CN204079986U - A kind of micro-nano-fluidic control device for cell migration research - Google Patents

Abstract

For a micro-nano-fluidic control device for cell migration research, mainly comprise: one deck of the most end is substrate of glass, and middle one deck is the PDMS layer that microfluidic channel is arranged at bottom, and the superiors are two glass cylinder liquid storage pools.The shaping microfluidic chamber of PDMS is divided into three independently regions by the nanoporous isolated gate that two hydrogels are shaping---the fluid channel of dual-side and centrocyte cultivation region.Add the solution of different concns in the ingress of two wing passages, a stable concentration gradient can be formed in centrocyte cultivation region by diffusion.Device have also been devised the pressure that the passage of two curved elongated and equilibrium area balance two side liquid passages, to guarantee repeatability and the stability of the concentration gradient in cell cultures district.This device architecture is simple and can realize easily reconfiguring, and by the thickness of adjustment hydrogel isolated gate, can realize concentration gradient and some other control affecting the parameter of cell migration to adapt to the needs of different research.

Description

A kind of micro-nano-fluidic control device for cell migration research

Technical field

The invention belongs to microfluidic field, relate to the manufacture method of a kind of micro-nano-fluidic control device for cell migration research and the described fluid device of formation.

Background technology

Cell migration is an important topic of current RESEARCH ON CELL-BIOLOGY, and scientists attempts the research by cell migration, in medical use such as prevention cancer metastasis, allo-skin graft etc., obtain larger achievement.Simultaneously also because of the motion characteristics that cell migration is exclusive, become the popular direction of biology at present research.Have the migration of more test to cell to be studied, early stage research device can be divided into Boyden cell, Zigmond room, Tang's grace room, and several structure such as microflowmeter.

But in these devices, cell is except being in concentration gradient, and be also exposed in the shearing stress because of flowing generation, these shearing forces directly can affect the migration of cell.In addition, also can be flushed away by most of signal factors (autocrine/paracrine) of emiocytosis, this can produce very adverse influence to research cell migration.Concentration gradient based on diffusion forms device, and their principle of work utilizes isolated gate or film by diffuseing to form and keeping concentration gradient.Therefore they can overcome some limitations of gradient forming device part based on flowing, but they also have the limitation of oneself: as they only can to non-adherent cell (such as, embryo, insect cell) or two dimension (2D) attached cell to cultivate and can not sustenticular cell epimatrix.In the function organized in vivo, the impact of extracellular matrix is vital.For this problem, some research groups using in three-dimensional (3D) material sorting to microfluidic device as cytostromatic supporting structure, but these equipment are often: 1) because the design of its complexity is difficult to manufacture; 2) controlled chemical concentration gradients cannot be provided; Or 3) cannot the maintenance concentration gradient of long period.

Summary of the invention

The problem that technology of the present invention solves is: in order to overcome above-mentioned restriction, and Design and Machining one microfluidic device, PEGDA gel is embedded the formation of assisting concentration gradient as semi-transparent isolated gate by this device, can be applicable to the research of cell migration.This device can provide repeatably, controlled, stable and can long period stable concentration gradient.

Solution of the present invention: this microfluidic device is made up of three parts: one deck of the most end is substrate of glass, and middle one deck is the PDMS layer that microfluidic channel is arranged at bottom, and the superiors are two glass cylinder, are used as the liquid storage pool of nutrient solution.The primary structure of device is a PDMS room, and this room is by two gel isolated gate formation spaced apart three independently passage---the fluid channel on both sides and centrocyte culture channel.When adding the solution of different concns in the ingress of two wing passages, solution can flow to wing passage under the force of gravity.With the effect of crossing diffusion, cultivate region at centrocyte and form a concentration gradient, can at this regional load and culturing cell.In order to ensure repeatability and the stability of concentration gradient, devise the pressure that two bending elongated passages and equilibrium area balance two side flow passages.By the thickness of adjustment hydrogel isolated gate, concentration gradient and some other control affecting the vital parameter of cell migration can be realized.

