CN104673669A - Microfluidics cell culture system based on micro-carrier and controlling method thereof - Google Patents
Microfluidics cell culture system based on micro-carrier and controlling method thereof Download PDFInfo
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Abstract
The invention discloses a microfluidics cell culture system based on a micro-carrier and a controlling method thereof. Two same triangular culture bins are built on a microfluidics chip; vertexes of the two culture bins are connected and a micro-valve is arranged at the connection part; the two culture bins are symmetrical to each other relative to the micro-valve; a magnetic micro-carrier for adsorbing cells are positioned in the two culture bins; each culture bin is connected with an injection pump through a liquid inlet pipe and connected with a vacuum pump through a liquid outlet pipe; a giant magnetoresistance sensor for probing the position of the magnetic micro-carrier is arranged around each culture bin; a magnetic field control module for controlling a motion track of the magnetic micro-carrier is arranged aside each culture bin. The micro-carrier is controlled by an external magnetic field to do circular motion or do back-and-forth constant-speed movement in the two culture bins, so that the accurate control of shear force and the rapid effective exchange of culture solutions in a cell culturing process can be realized, and the stability of the cell micro-environment is improved.
Description
Technical field
The present invention relates to a kind of cell culture technology, specifically based on the cell culture system of micro-fluidic chip and microcarrier, solve the shearing force control problem of puzzlement cell cultures.
Background technology
Cell cultures is one of foundation stone of the subjects such as modern biological project.Traditional cell cultures mainly builds the carrier of Growth of Cells in conjunction with substratum by the vessel such as culture dish or culturing bottle, built the microenvironment of Growth of Cells by incubator, provide necessary nutrient by the periodic replacement of nutritive medium for cell and discharge metabolic substd.
Microflow control technique is the function such as cultivation, separation, detection by realizing traditional biological, chemistry, medical laboratory to the control of the microfluid in micro-fluidic chip.Cultivate relative to tradition, micro-fluidic cultivation have closer to internal milieu, consume reagent few, highly customize, easily realize that perfusion is cultivated, easy and downstream analysis equipment integration and realize viable cell and the advantage such as to observe online.
But no matter be that tradition is cultivated or micro-fluidic cultivation, the control for cell shearing power is one of bottleneck of restriction cell long-period survival in vitro all the time.For plant and animal cell most absolutely, excessive shearing force can cause irreversible impact to the morphological structure of cell and physiological function, therefore needs the size controlling shearing force within certain scope.And for another part cell, as the endotheliocyte of blood vessel, only ability normal growth under certain shear action, therefore in culturing process, needs to maintain specific shearing force scope.In micro-current controlled cell is cultivated, speed generally by regulating and controlling the nutritive medium change of current carrys out the shearing force that regulating cell is subject to, and when then change of current speed is excessive, shearing force can cause certain injury to cell, when change of current speed is too small, has certain dead volume nutritive medium in cultivation storehouse and cannot participate in exchanging; Meanwhile, because the flow velocity by nutritive medium controls shearing force indirectly, when cultivating the cell to shearing force sensitivity such as vascular endothelial cell, be difficult to allow shearing force maintain accurately and stably in specific scope.
Summary of the invention
The object of the invention is for overcoming above-mentioned the deficiencies in the prior art, a kind of micro-current controlled cell culture systems based on microcarrier and control method thereof are provided, solve shearing force when changing liquid control and change the contradiction of liquid dead volume, realize the accurate control of shearing force in cell cultivation process, be particularly suitable for the cultivation of the cell to shearing force sensitivity.
