CN104762206B - In vitro cell culture device and culture method - Google Patents
In vitro cell culture device and culture method Download PDFInfo
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- CN104762206B CN104762206B CN201510194940.3A CN201510194940A CN104762206B CN 104762206 B CN104762206 B CN 104762206B CN 201510194940 A CN201510194940 A CN 201510194940A CN 104762206 B CN104762206 B CN 104762206B
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Abstract
The invention discloses an in vitro cell culture device. The in vitro cell culture device comprises a bioreactor, a double nutrient solution irrigating system, a loading device and a monitoring system. The invention also discloses a method for culturing the cell by using the in vitro cell culture device. The method comprises the following steps: biologically simulating a physiological status, establishing the double nutrient solution irrigating system, and supplying different nutrients to different tissue blocks; allocating two pipe systems for each double culture cup, taking the culture tissue as a spacer layer of two culture solutions, culturing by simultaneously using two culture solutions, automatically irrigating the culture solutions, timely and quantificationally changing the cell culture solution, regulating the irrigating speed of the culture solution and constantly providing various nutrients and stable pH value for the cell. A dynamic pressurized environment is adopted for the cell culture device and the cell culture method; a pressure is exerted on the culture tissue by exerting a controllable axial compressive deformation load; the pressure and the compressive deformation of each culture tissue can be monitored and analyzed in real time and the pressure can be adjusted.
Description
Technical field
The present invention relates to cell injuring model field, more particularly, to a kind of cell injuring model device and cultural method.
Background technology
The realization of the propagation of cell or tissue, differentiation and physiological function, the environment residing with it is closely related, these environment
Factor can be one or more of nutrient substance, pressure, temperature, ph etc..Have in human body a lot of tissues and cells survival in
Under dynamic load environment, such as chondrocyte and pneumonocyte etc., these tissues are cultivated, just with cell only under suitable pressure condition
Can normal proliferative, differentiation, generation and internal identical physiological function.Existing in vitro study shows: not under suitable pressure condition
The articular chondrocytes of In vitro culture, cell arrangement is irregular, and substrate produces less, and collagen fiber are thinner；Zooscopy simultaneously
Find, joint immobilization can make articular cartilage that degeneration occurs, and the especially synthesis to proteoglycan has an impact.On the contrary, certain machinery
Stress stimulation is to maintain growing environment necessary to chondrocyte eubolism function.
Pressurization culture technique is one of method of cell culture, applies one by means of different to the tissue of culture or cell
Fixed pressure loading, bears the environment of physiological loads to simulate it.Common pressurization culture technique has hydraulic pressure culture technique, centrifugation
Pipe culture technique and directly contact compression culture technique etc..Hydraulic pressure culture technique is that chondrocyte or cartilage block are placed in culture dish
In, wherein fill it up with complete culture solution, remove lid on culture dish and change pressure membrane into, after discharging air in ware with viscose glue make pressure membrane with
Ware is adjacent to it is ensured that noresidue bubble in ware, then culture dish is placed in pressure chamber, and pressure is indoor to be full of distilled water, pressure and
Frequency to be regulated and controled by the pressure valve under computer controls.Centrifuge culture technology is then that cell and culture medium are placed in centrifuge tube
Interior, pressurization is produced by centrifugal force, by regulating and controlling the speed of centrifugal speed, to adjust cell and to bear gravity size, biasing strength
Can not be adjusted flexibly, the condition of culture potentially unstable of cell in centrifugal process.Relatively there is generation in directly contact compression culture technique
Table be flexcell company cyto-mechanics culture products, this system ultimate principle (positive air pressure switch mode): utilize rubber
Glue sealing gasket forms closing chamber between Tissue Culture Plate basement membrane and pedestal, and this annular seal space is inserted dioxy into and out of trachea
Change in carbon incubator, this annular seal space put into CO2 gas incubator, extrude the piston in culture hole using closing chamber positive air pressure,
And then make the dimensional culture thing between piston and fixed station indirectly be under pressure to deform upon, adjust by computer control system
The pressure of gas is changing the deformation quantity of basement membrane.At present in several pressurization cultural methods, using hydraulic pressure or air pressure-loading mode,
Tissue is in relatively airtight culture environment it is difficult to realize the dynamic renewal to cell and tissue structrue liquid, need to be in experiment load
Culture fluid is changed in gap.
