CN103667054A - Integrated microfluidic cell culture chip and preparation method thereof - Google Patents
Integrated microfluidic cell culture chip and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an integrated microfluidic cell culture chip and a preparation method thereof. The microfluidic cell culture chip comprises a flexible polymer main structure layer, a bottom film and a supporting substrate which are sequentially bonded together, and a temperature control component, a micro pump, a micro valve and a control circuit board. The flexible polymer main structure layer comprises at least one culture solution/liquid medicine storage pool, a cell culture pool, a waste liquid pool, a microfluidic channel and a micro pump mounting area; the micro pump is arranged in the micro pump mounting area; a temperature control component mounting clamping groove is arranged on the supporting substrate; the temperature control component is arranged in the temperature control component mounting clamping groove; the micro valve and the chip control circuit are arranged on the control circuit board; and the chip is integrated due to assembly of the micro valve. The chip preparation process is mainly based on machining and flexible polymer composite molding. The invention provides a cell culture device which is integrated and simple in process, is provided with a closed inner cavity of the fluid channel and is particularly suitable for special fields of space on-orbit cytology research and the like.
Description
Technical field the present invention relates to a kind of integrated micro-flow control cell cultivation chip and preparation method thereof, and this chip is specially adapted to the cell cultures of the special dimensions such as space-orbit cytology research, belongs to micro-fluidic chip technical field.
The space-orbit cytology research of background technology becomes the important component part of space life science research gradually, and have or not applicable research plant and instrument, is the key factor that can this research be carried out smoothly.At present more existing can be used for device and the technology of cell cultures and morphologic observation under space environment, can meet cell and change liquid, dosing, video recording and the function such as fixing.But these operations of equipment are complicated, time-consuming; The volume ratio of most devices is larger; The a certain static response message constantly of cell can only be obtained, cytological Effect research cannot be under space environment, carried out real-time dynamicly.
Along with the progress of microflow control technique, micro-current controlled cell is cultivated chip and is studied new platform is provided for spatial cell.But current existing micro-current controlled cell is cultivated chip research achievement and is still existed some not to be suitable for the limitation of space-orbit cytology research: the relevant liquid reservoirs of cell cultures and microfluid driving governor part be as Micropump, micro-valve etc. is independent of outside micro-fluidic chip main body more, causes that whole chip system is more discrete and chip system overall volume is larger; Grand micro-interface between chip body and outside support equipment may be introduced and pollute for cell cultures; The manipulation require human interventions such as cell cultures fluid exchange, are not suitable for this class of manned space flight and lack a particular job environment that professional participates in; Cultivating chip internal flow passage structure has direct gas to contact with the external world, or cell cultures district is open architecture, thereby the weightlessness factor gas-liquid that causes in space coexists and may cause bubble to enter cell culture insert and affect the normal growth of cell; Chip manufacture complex process, cost are higher, and chip section assembly is difficult for assembling to be changed, and is unfavorable for disposable use etc.
Summary of the invention the object of this invention is to provide a kind of integrated micro-flow control cell cultivation chip and preparation method thereof, the micro-current controlled cell that processes is cultivated chip and is had that integrated, miniaturization, automatization, microfluidic channel inner chamber are airtight, chip section assembly is easily assembled the features such as replacing, suitable disposable use, be specially adapted to the cell cultures of the special dimensions such as space-orbit cytology research, preparation method's technique is simple, cost is lower.
Integrated micro-flow control cell cultivation chip provided by the invention, comprises the flexible polymer main structure layer, counterdie and the support base that are bonded together successively, and temperature-controlling module, Micropump, micro-valve and control circuit board etc.; Described flexible polymer main structure layer comprises at least 1 nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool, microfluidic channel, Micropump installing zone etc.; In described Micropump installing zone, described Micropump is installed; In described support base, there is temperature control assembly that draw-in groove is installed; Described temperature control assembly is installed in draw-in groove described temperature-controlling module is installed; On described control circuit board, be furnished with described micro-valve and chip controls circuit; The assembling of described micro-valve has realized the integration of chip simultaneously.
