CN104178423A - Micro-fluidic- technique-based living cell culture device - Google Patents
Micro-fluidic- technique-based living cell culture device Download PDFInfo
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- CN104178423A CN104178423A CN201410425640.7A CN201410425640A CN104178423A CN 104178423 A CN104178423 A CN 104178423A CN 201410425640 A CN201410425640 A CN 201410425640A CN 104178423 A CN104178423 A CN 104178423A
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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
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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
- C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
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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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
- C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/22—Testing for sterility conditions
Abstract
The invention discloses a micro-fluidic-technique-based living cell culture device which comprises a micro-fluidic culture chip set, a rotary joint, a rotary motor, a micromanipulation module and a fluidic pump, wherein the rotary joint is in butt joint with the bottom the micro-fluidic culture chip set, the rotary motor is arranged below the rotary joint, a rotary shaft is arranged on the rotary motor, the rotary shaft penetrates through and extends out of the rotary joint, the rotary motor is fixedly connected with the micro-fluidic culture chip set through the rotary shaft, the micromanipulation module is connected with the micro-fluidic culture chip set, and the fluidic pump is communicated with the micro-fluidic culture chip set by virtue of the rotary joint. The device disclosed by the invention can be used for simultaneously carrying out in vitro sequential culture of tens of cells/embryos and micro-intervening operation on the surrounding environment of a target and the living body.
Description
Technical field
The present invention relates to a kind of active somatic cell culture apparatus based on microflow control technique.
Background technology
Cell is minimum vital movement unit, difficult to the viviperception of cell, but meaning is huge.Can obtain the many multidate informations in life generation and growth course to embryo's vivo observation, be significant equally.Had to 2012 and exceed 4,000,000 tube babies in birth all over the world, and increasing with the speed that exceedes every year 100000; The whole world has in vitro fertilization (IVF) cycle that exceedes 1,000,000 examples to carry out every year; In the U.S., in ooecium slurry, the cycle life of microinjection (ICSI) accounts for 57.5% of full IVF cycle life.Latest survey shows, China's Infertile couples has also accounted for 15% of couple at child-bearing age's sum, at present, the medical expense in a conventional IVF cycle is about the tens thousand of left and right of Renminbi, if introducing ICSI treats expense also can increase, wherein main cost all concentrates on reagent, the consumptive material consumption of (general 3-5 days) in culturing process.And at present, this field mainly adopts traditional manual manipulation method, need at different times, target be proceeded in multiple different culture dish, technological operation very complicated, require high to operator's state of the art, it is longer that repeatedly jump operation causes embryo's residence time in external environment, the disadvantageous effect such as easily pollutes.Based on above-mentioned present situation and drawback, develop a integrated, automatization, the multichannel manipulation in vitro for cell and body early embryo and sequential culture, micrurgy experimental system, there is society, economy, scientific research and ethical value widely.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to provide a kind of active somatic cell culture apparatus based on microflow control technique, this device can be realized dozens of cell/embryo's the external order of passing through simultaneously and cultivate, micro-intervention and Real-Time Monitoring operation, and can realize in the situation of not changing culture dish and change nutrient solution.