Cartilage is a kind of connective tissue in people or vertebratess body.Osteoarthritis can cause the destruction of articular cartilage, its
His reason may also lead to cartilage defect, after cartilage destruction or defect, its own very difficult Regeneration and Repair, conventional homogenous cartilage transplanting,
Prosthese replacement and native cartilage cell transplantation etc. are treated, but the effect of these Therapeutic Method is not satisfactory.Scientists are just
In research, Regeneration and Repair is carried out to the cartilage of destruction or defect by tissue engineering.Tissue engineering refers to utilize biological activity
Material, by the method for In vitro culture or structure, reproduces or repairs the technology of organ and tissue.Organization engineered cartilage is
It is considered as gradually a kind of means being hopeful treatment full-thickness cartilage defects.The organizational project of cartilage is to digest articular cartilage
Afterwards, dissociate Primary chondrocyte, or directly utilize and can be planted in gathering that degradable absorbs to the stem cell of Chondrocyte Differentiation
On compound, such as polylactic acid (pla), polyglycolic acid (pga) etc. or natural carrier such as collagen fiber, during through one section of in vitro dimensional culture
Between after be transplanted to again in animal body, continue to generate new cartilage.Dimensional culture environmental benefits in the propagation of chondrocyte, differentiation,
Phenotype is stable and stromatin synthesizes.
Inside of human body packet is knitted or cell needs different nutrient substance in different parts, and these nutrient substance are for cell
Propagation, differentiation, phenotype be stable and stromatin synthesis etc. is also very important.With most representational cartilaginous tissue it is
Example is illustrating.Cartilage is a kind of connective tissue in people or vertebratess body.Articular cartilage tissue is in hierarchy, close
The part of articular cavity is articular cartilage top layer, and the part near subchondral bone is articular cartilage deep layer, is also called articular cartilage calcium
Change layer.Articular cartilage no blood vessel, no lymphatic vessel, impassivity, its source of nutrition has: 1. the blood vessel of subchondral bone tissue penetrates calcification
Layer, for should layer nutrition；2. the synovial membrane blood vessel of periarticular and synovial fluid carry out nutrition exchange；3. the synovial fluid mat of synovial membrane secretion oozes
Effect enters cartilage thoroughly, provides nutrition for articular cartilage top layer.Therefore, articular cartilage top layer and articular cartilage deep layer be not respectively by
Different nutriments with source of nutrition provide nutrition.Culture medium plays key to cell differentiation in chondrocyte dimensional culture
Property effect.But existing research not yet gives enough attention to the research of culture medium.In existing cartilaginous tissue or cell culture,
Structure differentiation is not carried out to cartilaginous tissue, only provide nutrition using a kind of culture medium.Not yet have using two kinds of culture medium pair at present
The report that cartilaginous tissue is cultivated, does not have that can to carry out 3 D stereo using two kinds of culture medium to chondrocyte external simultaneously
The device of culture.
When 3 D stereo In vitro culture being carried out to cell or tissues such as chondrocytes in extracellular medium matter, simulation
Its residing mechanics and/or nutrient environment are particularly important.Set up a set of external training that can simulate internal physiological environment
Foster device and method, is by the important means of the cell or tissue related basic research such as chondrocyte.
Content of the invention
In view of this it is necessary to dynamically irrigate for the layering not enabling two kinds of different culture fluid in existing cell culture
The problem of culture, provides a kind of cell injuring model device and cultural method.
