CN104893953A - Adherent cell scratch making and migration observing methods based on micro-fluidic chip - Google Patents
Adherent cell scratch making and migration observing methods based on micro-fluidic chip Download PDFInfo
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- CN104893953A CN104893953A CN201510050047.3A CN201510050047A CN104893953A CN 104893953 A CN104893953 A CN 104893953A CN 201510050047 A CN201510050047 A CN 201510050047A CN 104893953 A CN104893953 A CN 104893953A
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Abstract
The invention provides adherent cell scratch making and migration observing methods based on a micro-fluidic chip. The micro-fluidic chip is internally provided with a microchannel of a Y-shaped structure. The microchannel comprises a main channel body and two secondary channel bodies communicated with the main channel body. The main channel body is connected to a cell inlet; the two secondary channel bodies are connected to two drop inlets. The adherent cell scratch making method comprises the steps that on-chip adherent cell culture is carried out in the microchannel of the micro-fluidic chip through a complete medium corresponding to an adherent cell; solutions of the cell inlet and the two drop inlets of the micro-fluidic chip are sucked thoroughly; the complete medium corresponding to the adherent cell and a trypsin solution are injected into the two drop inlets of the micro-fluidic chip at the same time respectively; standing is performed for a preset time period; the solutions are sucked thoroughly from the cell inlet and the two drop inlets in sequence, and therefore a scratch is formed on the bottom wall of the microchannel of the micro-fluidic chip. A method more similar to the physiological environment is provided for the in-vitro migration of the adherent cell, and the experiment cost is reduced at the same time.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of attached cell cut based on micro-fluidic chip and make and migration observation procedure, this attached cell vascular smooth muscle cell typically.
Background technology
In atherosclerosis and iatrogenic vascular injuries (angioplasty, stenter to implant, organ transplantation etc.) complication forming process, vascular smooth muscle cell (vascular smooth muscle cell is called for short VSMC) is bred and is one of core mechanism to inner membrance migration.The factor affecting VSMC migration is a lot, comprises various somatomedin, chemokine, fluid stress etc.For exploring the mechanism of multifactor mediation VSMC migration, research method reproducible, with a high credibility is very necessary.
Tradition research VSMC migration depends on the classical technology such as cell chemotaxis cell (Boyden Chamber) and cell scratch method.
For cell chemotaxis chamber technique, for boundary with 8 μm of aperture filter membranes, VSMC is seeded to upper strata cell, lower room adds different inductor, after different time points taking-up filter membrane cotton swab wipes the cell in film front, film methyl alcohol is fixed, brazilwood extract dyeing, counts the cell count in the visual field, the film back side under the microscope.The process of fenestra can be striden across at electric Microscopic observation VSMC simultaneously.
For cell cut technology, be on orifice plate, strike off the cell of certain limit with aseptic liquid transfer gun head or blade when VSMC growth forms confluent individual layer, observation of cell is to the locomotory movement striking off cellular regions.General with the maximum migration distance of a certain cell mass for parameter characterization VSMC transfer ability, count the cell count of migration simultaneously.
But realizing in process of the present invention, applicant finds that above-mentioned VSMS migrating technology exists following technological deficiency:
(1) existing VSMC moves environment, and in cell chemotaxis chamber technique, the aperture of chemotactic cell is about 1mm; In cell cut technology, test and operate on porous plate, minimum-value aperture is also grade.In these two kinds of technology, the environment residing for cell is all open, and relative to the microsize of vessel wall in body and the metabolisable form of nutritive substance, difference is fairly obvious, is objectively difficult to the biological property truly reflecting physiological status VSMC.Therefore the cell migration apparatus simulating intravital blood vessel wall microenvironment is better needed;
(2) above-mentioned cell chemotaxis chamber technique, can not the directly form of observation of cell and displacement, loses the details of migration mechanism, the difference simultaneously can not distinguish cell attachment between different the intervention, sprawling, move;
(3) above-mentioned cell cut technology, its cut speed and dimensional uniformity poor; Secondly, in scoring processes, cell is subject to mechanical injuries, and release cells content is in surrounding environment, because cell injury degree is uncontrollable, causes migration results to present diversity.