Summary of the invention: for solving the problems of the technologies described above, the technology used in the present invention means are:
A kind of active somatic cell culture apparatus based on microflow control technique, comprise micro-fluidic cultivation chipset, swivel joint, rotating machine, micrurgy module and Flow Control pump, described swivel joint butts up against described micro-fluidic cultivation chipset below, described rotating machine is positioned at described swivel joint below, described rotating machine is provided with rotation axis, described rotation axis passes and stretches out described swivel joint, described rotating machine is fixedly connected with described micro-fluidic cultivation chipset by described rotation axis, described micrurgy module is connected with described micro-fluidic cultivation chipset, described Flow Control pump is connected with described micro-fluidic cultivation chipset by described swivel joint,
Wherein, described micro-fluidic cultivation chipset comprises top cover, culture layer group, Flow Control layer I, Flow Control layer II and sealing bottom successively, described top cover is provided with multiple passage mountings, the edge of described top cover is provided with draw-in groove I, described culture layer Zu center is circular area of knockout, the edge of described circular area of knockout is provided with the main culture channel that multiple and circular area of knockout is communicated with, the described main culture channel the other end with catch runner and be connected, described in to catch runner be runner mouth away from the port of center of circle end; Described Flow Control layer I lower surface center is provided with cylindrical protrusions, cylindrical protrusions is hollow structure, the internal surface of cylindrical protrusions is provided with internal thread, described Flow Control layer I lower surface is provided with annular channel I around cylindrical protrusions excircle, described annular channel I is communicated with multiple Flow Control runner I, the end of described Flow Control runner I is runner mouth I, and described runner mouth I is communicated with the main culture channel of culture layer group, and the edge of described Flow Control layer I is provided with multiple circles and gets through hole; Described Flow Control layer II center is provided with descending runner I, described Flow Control layer II lower surface is provided with annular channel II around descending runner I excircle, described annular channel II is communicated with many Flow Control runner II, the end of described Flow Control runner II is runner mouth II, described runner mouth II and the circle of Flow Control layer I are got through the runner mouth corresponding connection mutually of hole and culture layer group, and described cylindrical protrusions stretches in descending runner I; Described sealing bottom edge is provided with draw-in groove II, the draw-in groove I of described draw-in groove II and top cover corresponding connection mutually, and described sealing bottom center is provided with descending runner II, and described descending runner I is inserted in descending runner II;
Wherein, described swivel joint is provided with three layers of concentric tube wall from outside to inside successively, be respectively tube wall I, tube wall II and tube wall III, described rotating machine is provided with is with externally threaded rotation axis, described rotation axis is through swivel joint and stretch out swivel joint, the cylindrical protrusions of described micro-fluidic cultivation chip cooperatively interacts and is rotatably connected with the rotation axis projection of described rotating machine, the outer wall of described tube wall I and descending runner II connects, the outer wall of described tube wall II and descending runner I connects, the outer wall of described tube wall III and cylindrical protrusions connects, described tube wall I, between tube wall II and tube wall III, form successively from outside to inside two annular channels, the Flow Control runner II of described micro-fluidic cultivation chipset is communicated with the annular channel between described swivel joint tube wall I and tube wall II, the Flow Control runner I of described micro-fluidic cultivation chipset is communicated with the annular channel between described swivel joint tube wall II and tube wall III, described swivel joint is also provided with the outflow keyholed back plate road of both ends open, one of them outflow keyholed back plate road is communicated with the annular channel between tube wall I and tube wall II, another outflow keyholed back plate road is communicated with the annular channel between tube wall II and tube wall III, described outflow keyholed back plate road is connected with described Flow Control pump,
Wherein, described micrurgy module comprises stepper-motor I, stepper-motor II, guide rail I, guide rail II, steady arm, needle holder, microneedle and micro-injection pump, described stepper-motor I is positioned on described guide rail I, described steady arm drives motion vertically by stepper-motor I, described guide rail II is fixedly connected with described stepper-motor I, described stepper-motor II is positioned on described guide rail II, described needle holder drives motion vertically by stepper-motor II, one end of described needle holder is connected with micro-injection pump by flexible pipe, its relative the other end is provided with pin permanent seat, described microneedle is sealedly connected on pin permanent seat, described steady arm is positioned at the syringe needle place of described microneedle, the syringe needle of described microneedle can move forward and backward in steady arm, the front end of described steady arm is provided with projection II.
Further preferably, described top cover also comprises micrurgy lid, and the edge of described micrurgy lid is provided with draw-in groove, and described micrurgy Gai center is circular area of knockout, and the outer edge of circular area of knockout is a circle long strip shape area of knockout
Further preferably, described passage mounting is circular arc.
Further preferably, described micro-fluidic cultivation chipset adopts PMMA or the casting of PDMS material or etching to be prepared from.
Further preferably, described tube wall I, tube wall II and tube wall III are stainless material and are prepared from.
Further preferably, in described tube wall I, tube wall II and tube wall III, be equipped with application of anti-leakage layers, described application of anti-leakage layers annular is affixed on tube wall inner side.
Further preferably, described application of anti-leakage layers adopts graphite carbon, silicon carbide, wolfram varbide or stupalith to be prepared from.