In order to realize foregoing invention purpose, the present invention adopts the following technical scheme that
Cell injuring model device, including bioreactor and double culture fluid irrigation system；
Described bioreactor comprises cover plate, middle plate, base plate and dual culture cup, and described cover plate pairing is placed on middle plate
On, described base plate pairing is placed under middle plate, and described middle plate is provided with middle plate square hole, places dual culture cup, institute in described square hole
State the dual culture cup side of inclusion cup and be located at the round cup within square cup；Described circle cup comprises circle wall of cup and circle cup porous egative film,
Described circle cup is provided with round cup suction tube and circle cup tube for transfusion, described side's cup side of being provided with cup suction tube and square cup tube for transfusion；Described circle
It is provided with supporting construction between cup and square cup；
Described pair of culture fluid irrigation system includes two culture fluid apparatus for placing, two waste liquid recovery apparatus, two cultures
Liquid delivery pipe, two devil liquor recovery pipes and a multi-channel peristaltic pump, two culture fluid apparatus for placing pass through one respectively and cultivate
Liquid delivery pipe is connected with circle cup tube for transfusion or square cup tube for transfusion, and described circle cup suction tube and square cup suction tube respectively pass through a waste liquid
Recovery tube is connected with waste liquid recovery apparatus；Described multi-channel peristaltic pump is the conveying of culture fluid and the suction of waste liquid provides and moves
Power.
Preferably, described cell injuring model device also includes charger, and described charger includes motor, pressurization
Plate, pressured column and porous pressure sheet；Described increased pressure board is located on cover plate, and during described cover plate corresponds to, the position of plate square hole is provided with
Cover plate square hole, threadeded with increased pressure board in the upper end of described pressured column, pressured column pass through cover plate square hole, the lower end of pressured column with many
Hole pressure sheet is threaded；Described motor is connected with increased pressure board.
Preferably, described cell injuring model device also includes monitoring system, described monitoring system include pressure sensor piece,
Ultramicroscope and computer；Described pressure sensor piece is located on described base plate, and is correspondingly arranged with dual culture cup, described pressure
Sensing chip is connected with computer by data wire；Described ultramicroscope is located between cover plate and middle plate, by data wire and computer
Connect；Described computer is connected with multi-functional peristaltic pump and motor also by data wire.
Preferably, the bottom surface of described cover plate is provided with cover plate tube seat and cover plate ultramicroscope mounting groove, and described middle plate is just
Face is provided with middle plate tube seat and middle plate ultramicroscope mounting groove.
Preferably, described side's cup and circle cup are respectively equipped with piping fixing structure.
It is furthermore preferred that described dual culture cup is divided into, pressure carries cup and non-pressure carries cup；Described pressure carries cup
Square cup contains 3 piping fixing structures, and circle cup has 1 piping fixing structure, and circle cup suction tube passes through pressured column；Described non-pressure
The round cup of carrying cup contains 2 and contains piping fixing structure, and described side's cup contains 4 piping fixing structures.
Preferably, cell injuring model device contains 12 dual culture cups, is divided into 3 rows, often arranges 4, and middle 1 row is non-
Pressure carries cup, and remaining 2 row carries cup for pressure.
Preferably, described supporting construction is the support feet located at circle cup bottom.
A kind of cell injuring model method, is cultivated using above-mentioned cell injuring model device and two kinds of culture fluid, bag
Include following steps:
S1, the prefabricated liquid gel containing cell；
S2, it is placed in after bioreactor and charger assembling on super-clean bench, computer is connected motor, controls motor to adjust
Section pressurization Board position makes the gap having 1-4mm between porous pressure sheet and circle cup porous egative film, then by prefabricated liquid gel
Inject in this gap, so that liquid gel and the surface of porous pressure sheet and circle cup porous egative film is fully contacted, be placed in room temperature and treat liquid
State gel fully solidifies；
S3, in circle cup and square cup respectively plus the first appropriate culture fluid or second culture fluid；Again by biological respinse
Device is transferred to cell culture incubator, connects double culture fluid irrigation systems and monitoring system；Start drive motor and culture fluid is wriggled
Pump, applies the pressure loading setting, and maintains stable culture fluid environment to cultured tissue, the pressurization position of real-time monitoring increased pressure board
Shift one's love condition, and the stress size variation of tissue.
Preferably, cell injuring model device and cultural method are applied to survive under pressure condition in human body various groups
Knit the In vitro culture with cell, for example: gastric epithelial cell, enterocyte, cartilaginous tissue, intervertebral disc osseous tissue, tendon tissue,
Ligament tissue and the cell separated from muscle, lung, heart, blood vessel, skin, tendon, ligament, cartilage and bone.