For achieving the above object, the technical solution that a kind of micro-current controlled cell culture systems based on microcarrier of the present invention adopts is: micro-current controlled cell culture systems has a micro-fluidic chip, micro-fluidic chip constructs two identical leg-of-mutton cultivation storehouses, two summits of cultivating storehouse connect and are provided with a micro-valve in joint, cultivate storehouse relative to micro-valve symmetry for two, the magnetic microcarrier of adherent cell is positioned at two and cultivates storehouses; Each cultivation storehouse all connects a syringe pump by a liquid-inlet pipe, and each cultivation storehouse all connects a vacuum pump by a drain pipe, and each liquid-inlet pipe is provided with a liquid-inlet pipe opening-closing valve, and each drain pipe is provided with a drain pipe opening-closing valve; One is arranged in order to detect the giant magneto-resistance sensor of magnetic microcarrier position around each cultivation storehouse; At each cultivation storehouse side, the magnetic field control module that controls magnetic microcarrier movement locus is set; Micro-valve, two described liquid-inlet pipe opening-closing valves, two described drain pipe opening-closing valves, two described magnetic field control module, two described giant magneto-resistance sensors, two described vacuum pumps and two described syringe pumps are connected to controller.
The technical solution that the control method of a kind of micro-current controlled cell culture systems based on microcarrier of the present invention adopts is according to the following steps sequentially:
1) will add in syringe pump after the mixing of the nutrient solution of magnetic microcarrier and 37 degrees Celsius, controller controls to open corresponding liquid-inlet pipe opening-closing valve, to be injected into by mixed solution and to cultivate in storehouse, make to be full of mixed solution in cultivation storehouse by syringe pump; The corresponding liquid-inlet pipe opening-closing valve of closedown and syringe pump after completing; When cultivating the other giant magneto-resistance sensor in storehouse and sensing the existence of magnetic microcarrier to controller feedback high level, complete the sample introduction to cultivating storehouse;
2) controller 30 starts magnetic field control module, controls magnetic microcarrier make uniform circular motion by magnetic field control module in cultivation storehouse (6,7);
3) when cultivating the nutrient solution in storehouse and needing to change, micro-valve opened by controller, two cultivate storehouse is communicated with, and needs first magnetic microcarrier cultivated in storehouse changing nutritive medium to move by corresponding magnetic field control module control, cultivates in storehouse enter in second cultivation storehouse by micro-valve 5 from first; When first is cultivated the signal of the other huge magnetic impedance inductor block induction in storehouse less than magnetic microcarrier, export and jump to lower level by high level, cultivate the signal that the other huge magnetic impedance inductor block in storehouse senses magnetic microcarrier for second simultaneously, export by low transition to high level time, magnetic microcarrier complete from first cultivate storehouse to second cultivation storehouse transfer;
4) controller opens connects first drain pipe opening-closing valve on first drain pipe cultivating storehouse, start corresponding first vacuum pump, the nutrient solution that first is cultivated in storehouse is pumped, close first drain pipe opening-closing valve and first vacuum pump afterwards and open connection first and cultivate first liquid-inlet pipe opening-closing valve on the liquid-inlet pipe in storehouse, start corresponding first injection and penetrate pump, fresh nutrient solution is injected into first to cultivate in storehouse, after completing, closedown first liquid-inlet pipe opening-closing valve (14) and first injection penetrate pump.。
The present invention compares with prior art and has the following advantages:
1, the present invention is by the motion control of foreign field to the magnetic microcarrier of adherent cell, carries out circumferential motion or to cultivate in storehouse constant speed back and forth at two moving under making the effect of magnetic microcarrier outside magnetic field, realizes the accurate control to shearing force in cell cultivation process.
2, the present invention utilizes and independently cultivates storehouse by building two of being connected by micro-valve on micro-fluidic chip, and cultivate in storehouse and be filled with nutrient solution, two cultivation storehouses exchange cultivates, and solves when changing nutrient solution, the contradiction of shearing force and nutrient solution dead volume when nutrient solution exchanges.
3, the present invention utilizes vacuum drainage system, and the nutrient solution achieved rapidly and efficiently exchanges, and thoroughly solves the nutrient solution change of current dead volume problem that cannot overcome in cultivating for conventional microfluidic control chip, improves the stability of cell micro-environment.
4, the present invention passes through micro-channel structure more close to the environment of human body.The many advantages such as cultivate relative to tradition, micro-fluidic culture technique has that to consume reagent few, highly customizes, easily realizes perfusion and cultivate, easy and downstream analysis equipment integration.
5, the present invention uses transparent chip, can realize traditional active somatic cell that cannot realize of cultivating and observe online.