Beneficial effect: than prior art, active somatic cell culture apparatus based on microflow control technique of the present invention can carry out dozens of cell/embryo simultaneously externally passes through that order is cultivated and to target surrounding environment and intravital micro-intervention operation, as the interior microinjection of cell (ooecium slurry), embryonic cell biopsy, foreign gene cell transfecting, cell paracrine signal conduction studies, embryonic cell differentiation mechanism research etc., and can realize in the situation of not changing culture dish and change nutrient solution, apparatus of the present invention are rational in infrastructure, practical, overcome at different times and target need to have been proceeded to the loaded down with trivial details technological operation in multiple different culture dish, thereby and then avoid occurring because jump operation repeatedly causes embryo's long situation that embryo is polluted of the residence time in external environment.
Brief description of the drawings
Fig. 1 is the structural representation of micro-fluidic cultivation chipset entirety in apparatus of the present invention;
Fig. 2 is the structural representation of swivel joint in apparatus of the present invention;
The structural representation that in Fig. 3 apparatus of the present invention, rotating machine and swivel joint cooperatively interact;
Fig. 4 is 20 micro-fluidic culture channel schematic diagram that form after the micro-fluidic cultivation chipset assembling of apparatus of the present invention bonding;
Fig. 5 is the structural representation of each micro-fluidic culture channel;
Fig. 6 is the structural representation that difference is cultivated top cover;
Fig. 7 is the structural representation of micrurgy top cover;
Fig. 8 is the structural representation of culture layer I;
Fig. 9 is the structural representation of culture layer II;
Figure 10 is the structural representation of Flow Control layer I;
Figure 11 is the structural representation of Flow Control layer II;
Figure 12 is the structural representation of end sealing cover;
Figure 13 is the structural representation I of micrurgy module;
Figure 14 is the structural representation II of micrurgy module;
Figure 15 is the structural representation of apparatus of the present invention;
Figure 16 is the structural representation of micro-fluidic cultivation chipset split in apparatus of the present invention.
Embodiment
As shown in Fig. 1～16, active somatic cell culture apparatus based on microflow control technique of the present invention, comprise micro-fluidic cultivation chipset 1, swivel joint 2, rotating machine 3 and micrurgy module 4, rotating machine 3 is provided with is with externally threaded rotation axis 5, swivel joint is provided with through hole in the middle of 2 bottoms, rotation axis 5 is through this through hole and stretch out swivel joint 2, swivel joint 2 bottom rest parts are sealed structure, the top rotary moveable of swivel joint 2 butts up against micro-fluidic cultivation chipset 1 bottom, rotating machine 3 is positioned at swivel joint 2 belows, rotating machine 3 is fixedly connected with micro-fluidic cultivation chipset 1 by rotation axis 5, micrurgy module 4 is connected with micro-fluidic cultivation chipset 1, apparatus of the present invention also comprise Flow Control pump 6, Flow Control pump 6 is connected with micro-fluidic cultivation chipset 1 by swivel joint 2,
As shown in figure 16, wherein, micro-fluidic cultivation chipset 1 is transparent by PMMA or PDMS etc., have certain flexible material to form by casting or lithography, and micro-fluidic cultivation chipset 1 comprises top cover 7, culture layer I8, culture layer II9, Flow Control layer I10, Flow Control layer II11 and sealing bottom 12 successively; Top cover 7 divides again difference to cultivate cover 601 and 602 two kinds, micrurgy lid, and difference is cultivated and covered 601 cultivations of the differences for different runners, and micrurgy lid 602 is for micrurgy;
As shown in Figure 6, difference is cultivated and is covered the 601 circular arc passage mountings 701 that are provided with circle 20 deciles, the center of circle of circular arc passage mounting 701 and difference are cultivated and are covered 601 the center of circle and overlap, circular arc passage mounting 701 is corresponding with 20 main culture channel of culture layer group, difference cultivation is covered to 601 and combine rear salable main culture channel with culture layer group, difference is cultivated and is covered 601 edge and be provided with fixed card slot I702, fixed card slot I702 and fixed card slot II1201 cooperatively interact, and micro-fluidic cultivation chipset 1 is combined;
As shown in Figure 7, micrurgy lid 602 lid center have one with the circular area of knockout 801 of lid concentric, the outer edge of circular area of knockout 801 is the long strip shape area of knockout of circle 20 deciles, long strip shape area of knockout and the corresponding coincidence mutually of main culture channel, for the space of micrurgy needle movement is provided, micrurgy lid 602 is horizontally rotated to 9 °, can again seal main culture channel; The edge of micrurgy lid 602 is also provided with fixed card slot 802, and fixed card slot 802 cooperatively interacts with fixed card slot II1201, and micro-fluidic cultivation chipset 1 is combined;