The beneficial effect of cell culture system of the present invention and cultural method is specific as follows:
(1) cell culture system of the present invention and cultural method adopt bioreactor and double culture fluid irrigation system, bionical
Simulation physiological statuss, set up double culture fluid irrigation systems, supply different nutrients to the piece of tissue of different levels；Each dual training
Foster cup is equipped with 2 circuits systems, by the use of cultured tissue itself as the separate layer of two kinds of culture fluid, can utilize 2 kinds of trainings simultaneously
Nutrient solution is cultivated, culture fluid automatic irrigation, can regularly, quantitative replacing cell culture fluid, the rate of flooding of regulation culture liquid, energy
Various nutrients needed for constant offer cell and stable ph value.
(2) cell culture system of the present invention and cultural method adopt dynamic pressurized environment, by applying controlled axial direction pressure
Contracting deformation load, applies pressure to cultured tissue, and can each cultured tissue pressure size of Real Time Monitoring and compression
Deformation size, and pressure is adjusted.
(3) cell culture system of the present invention and cultural method adopt dimensional culture, replace traditional plane culture, so as to
Enough be conducive to maintaining cellular morphology, cell culture system of the present invention and cultural method to be applied to most of dimensional culture host materials
And timbering material.
(4) cell culture system of the present invention and cultural method are applied in human body and survive under pressure condition or/and different
There are the various tissues of different requirements and the In vitro culture of cell in position to nutrient substance, is particularly suited for gastric epithelial cell, on intestinal
Chrotoplast, cartilaginous tissue, intervertebral disc osseous tissue, tendon tissue, ligament tissue and from muscle, lung, heart, blood vessel, skin, flesh
The In vitro culture of the cell separated in tendon, ligament, cartilage and bone.
Brief description
Fig. 1 is the overall structure diagram of cell injuring model device of the present invention.
Fig. 2 is bioreactor construction schematic diagram.
Fig. 3 is dual culture cup structural representation.
Fig. 4 is that pressure carries cup structure schematic diagram.
Fig. 5 is that non-pressure carries cup structure schematic diagram.
Fig. 6 is covering plate structure schematic diagram.
Fig. 7 is middle plate structure schematic diagram.
Fig. 8 is base arrangement schematic diagram.
The each reference of in figure is:
Bioreactor 1, cover plate 11, middle plate 12, base plate 13, dual culture cup 14, cover plate square hole 111, cover plate tube seat
112, cover plate ultramicroscope mounting groove 113, middle plate square hole 121, middle plate tube seat 122, middle plate ultramicroscope mounting groove 123,
Square cup 141, circle cup 142, circle cup suction tube 144, circle cup tube for transfusion 145, square cup suction tube 146, square cup tube for transfusion 147, support
Structure 148, piping fixing structure 149, culture fluid apparatus for placing 21, waste liquid recovery apparatus 22, culture fluid delivery pipe 23, waste liquid returns
Closed tube 24, multi-channel peristaltic pump 25, motor 31, increased pressure board 32, pressured column 33, porous pressure sheet 34, pressure sensor piece 41, computer
42, pressure experience data wire subchannel 43, pressure experience data wire overall channel 44.
Specific embodiment
In order to the present invention is better described, it is described further with reference to the accompanying drawings and examples.
Embodiment 1
As shown in figure 1, a kind of cell injuring model device, including bioreactor 1, double culture fluid irrigation system, loading
Device and monitoring system.
As indicated with 2, described bioreactor 1 comprises cover plate 11, middle plate 12, base plate 13 and dual culture cup 14, described lid
Plate 11 pairing is placed on middle plate 12, and described base plate 13 pairing is placed under middle plate 12, and described middle plate 12 is provided with middle plate square hole
121, place dual culture cup 14 in described middle plate square hole 121.
As in Figure 3-5, described dual culture cup 14 side's of inclusion cup 141 and the round cup 142 within square cup 141.
