CN104911104B - In-vitro cell and organ 3-D culture device and artificial organ culture method - Google Patents
In-vitro cell and organ 3-D culture device and artificial organ culture method Download PDFInfo
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- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses an in-vitro cell and organ 3-D culture device and an artificial organ culture method, and relates to the technical field of microbiology. The in-vitro cell and organ 3-D culture device comprises a culture chamber, a 3-D moving shaking tank, a condition culture solution circulation system, a gas exchange system as well as a multi-freedom-degree moving mechanism, wherein the condition culture solution circulation system and the gas exchange system are respectively communicated with the culture chamber. The artificial organ culture method comprises a step of culturing an artificial organ by simulating a dynamic environment for growth and development of the human body organ by using the in-vitro cell and organ 3-D culture device. Due to the adoption of the 3-D moving shaking tank, the growth microenvironment that a fetus is grown or developed in a parent body from a human body cell or organ can be relatively really simulated, the efficiency that various stem cells are grown and differentiated into functional cells in in-vitro 3-D culture is improved, a simple, economic, gentle and three-dimensional growth microenvironment with comprehensive nutrition is provided for in-vitro cell 3-D culture, and the experiment shows that the method for in-vitro human body organ culture by using the device disclosed by the invention is feasible, and the device and the method can be clinically popularized and applied.
Description
Technical field
The invention belongs to technical field of microbiology, is related to a kind of cell and the external 3-D culture apparatuses of organ and artificial device
The breeding method of official.
Background technology
To avoid cell or tissue that the disadvantage of many basic stitch physiological features is gradually lost in two-dimentional (2-D) growth course
External three-dimensional (3-D) culture has been invented at end, people.The external 3-D culture apparatuses for adopting at present mainly adopt bioreactor
(Bioreactor, BR), refers to that the biological function having using enzyme or organism (such as microorganism) provides biological work in vitro
The apparatus system or engineering equipment of property environment.Mainly divided according to the different BR of cultured cells, culture carrier, culture fluid hybrid mode
For stirring-type, gas type, hollow fiber form, Clothoid type (space B R) etc..Combined for rotation technique according to the different BR of structure points
The body structure of common 37 DEG C of 5%CO2 cell culture incubators；Cyclic culture device and common 37 DEG C of 5%CO2 cell culture incubators
Each self-contained unit and Cyclic culture combine gas for body structure, Cyclic culture device and common 37 DEG C of 5%CO2 cell culture incubators
Body exchanges closed device.Additionally, the ancillary technique supporting with BR also has perfusion cultures formula, microcarrier formula, porous microsphere formula etc..
However, considering from economic benefit, these BR generally existing high costs, equipment complexity etc..For example stir from functional perspective
The shearing force for mixing the stirrer paddle generation of formula BR can cause to damage to cell；The culture environment heterogeneity of hollow fiber form BR, training
Foster technique is difficult to amplify, sterilize reuse difficulty etc.；Analyze from structural point, one apparatus cost of structure is expensive, bulky,
The difficulty bought to instrument and use is increased undoubtedly；Structure two devices are except involving great expense, bulky outer, 3-D trainings in device
Foster pipe is that fixation is present in incubator, it is impossible to rotated, it is impossible to realize cell or tissue similar to human development's mistake very well
The such a stereoscopic culture function of vacantly moving naturally growth of journey, causes artificial organ afunction or dysplasia；Structure
Although waving in three devices or rotary system can be such that cell 3-D cultivates in dynamic process, as 3-D culture tubes are placed in culture
Outside case, pollution probability is considerably increased, Cyclic culture bound gas exchange closed device and then require higher high-level of cost
Surrounding culture atmosphere.
To overcome the problems referred to above, urgent need that existing culture apparatuses are carried out with structure improvement, improved device can both simulate tire
Compound motion of the youngster in placenta materna amniotic fluid growth course, the circulation that can carry out metabolite again are exchanged, and both fully can be met
High cell densities increment needs, and ensures that cell with high efficient secretion, is that cell or organ in vitro cultivation provide a simple, Jing
The 3-D growth microenvironments of Ji, gentle, three-dimensional, comprehensive nutrition, promote cell regeneration and the generation of artificial organ.
The content of the invention
In view of this, an object of the present invention is to provide a kind of cell and the external 3-D culture apparatuses of organ, existing to solve
Human organ growth microenvironment cannot truly be simulated with the presence of technology causes artificial organ dysplasia, afunction, cultivates effect
The low problem of rate.The second object of the present invention is provided and carries out artificial culture using above-mentioned cell and the external 3-D culture apparatuses of organ
The method of organ.