Summary of the invention
(1) technical problem that will solve
In view of above-mentioned technical problem, the invention provides a kind of attached cell cut based on micro-fluidic chip and make and migration observation procedure, better to simulate intravital blood vessel wall microenvironment, the migration situation of observation of cell more accurately.
(2) technical scheme
According to an aspect of the present invention, a kind of attached cell cut making method based on micro-fluidic chip is provided.There is in this micro-fluidic chip micro-raceway groove of " Y " type structure, comprising: a tap drain road; And be positioned at this tap drain road side and be communicated with this tap drain road two from raceway groove, wherein, side, tap drain road is connected to cell entry, two are connected to the first droplet inlet and the second droplet inlet respectively from raceway groove, and cell entry, the first droplet inlet and the second droplet inlet are all higher than micro-raceway groove position.This attached cell cut making method comprises: steps A: in micro-raceway groove of micro-fluidic chip, adopt the perfect medium that this attached cell is corresponding to carry out attached cell cultivation on sheet; Step B: the solution of the cell entry of micro-fluidic chip, the first droplet inlet and the second droplet inlet is all exhausted; Step C: to the first droplet inlet of micro-fluidic chip and the second droplet inlet simultaneously, inject perfect medium corresponding to this attached cell and trypsin solution respectively; Step D: leave standstill Preset Time; And step e: in order from cell entry, the second droplet inlet, the first droplet inlet exhaustion solution, thus form cut on the diapire of the micro-raceway groove of micro-fluidic chip.
According to another aspect of the present invention, a kind of attached cell migration observation procedure of attached cell cut making method is additionally provided.This attached cell migration observation procedure comprises: carry out finishing to micro-raceway groove of micro-fluidic chip, be suitable for adherent cell growth to make this micro-raceway groove; Attached cell inoculation on sheet is carried out in micro-raceway groove of micro-fluidic chip; In micro-raceway groove of micro-fluidic chip, carry out attached cell on sheet cultivate and synchronization process; Above-mentioned attached cell cut making method is adopted to make cut; At cell entry and two droplet inlet, one of them adds the DMEM containing the cell migration factor arbitrarily, then micro-fluidic chip is put back to incubator; And after the preset lime, carry out sheet dyes to the attached cell in micro-raceway groove of micro-fluidic chip, to characterize migrating cell and non-migrating cell.
(3) beneficial effect
As can be seen from technique scheme, the attached cell cut that the present invention is based on micro-fluidic chip makes and moves observation procedure and has following beneficial effect:
(1) microflow control technique is combined with traditional VSMC migrating technology, cultivation on substrate modification, VSMC sheet, the migration of VSMC cut and VSMC dyeing is realized in micron-sized raceway groove, for the external migration of VSMC provides the method closer to physiological environment, reduce experimental cost simultaneously;
(2) with report depend on cell chemotaxis cell method compared with, the present invention can pass through microscope cell migration directly perceived, and spike is unicellular, and quantizes to dye after transfer parameter, and the quantity of information of acquisition is more comprehensively;
(3) compared with the traditional cut method reported, realize cell cut based on action of gravity, specification operational standard, reduce personal errors, improve the repeatability of cut size and cell damage situation, consistence, result can be compared.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that micro-fluidic chip designed in the embodiment of the present invention forms cut;
Fig. 2 A and Fig. 2 B is respectively vertical view and the side-view of the micro-fluidic chip adopted according to embodiment of the present invention attached cell cut making method;
Fig. 3 is the schematic diagram of the making processes according to the micro-fluidic chip adopted in the embodiment of the present invention;
Fig. 4 is the schema according to embodiment of the present invention attached cell cut making method;
Fig. 5 is the schema according to embodiment of the present invention attached cell migration observation procedure;
Fig. 6 be cultured in micro-raceway groove be full of individual layer VSMC after the schematic diagram of micro-raceway groove inner cell state and true micro-image;
Fig. 7 is the schematic diagram of micro-raceway groove inner cell state after making cut and recording cut state and true micro-image;
Fig. 8 is the schematic diagram of micro-raceway groove inner cell state after staining procedure and true micro-image.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.It should be noted that, in accompanying drawing or specification sheets describe, similar or identical part all uses identical figure number.The implementation not illustrating in accompanying drawing or describe is form known to a person of ordinary skill in the art in art.In addition, although herein can providing package containing the demonstration of the parameter of particular value, should be appreciated that, parameter without the need to definitely equaling corresponding value, but can be similar to corresponding value in acceptable error margin or design constraint.The direction term mentioned in embodiment, such as " on ", D score, "front", "rear", "left", "right" etc., be only the direction with reference to accompanying drawing.Therefore, the direction term of use is used to illustrate and is not used for limiting the scope of the invention.