Described circle cup 142 is made up of circle wall of cup and dismountable circle cup porous egative film, and described circle cup porous egative film can make culture fluid lead to
Cross.Described circle cup 142 is provided with round cup suction tube 144 and circle cup tube for transfusion 145, described side's cup 141 side's of being provided with cup suction tube 146 He
Square cup tube for transfusion 147.Circle cup tube for transfusion 145 and square cup tube for transfusion 147 are used for the culture fluid warp in culture fluid apparatus for placing 21
In culture fluid delivery pipe 23 input circle cup 142 or square cup 141, described circle cup suction tube 144 and square cup suction tube 146 are used for justifying
Culture fluid in cup 142 or square cup 141 exports to waste liquid recovery apparatus 22 through devil liquor recovery pipe 24.Described circle cup 142 and square cup
Being provided with supporting construction 148 between 141, so that leaving certain space between circle 142 glasss of bottoms of cup and square 141 glasss of bottoms of cup, being easy to deposit
Culture fluid in the side's of putting cup 141；Described supporting construction 148 is preferably provided at the support feet at round 142 glasss of bottoms of cup, and described support feet has
3 or 4.Using different culture fluid, can disclosure satisfy that in circle cup 142 and square cup 141 nutrition of cell different parts needs
Ask.Culture fluid in circle cup 142 and side's cup 141 can entirely different it is also possible to the content of only a certain component is different or gradient
Change.
As shown in figure 1, described pair of culture fluid irrigation system includes 21, two devil liquor recovery dresses of two culture fluid apparatus for placing
Put 23, two devil liquor recovery pipes 24 of 22, two culture fluid delivery pipes and a multi-channel peristaltic pump 25, two culture fluid hold dress
Put 21 to be connected with circle cup tube for transfusion 145 or square cup tube for transfusion 147 by a culture fluid delivery pipe 23 respectively, described circle cup suction
Pipe 144 is respectively connected with waste liquid recovery apparatus 22 by a devil liquor recovery pipe 24 with square cup suction tube 146；Described multichannel
Peristaltic pump 25 is the conveying of culture fluid and the suction of waste liquid provides power.Preferably, described culture fluid delivery pipe 23 and waste liquid return
Closed tube 24 is medical silicone tube.
Preferably, as shown in Figure 6 and Figure 7, described middle plate 12 front is provided with middle plate tube seat 122, is used for fixing culture fluid defeated
Send pipe 23 and devil liquor recovery pipe 24；Described cover plate 11 bottom surface is provided with cover plate tube seat 112, fixes for coordinating with middle plate tube seat 122
Culture fluid delivery pipe 23 and devil liquor recovery pipe 24.Described cover plate 11 is provided with cover plate square hole 111 corresponding with middle plate square hole 121.
When carrying cup using pressure, described pressured column 33 passes through cover plate by cover plate square hole 111.Described middle plate 12 front is additionally provided with middle plate
Ultramicroscope mounting groove 123, cover plate 11 bottom surface is additionally provided with cover plate ultramicroscope mounting groove 113, the two composition circular tube shaped
Ultramicroscope mounting groove, for installing ultramicroscope.
Preferably, as shown in Fig. 2 described cell injuring model device also comprises charger, described charger includes
Motor 31, increased pressure board 32, pressured column 33 and porous pressure sheet 34；Described increased pressure board 32 is located on cover plate 11, described pressured column
Being threadeded with increased pressure board 32 in 33 upper end, is threadeded with porous pressure sheet 34 in the lower end of pressured column 33.When motor 31 to plus
When pressing plate 32 applies pressure, increased pressure board 3 moves down, and drives connected pressured column 33 to move down, with pressured column simultaneously
The porous pressure sheet 34 of 33 connections also moves down, and the tissue to culture or cell apply pressure.Vice versa.
Preferably, the bottom of described pressured column 33 and porous pressure sheet 34 are located in described circle cup 142, described porous pressurization
The distance between piece 34 and porous egative film are 1-4mm, preferably 2mm.Described motor 31 is connected with increased pressure board 32, in order to drive pressurization
The displacement of plate 32.Described motor 31 is programmable linear progressive motor.
Preferably, described dual culture cup 14 is divided into pressure to carry cup and non-pressure carrying cup.It is furthermore preferred that described middle plate
Square hole 121 has 12, is divided into 3 rows, often arranges 4, places a dual culture cup in each square hole.Still more preferably, in
Between in a row plate square hole 121 place non-pressure and carry cup, in the row of both sides two, plate square hole 121 placement force carries cup.