An object of the present invention is achieved through the following technical solutions：
The present invention the external 3-D culture apparatuses of cell and organ, including culturing room, can do compound motion 3-D motion shake
Case, and the gentle body exchange system of CMC model fluid circulation for connecting with culturing room respectively, the culturing room is by elasticity
Part removably hang on 3-D motion shake in case, also including be arranged on 3-D motion shake case one or both sides multiple degrees of freedom fortune
Motivation structure.
Further, the culture environment of the culturing room：Temperature is 37 DEG C；Humidity is 95～98%；It is 5% containing volume fraction
CO2Air；Soda acid p H-number is 7.2-7.4.
Further, the movement mechanism with multiple degrees of freedom includes longitudinal rotating mechanism, horizontal rotary mechanism and is arranged on both
Between multi-functional attachment structure, the CMC model fluid circulation and gas exchange systems by multi-functional attachment structure with
Extraneous connection carries out culture fluid and gas exchange.
Further, longitudinal rotating mechanism includes longitudinal rotating shaft and buncher I, one end of the longitudinal rotating shaft
Shake case and fix with 3-D motions, the main shaft of the other end and buncher I is fixed.
Further, the horizontal rotary mechanism includes horizontal rotating frame, feathering axis and buncher II, the level
One end of swivel mount is rotated with one end of longitudinal rotating shaft and is connected, and the other end is rotated with one end of feathering axis and is connected, described
The main shaft of the other end of feathering axis and buncher II is fixed.
Further, the horizontal rotating frame is provided with multi-functional connection knot with the junction of longitudinal direction and feathering axis
Structure.
Further, the movement mechanism with multiple degrees of freedom is arranged on the both sides that case is shaken in 3-D motions, the multifreedom motion machine
Structure also includes being fixedly installed on 3-D motions and shakes longitudinal driven shaft of case opposite side, longitudinal driven shaft and horizontal rotating frame it
Between be provided with multi-functional attachment structure.
Further, the multi-functional attachment structure includes rotatable and sealing shroud loaded on longitudinal direction or feathering axis periphery
Movable sleeve, the annular seal space being arranged between movable sleeve and longitudinal direction or feathering axis, be arranged on movable sleeve and longitudinal direction or level
Rolling bearing between rotary shaft, and be arranged in feathering axis, horizontal rotating frame, longitudinal rotating shaft and longitudinal driven shaft
Conditioned medium circulation canal and gas crossover passage, the annular seal space passes through conditioned medium circulation canal and gas exchange
Passage is connected with culturing room respectively.
Further, the indoor uniform intervals of the culture are provided with baffle plate made by multiple elastomeric materials, the head of the baffle plate
Portion is arranged with the elastic buffer thin film at the top of circular arc.
Further, the elastomeric element is spring or rubber or other elastic components.
Further, the culturing room includes chamber body and chamber body can be sealed fixed chamber cap, and the chamber cap is provided with and room
The mutually chimeric snap-in structure of body phase, the chamber body are provided with the snap fit structure matched with snap-in structure.
Further, also include for this culture apparatus are carried out with PLC control system and the presentation of information system of Automatic Control
System.
The second object of the present invention is achieved through the following technical solutions：
The method that artificial organ is cultivated using cell in vitro as described above and organ 3-D culture apparatuses, using cell and device
The dynamic environment of the external 3-D culture apparatuses simulation human organ growth promoter of official cultivates artificial organ.
Further, the dynamic environment of the human organ growth promoter includes：Natural by seed cell goes the whole of cell
Hang in 3-D culture apparatuses body organ rack elasticity, be 5%CO in 37 DEG C, volume fraction2Under the conditions of cultivate stage by stage,
1～7 day first stage, second stage 8～14 days, 15～21 days phase IIIs, using 24h dynamic cultivations, daytime elder generation's level to
Left 360 ° of rotations, the 360 ° of rotations to the right of rear level, the compound 3-D rotations that combined in the evening using longitudinal direction rotation and horizontal rotation,
Daytime rotating speed be 20～40rpm/min, evening rotating speed be less than 20rpm/min, changed culture medium once per three days, per the stage change not
Same culture medium.