6, the present invention can be widely used in the cultivation of various cell, particularly can for providing suitable culture environment to the cell of shearing force sensitivity.
Accompanying drawing explanation
The structural representation of a kind of micro-current controlled cell culture systems based on microcarrier of Fig. 1 the present invention;
Fig. 2 is the enlarged diagram of micro-fluidic chip and flow passage structure thereof in Fig. 1;
Fig. 3 is that magnetic microcarrier shown in Fig. 2 cultivates transfer path schematic diagram between storehouse at two;
Fig. 4 is the control circui block diagram of Fig. 1;
Fig. 5 is the workflow diagram of the culture systems of micro-current controlled cell shown in Fig. 1.
In figure: 1. magnetic field control module; 2. micro-fluidic chip; 3. drain pipe opening-closing valve; 4. drain pipe opening-closing valve; 5. micro-valve; 6. cultivate storehouse; 7. cultivate storehouse; 8. drain pipe; 9. drain pipe; 10. vacuum pump; 11. vacuum pumps; 12. magnetic field control module; 13. temperature control unit assemblies; 14. liquid-inlet pipe opening-closing valves; 15. liquid-inlet pipe opening-closing valves; 16. liquid-inlet pipes; 17. liquid-inlet pipes; 18. syringe pumps; 19. syringe pumps; 20. giant magneto-resistance sensors; 21. giant magneto-resistance sensors; 22. magnetic microcarriers; 23. liquid outlets; 24. liquid outlets; 25 fluid inlets; 26. fluid inlets; 27. tracks; 28. tracks; 29. tracks; 30. controllers.
Embodiment
See Fig. 1 and Fig. 2, the present invention has a micro-fluidic chip 2, micro-fluidic chip 2 horizontal positioned.Micro-fluidic chip 2 material can be traditional micro-manufactured materials such as glass, silicon, can be also the polyester materials such as PDMS, PMMA, is a kind of transparent chip.Micro-fluidic chip 2 use conventional lithography or the polyester such as hot pressing, mechanical workout chip processing technology construct two leg-of-mutton cultivation storehouses 6,7, the structure in two leg-of-mutton cultivation storehouses 6,7 is identical, a summit of cultivating storehouse 6 and a summit of cultivating storehouse 7 connect, in joint, two summits, micro-valve 5 is installed, cultivate storehouses 6,7 for two to arrange symmetrically relative to micro-valve 5, micro-valve 5 is used for connection two and independently cultivates storehouse 6,7.The magnetic microcarrier 22 of adherent cell is positioned at cultivates storehouse 6,7, and magnetic microcarrier 22 is the magnetic microcarrier particle of micron dimension.Kernel is magnetic-particle, and skin is made up of biological affinity material, makes cell be easy in its surface adsorption, forms the microcarrier loading with bacterium.When micro-valve 5 closes, cultivate storehouse 6,7 for two to be communicated with, magnetic microcarrier 22 can be cultivated storehouse from one of them and transfer to another and cultivate storehouse and go, when micro-valve 5 closes, cultivate storehouse 6,7 separate for two, when substituting one of them nutrient solution cultivated in storehouse, another culture environment of cultivating in storehouse can not be affected.
