of cell factor, also the addition including cell factor with The change of time and the factor such as gel structure density and culture duration.
Step S223, will cultivate the multi-cellular structure for obtaining with simulation multi-cellular structure respectively according to identical conditions of mixture ratios pair Than extracting tendency information, to adjust the related exogenous cytokines parameter of addition.
In the present embodiment, exogenous cytokines are added to change the configuration of three-dimensional multi-cellular structure by uniform. When single exogenous cytokines are individually added, such as when Noggin, BMP2 or BMP4, the form of three-dimensional multi-cellular structure body In the presence of each foreign protein, it may appear that respective unique geometry feature.Although uniformly dividing in gel-type vehicle Influence of the exogenous cytokines of the outside addition of cloth to each cell is equivalent, but the motion of the cell of different zones rings Should but be different.When any exogenous cytokines are not added with, what blood vessel mescenchymal stem cell was formed is honey comb like three Dimension multi-cellular structure 11, now closely links together between cell, and weave in, the network-like many cells of formation Structure is tiny and sparse, and many hollow regions (Fig. 3) are still suffered between a large amount of cells.Add when according to certain sequential rule Plus after one or two exogenous factors, for example, Noggin is added in the way of being incremented by by day, or add in the way of being incremented by by day Plus after the mixture of noggin and a small amount of BMP2/BMP4, the aggregation extent of multi-cellular structure 12 is higher, and it is thick local connection occur Big feature, its network-like multi-cellular structure for being formed is relatively coarse and intensive (referring to Fig. 3)；It is thin with the addition of external source After intracellular cytokine BMP2 (200-500ng/ml), sample can be promoted to occur the three-dimensional ball that diameter is about 200 microns afterwards at more than ten days Shape multi-cellular structure 131, each spheroid is comprising compact arranged thousands of or even up to ten thousand between cell, and most of adjacent balls There is the strip multi-cellular structure 132,133 for exhaling to be connected.When reducing in gel the density of corsslinking molecular and add concentration and be After the BMP2 of 0.1-0.5 μ g/ml, mescenchymal stem cell can also form the multi-cellular structure with distributed aggregation (the micro- pattern 13 in similar Fig. 3).
Embodiment 2
On the basis of embodiment 1, the present embodiment 2 provides a kind of three-dimensional multi-cellular structure preparation system, including：
Preliminary culture unit, in vitro culture is carried out to stem cell by the three dimensional gel system for building, and forms a series of tools There is the multi-cellular structure of different geometry features；And experiment material unit, regulate and control multi-cellular structure form.
The experiment material unit is suitable to build diffusion-reaction model, and is guided by diffusion-reaction model, to three-dimensional Multi-cellular structure in gelinite carries out morphology control, to form required multi-cellular structure.
The mathematic(al) representation of diffusion-reaction model is as described in Example 1.
Under this in diffusion-reaction model by guiding in the present invention three-dimensional many cells are prepared by applying exogenous cytokines The flow embodiment of the method for structure and regulation and control multi-cellular structure Three Dimensions Structure is referring to Fig. 4.
When being prepared in hydrogel and regulating and controlling the shape or function of multi-cellular structure, the knowledge set up in early-stage Study first On the basis of storehouse and parameter, sample is made by set test operation program 21, and by the corresponding foreign cell of addition because The step of combination of sub or various exogenous cytokines 22, the external dimensional culture of cell is then carried out, and obtain many cells Structural series 23, if not obtaining desired multi-cellular structure 24 now；So a series of experimental results above is now combined, Above-mentioned diffusion-reaction model is reused 25 the step of carrying out Computer Simulation, the multi-cellular structure series 26 of simulation is drawn, now Useful tendency information is extracted from a series of multi-cellular structures for simulating and (changes certain or multiple exogenous cells The concentration of the factor may be such that current multi-cellular structure type is easier to be changed into expected structure), feed back to the front-end process of experiment Partly (add the operation part before cell factor), corresponding seriation is then carried out using improved test parameters how thin The dimensional culture of born of the same parents' structure；If not obtaining desired institute's eucaryotic cell structure 24 also afterwards, above-mentioned flow is repeated, proceed to imitate True simulation, excavates the qualitative relationships between the variation tendency and parameter value of multi-cellular structure from simulation model, and by this information Feed back to the leading portion flow elements of experiment.After circulation by said process several times, desired multi-cellular structure 14 is It is available.Then, by from it is above-mentioned it is repeated multiple times during extract key regularity information import knowledge base 27, according to renewal Knowledge base afterwards, is improved to test operation program 21 and Computer Simulation step 25.