Microflow control technique combines with traditional attached cell migrating technology by the present invention, realize based on the substrate modification of action of gravity, attached cell sheet are cultivated in microflow control technique, dyeing, and make based on the cut of lamina flow principle, and microscopic examination is utilized to move.When so-called laminar flow refers to that in pipeline, flow velocity is lower, fluid is a kind of flow state of laminar flow.
Fig. 1 is the schematic diagram that micro-fluidic chip designed in the embodiment of the present invention forms cut.As shown in Figure 1, in a Y type raceway groove, if inject different solution at two entrances, as long as flow rate of liquid is enough low, two kinds of liquid in tap drain road would not mix, but laminar flow.Utilize this principle, pass into Trypsin respectively and containing the substratum of serum at two entrances, Trypsin flows through in tap drain road region can form cut.
In one exemplary embodiment of the present invention, provide a kind of attached cell cut making method based on micro-fluidic chip.First, the micro-fluidic chip that the present embodiment adopts is described in detail.
Fig. 2 A and Fig. 2 B is respectively vertical view and the front view of the micro-fluidic chip adopted according to embodiment of the present invention attached cell cut making method.Please refer to Fig. 2 A and Fig. 2 B, the micro-raceway groove in this micro-fluidic chip is y-type structure, comprises a tap drain road; And be positioned at this tap drain road side and be communicated with this tap drain road two from raceway groove.Wherein, the length in tap drain road is 3mm, and width is 800 μm.Two is 4mm from the length of raceway groove, and width is 400 μm.This tap drain road and be rectangle from the shape of cross section of raceway groove, is highly about 100 μm.Each raceway groove port respectively has an opening, and side, tap drain road is connected to cell entry, is connected to two droplet inlet-the first droplet inlet and the second droplet inlet from raceway groove side, and each inlet aperture is 5mm.Further, this cell entry, the first droplet inlet and the second droplet inlet all exceed tap drain road and two and are about 8mm from channel top.
Below the making processes of this micro-fluidic chip is simply introduced.As shown in Figure 3, the making processes of this micro-fluidic chip is as follows:
Step S202: photoresist material SU8-5 makes Seed Layer:
Slide glass cleans successively in acetone, ethanol and deionized water, and dry rear surface Rotating with Uniform and apply one deck SU 8-5, exposure forms Seed Layer, sees subgraph A in Fig. 3.
Step S204: photoresist material SU8-2100 makes formpiston:
In Seed Layer, Rotating with Uniform applies one deck SU 8-2100 again, puts mask exposure, sees subgraph B in Fig. 3; Development, post bake obtain the micro-raceway groove formpiston of cell migration, see subgraph C in Fig. 3.
The cast of step S206:PDMS, turn over mould, bonding pillar, punching and with substrate of glass bonding:
Cell migration micro-raceway groove formpiston cast polydimethylsiloxane (polydimethylsiloxane is called for short PDMS) solidifies, turn over the PDMS that mould obtains the micro-raceway groove of cell migration turns over mould, sees subgraph D, E in Fig. 3; With PDMS pillar bonding, see subgraph F in Fig. 3; After the micro-raceway groove punching two ends of cell migration, see subgraph G in Fig. 3; Bonding PDMS and substrate of glass, complete chip manufacturing, sees subgraph H in Fig. 3.