Each cultivation storehouse 6,7 all connects a syringe pump 18,19 by a nutrient solution liquid-inlet pipe 16,17, and each cultivation storehouse 6,7 all connects a vacuum pump 10,11 by a drain pipe 8,9.Each nutrient solution liquid-inlet pipe 16,17 installs a liquid-inlet pipe opening-closing valve 14,15, each drain pipe 8,9 installs a drain pipe opening-closing valve 3,4.In order to improve nutritive medium exchange efficiency, when liquid-inlet pipe 16,17 is connected with micro-fluidic chip 2 with drain pipe 8,9, connect another two summits place in leg-of-mutton cultivation storehouse 6,7 respectively, second summit place of namely cultivating storehouse 6,7 at two all connects syringe pump 18,19 by liquid-inlet pipe 16,17, and the 3rd the summit place of cultivating storehouse 6,7 at two all connects vacuum pump 10,11 by drain pipe 8,9.Specifically: establish fluid inlet 25 at second summit place of cultivating storehouse 6, establish liquid outlet 23 at the 3rd summit place, cultivate storehouse 6 connects liquid-inlet pipe 16 by fluid inlet 25 one end at second summit place, liquid-inlet pipe 16 the other end connects syringe pump 18; Cultivate storehouse 6 connects drain pipe 8 by liquid outlet 23 one end at the 3rd summit place, the other end of drain pipe 8 connects vacuum pump 10.Cultivate storehouse 7 and fluid inlet 26 is set at second summit place, liquid outlet 24 is established at the 3rd summit place, cultivate storehouse 7 connects liquid-inlet pipe 17 one end by fluid inlet 26, the other end of liquid-inlet pipe 17 connects syringe pump 19, cultivate storehouse 7 connects drain pipe 9 one end by liquid outlet 24, the other end of drain pipe 9 connects vacuum pump 11.Liquid-inlet pipe 16 arranges liquid-inlet pipe opening-closing valve 14, liquid-inlet pipe 17 arranges liquid-inlet pipe opening-closing valve 15, liquid-inlet pipe opening-closing valve 14 and liquid-inlet pipe opening-closing valve 15 are all used to control cut-offfing of liquid-inlet pipe.Drain pipe 8 arranges drain pipe opening-closing valve 3, drain pipe 9 arranges drain pipe opening-closing valve 4, drain pipe opening-closing valve 3 and drain pipe opening-closing valve 4 are all used to control cut-offfing of drain pipe.
Be fixed with temperature control unit assembly 13 in the bottom of micro-fluidic chip 2, temperature control unit assembly 13 includes heating unit and temperature sensor, cultivates storehouses 6,7 suitable temperature for maintaining two.Because liquid independently can be changed, so independently temperature sensor and independently heating unit are arranged at bottom, each cultivation storehouse 6,7 in two cultivation storehouses 6,7.
Around each cultivation storehouse 6,7, arrange a giant magneto-resistance sensor 21,20, giant magneto-resistance sensor 21,20 is in order to detect the position of magnetic microcarrier 22.The useful range of giant magneto-resistance sensor 21,20 wants adjustment to just covering the cultivation storehouse 6,7 corresponding with it.Namely giant magneto-resistance sensor 21 is set around cultivation storehouse 6, giant magneto-resistance sensor 20 is set around cultivation storehouse 7.Giant magneto-resistance sensor 21,20 is for responding to the magnetic microcarrier 22 cultivated in storehouse 6,7 respectively.When magnetic microcarrier 22 near giant magneto-resistance sensor 21,20 time, giant magneto-resistance sensor 21,20 detects magnetic microcarrier 22 and just feeds back high level, when magnetic microcarrier 22 away from giant magneto-resistance sensor 21,20 time, giant magneto-resistance sensor 21,20 due to induction just feed back lower level less than magnetic microcarrier 22.The measurement of inductance scope of giant magneto-resistance sensor 21,20 is through adjustment.For giant magneto-resistance sensor 21, its useful range wants adjustment to cultivate storehouse 6 to just covering.When magnetic microcarrier 22 is when cultivating in storehouse 6, giant magneto-resistance sensor 21 feeds back high level to controller 30, and when magnetic microcarrier 22 leaves cultivation storehouse 6, giant magneto-resistance sensor 21 feeds back lower level to controller 30.
In each cultivation storehouse 6,7, side arranges a magnetic field control module 1,12, magnetic field control module 1,12 also claims magnetic field generator, be be made up of the coil winding of some different angles, be optionally energized to the coil winding of different angles, the magnetic field of different angles can be produced.Thus magnetic microcarrier 22 can be driven to have different movement locus corresponding cultivation in storehouse 6,7, realize cultivating the transfer between storehouse 6,7 at the circumferentially orbiting motion cultivated in storehouse 6,7 and at two.Magnetic field control module 1,12 connection control device 30, specifically variable magnetic field can be produced under the control of the controller 30, the magnetic field of different directions, varying strength can be there is, to control the different movement locus of magnetic microcarrier 22, make magnetic microcarrier 22 constant speed, orientation movement.That is: arrange magnetic field control module 1 at cultivation storehouse 6 side, magnetic field control module 1 produces specific magnetic field to control magnetic microcarrier 22 constant speed of cultivating in storehouse 6, orientation movement under the control of the controller 30; In cultivation storehouse 7, side arranges magnetic field control module 12, and magnetic field control module 12 produces specific magnetic field to control magnetic microcarrier 22 constant speed of cultivating in storehouse 7, orientation movement under the control of the controller 30.