Beneficial effects of the present invention：
1st, the external 3-D culture apparatuses of cell and organ of the invention, shake case simulation fetus in mother by arranging 3-D motions
Human development's compound motion in body Placenta Hominiss amniotic fluid growth course, and simulation culture in case is shaken by being arranged on for culturing room's elasticity
Simple harmonic motion of the thing during suspention in human body, more really simulates the growth of human body cell or organ in growth course
Microenvironment, improve various seed cells are divided into mature cell density and culture efficiency, are the external 3-D trainings of cell or organ
Support and a simple, economic, gentle, three-dimensional, growth microenvironment for comprehensive nutrition is provided, the outer artificial organ of acceleration bodies is generated, the dress
Put compared with prior art, with obvious cost advantage and promotional value.
2nd, relatively turnable purpose between medium input system and rotating shaft is realized by multi-functional attachment structure, then due to
Each medium channel is arranged in rotating shaft, 3-D motions are shaken case and realize medium needed for growing environment (culture fluid and 37 DEG C with culturing room
5%CO2Gas) connect and exchange with culturing room, solve the problems, such as input channel winding in multiple degrees of freedom rotation process.
3rd, the method that the present invention cultivates artificial organ, as the device truly simulates the growth promoter of human body cell or organ
Microenvironment, it is possible to achieve the in vitro cultivation and reconstruction to various human organs.The liver formed using method of the present invention culture
Sample tissue only needs 3 time-of-weeks, than recent report using external evoked DLS mescenchymal stem cells (the Meschymal stem of BR methods
Cells, MSC) liver sample tissue (need 4 time-of-weeks) that formed saves 1 time-of-week, and experiment shows artificial liver obtained in the present invention
With liver structure and function, can use as liver transplantation donor, be further characterized by human organ body being carried out using this device
The method of outer cultivation is feasible, can carry out clinical application, and have broad application prospects and higher social valency
Value.
Other advantages of the present invention, target and feature will be illustrated to a certain extent in the following description, and
And to a certain extent, based on being will be apparent to investigating hereafter to those skilled in the art, Huo Zheke
To be instructed from the practice of the present invention.The present invention target and other advantages can be realized by description below and
Obtain.
Description of the drawings
In order that the object, technical solutions and advantages of the present invention are clearer, below in conjunction with accompanying drawing the present invention is made into
The detailed description of one step, wherein：
Structural representation of the accompanying drawing 1 for the cell in vitro and organ 3-D culture apparatuses of the present invention；
Enlarged drawing of the accompanying drawing 2 for culturing room of the present invention；
Enlarged drawing of the accompanying drawing 3 for multi-functional attachment structure I of the invention；
Enlarged drawing of the accompanying drawing 4 for multi-functional attachment structure II of the invention；
Enlarged drawing of the accompanying drawing 5 for multi-functional attachment structure III of the invention.
Accompanying drawing 6 is the artificial liver cultivated with this device.A is cultivated the 9th day for external 3-D and is generated lobe of the liver spline structure；B is external
3-D is cultivated and is generated within the 21st day the full liver spline structure of class；C (a-b) tissue slice shows the lobules of liver sample knot similar to normal liver
Structure；C (c) secretes liver function albumen.
Accompanying drawing 7 is mammal liver development maturation schematic diagram.A is mice；B is the mankind.
Reference：1- culturing room；Case is shaken in 2-3-D motions；3- longitudinal rotating shafts；4- buncher I；5- is horizontally rotated
Frame；6- feathering axis；7- buncher II；8- longitudinal directions driven shaft；9- movable sleeves；10- rolling bearings；11- culture fluid is circulated
Passage；12- gas crossover passages；13- annular seal space I；14- annular seal space II；15- annular seal space III；16- annular seal space IV；17- is cultivated
Liquid pipe；18- tracheas；19- baffle plates；20- elastic buffer thin film；21- cultures；22- springs；23- chamber bodies；24- chamber caps；25- is convex
Rise；26- open slots；27- transfer tubes；28-PLC control systems；29- information display systems；Multi-functional attachment structures I of 30-；31-
Multi-functional attachment structure II；Multi-functional attachment structures III of 32-.
Specific embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.It should be appreciated that preferred embodiment
Only for the explanation present invention, rather than in order to limit the scope of the invention.