Preferably, described side's cup 141 and circle cup 142 are respectively equipped with piping fixing structure 149, are used for fixing tube for transfusion and take out
Suction pipe.In the present embodiment, described piping fixing structure 149 is recessed recess at circle cup 142 or square cup 141 cup edge.Described
The round cup 142 that pressure carries cup only has 1 piping fixing structure 149, for fixing circle cup tube for transfusion 145, circle cup suction tube 144
It is connected with its culture fluid delivery pipe 23 after the pressured column 33 then passing through hollow；Square cup 141 contains 3 piping fixing structures 149, its
In 2 piping fixing structures 149 be respectively used to fixation side's cup tube for transfusion 147 and square cup suction tube 146, another 1 pipeline fixed knot
Structure 149 is corresponding with the piping fixing structure 149 of circle cup, for fixing circle cup tube for transfusion 145.Described non-pressure carries the round cup of cup
Contain piping fixing structure 149 containing 2, be respectively used to fixing circle cup tube for transfusion 145 and circle cup suction tube 144；Described side's cup
141 contain 4 piping fixing structures 149, and wherein 2 piping fixing structures 149 are respectively used to fixation side's cup tube for transfusion 147 and side
Cup suction tube 146, another 2 piping fixing structures 149 are corresponding with 2 piping fixing structures 149 of circle cup, are respectively used to fixing circle
Cup tube for transfusion 145 and circle cup suction tube 144.
Described monitoring system 4 includes pressure sensor piece 41, ultramicroscope (not shown in FIG.) and computer 42；Described pressure
Sensing chip 41 is located at described base plate 13 upper surface, and carries cup and be correspondingly arranged with porous pressure sheet 34, pressure, described pressure sensitive
Piece 41 is connected with computer 42 by data wire；Described ultramicroscope be located between cover plate 11 and middle plate 12, by data wire with
Computer 42 is connected to the micrometric displacement of monitoring pressure post 33, and then the micrometric displacement of monitoring pressure plate 32, in real time display increased pressure board 32
Misalignment.Described computer 42 is connected with multi-functional peristaltic pump 25 and motor 31 also by data wire.
As shown in figure 8, the position that described base plate 13 corresponding pressure carries cup is provided with pressure sensor piece 41, described pressure sensitive
Piece 41 is connected with computer by pressure experience data wire.Base plate 13 is provided with pressure experience data wire subchannel 43 and pressure experience number
According to line overall channel 44, described pressure experience data wire subchannel 43 is used for the feeling of stress that fixation is directly drawn by pressure sensor piece 41
By data wire, pressure experience data wire overall channel 44 is used for the pressure experience data wire after fixation collects and draws base plate 13.?
In the present embodiment, because carrying cup for pressure in plate square hole 121 in both sides, in plate square hole 121 in centre, carry cup for non-pressure,
So, the passage opening up in the middle part of base plate 13 is pressure experience data wire overall channel 44, connects pressure sensor piece 41 and pressure experience
The passage of data wire overall channel 44 is pressure experience data wire subchannel 43.
Embodiment 2
In order to be better understood from the present invention, only, explanation uses the cell described in embodiment 1 taking chondrocyte as a example below
The method of In vitro culture device cultured cells, comprises the following steps:
S1, the prefabricated liquid gel containing chondrocyte；In the present embodiment dimensional culture is adopted to chondrocyte, permissible
Using any dimensional culture carrier of the prior art；
S2, it is placed in after bioreactor and charger assembling on super-clean bench, computer is connected motor, controls motor to adjust
Section pressurization Board position makes the gap having 1-4mm between porous pressure sheet and circle cup porous egative film, then by liquid prefabricated for step s1
State gel injects in this gap, so that liquid gel and the surface of porous pressure sheet and circle cup porous egative film is fully contacted, is placed in room
Temperature treats that liquid gel fully solidifies；
S3, in circle cup and square cup respectively plus the first appropriate culture fluid or second culture fluid, the culture in square cup
Liquid can nourish the bottom of cultured tissue in circle cup, the culture fluid direct Feeder culture tissue in circle cup through circle cup porous egative film infiltration
Top layer；Again bioreactor is transferred to cell culture incubator, connects double culture fluid irrigation systems and monitoring system；According to reality
Test design, start drive motor and culture fluid peristaltic pump according to program, cultured tissue is applied with the pressure loading setting, and maintains
Stable culture fluid environment, the pressurization misalignment of real-time monitoring increased pressure board, and the stress size variation of tissue.
