CN103667054B - A kind of integrated micro-flow control cell cultivation chip and preparation method thereof - Google Patents
A kind of integrated micro-flow control cell cultivation chip and preparation method thereof Download PDFInfo
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- CN103667054B CN103667054B CN201310426589.7A CN201310426589A CN103667054B CN 103667054 B CN103667054 B CN 103667054B CN 201310426589 A CN201310426589 A CN 201310426589A CN 103667054 B CN103667054 B CN 103667054B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/14—Pressurized fluid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
Abstract
The present invention relates to a kind of integrated micro-flow control cell cultivation chip and preparation method thereof.Described micro-current controlled cell cultivates chip, comprises the flexible polymer main structural, counterdie and the support base that are bonded together successively, and temperature-controlling module, Micropump, micro-valve and control circuit board etc.; Flexible polymer main structural comprises at least 1 nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool, microfluidic channel, Micropump installing zone etc.; In Micropump installing zone, Micropump is installed; Support base there is temperature control assembly draw-in groove is installed; Temperature control assembly is installed in draw-in groove and is provided with temperature-controlling module; Control circuit board is furnished with micro-valve and chip control circuit; The assembling of micro-valve achieves the integration of chip simultaneously.Chip preparing process mainly based on machining and flexible polymer molded.The invention provides the cell culture apparatus that a kind of integrated, technique is simple, fluid channel inner chamber is airtight, be specially adapted to the special dimensions such as space-orbit cytology research.
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 is the key factor that can this research be carried out smoothly with or without the research plant and instrument be suitable for.At present more existing can be used for device and the technology of cell cultures and morphologic observation under space environment, cell can be met 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 device is larger; The response message in cell a certain static moment can only be obtained, under space environment, cytological Effect research cannot be carried out real-time dynamicly.
Along with the progress of microflow control technique, it is that spatial cell research provides new platform that micro-current controlled cell cultivates chip.But current existing micro-current controlled cell is cultivated chip research achievement and still be there is the limitation that some are not suitable for space-orbit cytology research: cell cultures related liquid storage pool and micro fluid dynamcis control device as many in Micropump, micro-valve etc. independent of outside micro-fluidic chip body, cause whole chip system more discrete and chip system overall volume is larger; Grand micro-interface between chip body and outside support equipment may be introduced for cell cultures and pollute; The manipulation require human interventions such as cell cultures fluid exchange, are not suitable for this kind of a particular job environment lacking professional and participate in of manned space flight; Cultivating chip internal flow passage structure has direct gas to contact with the external world, or cell cultures district is open architecture, and the gas-liquid that thus the weightless factor in space causes coexists and bubble may be caused to enter cell culture insert and affect the normal growth of cell; Chip manufacture complex process, cost are higher, and chip section assembly not easily assembles replacing, is unfavorable for single 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 replacings, is suitable for the features such as single 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 structural, 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 structural 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; Described support base there is temperature control assembly draw-in groove is installed; Described temperature control assembly is installed in draw-in groove and is provided with described temperature-controlling module; Described control circuit board is furnished with described micro-valve and chip control circuit; The assembling of described micro-valve achieves the integration of chip simultaneously.
Integrated micro-flow control cell cultivation chip provided by the invention, its procedure of processing comprises:
(1) obtain the molded mould of described flexible polymer main structural by CNC numerically-controlled machine precision sizing, comprise the punch consistent with described main structural inner flow passage structure, and the die consistent with described main structural external form;
(2) assemble described punch and die, by flexible polymer in advance admittedly thing inject the molding space that described punch and die determine, solidification flexible polymer thing admittedly in advance, sloughs described die and punch, obtains described flexible polymer main structural;
(3) on silicon chip, spin coating flexible polymer obtains described counterdie, counterdie described in bonding and described flexible polymer main structural after consolidating thing, solidification in advance;
(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 described in bonding and counterdie;
(6) weld or connect described chip control circuit associated tracks, assembling described micro-valve;
(7) surface treatment is carried out to 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 described cell culture insert and replys based on the elastic deformation of cell culture insert roof.