As shown in Figure 4, based on above-mentioned micro-fluidic chip, the present embodiment attached cell cut making method comprises:
Step S302: adopt cultivation tomb DMEM completely to carry out VSMC on sheet in micro-raceway groove of micro-fluidic chip and cultivate;
Containing Dole uncle section improvement Yi Geer substratum (Dulbecco Modified Eagle Medium is called for short DMEM) 90% in this perfect medium, containing foetal calf serum 10%, hereinafter by perfect medium referred to as DMEM.
Step S304: the solution of cell entry and two droplet inlet is all exhausted with liquid-transfering gun;
Step S306: move liquid passage with two in multiple tracks liquid-transfering gun and draw 120 μ l DMEM solution and 120 μ l trypsin Trypsin respectively) solution, aim at two droplet inlet of micro-fluidic chip, also inject two kinds of solution respectively simultaneously, wherein, first droplet inlet injects DMEM solution, and the second droplet inlet injects Trypsin solution;
Wherein, inject the temperature that DMEM and the Trypsin demand fulfillment of two droplet inlet of micro-fluidic chip is certain, this will take out fresh DMEM and 0.25% trypsin Trypsin with regard to needs from refrigerator) adopt water-bath to carry out preheating.
In the present embodiment, attached cell is VSMC, and therefore, what inject two droplet inlet is DMEM and Trypsin solution, and for other attached cell, that injects droplet inlet should be other cells perfect medium used and Trypsin solution.
Step S308: leave standstill and wait for 2min;
Wherein, the time left standstill can according to factor settings such as micro-channel dimensions and flow rate of liquid.In the present embodiment, in view of micro-raceway groove mentioned above, utilize gravity that liquid is flowed in micro-raceway groove, the time forming stable laminar flow is about 5min.Therefore, the time of repose in this step is generally no more than 5min.
Step S310: in order from cell entry, the second droplet inlet, the first droplet inlet exhaustion solution, so far on the diapire of the micro-raceway groove of micro-fluidic chip, just can form cut.
The schematic diagram of the cut adopting the present embodiment method to make is as shown in subgraph A in Fig. 7, and microscope figure is as shown in subgraph B in Fig. 7.Visible, adopt the present embodiment method to define cut well.
It should be noted that, if after above-mentioned S304, S306, S308, S310 step, do not form clearly cut, this four steps can be repeated, till cut is clear.
So far, the present embodiment attached cell cut making method is introduced complete.
In another exemplary embodiment of the present invention, additionally provide a kind of attached cell based on above-described embodiment attached cell cut making method migration observation procedure.As shown in Figure 5, the present embodiment attached cell migration observation procedure comprises:
Step S402: carry out finishing to micro-raceway groove of micro-fluidic chip, is suitable for VSMC growth to make this micro-raceway groove;
This specifically comprises the step that micro-raceway groove of micro-fluidic chip carries out finishing:
Sub-step S402a: uv irradiating sterilizing is carried out to micro-fluidic chip;
Sub-step S402b: the cell entry aiming at micro-raceway groove with liquid-transfering gun, phosphate buffered saline buffer (phosphate buffered saline is made in micro-raceway groove, be called for short PBS), then fill up cell entry and two droplet inlet with PBS, micro-raceway groove and each entrance are fully wet out;
In order to ensure follow-up add other reagent time, reagent can rely on action of gravity flow into raceway groove, can at least 1 hour be left standstill after the present embodiment adds PBS, make hydrophilicity in micro-raceway groove good.
Sub-step S402c: by the whole sucking-off of PBS in cell entry, two droplet inlet;
Sub-step S402d: move liquid passage with two in multiple tracks liquid-transfering gun and draw the solution that 120 μ l contain decorating molecule respectively simultaneously, aim at two droplet inlet respectively, get solution, makes solution rely on action of gravity to flow into micro-raceway groove; Experimentally need, leave standstill for some time, make the decorating molecule in solution fall to micro-trench floor, form decorative layer.