Embodiment 1, cell in vitro and organ 3-D culture apparatuses
As Figure 1-5, case 2 is shaken including the motion of culturing room 1,3-D, and the culture fluid for being connected with culturing room 1 respectively is handed over
System and gas exchange systems are changed, in the culturing room 1, fetus development growth environment in placenta materna amniotic fluid can be simulated, including
The dynamic environment of the temperature environment, oxygen atmosphere and various compound movements that are adapted with Placenta Hominiss, simulates tire with culturing room 1
Disk, simulates the amniotic fluid in placenta materna with culture fluid, realizes temperature environment, oxygen atmosphere with gas exchange systems, moved with 3-D
Shake simulation human body compound motion of the fetus in placenta materna amniotic fluid growth course of case 2, the final nurturing an environment realized：Temperature is
37℃；Humidity is 95～98%；It is 5%CO containing volume fraction2Air；Soda acid p H-number is 7.2-7.4；Including by culturing room 1
Shaken in case 2 by the 3-D motions of being fixed on that elastomeric element flexibly can be dismantled, make various irregular movements, the dynamic environment of realization.
The present embodiment shakes case simulation people bluk recombination of the fetus in placenta materna amniotic fluid growth course by arranging 3-D motions
Motion, and simple harmonic motion of the simulated organ during the suspention in human body in case is shaken by being arranged on for culturing room's elasticity, compared with
The human body cell or organ real growing environment in human body is really simulated, being divided into into for various seed stem cell is improved
Ripe cell density level and effectively facilitate 3-D cell culture efficiencies, for vitro in organ growth provides one it is simple, economical, warm
With the growing environment of, three-dimensional, comprehensive nutrition, accelerate cell regeneration and orga- nogenesis, the device compared with prior art, with bright
Aobvious cost advantage and promotional value.
As the improvement of the present embodiment, also including the multifreedom motion machine for being arranged on 3-D motions and shaking case one or both sides
Structure, the present embodiment are arranged on the both sides that case 2 is shaken in 3-D motions, and the movement mechanism with multiple degrees of freedom includes longitudinal rotating mechanism, level
Rotating mechanism and the multi-functional attachment structure being arranged between, realize that the rotation of the level and longitudinal direction of case 2 is shaken in 3-D motions
Turn, the culture fluid exchange system and gas exchange systems by multi-functional attachment structure with it is extraneous connect exchange, realization with it is outer
37 DEG C of boundary 5%CO2The exchange of gas exchange and culture fluid, reaches cell and the suitable temperature environment of organ growth, oxygen ring
Border and amniotic fluid environment.
Used as the improvement of the present embodiment, longitudinal rotating mechanism includes longitudinal rotating shaft 3 and buncher I 4, described
One end of longitudinal rotating shaft 3 and 3-D motion is shaken case 2 and is fixed, and the main shaft of the other end and buncher I 4 is fixed, in buncher I
Under 4 driving, 3-D motions are made to shake the left and right rotary motion that case 2 produces longitudinal direction.
Used as the improvement of the present embodiment, the horizontal rotary mechanism includes horizontal rotating frame 5, feathering axis 6 and speed governing
Motor II 7, one end of the horizontal rotating frame 5 are rotated with one end of longitudinal rotating shaft 3 and be connecteds, the other end and feathering axis 6
One end rotate connection, the other end of the feathering axis 6 is fixed with the main shaft of buncher II 7, in buncher II 7
Driving under, drive feathering axis 6 and horizontal rotating frame 5 to rotate, finally order about 3-D motions and shake case 2 producing horizontal direction
Left and right rotary motion.