See Fig. 3, under the foreign field of magnetic field control module 1 controls, make magnetic microcarrier 22 circumferentially track 27 uniform motion in cultivation storehouse 6, the speed of motion by foreign field accuracy controlling, to meet the requirement of the shearing force of cell cultures needs.When magnetic microcarrier 22 is transferred in cultivation storehouse 7 from cultivation storehouse 6, magnetic microcarrier 22 moves along the motion track 28 towards micro-valve 5 in cultivation storehouse 6, after magnetic microcarrier 22 enters and cultivates storehouse 7, under the foreign field of magnetic field control module 12 controls, make magnetic microcarrier 22 circumferentially track 29 uniform motion in cultivation storehouse 7, motion speed by foreign field accuracy controlling, to meet the requirement of the shearing force of cell cultures needs.Viscosity due to fluid is constant, the shearing force that the movement velocity of magnetic microcarrier 22 and the cell of its glued on top are subject to is directly proportional, and the present invention can realize the regulation and control to shearing force scope suffered by cell by the movement velocity that magnetic field control module 1,12 controls magnetic microcarrier 22.
See Fig. 4, micro-valve 5, two drain pipe opening-closing valves 3,4, two liquid-inlet pipe opening-closing valves 14,15, two magnetic field control module 1,12, two vacuum pumps 10,11, two syringe pumps 18,19 and temperature control unit assembly 13 are connected to controller 30 respectively by respective control line, carry out work under the control of the controller 30.Giant magneto-resistance sensor 20,21 and temperature control unit assembly 13 are connected to controller 30 respectively by respective signal wire.Giant magneto-resistance sensor 20,21, for feeding back the particular location of magnetic microcarrier 22, after it senses magnetic microcarrier 22, feeds back high level to controller 30; When not sensing magnetic microcarrier 22, feed back lower level to controller 30.Temperature control unit assembly 13 controls its heating unit work by controller 3, and the temperature sensor of temperature control unit assembly 13 in order to detect the real time temperature cultivated in storehouse 6,7, and sends real time temperature to controller 30.
See Fig. 1,2,3,4,5, be divided into sample introduction, cultivation, microcarrier transfer during cell culture system work, change liquid four-stage.Specific as follows:
1) the sample introduction stage
Mixed solution is formed by after the mixing of the nutrient solution of the magnetic microcarrier 22 of adherent cell and 37 degrees Celsius, mixed solution is added in syringe pump 18, controller 30 controls liquid-inlet pipe opening-closing valve 14 and opens, and control syringe pump 18 and work, magnetic microcarrier 22 and nutrient solution mixed solution being injected into lentamente by syringe pump 18 cultivates in storehouse 6, the volume of the mixed solution injected and the volume in cultivation storehouse 6 match, and make to be full of mixed solution in cultivation storehouse 6,7.After injection completes, controller 30 controls liquid-inlet pipe opening-closing valve 14 and cuts out, and control corresponding syringe pump 18 and quit work, now, huge magnetic impedance inductor block 21 works, when the huge magnetic impedance inductor block 21 on side, cultivation storehouse 6 senses existing of magnetic microcarrier 22, feed back high level to controller 30, the cultivation storehouse 6 sample introduction stage is completed.