Culture layer group is made up of culture layer I8 and culture layer II9, and one deck tips upside down on another layer, encapsulates, and forms 20 Flow Controls and cultivates unit;
Culture layer Zu center is for being communicated with pond 902, being communicated with pond 902 is a circular area of knockout, top can be cultivated and be covered 601 sealings by difference, be communicated with the edge in pond 902 and be provided with multiple main culture channel 901 with being communicated with pond 902 and being communicated with, main culture channel 901 is divided in two parts, larger near the part that is communicated with pond 902, less away from the part that is communicated with pond 902, away from the port that is communicated with pond 902 with catch runner 903 and be connected, catch the size of runner 903 and can process adjusting according to target cell/embryo's size, catching runner 903 is runner mouth 904 away from center of circle end, runner mouth 904 is that a circle is got through hole, for being communicated with lower one deck chip,
As shown in Figure 5, main culture channel 901 is runner mouth 101 with the junction that is communicated with pond 902, and runner mouth 101 is for target cell/embryo is put into, or shifts out chip (docking with microinjection mouth); Main culture channel 901 is communicated with the runner mouth I1001 of Flow Control layer I, and runner mouth 1001 is for removing ovum or embryo granulosa cell around; Main culture channel 901 is runner mouth 103 with the junction of catching runner 903, and runner mouth 103, for absorption, is caught and fixing Object of Development, to it is carried out to micrurgy;
As shown in Figure 8, culture layer I8 center is circular area of knockout I, the edge of circular area of knockout I is provided with the long strip shape area of knockout I that multiple and circular area of knockout I is communicated with, long strip shape area of knockout I is divided in two parts, part near circular area of knockout I is larger, part away from circular area of knockout I is less, and the edge of culture layer I8 is provided with multiple circles and gets through hole 905;
As shown in Figure 9, culture layer II9 center is circular area of knockout II, the edge of circular area of knockout II connects long strip shape area of knockout II multiple and that circular area of knockout II is communicated with, long strip shape area of knockout II is divided in two parts, part near circular area of knockout II is larger, part away from circular area of knockout II is less, long strip shape area of knockout II away from circular area of knockout II end with catch runner 903 and be connected, catch runner 903 and be provided with circle away from center of circle end and get through hole 904 (catching the runner mouth 904 of runner); The circle of culture layer I8 is got through hole 905 and is got through the mutual corresponding connection in hole 904 with the circle of culture layer II9;
As shown in figure 10, Flow Control layer I10 chip monolithic molding, when encapsulation, be positioned at the below of culture layer group, Flow Control layer I10 is provided with circle and gets through hole 1010 (runner mouth), this circle is got through hole 1010 and is got through hole 904 with the circle of culture layer group, 905 are connected, the circle of Flow Control layer I10 is got through hole 1010 and is also got through hole 1110 (runner mouth II) with the circle of Flow Control layer II11 and be connected, Flow Control layer I10 center is provided with the cylindrical protrusions 1004 of stretching out downwards, cylindrical protrusions 1004 is hollow structure, the internal surface of cylindrical protrusions 1004 is provided with internal thread, be fixedly connected with for cooperatively interacting to rotate with the rotation axis 5 of below rotating machine 3, Flow Control layer I10 lower surface is provided with annular channel I1003 around cylindrical protrusions 1004 excircles, Flow Control runner I1002 is positioned at Flow Control layer I lower surface, size can be as required---and the size that is mainly target cell/embryo is processed adjusting, Flow Control runner I1002 one end is connected with annular channel I1003, the other end is runner mouth I1001, runner mouth I1001 is positioned at the main culture channel of culture layer group 901 belows, and is communicated with main culture channel 901,