Used as the improvement of the present embodiment, the movement mechanism with multiple degrees of freedom also includes being fixedly installed on 3-D motions that to shake case 2 another
Longitudinal driven shaft 8 of side, is provided with multi-functional attachment structure I 30, institute between longitudinal driven shaft 8 and horizontal rotating frame 5
The junction that horizontal rotating frame 5 is stated with longitudinal rotating shaft 3 is provided with multi-functional attachment structure II 31, horizontal rotating frame 5 and level
The junction of rotary shaft 6 is provided with multi-functional attachment structure III 32, and the multi-functional attachment structure includes rotatable and seals
Be set in the movable sleeve 9 of 6 periphery of longitudinal rotating shaft 3 or feathering axis, movable sleeve 9 is arranged on longitudinal rotating shaft 3 or water
Annular seal space, the rolling bearing being arranged between movable sleeve 9 and longitudinal rotating shaft 3 or feathering axis 6 between flat rotary shaft 6
10, and the culture fluid circulation being arranged in feathering axis 6, horizontal rotating frame 6, longitudinal rotating shaft 3 and longitudinal driven shaft 8 is logical
Road 11 and gas crossover passage 12, the annular seal space by with culture fluid circulation canal 11 and gas crossover passage 12 respectively with training
Foster room 1 connects, and the annular seal space is set to loop configuration, and multi-functional attachment structure III 32 has two separate sealings
Chamber I, II (13,14), multi-functional attachment structure I 30 have an annular seal space III 15, and multi-functional attachment structure II 31 has
One annular seal space IV 16, the culture fluid exchange system include the annular seal space I 13, feathering axis being sequentially communicated and level rotation
The culture fluid circulation canal 11 of pivoted frame part, annular seal space III 15, the culture fluid circulation canal 11 of the driven shaft portion in longitudinal direction, and training
The culture liquid pipe 17 of nutrient solution circulation canal connection, the annular seal space I 13 are connected with extraneous culture fluid source, the culture liquid pipe 17
Case 2 is shaken through 3-D motions to connect with culturing room 1；The gas exchange systems include the annular seal space II 14, level being sequentially communicated
The gas crossover passage 12 of rotary shaft and horizontal rotating frame part, annular seal space IV 16, the gas exchange of longitudinal rotating shaft part lead to
Road 12, the trachea 18 connected with gas crossover passage 12, the annular seal space II 14 and 37 DEG C of 5%CO in the external world2Source of the gas is connected, institute
State trachea 18 to shake case 2 and connect with culturing room 1 through 3-D motions.
Used as the improvement of the present embodiment, above-mentioned culture fluid exchange system and gas exchange systems can be adjusted specifically as needed
Through passage, select suitable annular seal space I, annular seal space II, annular seal space III and annular seal space IV.
Used as the improvement of the present embodiment, in the culturing room 1, uniform intervals are provided with baffle plate made by multiple elastomeric materials
19, can be silica gel etc., the head setting of the baffle plate 19 is with the elastic buffer thin film 20 at the top of circular arc.In the effect of baffle plate 19
Under, culture 21 can make various irregular movements, simulate more complicated human motion, it is possible to culture 21 is formed and is supported,
Realize the growth conditions for suspending；Uniform intervals are arranged to suitable density, can simulate the chorion in placenta materna to culture
21 form protection, can avoid the injury to culture 20, in addition, arranging with dome elastic buffer thin film 20, effectively can subtract
The contact area of few and culture 21, beneficial to being fully contacted with culture medium, promotes the growth which is good.
Used as the improvement of the present embodiment, the elastomeric element is spring or rubber or other elastic components, and the present embodiment is preferred
Spring 22, can shake in 3-D motions and make under case effect simple harmonic oscillation or swing, compound motion during simulation suspention.
Used as the improvement of the present embodiment, the culturing room 1 includes chamber body 23 and chamber body 23 can be sealed fixed chamber cap 24,
The chamber cap 24 is provided with and 23 mutually chimeric snap-in structure of chamber body, and the chamber body is provided with the card matched with snap-in structure
Fit structure is closed, the snap-in structure of the present embodiment is the projection 25 being arranged on chamber cap, the snap fit structure is and projection
25 close-fitting open slots 26, during use load culture after culturing room, and raised with chamber cap loads close in open slot 26
Then both fixed by envelope again with bolt or screw or quick-changing mechanism.
As the improvement of the present embodiment, between the annular seal space I and extraneous culture fluid source, the annular seal space II with it is extraneous
37 DEG C of 5%CO2Transfer tube 27 is provided between source of the gas.
As the improvement of the present embodiment, also include for this culture apparatus are carried out with the PLC control system of Automatic Control
28 and information display system 29, be conducive to complete monitoring and management being carried out to incubation, improve its culture efficiency and quality.
Embodiment 2, there is organ structure and functional artificial liver using cell and the external 3-D culture apparatuses culture of organ
Dirty method
As shown in Figure 6.The device and test kit material that the present embodiment is used is as follows：Cell in vitro and organ 3-D culture dresses
Put that (case is shaken in the 3-D of 0～50rmp/min of rotating speed motion；37 DEG C, 5%CO2Culturing room), conditioned medium (CM1, CM2, CM3),
100U/mL penicillins, 100 μ g/mL streptomycins (Cellgro, cat.no.30-001-CI), 5000U/mL penicillins, 5000 μ g/
ML streptomycins, DMEM/F12 (Gibco-BRL, cat.no.11330), VEGF (Pepnotech, cat.no.100-20C), HGF
(R＆D, cat.no.294-HG-005/CF), bFGF (Pepnotech, cat.no.100-188), bovine insulin (Sigma,
Cat.no.I6634), human transferrin (Sigma, cat.no.T1147), levothyrocine (Sigma,
Cat.no.T6397), sodium selenite (Sigma, cat.no.S9133), putrescine (Sigma, cat.no.P5780), progestogen
(Sigma, cat.no.7556), Oncostatin M, dexamethasone, non essential amino acid, L-glutaminate.