To cultivating the sample introduction process in storehouse 7 and similar to cultivating the sample introduction process in storehouse 6: will add in syringe pump 19 after the nutrient solution mixing of the magnetic microcarrier 22 of adherent cell and 37 degrees Celsius, controller 30 controls liquid-inlet pipe opening-closing valve 15 and to open and syringe pump 19 works, injection completes, close liquid-inlet pipe opening-closing valve 15 and syringe pump 19, now, huge magnetic impedance inductor block 20 senses the existence of magnetic microcarrier 22, feeds back high level to controller 30, completes the cultivation storehouse 7 sample introduction stage.
2) cultivation stage
Controller 30 driving magnetic field control module 1,12 starts, controlling magnetic field control module 1,12 modulates the foreign field of coupling, accurate control is realized to magnetic microcarrier 22 movement velocity, makes magnetic field control module 1,12 control magnetic microcarrier 22 and make uniform circular motion in cultivation storehouse 6,7.Because the circumferential motion speed of magnetic microcarrier 22 is determined, so magnetic field control module 1,12 accurately can control the shearing force size in its culturing process by the best shearing force scope of cell tolerance.
After entering cultivation stage, controller 30 control temperature control unit assembly 13 starts, and the temperature sensor cultivated below storehouse 6,7 starts to collect temperature information, and Real-time Feedback is to controller 30.Controller 30 is according to the temperature information received, the temperature information cultivating storehouse 6,7 is fed back to controller 30 by temperature sensor, when cultivating the temperature in storehouse 6,7 lower than 37 degrees Celsius, controller 30 controls heating unit work, the nutrient solution cultivated in storehouse 6,7 is heated up, make the culture-liquid temp in cultivation storehouse 6,7 remain on the optimum temps 37 degrees Celsius of cell cultures, this is the optimum temps of cell cultures.
3) microcarrier transition phase
Nutrient solution in the cultivation storehouse 6,7 at magnetic microcarrier 22 place, due to the metabolism of cell, causes nutritive substance consumption, and meta-bolites is assembled, and when being no longer applicable to Growth of Cells, changes with regard to needing the nutrient solution to cultivating in storehouse 6,7.With traditional micro-current controlled cell cultivate chip unlike, present invention employs two cultivation storehouses 6,7 alternate culture method, replaced during traditional microfluidic chip is cultivated the mode being changed nutritive medium by perfusion by mobile magnetic microcarrier 22 to the way in cultivation storehouse of perfusion fresh medium, ensure that the shearing force in whole culturing process suffered by cell is all in accurate slave mode.
In microcarrier transition phase, controller 30 controls micro-valve 5 and opens, two cultivate storehouse 6,7 is communicated with, under the control of magnetic field control module 1, magnetic microcarrier 22 enters another along track 28 by micro-valve 5 and cultivates in storehouse 7 in the cultivation storehouse 6 needing replacing nutrient solution, movement velocity is optimized through controller 30, guarantees that shearing force suffered on magnetic microcarrier 22 is identical with the shearing force moved in a circle in cultivation storehouse 6.When the huge magnetic impedance inductor block 21 cultivating side, storehouse 6 responds to the signal less than magnetic microcarrier 22, export and jump to lower level by high level.Cultivate the signal that the other huge magnetic impedance inductor block 20 in storehouse 7 senses magnetic microcarrier 22 simultaneously, when to export by low transition to high level, represent magnetic microcarrier 22 and complete from cultivating storehouse 6 to the transfer of cultivating storehouse 7, the micro-valve 5 of closedown after transfer.After magnetic microcarrier 22 transfers to the cultivation storehouse 7 on opposite, magnetic microcarrier 22 still moves in a circle along track 29, the speed of motion by foreign field magnetic field control module 12 accuracy controlling, to adapt to the needs of specific cells to shearing force.
Magnetic microcarrier 22, from cultivating storehouse 7 to cultivating the transfer control process in storehouse 6 with similar to the transfer control process of cultivating storehouse 7 from cultivating storehouse 6, repeats no more.