Described counterdie is one deck flexible polymer film.
Described support base can be polymethylmethacrylate (PMMA) plate or glass-fiber-plate.
Described flexible polymer main structural, counterdie and support base in corresponding position with the bolt mounting holes of same shape and size and wire punched out.
Described Micropump is imported and exported after the Micropump inserting in described Micropump installing zone respectively imports and exports mounting groove and is needed import and export mounting groove to seal.
Described temperature-controlling module comprises Heating element ITO conductive glass and sticky 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 draws 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 one section in aforementioned microfluidic channel between any 2 liquid storage tanks needing to control its internal flow break-make, described electromagnetic drive mechanism is positioned at below under described micro-valve open and close controlling microchannel, be fixed on described control circuit board, micro-valve regulation nut described in a pair is symmetrical in described electromagnetic drive mechanism and is fixed on described control circuit board.
Change nutrient solution/liquid number of times needed for described nutrient solution/liquid medicine storage pond volume and the ratio of described cell culture insert volume are greater than, pool wall thickness is 0mm-0.3mm, but is not 0.
Described cell culture insert bottom surface can be 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 thickness that described flexible polymer main structural only comprises microfluidic channel part is 0.2-0.5mm.
The thickness that described counterdie only comprises microfluidic channel part is 0-0.3mm, but is not 0.
The path shape of described microfluidic channel described in each between nutrient solution/liquid medicine storage pond to described cell culture insert can be identical, 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.
The inner chamber that described flexible polymer main structural and counterdie bonding are formed is not communicated with the external world.
The described Micropump pump housing there is Flexible Control plug, with Micropump described in the Micropump adapter grafting rear drive in described chip control circuit.
Described control circuit board is positioned at place immediately below described cell culture insert and has cell culture insert view port.
There is golden finger described control circuit board side, and the outer computer interface of pluggable chip or special power supply interface are that described chip control circuit is powered, and computer communication outer with chip.
Described chip control circuit service voltage is 5-12V.
Described nutrient solution/liquid medicine storage pond and waste liquid pool can on described flexible polymer main structural integrated molding, key and on the flexible polymer main structural not comprising described nutrient solution/liquid medicine storage pond and waste liquid pool again after also first can processing single liquid storage tank respectively.
Surface treatment is carried out to described cell culture insert bottom surface, bag quilt can be carried out with any one solution in poly-Dopamine HCL (PDA), poly-lysine (PLL) or rat tail collagen protein I type.
Integrated micro-flow control cell cultivation chip working process provided by the invention is: when starting culturing cell, with syringe, nutrient solution/liquid, cell suspension are injected nutrient solution/liquid medicine storage pond and cell culture insert respectively successively after removing the gas in the inner chamber of flexible polymer main structural and counterdie key and formation, the golden finger of control circuit board side inserts the outer computer interface of chip or special power supply interface is that chip control circuit is powered, cell culture insert internal temperature is stable at preset temp, because micro-valve is normally closed, in inner chamber, there is no liquid-flow.Cell starts adherent and grows.Need when more changing to new nutrient solution/liquid in cell culture insert, the micro-valve between Micropump and Micropump to waste liquid pool is opened, 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 reach predetermined extent time, micro-valve between Micropump and Micropump to waste liquid pool cuts out, micro-valve between nutrient solution/liquid medicine storage pond to cell culture insert is opened, 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 nutrient solution/subsiding of liquid medicine storage pond pool wall.After new nutrient solution/liquid is full of cell culture insert, the micro-valve between nutrient solution/liquid medicine storage pond to cell culture insert cuts out, and starts the cell static cultivation of a new round, until next round more renew nutrient solution/liquid time, repeat the micro-valve work schedule of above-mentioned Micropump.