Preferably, described chondrocyte extracorporeal culturing method also includes applying dynamic pressure load to chondrocyte, described
Dynamic pressure load is 10% compressive strain load, and frequency 1hz gave dynamic pressurized 1 hour at interval of 1 hour.
Cell injuring model device of the present invention improves chondrocyte vitro culture conditions, beneficial to maintenance chondrocyte table
Type, promotes cartilage cell epimatrix to produce and reinvent, sets up the extracorporeal culturing method of more advanced tissue engineering bone/cartilage.This
Clear-cellss culture apparatuses and cultural method adopt bioreactor and double culture fluid irrigation system, and human simulation cartilaginous tissue is being given birth to
Under reason state, articular cartilage surface serum-free environment, the Nutrient Characteristic of cartilage bottom same level, set up double culture fluid and irrigate system
System, supplies different nutrients to the piece of tissue of different levels；Each dual culture cup is equipped with 2 circuits systems, using culture
Tissue itself, as the separate layer of two kinds of culture fluid, can be cultivated using 2 kinds of culture fluid, culture fluid automatic irrigation simultaneously, can
Regularly, quantitation changes cell culture fluid, the rate of flooding of regulation culture liquid, constant can provide various nutrients needed for cell and stablize
Ph value.
Embodiment described above only have expressed the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformation can also be made and improve, these broadly fall into the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. cell injuring model device it is characterised in that: include bioreactor and double culture fluid irrigation systems；
Described bioreactor comprises cover plate, middle plate, base plate and dual culture cup, and described cover plate pairing is placed on middle plate, institute
State base plate pairing to be placed under middle plate, described middle plate is provided with square hole, in described square hole, place dual culture cup, described dual culture
Cup inclusion side's cup and the round cup within square cup；Described circle cup comprises circle wall of cup and circle cup porous egative film, and described circle cup sets
There are round cup suction tube and circle cup tube for transfusion, described side's cup side of being provided with cup suction tube and square cup tube for transfusion；Described circle cup and square cup it
Between be provided with supporting construction；
It is defeated that the described pair of culture fluid irrigation system includes two culture fluid apparatus for placing, two waste liquid recovery apparatus, two culture fluid
Send pipe, two devil liquor recovery pipes and a multi-channel peristaltic pump, two culture fluid apparatus for placing are defeated by a culture fluid respectively
Guan Yuyuan cup tube for transfusion or square cup tube for transfusion is sent to be connected, described circle cup suction tube and square cup suction tube respectively pass through a devil liquor recovery
Guan Yuyi waste liquid recovery apparatus are connected；Described multi-channel peristaltic pump is the conveying of culture fluid and the suction of waste liquid provides power.
2. cell injuring model device according to claim 1 it is characterised in that: described cell injuring model device also wraps
Include charger, described charger includes motor, increased pressure board, pressured column and porous pressure sheet；Described increased pressure board is located at cover plate
On, during described cover plate corresponds to, the position of plate square hole is provided with cover plate square hole, and the upper end of described pressured column is with increased pressure board screw thread even
Connect, pressured column passes through cover plate square hole, is threadeded with porous pressure sheet in lower end；Described motor is connected with increased pressure board, in order to drive
The displacement of increased pressure board.
3. cell injuring model device according to claim 1 it is characterised in that: described cell injuring model device also wraps
Include monitoring system, described monitoring system includes pressure sensor piece, ultramicroscope and computer；Described pressure sensor piece is located at described
On base plate, and it is correspondingly arranged with dual culture cup, described pressure sensor piece is connected with computer by data wire；Described electron microscopic
Mirror is located between cover plate and middle plate, is connected with computer by data wire, for the micrometric displacement of monitoring pressure plate；Described computer is also logical
Cross data wire to be connected with multi-functional peristaltic pump and motor.