Integrated micro-flow control cell cultivation chip provided by the invention, its procedure of processing comprises:
(1) by CNC numerically-controlled machine precision sizing, make the mould of the molded use of described flexible polymer main structure layer, comprise the punch consistent with described main structure layer inner flow passage structure, and the die consistent with described main structure layer external form;
(2) assemble described punch and die, flexible polymer is consolidated to thing in advance and inject described punch and the definite molding space of die, curing flexible polymer is consolidated thing in advance, sloughs described die and punch, obtains described flexible polymer main structure layer;
(3) on silicon chip spin coating flexible polymer in advance admittedly thing, obtain described counterdie after solidifying, counterdie and described flexible polymer main structure layer described in bonding;
(4) described Micropump is installed in described Micropump installing zone, described temperature-controlling module is installed and in described temperature control assembly, draw-in groove is installed;
(5) support base and counterdie described in bonding;
(6) weld or connect described chip controls circuit associated tracks, assembling described micro-valve;
(7) surface treatment is carried out in described cell culture insert bottom surface.
Described cell culture insert roof thickness and the thick Design and Machining according to a certain percentage of described nutrient solution/liquid medicine storage pool wall, nutrient solution/liquid enters the elastic deformation of described cell culture insert based on cell culture insert roof and replys.
Described counterdie is one deck flexible polymer film.
Described support base can be polymethylmethacrylate (PMMA) plate or glass-fiber-plate.
Bolt mounting holes and the wire with identical shaped and size passes hole in corresponding position for described flexible polymer main structure layer, counterdie and support base.
Described Micropump after importing and exporting the Micropump import and export mounting groove that inserts in respectively described Micropump installing zone needs import and export mounting groove to seal.
Described temperature-controlling module comprises Heating element ITO conductive glass and glues temperature-sensing element RTD Pt100 or Pt1000 thereon, the pin of described RTD is drawn temperature sensing element and is connected wire, described ITO conductive glass both sides are coated with gold electrode, and described gold electrode is drawn ito glass electric power conductor.
Described micro-valve is the normally closed micro-valve of electromagnetism, mainly comprise micro-valve open and close controlling microchannel, electromagnetic drive mechanism and micro-valve regulation nut, described micro-valve open and close controlling microchannel is at one section that need to control between any 2 liquid storage tanks of its internal flow break-make in aforementioned microfluidic channel, described electromagnetic drive mechanism is positioned at below under described micro-valve open and close controlling microchannel, be fixed on described control circuit board, a pair of described micro-valve regulation nut is symmetrical in described electromagnetic drive mechanism and is fixed on described control circuit board.
Described nutrient solution/liquid medicine storage pond volume is greater than required replacing nutrient solution/liquid number of times with the ratio of described cell culture insert volume, and pool wall thickness is 0mm-0.3mm, but is not 0.
Described cell culture insert bottom surface can be for plane or with cell capture microstructure, described cultivation pool near described Micropump inlet side with latticed or strip fence.
Described waste liquid pool volume is greater than the summation of described cell culture insert, nutrient solution/liquid medicine storage pond volume, and pool wall thickness is 0mm-0.3mm, but is not 0.
The wide 0.5-1.2mm of described microfluidic channel, maximum height 0.1-0.2mm.
The bed thickness that described flexible polymer main structure layer only comprises microfluidic channel part is 0.2-0.5mm.
The bed thickness that described counterdie only comprises microfluidic channel part is 0-0.3mm, but is not 0.
Nutrient solution/liquid medicine storage pond can be identical to the path shape between described cell culture insert described in each for described microfluidic channel, or select suitable path to make the flow resistance of some storage pool respective branch be greater than other storage pool respective branch flow resistances.
Described flexible polymer main structure layer is not communicated with the external world with the inner chamber that counterdie bonding forms.
On the described Micropump pump housing, there is Flexible Control plug, with Micropump described in Micropump adapter grafting rear drive in described chip controls circuit.
Described control circuit board locates to have cell culture insert view port under being positioned at described cell culture insert.
Described control circuit board one side has golden finger, and the outer computer interface of the chip of can pegging graft or special-purpose power supply interface are described chip controls circuit supply, and with the outer computer communication of chip.
Described chip controls circuit supply voltage is 5-12V.
Described nutrient solution/liquid medicine storage pond and waste liquid pool can be on described flexible polymer main structure layer integrated molding, also can first process respectively after single liquid storage tank again key and to not comprising on the flexible polymer main structure layer of described nutrient solution/liquid medicine storage pond and waste liquid pool.
Surface treatment is carried out in described cell culture insert bottom surface, can be coated with any one solution in poly-Dopamine HCL (PDA), poly-lysine (PLL) or mouse tail collagen protein I type.