The distinguishing feature of the integrated micro-flow control cell cultivation chip that the present invention proposes and preparation method thereof is: all liquid storage tanks such as nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool and the micro fluid dynamcis such as Micropump, micro-valve control device are all integrated into micro-current controlled cell and cultivate in chip, chip overall volume is much smaller than the traditional microfluidic cell cultivation chip needing outside support equipment, and the grand micro-interface avoided between traditional microfluidic cell cultivation chip chips main body and outside support equipment pollutes; The work schedule that liquid in cell culture insert is changed by controlling the micro-valve of Micropump realizes, and chip fully automated works, without the need to human intervention; The inner chamber that flexible polymer main structural is formed with counterdie bonding is not communicated with the external world, avoids ambient atmos and enters the normal growth that cell culture insert affects cell; Based on the input mode that the elastic deformation of cell culture insert item wall is replied, the original waste liquid of cell culture insert inside is discharged more thorough, decrease the time of powering for micro fluid dynamcis parts simultaneously, and then reduce 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, reduce cost of manufacture; Aforesaid integrated, miniaturization, automatization, microfluidic channel inner chamber are airtight, the feature such as replacing, suitable single use easily assembled by chip section assembly, 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 provided by the invention assembling
Fig. 3 be flexible polymer main structural of the present invention molded time use die punch three-dimensional structure schematic diagram
The female die of die three-dimensional structure schematic diagram used when Fig. 4 flexible polymer of the present invention main structural is molded
Rear structural representation assembled by the die punch used when Fig. 5 flexible polymer of the present invention main structural is molded and die.
Fig. 6 is that the present invention adopts the liquid storage tank three-dimensional structure biopsy cavity marker devices schematic diagram processed first separately during non-integral moulding process when preparing flexible polymer main structural.
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 structural 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 structural 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, flexible polymer main structural 1, counterdie 2 and support base 3 in corresponding position with the bolt mounting holes 23 of same shape and size and wire punched out 24, be provided with Micropump 9 in Micropump installing zone 8, its import and export insert in the Micropump import and export mounting groove of Micropump installing zone 8 respectively and import and export mounting groove needs sealing, support base 3 there is temperature control assembly 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 sticky 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, control circuit board 4 is furnished with chip control circuit 20, temperature sensing element connection wire 21 and ito glass electric power conductor 15 are through the corresponding position being welded to chip control circuit 20 after wire punched out 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 one section in aforementioned microfluidic channel 7 between any 2 liquid storage tanks needing to control its internal flow break-make, electromagnetic drive mechanism 16 is positioned at immediately below micro-valve open and close controlling microchannel 7, be fixed on control circuit board 4, a pair micro-valve regulation nut 17 is symmetrical in electromagnetic drive mechanism 16 and is fixed on control circuit board 4, when 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.
Change nutrient solution/liquid number of times needed for the volume in nutrient solution/liquid medicine storage pond 5 and the ratio of the volume of cell culture insert 6 are greater than, pool wall thickness is 0mm-0.3mm, but is not 0.Cell culture insert 6 bottom surface can be plane or with cell capture microstructure, near described Micropump 9 inlet side with latticed or strip fence.The wall thickness Design and Machining according to a certain percentage in the roof thickness of cell culture insert 6 and nutrient solution/liquid medicine storage pond 5, making nutrient solution/liquid enter cell culture insert 6 is reply based on the elastic deformation of 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 volume, 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 thickness only comprising microfluidic channel 7 part in flexible polymer main structural 1 is 0.2-0.5mm.The thickness only comprising microfluidic channel 7 part in counterdie 2 is 0-0.3mm, but is not 0.The path shape of microfluidic channel 7 between different nutrient solution/liquid medicine storage pond 5 to cell culture insert 6 can be selected as depicted in figs. 1 and 2, make the flow resistance of liquid medicine storage pond respective branch be greater than the flow resistance of nutrient solution storage pool respective branch, also can the path shape of all branch roads identical.The inner chamber that flexible polymer main structural 1 is formed with counterdie 2 bonding is not communicated with the external world.Micropump 9 pump housing there is Flexible Control plug 10, with the Micropump adapter 18 grafting rear drive Micropump 9 in chip control circuit 20.RTD 13 can be Pt100 or Pt1000.Control circuit board 4 is positioned at place immediately below cell culture insert 6 and has cell culture insert view port 19.There is golden finger 22 control circuit board 4 side, and the outer computer interface of pluggable chip or special power supply interface are that chip control circuit 20 is powered, and computer communication outer with chip.Chip control circuit 20 service voltage is 5-12V.Nutrient solution/liquid medicine storage pond 5 and waste liquid pool 12 can based on the integrated moldings on flexible polymer main structural 1 of mould shown in Fig. 3, key and on the flexible polymer main structural not comprising nutrient solution/liquid medicine storage pond and waste liquid pool again after also first can processing the single liquid storage tank shown in Fig. 6 respectively.