Test operation program 21 in above-mentioned flow includes propagation, the precursor solution of gel and the corsslinking molecular solution of cell Preparation, the operating process of gel-forming and cell parcel.The step of Computer Simulation being carried out according to Reaction-Diffusion Models (activator saturation rate, the generation of retardance or degradation rate and matrix are raw for 25 settings for including model and the setting of parameter Into or degraded speed etc.), and combine the improvement that above a series of experimental results is carried out to model.Knowledge base 27 includes：It is living At the beginning of diffusivity, degradation rate and cell chemotaxis mobility, gel density and the uniformity, the cell of beggar, retardance or matrix The affecting laws of beginning concentration distribution and additional cell factor to three-dimensional multi-cellular structure pattern；Other known physical chemistry and life Saturation history, the Chemotaxis and rule of proliferation of cell when thing rule, such as activator self-catalysis are generated；It is specific in model Relation between parameter value and the entirety that draws of simulation or partial structurtes characteristic size etc..
In the present embodiment 1 and embodiment 2, hydrogel is such as, but not limited to selection fibrin gel, basement membrane matrix and coagulates Glue or hyaluronic acid derivatives, or other kinds of it is suitable to the hydrogel of cell growth.Stem cell species can select preliminary calcium The mescenchymal stem cell of change or its combination with epithelial cell or endothelial cell.
With above-mentioned according to desirable embodiment of the invention as enlightenment, by above-mentioned description, relevant staff is complete Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention Property scope is not limited to the content on specification, it is necessary to its technical scope is determined according to right.

Claims (10)

1. a kind of preparation method of three-dimensional multi-cellular structure, it is characterised in that comprise the following steps：
Step S1, in vitro culture is carried out by the three dimensional gel system for building to stem cell；And
Step S2, regulates and controls multi-cellular structure form.
2. preparation method according to claim 1, it is characterised in that
Three dimensional gel system in the step S1 by building comprises the following steps to the method that stem cell carries out in vitro culture：
Step S11, exogenous cytokines, i.e. activator, retardance, matrix are mixed according to a certain percentage, and dispersed In cell culture medium；
Step S12, adds a number of stem cell and gel compositions formulated to be mixed in cell culture medium, makes Gel precursors solution；
Step S13, the gel premise solution is mixed in certain proportion with corsslinking molecular solution, by incubating for certain hour Educate, realize gelation；
Step S14, appropriate culture medium is added in the top of three dimensional gel body, is cultivated, and constitutes a multi-cellular structure.
3. preparation method according to claim 2, it is characterised in that
Three dimensional gel system in the step S1 by building also includes to the method that stem cell carries out in vitro culture：
Repeat the above steps S11 to step S14, the cell culture medium of the exogenous cytokines containing different ratio is configured, with structure Into a series of multi-cellular structures with different geometry features.
4. preparation method according to claim 3, it is characterised in that
The method for regulating and controlling multi-cellular structure form in the step S2 comprises the following steps：
Step S21, builds diffusion-reaction model；
Step S22, is guided by diffusion-reaction model, morphology control is carried out to the multi-cellular structure in three dimensional gel body, with shape Into required multi-cellular structure.