Sub-step S402e: recycle the method that above-mentioned sub-step S402c ~ S402d is same, is replaced by DMEM, hold over night by modifying solution in micro-raceway groove, so that the inoculation of later stage VSMC.
Step S404: carry out VSMC inoculation on sheet in micro-raceway groove of micro-fluidic chip;
By attached cell suspension adjustment concentration to 5 × 10
6individual/ml, the DMEM of sucking-off cell entry and two droplet inlet, with two liquid-transfering guns-common liquid-transfering gun in cell entry side, droplet inlet side multiple tracks liquid-transfering gun, draws VSMC suspension, droplet inlet side 25 μ l respectively, cell entry side 30 μ l, inject VSMC suspension simultaneously, leave standstill 5min, until each entrance level balance.After balance, chip is put in density and the situation such as degree of uniformity of the inoculation of basis of microscopic observation record.
Step S406: carry out VSMC on sheet and cultivate and synchronization process in micro-raceway groove of micro-fluidic chip;
In the present embodiment, at interval of 24 hours, just use the DMEM at liquid-transfering gun sucking-off cell entry and each droplet inlet place, and with liquid-transfering gun at each entrance-cell entry and two droplet inlet, instill the fresh DMEM of 60 μ l simultaneously, for VSMC provides necessary nutritive substance.The object simultaneously adding identical liquid measure DMEM is that elimination micro-raceway groove two ends liquid level is poor, and ensure do not have liquid-flow in micro-raceway groove, namely VSMC can not be affected growth by hydrodynamic shear effect.
Be cultured to after being full of individual layer VSMC in micro-raceway groove, cell state schematic diagram is shown in Fig. 6 shown in subgraph A, and micro-image to be shown in Fig. 6 shown in subgraph B.After cell covers with micro-raceway groove, use serum-free DMEM instead and cultivate 24 hours to VSMC synchronization process.
Step S408: carry out VSMC cut on sheet and make in micro-raceway groove of micro-fluidic chip;
On this sheet, the method for VSMC cut migration is identical with the associated viscera of a upper embodiment, specifically comprises:
Sub-step S408a: adopt perfect medium DMEM to carry out VSMC on sheet in micro-raceway groove of micro-fluidic chip and cultivate;
Containing Dole uncle section improvement Yi Geer substratum (Dulbecco Modified Eagle Medium is called for short DMEM) 90% in this perfect medium, containing foetal calf serum 10%, hereinafter by perfect medium referred to as DMEM.
Sub-step S408b: the solution of cell entry and two droplet inlet is all exhausted with liquid-transfering gun;
Sub-step S408c: move liquid passage with two in multiple tracks liquid-transfering gun and draw 120 μ l DMEM solution and 120 μ l trypsin Trypsin respectively simultaneously) solution, aim at two droplet inlet of micro-fluidic chip, also inject two kinds of solution respectively simultaneously, wherein, first droplet inlet injects DMEM solution, and the second droplet inlet injects Trypsin solution;
Wherein, inject the temperature that DMEM and the Trypsin demand fulfillment of two droplet inlet of micro-fluidic chip is certain, this will take out fresh DMEM and 0.25% trypsin Trypsin with regard to needs from refrigerator) adopt water-bath to carry out preheating.
In the present embodiment, attached cell is VSMC, and therefore, what inject two droplet inlet is DMEM and Trypsin solution, and for other attached cell, that injects droplet inlet should be other cells perfect medium used and Trypsin solution.
Step S408d: leave standstill and wait for 2min;
Wherein, the time left standstill can according to factor settings such as micro-channel dimensions and flow rate of liquid.In the present embodiment, in view of micro-raceway groove mentioned above, this time of repose is generally no more than 5min.
Step S408e, in order from cell entry, the second droplet inlet, the first droplet inlet exhaustion solution, thus forms cut on the diapire of the micro-raceway groove of micro-fluidic chip.