4) the liquid stage is changed
When magnetic microcarrier 22 is cultivated storehouse 6 be transferred to after second of not needing to change nutrient solution cultivate storehouse 7 from being needed to change first of nutrient solution, the nutrient solution cultivated in storehouse 6 is changed.The working process changing nutrient solution is as follows: controller 30 controls drain pipe opening-closing valve 3 and opens, and starts corresponding vacuum pump 10, is pumped out in vacuum pump 10 by the nutrient solution cultivated in storehouse 6.After nutrient solution pumps completely, close drain pipe opening-closing valve 3, close vacuum pump 10 subsequently.After imbibition end of processing, the liquid-inlet pipe opening-closing valve 14 cultivated with first that needs to change nutrient solution on liquid-inlet pipe 16 that storehouse 6 is connected opened by controller 30, starts corresponding syringe pump 18, is injected into by fresh nutrient solution cultivates in storehouse 6 by liquid-inlet pipe 16.The volume and cultivation storehouse 6 volume that inject liquid match.After nutrient solution has been injected, close liquid-inlet pipe opening-closing valve 14, syringe pump 18 quits work subsequently.Changing the liquid stage completes.
After magnetic microcarrier 22 transfers to cultivation storehouse 6 from cultivation storehouse 7, same needs is changed the liquid-inlet pipe liquid in cultivation storehouse 7, and substitute mode is similar with to the mode of the nutrient solution replacing of cultivating in storehouse 6, repeats no more.
Exchange relative to traditional perfusion mode nutritive medium, the present invention adopts vacuum pump negative pressure drainage system, first by vacuum pump 10,11, the nutritive medium cultivated in storehouse 6,7 is exhausted, then utilize syringe pump 18,19 to be injected into by fresh nutrient solution and cultivate in storehouse 6,7, thoroughly solve the dead volume problem in commutation course, ensure that property and the stability all of nutrient solution composition.
Generally, connect the two micro-valves 5 cultivated between storehouse 6,7 and be in closing condition, under the effect of magnetic microcarrier 22 outside magnetic field of adherent cell, in a certain cultivation storehouse, carry out circumferential motion with specific speed.The shearing force that the speed that magnetic microcarrier 22 moves can be tolerated by cell determines, realizes the accuracy controlling to cell shearing power by the accurate control of foreign field to magnetic microcarrier 22 movement velocity.When nutrient solution changed by needs, the two micro-valves 5 cultivated between storehouse 6,7 are just opened, and extracorporeal magnetic driving magnetic microcarrier 22 moves to another and is full of in the cultivation storehouse of fresh medium, continues to move in a circle.Micro-valve 5 cuts out, and utilizing vacuum drainage system to pump needing the nutrient solution of discharging, utilizing syringe pump to inject fresh nutritive medium.Complete the Renewal process of a nutrient solution.
Claims (7)
1. the micro-current controlled cell culture systems based on microcarrier, there is a micro-fluidic chip (2), it is characterized in that: on micro-fluidic chip (2), construct two identical leg-of-mutton cultivation storehouses (6,7), two summits of cultivating storehouse (6,7) connect and are provided with a micro-valve (5) in joint, two cultivations storehouse (6,7) are symmetrical relative to micro-valve (5), and the magnetic microcarrier (22) of adherent cell is positioned at two and cultivates storehouse (6,7); Each cultivation storehouse (6,7) all connects a syringe pump (18,19) by a liquid-inlet pipe, each cultivation storehouse (6,7) all connects a vacuum pump (10,11) by a drain pipe, each liquid-inlet pipe is provided with a liquid-inlet pipe opening-closing valve (14,15), each drain pipe is provided with a drain pipe opening-closing valve (3,4); One is arranged in order to detect the giant magneto-resistance sensor (21,20) of magnetic microcarrier (22) position around each cultivation storehouse (6,7); At each cultivation storehouse (6,7) side, the magnetic field control module (1,12) that controls magnetic microcarrier (22) movement locus is set; Micro-valve (5), two described liquid-inlet pipe opening-closing valves (14,15), two described drain pipe opening-closing valves (3,4), two described magnetic field control module (1,12), two described giant magneto-resistance sensors (21,20), two described vacuum pumps (10,11) and two described syringe pumps (18,19) are connected to controller (30).