Prepare three kinds of conditioned mediums (Conditioned mediums, CMs), respectively CM1 culture medium, CM2 cultures
Base, CM3 culture medium, comprises the following steps that：
1) remove stem cell and the ancester cell in embryonic-period mice liver：
A. 7 to 15 days embryonic-period mice (E7-E15) liver sample tissues, each issue five embryos are taken out under anatomic microscope respectively
Mice, implements slow grinding with homogenizer after mixing, filters, collect filtrate；
B. multigelation 3 times under the conditions of -80 DEG C and 39 DEG C by filtrate, each each 30mim, then with 0.45 μm of filter membrane mistake
Filter, collects filtrate；
C. with OX43 and OX44 Positive Hematopoietic Stem Cells, Thy-1 positive hepatic embryos in immunofluorescence method detection filtrate
Fetal liver cell, if testing result is negative, shows that the stem cell in embryonic-period mice liver and ancester cell are removed；
2) using protein reagent box detection protein content (50-100mg/mL)
3) CM1, CM2 and CM3 culture medium are prepared
CM1 culture medium：To the embryonic-period mice liver filtrate and the DMEM complete culture solutions that remove stem cell and ancester cell
Mixed solution (1:4) the final concentration of 20ng/ml of VEGF to VEGF are added in；
CM2 culture medium：To the embryonic-period mice liver filtrate and the DMEM complete culture solutions that remove stem cell and ancester cell
Mixed solution (1:4) in add HGF, bFGF, bovine insulin, human transferrin, levothyrocine, sodium selenite, putrescine,
Progestogen turn ferrum to the final concentration of 10ng/mL of HGF, the final concentration of 5ng/mL of bFGF, the final concentration of 0.5mg/mL of bovine insulin, people
Final concentration of protein is 0.5mg/mL, the final concentration of 40ug/mL of levothyrocine, the final concentration of 34ug/mL of sodium selenite, putrescine
Final concentration of 0.5ug/mL, the final concentration of 6ug/mL of progestogen (each component concentration is how many, please be supplemented)；
CM3 culture medium：To the embryonic-period mice liver filtrate and the DMEM complete culture solutions that remove stem cell and ancester cell
Mixed solution (1:4) HGF, bFGF, Oncostatin M, dexamethasone, non essential amino acid and L-glutaminate are added in
To the final concentration of 100ng/mL of HGF, the final concentration of 50ng/mL of bFGF, the final concentration of 20ng/ml of Oncostatin M, ground plug rice
Pine is final concentration of 0.1 μM, and non essential amino acid mass fraction is 1% and the final concentration of 5mM of L-glutaminate.
Then using the CM1 for preparing, the outer 3-D cultures of CM2 and CM3 medium bodies are with organ structure and functional artificial
Liver, concrete grammar are as follows：
First under computer system monitoring, cell is included into seed from portal vein and postcava respectively using three-step approach
Stem cell is slowly injected in support, and every time at least 3～5 × 105, 15-30min is spaced, is injected 2-3 time；Then by seed injection
WDLS (the hereinafter referred to as WDLS-NG2 of stem cell+HSC) it is put in culturing room, and culturing room is flexibly hung on 3-D motions
Shake in case, Primary culture liquid exchange system and gas exchange systems, add 500 μ l CM1 culture medium to carry out external 3-D cultures one
All (the 1st～7 day), starts horizontal rotary mechanism daytime, and first level rotates 8h for 360 ° to the left, and rear level rotates 8h for 360 ° to the right,
Start longitudinal rotating mechanism and horizontal rotary mechanism in the evening, carries out the compound 3-D rotations 8h that longitudinal direction and horizontal rotation combine, often
Add within two days 200 μ L CM1 culture medium；Culture changes 500 μ LCM2 culture medium into (the 8th day) after second week, training method and
Condition (the 8-14 days) identical with first week；Culture changes 500 μ L CM3 culture medium, culture into (the 15th day) after the 3rd week
Mode and condition are identical with first week, and dosage is identical with first week with mode, continue 7 days (8-14 days)；Culture was into the 3rd week
Afterwards (the 15th day), 500 μ L CM3 culture medium, dosage (the 15th～21 day) identical with first week with mode, after cultivating 21 days are changed
Terminate culture, obtain and there is organ structure and functional artificial liver, then the tissues observed under white light and fluorescence microscope
Structure is learned, and analyzes supernatant liver function albumen.As a result show, cultivate to the 9th day and define lobe of the liver sample profile (Fig. 6 A), cultivate
Full liver sample profile (Fig. 6 B) was defined to the 21st day, the cultures of 21 days are carried out with H＆E dyeing and culture supernatant functional protein
Analysis, show culture with normal liver tissue (Fig. 6 Ca) similar structures (Fig. 6 Cb) if any central veins of hepatic lobules (the
Central vein, CV) formed (shown in arrow), with the prolongation of incubation time, (Alb is represented culture supernatant functional protein
Liver albumin) secretion increase (Fig. 6 Cc), show that seed stem cell can be formed with organ structure in specific culture microenvironment
With the artificial liver of function, the seed cell for there are very much clinical practice potentiality is demonstrated by.