Before culturing cell, first whole cell cultivation chip is placed 24h dry sterilization in 60 DEG C of thermostatic drying chambers, then with any one solution in poly-Dopamine HCL (PDA), poly-lysine (PLL) or rat tail collagen protein I type, surface treatment is carried out to cell culture insert 6 bottom surface.
When preparing integrated micro-flow control cell cultivation chip provided by the invention, first the molded mould of flexible polymer main structural shown in Fig. 3 ~ Fig. 5 is obtained by CNC numerically-controlled machine precision sizing, comprise the punch consistent with main structural inner flow passage structure as shown in Figure 3, and the die consistent with main structural external form as shown in Figure 4; Assembling punch and die obtain the full die shown in Fig. 5, by flexible polymer in advance admittedly thing inject the molding space that punch and die determine, solidification flexible polymer thing admittedly in advance, sloughs die and punch, obtains flexible polymer main structural 1; On silicon chip, spin coating flexible polymer obtains counterdie 2 after consolidating thing, solidification in advance, bonding counterdie 2 and flexible polymer main structural 1; Micropump 9 is installed on Micropump installing zone 8, then temperature-controlling module is installed on temperature control assembly installation draw-in groove 25; Bonding support base 3 and counterdie 2; Temperature sensing element connection wire 21 and ito glass electric power conductor 15 are welded to the corresponding position of chip control circuit 20; Assemble micro-valve, complete whole cell cultivation chip simultaneously and integrate; By Micropump Flexible Control plug 10 and Micropump adapter 18 grafting; Finally surface treatment is carried out to cell culture insert 6 bottom surface.
When starting culturing cell, first remove the gas in the inner chamber of flexible polymer main structural 1 and counterdie 2 key and formation, then with syringe, nutrient solution/liquid, cell suspension are injected nutrient solution/liquid medicine storage pond 5 and cell culture insert 6 respectively successively; It is that chip control circuit 20 is powered that the golden finger 22 of control circuit board 4 side is inserted computer interface or special power supply interface outside chip, cell culture insert 6 internal temperature is stable at preset temp, because micro-valve is normally closed, does not have liquid-flow in inner chamber.Cell starts adherent growth.When needing more to change to new nutrient solution/liquid in cell culture insert 6, open the micro-valve between Micropump 9 and Micropump 9 to waste liquid pool 12, 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 reach predetermined extent time, micro-valve that Micropump 9 and Micropump 9 arrive between waste liquid pool 12 cuts out, micro-valve between nutrient solution/liquid medicine storage pond 5 to cell culture insert 6 is opened, 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/subsiding of pool wall of liquid medicine storage pond 5.After new nutrient solution/liquid is full of cell culture insert 6, micro-valve between nutrient solution/liquid medicine storage pond 5 to cell culture insert 6 cuts out, start the cell static cultivation of a new round, until next round more renew nutrient solution/liquid time, 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 namely between cell culture insert 6 and each nutrient solution/liquid medicine storage pond can not be opened simultaneously.
Claims (13)
1. an integrated micro-flow control cell cultivation chip, is characterized in that: comprise the flexible polymer main structural, counterdie and the support base that are bonded together successively, and temperature-controlling module, Micropump, micro-valve and control circuit board; Described flexible polymer main structural comprises at least 1 nutrient solution/liquid medicine storage pond, cell culture insert, waste liquid pool, microfluidic channel, Micropump installing zone; In described Micropump installing zone, described Micropump is installed; Described support base there is temperature control assembly draw-in groove is installed; Described temperature control assembly is installed in draw-in groove and is provided with described temperature-controlling module; Described control circuit board is furnished with described micro-valve and chip control circuit; The assembling of described micro-valve achieves the integration of chip simultaneously;
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 described cell culture insert and replys based on the elastic deformation of cell culture insert roof.