2. according to claim 1 based on the micro-current controlled cell culture systems of microcarrier, it is characterized in that: be fixed with the temperature control unit assembly (13) comprising heating unit and temperature sensor in the bottom of micro-fluidic chip (2), temperature control unit assembly (13) connection control device (30).
3. according to claim 1 based on the micro-current controlled cell culture systems of microcarrier, it is characterized in that: respectively establish fluid inlet (25) at second summit place of leg-of-mutton cultivation storehouse (6,7), the 3rd summit place establish liquid outlet (23), liquid-inlet pipe connects fluid inlet (25), and drain pipe connects liquid outlet (23).
4. according to claim 1 based on the micro-current controlled cell culture systems of microcarrier, it is characterized in that: magnetic field control module (1,12) is made up of the coil winding of some different angles, controller (30) with the magnetic field producing different angles, drives magnetic microcarrier (22) in cultivation storehouse (6,7) circumferentially orbiting motion and transfer between two cultivations storehouse (6,7) to the energising of the coil winding of different angles.
5. according to claim 1 based on the micro-current controlled cell culture systems of microcarrier, it is characterized in that: micro-fluidic chip (2) horizontal positioned, micro-fluidic chip (2) is transparent chips.
6., as claimed in claim 1 based on a control method for the micro-current controlled cell culture systems of microcarrier, it is characterized in that sequentially according to the following steps:
1) will add in syringe pump (18,19) after the mixing of the nutrient solution of magnetic microcarrier (22) and 37 degrees Celsius, controller (30) controls to open corresponding liquid-inlet pipe opening-closing valve (14,15), by syringe pump (18,19), mixed solution is injected in cultivation storehouse (6,7), makes to be full of mixed solution in cultivation storehouse (6,7); The corresponding liquid-inlet pipe opening-closing valve of closedown (14,15) and syringe pump (18,19) after completing; When cultivating the other giant magneto-resistance sensor (21,20) in storehouse (6,7) and sensing the existence of magnetic microcarrier (22) to controller (30) feedback high level, complete the sample introduction to cultivating storehouse (6,7);
2) controller 30 starts magnetic field control module (1,12), controls magnetic microcarrier (22) make uniform circular motion by magnetic field control module (1,12) in cultivation storehouse (6,7);
3) when cultivating the nutrient solution in storehouse (6,7) and needing to change, micro-valve (5) opened by controller (30), two cultivate storehouse (6,7) is communicated with, first magnetic microcarrier (22) cultivated in storehouse (6) being controlled to need to change nutritive medium by corresponding magnetic field control module (1) moves, and enters second cultivate in storehouse (7) in first cultivation storehouse (6) by micro-valve 5; When first is cultivated the signal of other huge magnetic impedance inductor block (21) induction in storehouse (6) less than magnetic microcarrier (22), export and jump to lower level by high level, cultivate the signal that the other huge magnetic impedance inductor block (20) in storehouse (7) senses magnetic microcarrier (22) for second simultaneously, export by low transition to high level time, magnetic microcarrier (22) complete from first cultivate storehouse (6) to second cultivation storehouse (7) transfer;
4) first drain pipe opening-closing valve (3) on the drain pipe of connection first cultivation storehouse (6) opened by controller (30), start corresponding first vacuum pump (10), the nutrient solution that first is cultivated in storehouse (6) is pumped, close first drain pipe opening-closing valve (3) and first vacuum pump (10) afterwards and open first liquid-inlet pipe opening-closing valve (14) on the liquid-inlet pipe of connection first cultivation storehouse (6), start corresponding first injection and penetrate pump (18), fresh nutrient solution being injected into first cultivates in storehouse (6), after completing, closedown first liquid-inlet pipe opening-closing valve (14) and first injection penetrate pump (18).
7. control method according to claim 6, it is characterized in that: step 2) in, controller (30) is enabled in the temperature control unit assembly (13) of micro-fluidic chip (2) bottom, the temperature information cultivating storehouse (6,7) is fed back to controller (30) by temperature sensor, when temperature is lower than 37 degrees Celsius, controller (30) controls heating unit work, makes the temperature in cultivation storehouse (6,7) remain on the optimum temps 37 degrees Celsius of cell cultures.
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