2 points are illustrated finally：(1) preparation of three kinds of conditioned mediums (CM1, CM2, CM3 -) that we develop is according to the food in one's mouth
Newborn class animal liverss fully-developed theoretical basiss (see Fig. 7), spending a large amount of time carries out scientific research proof and obtains,
Related article will be delivered；(2) preferred embodiment above is only to illustrate technical scheme and unrestricted, although passing through
Above preferred embodiment is described in detail to the present invention, it is to be understood by those skilled in the art that can be in shape
Various changes are made in formula and to which in details, without departing from claims of the present invention limited range.
Claims (12)
1. cell and the external 3-D culture apparatuses of organ, it is characterised in that：Including culturing room, can do compound motion 3-D motion shake
Case, and the gentle body exchange system of CMC model fluid circulation for connecting with culturing room respectively, the culturing room is by elasticity
Part removably hang on 3-D motion shake in case, also including be arranged on 3-D motion shake case one or both sides multiple degrees of freedom fortune
Motivation structure；
The movement mechanism with multiple degrees of freedom includes longitudinal rotating mechanism, horizontal rotary mechanism and the connection knot being arranged between
Structure, the attachment structure are mainly made up of movable sleeve and rolling bearing, and longitudinal rotating mechanism and horizontal rotary mechanism are distinguished
It is connected with movable sleeve by rolling bearing, in the movable sleeve, is additionally provided with annular seal space, the CMC model fluid circulation and gas
Body exchange system is connected with the external world by the annular seal space in attachment structure carries out culture fluid and gas exchange.
2. cell according to claim 1 and the external 3-D culture apparatuses of organ, it is characterised in that：The training of the culturing room
Foster environment：Temperature is 37 DEG C；Humidity is 95～98%；It is 5%CO containing volume fraction2Air；Soda acid pH value is 7.2-7.4.
3. cell according to claim 1 and the external 3-D culture apparatuses of organ, it is characterised in that：Longitudinal whirler
Structure includes longitudinal rotating shaft and buncher I, and one end of the longitudinal rotating shaft and 3-D motions are shaken case and fixed, the other end with adjust
The main shaft of speed motor I is fixed.
4. cell according to claim 3 and the external 3-D culture apparatuses of organ, it is characterised in that：The horizontal rotation machine
Structure includes horizontal rotating frame, feathering axis and buncher II, one end of the horizontal rotating frame and the one of longitudinal rotating shaft
End rotates connection, and the other end is rotated with one end of feathering axis and is connected, the other end and buncher of the feathering axis
The main shaft of II is fixed.
5. cell according to claim 4 and the external 3-D culture apparatuses of organ, it is characterised in that：The horizontal rotating frame
Attachment structure is provided with the junction of longitudinal direction and feathering axis.
6. cell according to claim 5 and the external 3-D culture apparatuses of organ, it is characterised in that：The multiple degrees of freedom fortune
Motivation structure is arranged on the both sides that case is shaken in 3-D motions, and the movement mechanism with multiple degrees of freedom also includes that being fixedly installed on 3-D motions shakes case
Longitudinal driven shaft of opposite side, is provided with attachment structure between longitudinal driven shaft and horizontal rotating frame.