2. the preparation method of integrated micro-flow control cell cultivation chip as claimed in claim 1, it is characterized in that, its procedure of processing comprises:
(1) obtain the molded mould of described flexible polymer main structural by CNC numerically-controlled machine precision sizing, comprise the punch consistent with described main structural inner flow passage structure, and the die consistent with described main structural external form;
(2) assemble described punch and die, by flexible polymer in advance admittedly thing inject the molding space that described punch and die determine, solidification flexible polymer thing admittedly in advance, sloughs described die and punch, obtains described flexible polymer main structural;
(3) on silicon chip, spin coating flexible polymer obtains described counterdie, counterdie described in bonding and described flexible polymer main structural after consolidating thing, solidification in advance;
(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 described in bonding and counterdie;
(6) weld or connect described chip control circuit associated tracks, assembling described micro-valve;
(7) surface treatment is carried out to described cell culture insert bottom surface.
3., by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, described counterdie is one deck flexible polymer film; Described support base is polymethylmethacrylate plate or glass-fiber-plate; Described flexible polymer main structural, counterdie and support base in corresponding position with the bolt mounting holes of same shape and size and wire punched out.
4. by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, described temperature-controlling module comprises Heating element ITO conductive glass and sticky 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 draws ito glass electric power conductor.
5. by integrated micro-flow control cell cultivation chip according to claim 1, 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 one section in aforementioned microfluidic channel between any 2 liquid storage tanks needing to control its internal flow break-make, described electromagnetic drive mechanism is positioned at immediately below described micro-valve open and close controlling microchannel, be fixed on described control circuit board, micro-valve regulation nut described in a pair is symmetrical in described electromagnetic drive mechanism and is fixed on described control circuit board.
6. by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, change nutrient solution/liquid number of times needed for described nutrient solution/liquid medicine storage pond volume and the ratio of described cell culture insert volume are greater than, 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.
7. by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, described cell culture insert bottom surface is plane or with cell capture microstructure, described cell culture insert near described Micropump inlet side with latticed or strip fence.
8., by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, the wide 0.5-1.2mm of described microfluidic channel, maximum height 0.1-0.2mm; The thickness that flexible polymer main structural only comprises microfluidic channel part is 0.2-0.5mm; The thickness that counterdie only comprises microfluidic channel part is 0-0.3mm, but is not 0.
9. by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, described microfluidic channel is identical to the path shape between described cell culture insert in different described nutrient solution/liquid medicine storage ponds, or selects suitable path to make the flow resistance of some liquid medicine storage pond respective branch be greater than other liquid medicine storage pond respective branch flow resistances.
10., by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, the inner chamber that described flexible polymer main structural is formed with described counterdie bonding is not communicated with the external world.
11. by integrated micro-flow control cell cultivation chip according to claim 1, and it is characterized in that, described control circuit board is positioned at place immediately below described cell culture insert and has cell culture insert view port; There is golden finger control circuit board side, and the outer computer interface of pluggable chip or special power supply interface are that described chip control circuit is powered, and computer communication outer with chip; Chip control circuit service voltage is 5-12V.
12. by integrated micro-flow control cell cultivation chip according to claim 1, it is characterized in that, described nutrient solution/liquid medicine storage pond and waste liquid pool integrated molding on described flexible polymer main structural, or key and on the flexible polymer main structural not comprising described nutrient solution/liquid medicine storage pond and waste liquid pool again after first processing single liquid liquid storage tank respectively.
13., by integrated micro-flow control cell cultivation chip according to claim 1, is characterized in that, carry out surface treatment to described cell culture insert bottom surface, carry out bag quilt with any one solution in poly-Dopamine HCL, poly-lysine or rat tail collagen protein I type.
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