As shown in figure 11, Flow Control layer II11 chip monolithic molding, when encapsulation, be positioned at Flow Control layer I10 below, Flow Control layer II11 center is provided with the descending runner I1101 stretching out downwards, descending runner I1101 adopts hollow structure, the cylindrical protrusions 1004 of Flow Control layer I10 can be inserted in it, and the runner forming between cylindrical protrusions 1004 and descending runner I1101 is connected with the annular channel between tube wall II1302 and the tube wall II1303 of swivel joint 2; Flow Control layer II11 lower surface is provided with annular channel II1102 around descending runner I1101 excircle, annular channel II1102 is communicated with many Flow Control runner II1103, the end away from center of circle end of Flow Control runner II1103 is runner mouth II1110, and Flow Control runner II1103 is communicated with runner mouth II1110 and annular channel II1102;
As shown in figure 12, sealing bottom 12 diagonal angles, edge are provided with two draw-in groove II1201, the draw-in groove of draw-in groove II1201 and top cover 7 (difference is cultivated and cover 601 draw-in groove I702 or the draw-in groove 802 of micrurgy lid 602) corresponding connection mutually, the draw-in groove of draw-in groove II1201 and top cover 7 mutually combine rotate stuck for whole micro-fluidic cultivation chipset 1 is tightly fixed together; Sealing bottom 12 centers are provided with descending runner II1202, descending runner II1202 adopts hollow structure, descending runner I1101 is inserted in descending runner II1202, and the runner forming between descending runner I1101 and descending runner II1202 is connected with the annular channel between tube wall I1301 and the tube wall II1302 of swivel joint 2;
As shown in Fig. 2～3, swivel joint 2 location butt up against micro-fluidic cultivation chipset 1 bottom, micro-fluidic cultivation chipset 1 rotary movable on swivel joint 2, and do not link with swivel joint 2, in the time that swivel joint 2 is fixed, micro-fluidic cultivation chipset 1 still can be thereon by external force driven rotary, and swivel joint 2 is connected with Flow Control pump 6 by outflow keyholed back plate road 1305 and outflow keyholed back plate road 1306; Rotating machine 3 is positioned swivel joint 2 bottoms, and rotating machine 3 is fixedly connected with micro-fluidic cultivation chipset 1 by rotation axis, thereby micro-fluidic cultivation chipset 1 rotates together with rotating machine 3;
Swivel joint 2 is provided with three layers of concentric tube wall from outside to inside successively, be respectively tube wall I1301, tube wall II1302 and tube wall III1303, tube wall I1301, be positioned at swivel joint 2 outermost, top is expanded region and is connected with the outer wall of sealing bottom 12 descending runner II1202, in tube wall I1301, is provided with application of anti-leakage layers, and application of anti-leakage layers ring attaching is loaded on tube wall I1301 inner side, bottom is slightly narrow, and sidewall opening is connected with outflow keyholed back plate road 1306, tube wall II1302, is positioned at slightly inner side of joint, and top is expanded the descending runner I1101 outer wall of region and Flow Control layer II11 and connected, and in tube wall II1302, has application of anti-leakage layers, and application of anti-leakage layers ring attaching is loaded on inside tube wall, tube wall II1302 bottom is slightly narrow, and sidewall opening is connected with outflow keyholed back plate road 1305, in tube wall III1303, have application of anti-leakage layers, application of anti-leakage layers ring attaching is loaded on tube wall III1303 inner side and connects with cylindrical protrusions 1004 outer walls of Flow Control layer I10, rotating machine 3 is provided with is with externally threaded rotation axis 5, rotation axis 5 is through swivel joint 2 and stretch out swivel joint 2, being used for cooperatively interacts to rotate with the cylindrical protrusions 1004 of micro-fluidic cultivation chipset 1 is fixedly connected with, thereby together with rotating machine 3 secure bond, next plays rotation in the drive of rotating machine 3 to make micro-fluidic cultivation chipset 1 by micro-fluidic cultivation chipset 1, the outer wall of tube wall I1301 and descending runner II1202 connects, the outer wall of tube wall II1302 and descending runner I1101 connects, the outer wall of tube wall III1303 and cylindrical protrusions 1004 connects, tube wall I1301, between tube wall II1302 and tube wall III1303, form successively from outside to inside two annular channels, the Flow Control runner II1103 of micro-fluidic cultivation chipset 1 is communicated with the annular channel between tube wall I1301 and tube wall II1302, the Flow Control runner I1002 of micro-fluidic cultivation chipset 1 is communicated with the annular channel between tube wall II1302 and tube wall III1303, swivel joint 2 is also provided with the outflow keyholed back plate road of both ends open, one of them outflow keyholed back plate road 1306 is communicated with the annular channel between tube wall I1301 and tube wall II1302, another outflow keyholed back plate road 1305 is communicated with the annular channel between tube wall II1302 and tube wall III1303, outflow keyholed back plate road 1305 is all connected with Flow Control pump 6 with outflow keyholed back plate road 1306, swivel joint 2 tube walls adopt stainless material, and application of anti-leakage layers can adopt the materials such as graphite carbon, silicon carbide, wolfram varbide, pottery to carry out leakproof, swabbing,