The schematic diagram of the cut adopting the present embodiment method to make is as shown in subgraph A in Fig. 7, and microscope figure is as shown in subgraph B in Fig. 7.Visible, adopt the present embodiment method to define cut well.
Step S410: one of them adds the DMEM containing the cell migration factor arbitrarily at cell entry and two droplet inlet, then micro-fluidic chip is put back to incubator.
Empirically need, the state (subgraph C in Fig. 7 is shown in by schematic diagram, and microgram is shown in subgraph D in Fig. 7) of Taking Pictures recording cell migration.
Due to the record of cell migration result, use microscope Taking Pictures recording.So experimenter can in office imaginary observe migration timing node, chip is taken out from incubator and carries out observation of taking pictures.Further, for recorded experimental result, analyzing and processing can also be carried out by any methods such as traditional method image procossing used.
Step S412, the VSMC on sheet that carries out in micro-raceway groove of micro-fluidic chip dyes, to characterize migrating cell and non-migrating cell.
On this sheet, VSMS dyeing is specially: after cut 24 hours, the VSMC in micro-raceway groove is carried out to the cellular immunization dyeing of alpha Actinin (α-actin), to characterize migrating cell and non-migrating cell.
α-actin is the differential protein differentiating smooth muscle cell, and in the smooth muscle cell being in synthesis type, expression amount is higher than the smooth muscle cell of shrinkage type.And the smooth muscle cell moved synthesis type cell just, the smooth muscle cell do not moved is shrinkage type cell.So α-actin dyeing can be used for characterizing migration and the cell do not moved, and its staining procedure and parameter comprise:
Dyeing sub-step S410a:4% paraformaldehyde fixes 15min;
Dyeing sub-step S410b: with the hydrogen peroxide confining liquid of methyl alcohol configuration 3%, close 15min;
Dyeing sub-step S410c: with the triton x-100 permeable membrane liquid permeable membrane 15min of PBS configuration 0.3%;
Dyeing sub-step S410d: configure 10% lowlenthal serum with PBS, hatch 60min for 37 DEG C;
Dyeing sub-step S410e: primary antibodie: PBS=1: 100, hatches primary antibodie 60min for 37 DEG C;
Dye sub-step S410f:37 DEG C and hatch two anti-60min;
Dyeing sub-step S410g: diaminobenzidine four hydrochloric acid colour developing 5min.
Above-mentioned dyeing sub-step S410a-S410g, originally has following 2 key illustration:
Illustrate 1: each dyeing sub-step above-mentioned all relies on action of gravity to realize.Method when each dyeing sub-step changes liquid is: all solution in first sucking-off cell entry and two drop gangways, draw target solution 20 μ l with multiple tracks liquid-transfering gun again and aim at two droplet inlet, common liquid-transfering gun is drawn target solution 40 μ l and is aimed at cell entry, instill simultaneously and stablize 2min, as shown in subgraph A in Fig. 8.Repeat above-mentioned steps again, in sucking-off hole, solution is added with once., the sub-step 5 that wherein dyes is added with twice, ensures that the concentration of institute's reagent adding is not diluted.After dyeing, the microscope figure of VSMC cell is as shown in subgraph B in Fig. 8.
Illustrate 2: except dyeing sub-step 5, other each dyeing sub-steps are all rinsed by PBS solution before carrying out.Rinse 3 times before the sub-step 6 that wherein dyes, all the other rinse 2 times.Purging method changes liquid method with described in explanation 1, and each flushing leaves standstill 5min.
Immunostaining is dyeing process comparatively complicated in cell dyeing, and VSMC before dyeing and after dyeing, form does not change substantially, and therefore, the present embodiment can also be used in other a variety of dyeing of relevant VSMC.
In the present embodiment, after cell cut, carrying out α-actin immunostaining, migration VSMC can being observed different from not moving the VSMC degree that develops the color.