5. preparation method according to claim 4, it is characterised in that
The mathematic(al) representation of the diffusion-reaction model includes：
∂ U ∂ t = D ▿ 2 U + γ [ nU 2 V ( 1 + kU 2 ) - c U - K U V ] + E x U - - - ( 1 )
∂ V ∂ t = ▿ 2 V + γ [ bnU 2 - e V - K U V ] + E x V - - - ( 2 )
∂ n ∂ t = q ▿ 2 n - χ [ ▿ · ( n ( k n + U ) 2 ▿ U ) ] + r n n ( 1 - n ) - - - ( 3 )
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output coefficient that retardance is produced by activator；With And
In formula (3)To be suitable to represent chemotactic of the cell to chemoattractant concentration regional movement higher Effect item；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
6. preparation method according to claim 5, it is characterised in that
Guided by diffusion-reaction model in the step S22, form tune is carried out to the multi-cellular structure in three dimensional gel body Control, comprises the following steps in the method for the multi-cellular structure needed for being formed：
A series of multi-cellular structures are taken pictures by step S221 by Laser Scanning Confocal Microscope,
Step S222, is simulated by diffusion-reaction model and is similarly configured under Parameter Conditions with a series of multi-cellular structures Simulation multi-cellular structure series；
Step S223, will cultivate the multi-cellular structure for obtaining with simulation multi-cellular structure respectively according to similarly configuring Parameter Conditions pair Than extracting tendency information, to adjust the related exogenous cytokines parameter of addition.
7. a kind of diffusion-reaction model, it is characterised in that
The mathematic(al) representation of the diffusion-reaction model includes：
∂ U ∂ t = D ▿ 2 U + γ [ nU 2 V ( 1 + kU 2 ) - c U - K U V ] + E x U - - - ( 1 )
∂ V ∂ t = ▿ 2 V + γ [ bnU 2 - e V - K U V ] + E x V - - - ( 2 )
∂ n ∂ t = q ▿ 2 n - χ [ ▿ · ( n ( k n + U ) 2 ▿ U ) ] + r n n ( 1 - n ) - - - ( 3 )
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output coefficient that retardance is produced by activator；With And
In formula (3)To be suitable to represent chemotactic of the cell to chemoattractant concentration regional movement higher Effect item；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
8. a kind of three-dimensional multi-cellular structure preparation system, it is characterised in that including：
Preliminary culture unit, in vitro culture is carried out to stem cell by the three dimensional gel system for building, and is formed a series of with not With the multi-cellular structure of geometry feature；
Experiment material unit, regulates and controls multi-cellular structure form.
9. three-dimensional multi-cellular structure preparation system according to claim 8, it is characterised in that
The experiment material unit is suitable to build diffusion-reaction model, and is guided by diffusion-reaction model, to three dimensional gel Multi-cellular structure in body carries out morphology control, to form required multi-cellular structure.
10. three-dimensional multi-cellular structure preparation system according to claim 9, it is characterised in that
The mathematic(al) representation of the diffusion-reaction model includes：
∂ U ∂ t = D ▿ 2 U + γ [ nU 2 V ( 1 + kU 2 ) - c U - K U V ] + E x U - - - ( 1 )
∂ V ∂ t = ▿ 2 V + γ [ bnU 2 - e V - K U V ] + E x V - - - ( 2 )
∂ n ∂ t = q ▿ 2 n - χ [ ▿ · ( n ( k n + U ) 2 ▿ U ) ] + r n n ( 1 - n ) - - - ( 3 )
In above formula (1)-(3)
U, V and n are respectively the dimensionless concentration of activator, retardance and matrix；
WithThe diffusion of activator, retardance and base proton concentration is described respectively；
C is the degradation rate of activator, and e is the degradation rate of retardance, and b is the output coefficient that retardance is produced by activator；With And
In formula (3)To be suitable to represent chemotactic of the cell to chemoattractant concentration regional movement higher Effect item；
K represents U and V by the bonded coefficient for carrying out non-linear degraded of physics；
D=DU/DVAnd q=Dn/DVRespectively activator is to retardance, and cell is to blocking the ratio between sub diffusion coefficient；
γ be with computational fields yardstick, biosynthesis time scale and block sub- diffusivity associate scale factor；
T is the time of simulation system, and t=DVT/L2, wherein
T is the time of cell culture in experiment, and L is the dimensionless unit length of the characteristic dimension of involved culture；
rnIt is the Dimensionless Form of cell largest value-added rate；
ExU, ExV are respectively activator, the outer source item of retardance.
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