Integrated micro-flow control cell cultivation chip working process provided by the invention is: while starting culturing cell, after gas in the inner chamber of removal flexible polymer main structure layer and counterdie key and formation, with syringe, nutrient solution/liquid, cell suspension are injected respectively to nutrient solution/liquid medicine storage pond and cell culture insert successively, the golden finger of control circuit board one side inserts the outer computer interface of chip or special-purpose power supply interface is chip controls circuit supply, cell culture insert internal temperature is stable at preset temp, normally closed because of micro-valve, in inner chamber, there is no liquid-flow.Cell starts adherent and growth.Need to be when more changing to new nutrient solution/liquid in cell culture insert, Micropump and Micropump are opened to the micro-valve between waste liquid pool, and in cell culture insert, original waste liquid is discharged in waste liquid pool, and cell culture insert roof subsides to bottom simultaneously.When waste liquid output and cell culture insert roof subside while reaching predetermined extent, Micropump and Micropump are closed to the micro-valve between waste liquid pool, open to the micro-valve between cell culture insert in nutrient solution/liquid medicine storage pond, and the nutrient solution/liquid in nutrient solution/liquid medicine storage pond enters cell culture insert along with the elastic deformation reply of cell culture insert roof and subsiding of nutrient solution/liquid medicine storage pond pool wall.New nutrient solution/liquid is full of after cell culture insert, and nutrient solution/liquid medicine storage pond is closed to the micro-valve between cell culture insert, starts the cell static cultivation of a new round, until during nutrient solution/liquid that next round more renews, repeat the micro-valve work schedule of above-mentioned Micropump.
The distinguishing feature of integrated micro-flow control cell cultivation chip that the present invention proposes and preparation method thereof is: the microfluid driving governor parts such as all liquid storage tanks such as nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool and Micropump, micro-valve are all integrated into micro-current controlled cell and cultivate in chip, chip overall volume is cultivated chip much smaller than traditional micro-current controlled cell of the outside support equipment of needs, and grand micro-interface of having avoided traditional micro-current controlled cell to cultivate between chip chips main body and outside support equipment pollutes; Liquid changing in cell culture insert is realized by controlling the work schedule of the micro-valve of Micropump, and the work of chip fully automated, without human intervention; Flexible polymer main structure layer is not communicated with the external world with the inner chamber that counterdie bonding forms, and has avoided ambient atmos to enter the normal growth that cell culture insert affects cell; The input mode of replying based on the elastic deformation of cell culture insert item wall, makes original waste liquid discharge of cell culture insert inside more thorough, has reduced the time for the power supply of microfluid drive element simultaneously, and then has reduced the power consumption of full culture cycle; The main process of chip is that machining and flexible polymer are molded, simple to operate; Micro-valve and Micropump can reclaim, and are assembled in new cell cultivation chip, have reduced cost of manufacture; Aforesaid integrated, miniaturization, automatization, microfluidic channel inner chamber are airtight, chip section assembly is easily assembled the features such as replacing, suitable disposable use, are specially adapted to the cell cultures of the special dimensions such as space-orbit cytology research.
Accompanying drawing explanation
Fig. 1 is integrated micro-flow control cell cultivation chip structure three-dimensional decomposing schematic representation provided by the invention
Fig. 2 is the three-dimensional structure schematic diagram after integrated micro-flow control cell cultivation chip assembling provided by the invention
Fig. 3 is the mould punch three-dimensional structure schematic diagram that flexible polymer main structure layer of the present invention is used when molded
The mould die three-dimensional structure schematic diagram using when Fig. 4 flexible polymer main structure of the present invention layer is molded
Structural representation after the mould punch using when Fig. 5 flexible polymer main structure of the present invention layer is molded and die assembling.
Fig. 6 is the liquid storage tank three-dimensional structure biopsy cavity marker devices schematic diagram that the present invention processes first separately while adopting non-integral moulding process while preparing flexible polymer main structure layer.
Embodiment
Below in conjunction with accompanying drawing, the present invention will be further described.