So far, by reference to the accompanying drawings the present embodiment has been described in detail.Describe according to above, those skilled in the art should make the attached cell cut that the present invention is based on micro-fluidic chip and migration observation procedure have and is clearly familiar with.
In addition, the above-mentioned definition to each element and method is not limited in various concrete structures, shape or the mode mentioned in embodiment, and those of ordinary skill in the art can change simply it or replace, such as:
(1) width of cell migration micro-channel cross-sections rectangle can between hundreds of micron within several millimeter, highly can within some tens of pm to hundreds of micron;
(2) density of cell inoculation is not limited to 5 × 10
6individual/ml, can replace with the cell density of any experiment demand;
(3) in VSMC culturing process, changing the liquid time is not limited to 24 hours, the arbitrary time span that can need for experiment;
(4), after cell cut, other intervention factors can also be added from droplet inlet, to observe the impact of its on cell migration, such as, serum-free DMEM, Thr6 PDGF BB, the tumour bad factor-α, transforming growth factor-beta etc.;
(5) migration is dyeed can the cellular immunization of α-actin be replaced with the dyeing that other any experiments need to dye with the difference of not moving VSMC, as: osteopontin immunostaining, calponin dyeing etc.;
(6) above-described embodiment is all described for VSMC, but the present invention is not as limit, any attached cell, method of the present invention all can be adopted to carry out cut migration, no longer repeat herein.
In sum, microfluidic chip technology is combined with traditional VSMC migrating technology by the present invention, simulates the microenvironment of VSMC in vitro to a certain extent.In addition, the present invention does not need to use syringe pump and conduit, completes the whole process of VSMC cut on sheet based on gravity method, and specification experimental implementation, reduces experimental cost.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1., based on an attached cell cut making method for micro-fluidic chip, it is characterized in that:
There is in described micro-fluidic chip micro-raceway groove of " Y " type structure, comprising: a tap drain road; And be positioned at this tap drain road side and be communicated with this tap drain road two from raceway groove, wherein, tap drain road is connected to cell entry, two are connected to the first droplet inlet and the second droplet inlet respectively from raceway groove, and cell entry, the first droplet inlet and the second droplet inlet are all higher than described micro-raceway groove position;
This attached cell cut making method comprises:
Steps A: adopt the perfect medium that this attached cell is corresponding to carry out attached cell cultivation on sheet in micro-raceway groove of micro-fluidic chip;
Step B: the solution of the cell entry of micro-fluidic chip, the first droplet inlet and the second droplet inlet is all exhausted;
Step C: to the first droplet inlet of micro-fluidic chip and the second droplet inlet simultaneously, inject perfect medium corresponding to this attached cell and trypsin solution respectively;
Step D: leave standstill Preset Time; And
Step e: in order from cell entry, the second droplet inlet, the first droplet inlet exhaustion solution, thus form cut on the diapire of the micro-raceway groove of micro-fluidic chip.
2. attached cell cut making method according to claim 1, is characterized in that, in described step e, is less than laminar flow holds time from the operating time of cell entry, the second droplet inlet, the first droplet inlet exhaustion solution.
3. attached cell cut making method according to claim 1, it is characterized in that, described step C comprises:
Adopt in multiple tracks liquid-transfering gun two to move liquid passage and draw perfect medium corresponding to this attached cell and trypsin solution respectively;
In multiple tracks liquid-transfering gun two are moved the first droplet inlet and the second droplet inlet that liquid passage aims at micro-fluidic chip respectively, respectively and inject perfect medium corresponding to this attached cell and trypsin solution simultaneously.
4. attached cell cut making method according to claim 1, is characterized in that, in described step B, adopts liquid-transfering gun all to be exhausted by the solution of the cell entry of micro-fluidic chip, the first droplet inlet and the second droplet inlet.
5. attached cell cut making method according to claim 1, is characterized in that, described attached cell is vascular smooth muscle cell, and perfect medium corresponding to this vascular smooth muscle cell is DMEM solution.