7. cell according to claim 6 and the external 3-D culture apparatuses of organ, it is characterised in that：The attachment structure bag
Include movable sleeve of the rotatable and sealing shroud loaded on longitudinal rotating shaft or the periphery of feathering axis or longitudinal driven shaft, be arranged on
Annular seal space between movable sleeve and longitudinal rotating shaft or feathering axis or longitudinal driven shaft, movable sleeve is arranged on longitudinal direction rotation
Rolling bearing between axle or feathering axis or longitudinal driven shaft, and it is arranged on feathering axis, horizontal rotating frame, longitudinal direction
Conditioned medium circulation canal and gas crossover passage in rotary shaft and longitudinal driven shaft, the annular seal space pass through CMC model
Liquid circulation canal and gas crossover passage are connected with culturing room respectively.
8. cell according to claim 1 and the external 3-D culture apparatuses of organ, it is characterised in that：The culture is indoor
Even to be arranged at intervals with baffle plate made by multiple elastomeric materials, the head of the baffle plate arranges thin with the elastic buffer at the top of circular arc
Film.
9. cell according to claim 1 and the external 3-D culture apparatuses of organ, it is characterised in that：The elastomeric element is
Spring or rubber or other elastic components.
10. cell according to claim 1 and the external 3-D culture apparatuses of organ, it is characterised in that：The culturing room includes
Chamber body be provided with the snap-in structure mutually chimeric with chamber body, the chamber body with the chamber cap that chamber body can be sealed fixation, the chamber cap
It is provided with the snap fit structure matched with snap-in structure.
11. according to the arbitrary described cell of claim 1-10 and the external 3-D culture apparatuses of organ, it is characterised in that：Also include
For this culture apparatus are carried out with the PLC control system and information display system of Automatic Control.
12. adoptSuch as rightIt is required that the arbitrary described cells of 1-11 and the external 3-D culture apparatuses of organ cultivate the side of artificial liver
Method, it is characterised in that：Cultivated using the dynamic environment of cell and the external 3-D culture apparatuses simulation artificial liver growth promoter of organ
Artificial liver；The dynamic environment of the artificial liver growth promoter includes：By the natural overall organ for removing cell of seed cell
Hung in 3-D culture apparatuses rack elasticity, be 5%CO in 37 DEG C, volume fraction2Under the conditions of cultivate stage by stage, the first rank
Section 1～7 day, second stage 8～14 days, 15～21 days phase IIIs, using 24h dynamic cultivations, daytime 360 ° to the left of level of elder generation
Rotation, the 360 ° of rotations to the right of rear level, the compound 3-D rotations that combined in the evening using longitudinal direction rotation and horizontal rotation turn daytime
Speed is 20～40r/min, and evening rotating speed is less than 20r/min, changed culture medium once per three days, and different cultures are changed per the stage
Base.
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| CN2106834U (en) * | 1991-11-06 | 1992-06-10 | 刘新利 | Tissue cultuer bottle |
| JP2003070464A (en) * | 2001-09-04 | 2003-03-11 | Mitsubishi Heavy Ind Ltd | Method for culturing cell, method for producing artificial organ and the resultant artificial organ |
| WO2004061092A1 (en) * | 2002-12-27 | 2004-07-22 | Mitsubishi Heavy Industries, Ltd. | Method of culturing pluripotent stem cells and culture apparatus therefor |
| JP4817362B2 (en) * | 2003-08-19 | 2011-11-16 | 独立行政法人国立循環器病研究センター | Apparatus and method for uniform cell seeding on a substrate having a three-dimensional surface |
| GB0520021D0 (en) * | 2005-10-01 | 2005-11-09 | Univ Liverpool | A reactor |
| CN102086438B (en) * | 2009-12-07 | 2012-12-19 | 韩春茂 | Device and method for biological culture of cell or tissue engineering |
| CN102876573B (en) * | 2012-10-26 | 2013-11-20 | 黑龙江八一农垦大学 | Mammal embryo in-vitro culture device and culture method thereof |
| CN104611225A (en) * | 2015-02-16 | 2015-05-13 | 昆明市第一人民医院 | Bioreactor for constructing tissue engineering liver by using in-vitro circulatory perfusion |
| CN104694390B (en) * | 2015-03-17 | 2016-08-24 | 天津开发区合普工贸有限公司 | A kind of equilibrium shift experiments Embryo Gallus domesticus cultivates instrument automatically |
| CN204714823U (en) * | 2015-06-23 | 2015-10-21 | 中国人民解放军第三军医大学第一附属医院 | Cell and the external 3-D culture apparatus of organ |
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