Integrated micro-flow control cell cultivation chip structure provided by the invention as depicted in figs. 1 and 2, comprises the flexible polymer main structure layer 1, counterdie 2 and the support base 3 that are bonded together successively, and temperature-controlling module, Micropump 9, micro-valve and control circuit board 4 etc., flexible polymer main structure layer 1 comprises at least 1 nutrient solution/liquid medicine storage pond 5, cell culture insert 6, waste liquid pool 12, microfluidic channel 7, Micropump installing zone 8 etc., counterdie 2 is one deck flexible polymer films, support base 3 can be polymethylmethacrylate (PMMA) plate or glass-fiber-plate, bolt mounting holes 23 and the wire with identical shaped and size passes hole 24 in corresponding position for flexible polymer main structure layer 1, counterdie 2 and support base 3, Micropump 9 is installed in Micropump installing zone 8, and it imports and exports to insert in respectively the Micropump import and export mounting groove of Micropump installing zone 8 and import and export mounting groove needs sealing, in support base 3, there is temperature control assembly that draw-in groove 25 is installed, its inside is provided with temperature-controlling module, temperature-controlling module comprises Heating element ITO conductive glass 14 and glues temperature-sensing element RTD 13 thereon, ITO conductive glass 14 both sides are coated with gold electrode, and draw ito glass electric power conductor 15, the pin of RTD 13 is drawn temperature sensing element and is connected wire 21, on control circuit board 4, be furnished with chip controls circuit 20, temperature sensing element connects wire 21 and ito glass electric power conductor 15 and through wire, passes the corresponding position that is welded to chip controls circuit 20 behind hole 24, micro-valve is the normally closed micro-valve of electromagnetism, mainly comprise micro-valve open and close controlling microchannel 7, electromagnetic drive mechanism 16 and micro-valve regulation nut 17, micro-valve open and close controlling microchannel 7 is at one section that need to control between any 2 liquid storage tanks of its internal flow break-make in aforementioned microfluidic channel 7, electromagnetic drive mechanism 16 is positioned under micro-valve open and close controlling microchannel 7, be fixed on control circuit board 4, a pair of micro-valve regulation nut 17 is symmetrical in electromagnetic drive mechanism 16 and is fixed on control circuit board 4, while assembling micro-valve, micro-valve holding bolt 11 coordinates with micro-valve regulation nut 17 through bolt mounting holes 23, realize the integration of whole cell cultivation chip simultaneously.
The volume in nutrient solution/liquid medicine storage pond 5 is greater than required replacing nutrient solution/liquid number of times with the ratio of the volume of cell culture insert 6, and pool wall thickness is 0mm-0.3mm, but is not 0.Cell culture insert 6 bottom surfaces can be for plane or with cell capture microstructure, near described Micropump 9 inlet sides with latticed or strip fence.The wall thickness in the roof thickness of cell culture insert 6 and nutrient solution/liquid medicine storage pond 5 is Design and Machining according to a certain percentage, and making nutrient solution/liquid enter cell culture insert 6 is the elastic deformation replies based on cell culture insert roof.The volume of waste liquid pool 12 is greater than the summation of cell culture insert 5, nutrient solution/liquid medicine storage pond 6 volumes, and pool wall thickness is 0mm-0.3mm, but is not 0.The wide 0.5-1.2mm of microfluidic channel 7, maximum height 0.1-0.2mm.The bed thickness that only comprises microfluidic channel 7 parts in flexible polymer main structure layer 1 is 0.2-0.5mm.The bed thickness that only comprises microfluidic channel 7 parts in counterdie 2 is 0-0.3mm, but is not 0.Microfluidic channel 7 can be selected as depicted in figs. 1 and 2 to the path shape between cell culture insert 6 in different nutrient solution/liquid medicine storages pond 5, make the flow resistance of liquid medicine storage pond respective branch be greater than the flow resistance of nutrient solution storage pool respective branch, path shape that also can all branch roads is identical.Flexible polymer main structure layer 1 is not communicated with the external world with the inner chamber that counterdie 2 bondings form.On Micropump 9 pump housings, there is Flexible Control plug 10, with the Micropump adapter 18 grafting rear drive Micropumps 9 in chip controls circuit 20.RTD 13 can be Pt100 or Pt1000.Control circuit board 4 locates to have cell culture insert view port 19 under being positioned at cell culture insert 6.Control circuit board 4 one sides have golden finger 22, and the outer computer interface of the chip of can pegging graft or special-purpose power supply interface are 20 power supplies of chip controls circuit, and with the outer computer communication of chip.Chip controls circuit 20 service voltages are 5-12V.Nutrient solution/liquid medicine storage pond 5 and waste liquid pool 12 can be based on the integrated moldings on flexible polymer main structure layer 1 of mould shown in Fig. 3, also can first process respectively after the single liquid storage tank shown in Fig. 6 key and to not comprising on the flexible polymer main structure layer of nutrient solution/liquid medicine storage pond and waste liquid pool again.
Before culturing cell, first whole cell cultivation chip is placed to 24h dry sterilization in 60 ℃ of thermostatic drying chambers, then with any one solution in poly-Dopamine HCL (PDA), poly-lysine (PLL) or mouse tail collagen protein I type, surface treatment is carried out in cell culture insert 6 bottom surfaces.