The principle of the invention: the design of this device is biocompatible based on PEGDA gel, simultaneously again can not be contaminated in complex environment.In this device, PEGDA gel is used as the porous-film of high flow resistance, and it both can help the quick formation of concentration gradient, the transverse flow of the fluid that the pressure imbalance between simultaneously can limiting again because of two access roades causes.For small molecules, the resistance of film does not affect its diffusion transport.In two entrance liquid pools, add the solution of different concns, under the driving of gravity, the two fluids that concentration is different can inflow side passage.Under the effect of diffusion, a concentration gradient can be formed in centrocyte cultivation region.In order to maintain the stable of the concentration gradient of cell culture area, then need the pressure equilibrium of maintenance two wing passage S1 and S2.In theory, can be solved, but in fact, this method is being problematic in microfluid system by the solution adding same volume in the fluid pool of two entrances, the level height of liquid level is difficult to accurately control.Capillary impact, also may cause the flowing of fluid.Therefore, two elongated bending wing passages and an equilibrium area is added in systems in which.We know that two wing passages are be directly proportional to the pressure difference between two streams (the Q=Δ P/R) that are inversely proportional to the resistance of stream at the through-current capacity of zone of action.Bending elongated sides passage, contributes to the fluid resistance (namely along with the increase of R, then Q reduces) of increase system thus controls the transverse flow of the fluid caused because of both sides channel pressure imbalance.Two bending wing passages, before access cell cultures district, are first connected to same fluid cushion district via two thin interconnections.This thin interconnection, can allow the circulation of fluid, can reduce the interchannel diffusion in both sides when both sides channel pressure reaches balance.When two fluids flow through elongated sides channel region after equilibrium area meets, substantially with identical pressure current through cell cultures district.In the manufacturing processed of device, because surface effects, always have space to exist between the wall of hydrogel and PDMS, that can not fit is very good.In order to address this problem, when structure design, devise groove structure at hydrogel and PDMS sidewall contact district, thoroughly can solve end gap Problems existing like this.

Advantage of the present invention: in this microfluidic device, the driving of fluid adopts gravity to drive, and can realize device operation so simple, and structure is enough compact can be placed in culture dish, this is very easily to the research of cell.In addition, the structure design of this device can realize reconfiguring easily, to adapt to the needs of different applied researcies.As concentration gradient just can be able to be realized easily by the thickness changing centrocyte cultivation region width or isolated gate: the thickness reducing central culture channel width or isolated gate, a precipitous concentration gradient can be set up fast.Utilize the handiness of this equipment, very simply can realize the detect delay of different cell migrations.

Accompanying drawing explanation

Fig. 1 is microfluidic device schematic diagram.

Fig. 2 is the schematic diagram of hydrogel isolated gate manufacturing processed.

Specific implementation method

In order to form the microtexture of hydrogel on glass basic surface, effects on surface has carried out processing the sticking power improving hydrogel.Be exactly that surface organosilane (organosilane) carries out reason and produces and fasten methyl methacrylate group (tethered methacrylate groups) with in the course of the polymerization process by covalent linkage and hydrogel bonding in simple terms.First substrate is immersed in the hydrogen peroxide and the vitriol oil that concentration is 30%w/v, carries out the clean of surface and hydroxylation (hydroxylate) substrate surface in " piranha " solution of the ratio mixing according to 1: 4.Through hydroxylated surface, be immersed in 3-(trichlorosilyl) propyl methacrylate (TPM that concentration is 1mM again, Sigma-Aldrich, St.Louis, MO) be in the mixing solutions of 80%/20% (V/V) 5 minutes in heptane (heptane)/carbon tetracol phenixin (carbon tetrachloride) ratio, network surface tissue and suspention methyl methacrylate (pendant methacrylate) of the Si-O-Si key of a densification can be formed like this at substrate surface.