As shown in Figure 13～14, micrurgy module 4 comprises stepper-motor I1501, stepper-motor II1504, guide rail I1502, guide rail II1503, steady arm 1508, needle holder 1505, microneedle 1507 and syringe pump 1509, stepper-motor I1501 is positioned on guide rail I1502, guide rail II1503 is fixedly connected with stepper-motor I1501, stepper-motor II1504 is positioned on guide rail II1503, micrurgy module 4 entirety are driven by stepper-motor I1501, upper along guide rail I1502 tangential movement at one axial (Z axis), stepper-motor II1504 drives needle holder 1505 to do tangential movement along guide rail II1503 together, one end of needle holder 1505 is connected with syringe pump 1509 by flexible pipe, the relative the other end of needle holder 1505 is provided with pin permanent seat 1506, microneedle 1507 is sealedly connected on pin permanent seat 1506, needle holder 1505 is interconnected with microneedle 1507, microneedle 1507 can disassemble from pin permanent seat 1506, change other entry needle, steady arm 1508 is positioned at the syringe needle place of microneedle 1507, the syringe needle of microneedle 1507 can move forward and backward in steady arm 1508, the front end of steady arm 1508 is provided with projection II, microneedle 1507 is combined by sleeve with steady arm 1508, cylinder inwall has lubricating substance, microneedle 1507 can seesaw along Z axis in sleeve, complete the puncture procedure of microinjection, steady arm 1508 can dock by front projection II (being the front end that the projection II of steady arm 1508 inserts main culture channel 901) with the main culture channel 901 of culture layer group in micro-fluidic cultivation chipset 1, complete location, drive steady arm 1508 to carry out axial motion by stepper-motor I1501 again, realize steady arm 1508 advance and retreat operations.
Apparatus of the present invention are carried out micro-manipulation by Flow Control pump 6 to the fluid in runner in culturing process, change liquid and goal displacement to realize in micro-fluidic cultivation chipset 1; In conjunction with rotating machine 3 and swivel joint 2, can realize micro-fluidic cultivation chipset 1 rotatablely move and rotary course in flow-control operation; Can realize the micro-intervention operation to target in conjunction with micrurgy module 4 again.
Obviously, above-described embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And these belong to apparent variation that spirit of the present invention extended out or variation still among protection scope of the present invention.
Claims (7)
1. the active somatic cell culture apparatus based on microflow control technique, is characterized in that: comprise micro-fluidic cultivation chipset, swivel joint, rotating machine, micrurgy module and Flow Control pump; Described swivel joint butts up against described micro-fluidic cultivation chipset below, described rotating machine is positioned at described swivel joint below, described rotating machine is provided with rotation axis, described rotation axis passes and stretches out described swivel joint, described rotating machine is fixedly connected with described micro-fluidic cultivation chipset by described rotation axis, described micrurgy module is connected with described micro-fluidic cultivation chipset, and described Flow Control pump is connected with described micro-fluidic cultivation chipset by described swivel joint;
Wherein, described micro-fluidic cultivation chipset comprises top cover, culture layer group, Flow Control layer I, Flow Control layer II and sealing bottom successively, described top cover is provided with multiple passage mountings, the edge of described top cover is provided with draw-in groove I, described culture layer Zu center is circular area of knockout, the edge of described circular area of knockout is provided with the main culture channel that multiple and circular area of knockout is communicated with, the described main culture channel the other end with catch runner and be connected, described in to catch runner be runner mouth away from the port of center of circle end; Described Flow Control layer I lower surface center is provided with cylindrical protrusions, cylindrical protrusions is hollow structure, the internal surface of cylindrical protrusions is provided with internal thread, described Flow Control layer I lower surface is provided with annular channel I around cylindrical protrusions excircle, described annular channel I is communicated with multiple Flow Control runner I, the end of described Flow Control runner I is runner mouth I, and described runner mouth I is communicated with the main culture channel of culture layer group, and the edge of described Flow Control layer I is provided with multiple circles and gets through hole; Described Flow Control layer II center is provided with descending runner I, described Flow Control layer II lower surface is provided with annular channel II around descending runner I excircle, described annular channel II is communicated with many Flow Control runner II, the end of described Flow Control runner II is runner mouth II, described runner mouth II and the circle of Flow Control layer I are got through the runner mouth corresponding connection mutually of hole and culture layer group, and described cylindrical protrusions stretches in descending runner I; Described sealing bottom edge is provided with draw-in groove II, the draw-in groove I of described draw-in groove II and top cover corresponding connection mutually, and described sealing bottom center is provided with descending runner II, and described descending runner I is inserted in descending runner II;