4. cell injuring model device according to claim 1 it is characterised in that: the bottom surface of described cover plate is provided with cover plate pipe
Groove and cover plate ultramicroscope mounting groove, the front of described middle plate is provided with middle plate tube seat and middle plate ultramicroscope mounting groove.
5. cell injuring model device according to claim 1 it is characterised in that: described side's cup and circle cup be respectively equipped with pipe
Road fixed structure.
6. cell injuring model device according to claim 5 it is characterised in that: described dual culture cup is divided into pressure to hold
Carry cup and non-pressure carries cup；The square cup that described pressure carries cup contains 3 piping fixing structures, and circle cup has 1 pipeline to fix
Structure, circle cup suction tube passes through pressured column；The round cup that described non-pressure carries cup contains 2 piping fixing structures, described side's cup
Containing 4 piping fixing structures.
7. cell injuring model device according to claim 6 it is characterised in that: cell injuring model device contains 12
Dual culture cup, is divided into 3 rows, often arranges 4, and middle 1 row carries cup for non-pressure, and remaining 2 row carries cup for pressure.
8. cell injuring model device according to claim 1 it is characterised in that: described supporting construction be located at circle cup bottom
The support feet in portion.
9. a kind of cell injuring model method is it is characterised in that usage right requires the cell injuring model described in any one of 1-8
Device and two kinds of culture fluid are cultivated, and comprise the following steps:
S1, the prefabricated liquid gel containing cell；
S2, be placed in after bioreactor and charger assembling on super-clean bench, computer connected motor, control motor to adjust plus
Press plate position makes the distance between the porous pressure sheet and circle cup porous egative film gap for 1-4mm, then coagulates prefabricated liquid
Glue injects in this gap, so that liquid gel and the surface of porous pressure sheet and circle cup porous egative film is fully contacted, is placed in room temperature and treats
Liquid gel fully solidifies；
S3, in circle cup and square cup respectively plus the first appropriate culture fluid or second culture fluid；Again bioreactor is turned
Move to cell culture incubator, connect double culture fluid irrigation systems and monitoring system；Start drive motor and culture fluid peristaltic pump, right
Cultured tissue applies the pressure loading setting, and maintains stable culture fluid environment, the pressurization displacement feelings of real-time monitoring increased pressure board
Condition, and the stress size variation of tissue.
10. according to claim 9 cell injuring model method it is characterised in that being suitable to gastric epithelial cell, enterocyte
And the In vitro culture of the cell separated from muscle, lung, heart, blood vessel, skin, tendon, ligament, cartilage and bone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510194940.3A CN104762206B (en) | 2015-04-22 | 2015-04-22 | In vitro cell culture device and culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510194940.3A CN104762206B (en) | 2015-04-22 | 2015-04-22 | In vitro cell culture device and culture method |
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| CN105670930B (en) * | 2016-03-25 | 2018-09-11 | 苏州诺普再生医学有限公司 | A kind of bioreactor of the pressure controllable of biometric print cartilage |
| CN108085254A (en) * | 2018-02-09 | 2018-05-29 | 中国人民解放军第四军医大学 | Tissue engineering product culture apparatus |
| EP3647407B1 (en) * | 2018-02-15 | 2023-06-07 | Fullstem Co., Ltd. | Cell culture device |
| AU2020349580B2 (en) * | 2019-09-22 | 2022-06-02 | Meatech 3D, Ltd. | Physical manipulation of cultured muscle tissue |
| CN111705034A (en) * | 2020-06-30 | 2020-09-25 | 山东省医药生物技术研究中心（山东省病毒研究所） | Method for efficiently promoting chondrocyte proliferation |
| CN111925939B (en) * | 2020-09-08 | 2023-07-04 | 华中科技大学同济医学院附属协和医院 | Application method of dynamic pressurization tissue engineering valve cell inoculator |
| CN113046244B (en) * | 2021-03-30 | 2023-01-06 | 上海睿钰生物科技有限公司 | Culture device and culture method using same |
| CN113355238A (en) * | 2021-06-10 | 2021-09-07 | 上海睿钰生物科技有限公司 | Culture device and culture method based on culture device |
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