Microfluidic chamber utilizes MEMS technology to be made.Mainly comprise SU-8 mould process and PDMS moulding process.Be exactly, after obtaining SU-8 mould, obtained by PDMS shaping on mould (weight ratio of cured body and performed polymer is 1: 10, Ellsworth Adhesives, Germantown, WI) in simple terms.PDMS layer after degassed continues solidification 2 hours under the condition of 65 DEG C on mould.PDMS layer after solidification takes off from its mould, and is used as fluid pool or is used for carrying out fluid interconnected with stainless steel tube punching.PDMS layer is by air plasma (Plasma cleaner Model PDC-32G, Harrick Plasma, Ithaca, NY) rear glass cover-slip (the VWR Vista Vision with processing of process, Suwanee, GA) bonding is fixed together, and forms microfluidic chamber.Finally glass cylinder (Fisher Scientific, Pittsburg, PA) is cemented in fluid inlet place and form liquid storage pool thus the making completing microfluidic device.

The preparation of hydrogel isolated gate is according to the needs of soluble factor, and the processing thickness of hydrogel isolated gate can change between 30-500 μm.By the restriction of hydrogel intensity, maximum ga(u)ge is subject to the restriction of effectively diffusibleness in hydrogel to the minimum thickness of hydrogel isolated gate.The research such as Michael shows, cell is in the scope of 0 to 600 μm at distance nutritive substance transfer passage in hydrogel, can keep stronger viability in 72 hours.The making processes of hydrogel is exactly in simple terms, first to surface light trigger methyl phenyl ketone (the photoinitiator acetophenone (2 of PDMS room, 2-dimethoxy-2-phenyl acetophenone in n-vinylpyrrolidone, 300mg/mL, Sigma) process, to improve the polymerization of PEGDA pre-polymer solution at interface of surface free radical induction.Too much light trigger first with purity be after 100% ethanol fully rinses again with and deionized water fully rinse, finally dry up with nitrogen.Then by polyethyleneglycol diacrylate (PEG-diacrylate) (PEG-DA, MW 575, Sigma-Aldrich, Polysciences, Warrington, PA) and concentration be 0.25% (W/W) Darocur 1173 (1-phenyl-2-hydroxy-2-methyl-1-propanone, Ciba Specialty Chemicals, Tarrytown, NY) being mixed to form Gel Pre polymers soln is injected in microfluidic chamber, the process injected, in order to avoid the generation of bubble, injection speed is very slow.After injection, before exposure in order to avoid pre-polymer solution overflows, the fluid pool of liquid in-out mouth all seals with scotch tape.Then with mask, fluid chamber is covered from bottom, with UV lamp (365nm, the 20mW/cm of longer wavelength ²) irradiate and be polymerized for 20 seconds, as shown in accompanying drawing 2 (f).Only exposure region there occurs free yl induction gelation, forms the structure being insoluble to PEG solvent.The pre-polymer solution of gelation is not had to utilize deionized water rinsing to fall.The speed of washing fluid to be controlled in the process of rinsing, avoid the destruction to hydrogel structure.The technical process of element manufacturing as shown in Figure 2.

Claims (3)

1. the micro-nano-fluidic control device for cell migration research, it is characterized in that: this micro-nano-fluidic control device described is made up of three parts: one deck of the most end is substrate of glass (1), middle one deck is the PDMS layer (2) that microfluidic channel is arranged at bottom, the superiors are two glass cylinder (3), are used as the liquid storage pool of nutrient solution; Wherein PDMS room (4) are by two gel isolated gate (5) formation spaced apart three independently passage---the fluid channel (6) on both sides and centrocyte culture channel (7); When adding the solution of different concns in the ingress (3) of two wing passages, solution can flow to wing passage under the force of gravity; With the effect of crossing diffusion, cultivate region (7) at centrocyte and form a concentration gradient; In order to ensure repeatability and the stability of concentration gradient, devise the pressure that two bending elongated passages (8) and equilibrium area (9) balance two side flow passages; By the thickness of adjustment hydrogel isolated gate (5), concentration gradient and some other control affecting the vital parameter of cell migration can be realized.

2. micro-nano-fluidic control device according to claim 1, is characterized in that: liquid storage pool (3) is communicated with equilibrium area (9) by curved elongated passage (8) with PDMS room (4).

3. micro-nano-fluidic control device according to claim 1, is characterized in that: PDMS room (4) are isolated into three regions by two broad-adjustable hydrogel isolated gates (5).