Wherein, described swivel joint is provided with three layers of concentric tube wall from outside to inside successively, be respectively tube wall I, tube wall II and tube wall III, described rotating machine is provided with is with externally threaded rotation axis, described rotation axis is through swivel joint and stretch out described swivel joint, the cylindrical protrusions of described micro-fluidic cultivation chip cooperatively interacts and is rotatably connected with the rotation axis projection of described rotating machine, the outer wall of described tube wall I and descending runner II connects, the outer wall of described tube wall II and descending runner I connects, the outer wall of described tube wall III and cylindrical protrusions connects, described tube wall I, between tube wall II and tube wall III, form successively from outside to inside two annular channels, the Flow Control runner II of described micro-fluidic cultivation chipset is communicated with the annular channel between described swivel joint tube wall I and tube wall II, the Flow Control runner I of described micro-fluidic cultivation chipset is communicated with the annular channel between described swivel joint tube wall II and tube wall III, described swivel joint is also provided with the outflow keyholed back plate road of both ends open, one of them outflow keyholed back plate road is communicated with the annular channel between tube wall I and tube wall II, another outflow keyholed back plate road is communicated with the annular channel between tube wall II and tube wall III, described outflow keyholed back plate road is all connected with described Flow Control pump,
Wherein, described micrurgy module comprises stepper-motor I, stepper-motor II, guide rail I, guide rail II, steady arm, needle holder, microneedle and micro-injection pump, described stepper-motor I is positioned on described guide rail I, described steady arm drives motion vertically by stepper-motor I, described guide rail II is fixedly connected with described stepper-motor I, described stepper-motor II is positioned on described guide rail II, described needle holder drives motion vertically by stepper-motor II, one end of described needle holder is connected with micro-injection pump by flexible pipe, its relative the other end is provided with pin permanent seat, described microneedle is sealedly connected on pin permanent seat, described steady arm is positioned at the syringe needle place of described microneedle, the syringe needle of described microneedle can move forward and backward in steady arm, the front end of described steady arm is provided with projection II.
2. the active somatic cell culture apparatus based on microflow control technique according to claim 1, it is characterized in that: described top cover also comprises micrurgy lid, the edge of described micrurgy lid is provided with draw-in groove, described micrurgy Gai center is circular area of knockout, and the outer edge of circular area of knockout is a circle long strip shape area of knockout.
3. the active somatic cell culture apparatus based on microflow control technique according to claim 1, is characterized in that: described passage mounting is circular arc.
4. the active somatic cell culture apparatus based on microflow control technique according to claim 1, is characterized in that: described micro-fluidic cultivation chipset adopts PMMA or the casting of PDMS material or etching to be prepared from.
5. the active somatic cell culture apparatus based on microflow control technique according to claim 1, is characterized in that: tube wall I, the tube wall II of described swivel joint and tube wall III are stainless material and are prepared from.
6. the active somatic cell culture apparatus based on microflow control technique according to claim 1, is characterized in that: in tube wall I, the tube wall II of described swivel joint and tube wall III, be equipped with application of anti-leakage layers, described application of anti-leakage layers annular is affixed on tube wall inner side.
7. the active somatic cell culture apparatus based on microflow control technique according to claim 6, is characterized in that: described application of anti-leakage layers adopts graphite carbon, silicon carbide, wolfram varbide or stupalith to be prepared from.
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