While preparing integrated micro-flow control cell cultivation chip provided by the invention, first by CNC numerically-controlled machine precision sizing, make the flexible polymer main structure layer shown in Fig. 3～Fig. 5 molded with mould, comprise the punch consistent with main structure layer inner flow passage structure as shown in Figure 3, and the die consistent with main structure layer external form as shown in Figure 4; Assembling punch and die obtain the full die shown in Fig. 5, flexible polymer is consolidated to thing in advance and inject punch and the definite molding space of die, and curing flexible polymer is consolidated thing in advance, sloughs die and punch, obtains flexible polymer main structure layer 1; On silicon chip spin coating flexible polymer in advance admittedly thing, obtain counterdie 2, bonding counterdie 2 and flexible polymer main structure layer 1 after solidifying; Micropump 9 is installed on to Micropump installing zone 8, then temperature-controlling module is installed on to temperature control assembly installation draw-in groove 25; Bonding support base 3 and counterdie 2; Temperature sensing element is connected to the corresponding position that wire 21 and ito glass electric power conductor 15 are welded to chip controls circuit 20; Assemble micro-valve, complete whole cell cultivation chip simultaneously and integrate; Micropump Flexible Control plug 10 and Micropump adapter 18 are pegged graft; Finally surface treatment is carried out in cell culture insert 6 bottom surfaces.
While starting culturing cell, first remove the gas in the inner chamber of flexible polymer main structure layer 1 and counterdie 2 keys and formation, then with syringe, nutrient solution/liquid, cell suspension are injected respectively to nutrient solution/liquid medicine storage pond 5 and cell culture insert 6 successively; The golden finger 22 outer computer interfaces of insertion chip or the special-purpose power supply interface of control circuit board 4 one sides, be 20 power supplies of chip controls circuit, cell culture insert 6 internal temperatures are stable to preset temp, normally closed because of micro-valve, in inner chamber, there is no liquid-flow.Cell starts adherent growth.Need to open Micropump 9 and Micropump 9 to the micro-valve between waste liquid pool 12 when more changing to new nutrient solution/liquid in cell culture insert 6, in cell culture insert 6, original waste liquid is discharged in waste liquid pool 12, and cell culture insert roof subsides to bottom simultaneously.When waste liquid output and cell culture insert roof subside while reaching predetermined extent, Micropump 9 and Micropump 9 are closed to the micro-valve between waste liquid pool 12, open to the micro-valve between cell culture insert 6 in nutrient solution/liquid medicine storage pond 5, and the nutrient solution/liquid in nutrient solution/liquid medicine storage pond 5 enters cell culture insert 6 along with the bounce-back of cultivation pool roof and nutrient solution/liquid medicine storage pond 5 subsiding of pool wall.New nutrient solution/liquid is full of after cell culture insert 6, nutrient solution/liquid medicine storage pond 5 is closed to the micro-valve between cell culture insert 6, start the cell static cultivation of a new round, until during nutrient solution/liquid that next round more renews, repeat the micro-valve work schedule of above-mentioned Micropump.Nutrient solution/liquid medicine storage pond 5 and corresponding fluid channel branch road, micro-valve quantity are at least 1, when quantity is greater than 1, the replacing of each road nutrient solution/liquid is generally carried out respectively, and each the micro-valve between cell culture insert 6 and each nutrient solution/liquid medicine storage pond can not opened simultaneously.
Claims (14)
1. integrated micro-flow control cell cultivation chip and preparation method thereof, described micro-current controlled cell is cultivated chip, comprise the flexible polymer main structure layer, counterdie and the support base that are bonded together successively, and temperature-controlling module, Micropump, micro-valve and control circuit board etc.; Described flexible polymer main structure layer comprises at least 1 nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool, microfluidic channel, Micropump installing zone etc.; In described Micropump installing zone, described Micropump is installed; In described support base, there is temperature control assembly that draw-in groove is installed; Described temperature control assembly is installed in draw-in groove described temperature-controlling module is installed; On described control circuit board, be furnished with described micro-valve and chip controls circuit; The assembling of described micro-valve has realized the integration of chip simultaneously.