6. attached cell cut making method according to claim 1, is characterized in that, described micro-fluidic chip is prepared by polydimethylsiloxane and substrate of glass;
In described micro-fluidic chip, the length in tap drain road is 3mm, and described two is 4mm from the length of raceway groove; Described tap drain road and be rectangle from the cross section of raceway groove; The width in described tap drain road is 800 μm, is highly 100 μm; The described width from raceway groove is 400 μm, is highly 100 μm; Further, the aperture of described cell entry, the first droplet inlet and the second droplet inlet is 5mm, and three exceeds described micro-channel top 8mm.
7. attached cell cut making method according to claim 6, is characterized in that, in described step D, the Preset Time left standstill is no more than 5min.
8., based on an attached cell migration observation procedure for the attached cell cut making method according to any one of claim 1 to 7, it is characterized in that, comprising:
Finishing is carried out to micro-raceway groove of micro-fluidic chip, is suitable for described adherent cell growth to make this micro-raceway groove;
Attached cell inoculation on sheet is carried out in micro-raceway groove of micro-fluidic chip;
In micro-raceway groove of micro-fluidic chip, carry out attached cell on sheet cultivate and synchronization process;
The attached cell cut making method according to any one of claim 1 to 8 is adopted to make cut;
At cell entry and two droplet inlet, one of them adds the DMEM containing the cell migration factor arbitrarily, then micro-fluidic chip is put back to incubator; And
After the preset lime, carry out sheet dyes to the attached cell in micro-raceway groove of micro-fluidic chip, to characterize migrating cell and non-migrating cell.
9. attached cell according to claim 8 migration observation procedure, is characterized in that, describedly in micro-raceway groove of micro-fluidic chip, carries out attached cell inoculation on sheet comprise:
Attached cell suspension is adjusted to preset concentration;
The perfect medium that this attached cell of sucking-off cell entry, the first droplet inlet and the second droplet inlet is corresponding;
Inject attached cell suspension to cell entry, the first droplet inlet and the second droplet inlet simultaneously, and leave standstill until the level balance of cell entry, the first droplet inlet and the second droplet inlet.
10. attached cell migration observation procedure according to claim 8, is characterized in that, carries out attached cell cultivation on sheet and comprise in micro-raceway groove of micro-fluidic chip:
Every Preset Time, the perfect medium solution of the attached cell of sucking-off cell entry, the first droplet inlet and the second droplet inlet;
Simultaneously to the perfect medium solution of the fresh attached cell of the liquid measures such as cell entry, the first droplet inlet and the second droplet inlet injection, to provide nutritive substance to attached cell.
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| CN108795759A (en) * | 2018-07-03 | 2018-11-13 | 大连理工大学 | Microfluidic system and method for shear stress and the experiment of chemotactic factor (CF) quantitatively regulating and controlling cell scratch removal |
| CN109593646A (en) * | 2018-12-17 | 2019-04-09 | 中国科学院电子学研究所 | It is a kind of for observing the big Array Substrate and its making and use method of cell migration |
| CN112358968A (en) * | 2020-11-17 | 2021-02-12 | 辽宁中医药大学 | Micro-fluidic chip for tumor cell migration research and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795759A (en) * | 2018-07-03 | 2018-11-13 | 大连理工大学 | Microfluidic system and method for shear stress and the experiment of chemotactic factor (CF) quantitatively regulating and controlling cell scratch removal |
| CN108795759B (en) * | 2018-07-03 | 2021-08-06 | 大连理工大学 | Microfluidic system and method for cell scratch repair experiment with shear stress and chemotactic factor quantitative regulation |
| CN109593646A (en) * | 2018-12-17 | 2019-04-09 | 中国科学院电子学研究所 | It is a kind of for observing the big Array Substrate and its making and use method of cell migration |
| CN112358968A (en) * | 2020-11-17 | 2021-02-12 | 辽宁中医药大学 | Micro-fluidic chip for tumor cell migration research and preparation method thereof |
| CN112358968B (en) * | 2020-11-17 | 2023-03-31 | 辽宁中医药大学 | Micro-fluidic chip for tumor cell migration research and preparation method thereof |
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