2. by integrated micro-flow control cell cultivation chip claimed in claim 1 and preparation method thereof, it is characterized in that, its procedure of processing comprises:
(1) by CNC numerically-controlled machine precision sizing, make the mould of the molded use of described flexible polymer main structure layer, comprise the punch consistent with described main structure layer inner flow passage structure, and the die consistent with described main structure layer external form;
(2) assemble described punch and die, flexible polymer is consolidated to thing in advance and inject described punch and the definite molding space of die, curing flexible polymer is consolidated thing in advance, sloughs described die and punch, obtains described flexible polymer main structure layer;
(3) on silicon chip spin coating flexible polymer in advance admittedly thing, obtain described counterdie after solidifying, counterdie and described flexible polymer main structure layer described in bonding;
(4) described Micropump is installed in described Micropump installing zone, described temperature-controlling module is installed and in described temperature control assembly, draw-in groove is installed;
(5) support base and counterdie described in bonding;
(6) weld or connect described chip controls circuit associated tracks, assembling described micro-valve;
(7) surface treatment is carried out in described cell culture insert bottom surface.
3. by integrated micro-flow control cell cultivation chip described in claim 1 or 2 and preparation method thereof, it is characterized in that, described cell culture insert roof thickness and the thick Design and Machining according to a certain percentage of described nutrient solution/liquid medicine storage pool wall, nutrient solution/liquid enters the elastic deformation of described cell culture insert based on cell culture insert roof and replys.
4. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described counterdie is one deck flexible polymer film; Described support base can be polymethylmethacrylate (PMMA) plate or glass-fiber-plate; Bolt mounting holes and the wire with identical shaped and size passes hole in corresponding position for described flexible polymer main structure layer, counterdie and support base.
5. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described temperature-controlling module comprises Heating element ITO conductive glass and glues temperature-sensing element RTD Pt100 or Pt1000 thereon, the pin of described RTD is drawn temperature sensing element and is connected wire, described ITO conductive glass both sides are coated with gold electrode, and described gold electrode is drawn ito glass electric power conductor.
6. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described micro-valve is the normally closed micro-valve of electromagnetism, mainly comprise micro-valve open and close controlling microchannel, electromagnetic drive mechanism and micro-valve regulation nut, described micro-valve open and close controlling microchannel is at one section that need to control between any 2 liquid storage tanks of its internal flow break-make in aforementioned microfluidic channel, described electromagnetic drive mechanism is positioned under described micro-valve open and close controlling microchannel, be fixed on described control circuit board, a pair of described micro-valve regulation nut is symmetrical in described electromagnetic drive mechanism and is fixed on described control circuit board.
7. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described nutrient solution/liquid medicine storage pond volume is greater than required replacing nutrient solution/liquid number of times with the ratio of described cell culture insert volume, pool wall thickness is 0mm-0.3mm, but is not 0; Described waste liquid pool volume is greater than the summation of described cell culture insert, nutrient solution/liquid medicine storage pond volume, and pool wall thickness is 0mm-0.3mm, but is not 0.
8. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described cell culture insert bottom surface can be for plane or with cell capture microstructure, described cultivation pool near described Micropump inlet side with latticed or strip fence.
9. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that the wide 0.5-1.2mm of described microfluidic channel, maximum height 0.1-0.2mm; The bed thickness that flexible polymer main structure layer only comprises microfluidic channel part is 0.2-0.5mm; The bed thickness that counterdie only comprises microfluidic channel part is 0-0.3mm, but is not 0.
10. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described microfluidic channel can be identical to the path shape between described cell culture insert in different described nutrient solution/liquid medicine storage ponds, or select suitable path to make the flow resistance of some storage pool respective branch be greater than other storage pool respective branch flow resistances.
11. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described flexible polymer main structure layer is not communicated with the external world with the inner chamber that described counterdie bonding forms.
12. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described control circuit board locates to have cell culture insert view port under being positioned at described cell culture insert; Control circuit board one side has golden finger, and the outer computer interface of the chip of can pegging graft or special-purpose power supply interface are described chip controls circuit supply, and with the outer computer communication of chip; Chip controls circuit supply voltage is 5-12V.
13. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, described nutrient solution/liquid medicine storage pond and waste liquid pool can be on described flexible polymer main structure layer integrated molding, also can first process respectively after single liquid storage tank again key and to not comprising on the flexible polymer main structure layer of described nutrient solution/liquid medicine storage pond and waste liquid pool.
14. by arbitrary described integrated micro-flow control cell cultivation chip and preparation method thereof in claim 1-3, it is characterized in that, surface treatment is carried out in described cell culture insert bottom surface, can be coated with any one solution in poly-Dopamine HCL (PDA), poly-lysine (PLL) or mouse tail collagen protein I type.
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