CN109289947B - Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof - Google Patents
Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof Download PDFInfo
- Publication number
- CN109289947B CN109289947B CN201811145368.1A CN201811145368A CN109289947B CN 109289947 B CN109289947 B CN 109289947B CN 201811145368 A CN201811145368 A CN 201811145368A CN 109289947 B CN109289947 B CN 109289947B
- Authority
- CN
- China
- Prior art keywords
- crosslinking
- gel
- hydrogel
- chip
- pieces
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004132 cross linking Methods 0.000 title claims abstract description 86
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 27
- 239000000017 hydrogel Substances 0.000 claims abstract description 81
- 239000000499 gel Substances 0.000 claims abstract description 76
- 238000000034 method Methods 0.000 claims abstract description 41
- 239000002243 precursor Substances 0.000 claims abstract description 24
- 238000010146 3D printing Methods 0.000 claims abstract description 14
- 238000004512 die casting Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 29
- 230000002792 vascular Effects 0.000 claims description 23
- 108010010803 Gelatin Proteins 0.000 claims description 18
- 239000008273 gelatin Substances 0.000 claims description 18
- 229920000159 gelatin Polymers 0.000 claims description 18
- 235000019322 gelatine Nutrition 0.000 claims description 18
- 235000011852 gelatine desserts Nutrition 0.000 claims description 18
- 239000000661 sodium alginate Substances 0.000 claims description 16
- 229940005550 sodium alginate Drugs 0.000 claims description 16
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 14
- 235000010413 sodium alginate Nutrition 0.000 claims description 14
- 210000002889 endothelial cell Anatomy 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 2
- 229910001424 calcium ion Inorganic materials 0.000 claims description 2
- 159000000007 calcium salts Chemical class 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 claims 1
- 230000000977 initiatory effect Effects 0.000 claims 1
- 231100000252 nontoxic Toxicity 0.000 abstract 1
- 230000003000 nontoxic effect Effects 0.000 abstract 1
- 239000002131 composite material Substances 0.000 description 19
- 239000000463 material Substances 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 238000010586 diagram Methods 0.000 description 9
- 238000009792 diffusion process Methods 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 8
- 238000003125 immunofluorescent labeling Methods 0.000 description 7
- 238000000016 photochemical curing Methods 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 230000000638 stimulation Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 210000004204 blood vessel Anatomy 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000007639 printing Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- GJKGAPPUXSSCFI-UHFFFAOYSA-N 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone Chemical compound CC(C)(O)C(=O)C1=CC=C(OCCO)C=C1 GJKGAPPUXSSCFI-UHFFFAOYSA-N 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000003999 initiator Substances 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 102000008790 VE-cadherin Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 108010018828 cadherin 5 Proteins 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000011960 computer-aided design Methods 0.000 description 2
- 238000001723 curing Methods 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 210000001650 focal adhesion Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000003754 machining Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 208000035868 Vascular inflammations Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- -1 gelatin-GelMA Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- SYJCUYXTMQSJLM-UHFFFAOYSA-N phenylphosphanyl-(2,4,6-trimethylphenyl)methanone Chemical compound CC1=CC(C)=CC(C)=C1C(=O)PC1=CC=CC=C1 SYJCUYXTMQSJLM-UHFFFAOYSA-N 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000007514 turning Methods 0.000 description 1
- 230000004218 vascular function Effects 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Dispersion Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a manufacturing method of a gel-based micro-fluidic chip based on secondary crosslinking, which comprises the following steps: (1) matching the hydrogel precursor solution with a primary crosslinking condition by a three-dimensional printing method or a die casting method to obtain two pieces of partially crosslinked hydrogel with a partial runner structure; (2) and (3) attaching two pieces of partially crosslinked hydrogel, butting flow channel structures of the two pieces of partially crosslinked hydrogel, applying a secondary crosslinking condition, bonding the two pieces of hydrogel and completely crosslinking the two pieces of hydrogel to obtain the gel-based microfluidic chip based on secondary crosslinking. The manufacturing method of the gel-based micro-fluidic chip based on secondary crosslinking is simple and convenient in manufacturing process, non-toxic and harmless and wide in application range. The gel-based micro-fluidic chip prepared by the method is stable and reliable, has various forms and good biocompatibility.
Description
Technical Field
The invention relates to the technical field of micro-fluidic chips, in particular to a method for manufacturing a gel-based micro-fluidic chip with an internal flow channel in a specific shape based on a secondary crosslinking principle and the prepared gel-based micro-fluidic chip.
Background
With the development of micro-fabrication technology, microfluidic chips are becoming an emerging platform for cell culture. The manipulation and analysis of cells on microfluidic chips has many advantages over current methods commonly used in laboratories: the size of the micro-channel is equivalent to that of the cell, and the perfusion culture mode used by the chip is closer to the physiological state in vivo, so that the biological characteristics of the cell in the physiological state can be reflected more truly; the microenvironment of the cells can be controlled more effectively; the chip has small volume, saves the consumption of cell solution and other reagents, and greatly reduces the analysis time; the micro-pipeline has rapid heat and mass transfer, and can obtain larger electric field intensity by using smaller voltage; the chip can be flexibly combined by various unit technologies and integrated in scale. Therefore, many studies on microfluidic chips for cell culture have been conducted.
The existing manufacturing method of the gel-based microfluidic chip mainly manufactures the gel-based microfluidic chip with a flow channel in a specific shape by methods of three-dimensional printing, a sacrificial layer process, a bonding process and the like. The three-dimensional printing generally adopts a coaxial nozzle to print the flow channel, and the method cannot realize the structure of the flow channel with a branched structure; other substances are introduced by the sacrificial layer process and the bonding process; the bonding process is not high in strength, and the bonding interface is easy to break.
Disclosure of Invention
The invention provides a manufacturing method of a gel-based micro-fluidic chip with various flow channel forms based on a secondary crosslinking principle, which can conveniently and non-toxically manufacture the gel-based micro-fluidic chip.
The invention also provides the gel-based micro-fluidic chip prepared by the method, and the gel-based micro-fluidic chip is stable and reliable, has various forms and good biocompatibility.
A manufacturing method of a gel-based micro-fluidic chip based on secondary crosslinking comprises the following steps:
(1) matching the hydrogel precursor solution with a primary crosslinking condition by a three-dimensional printing method or a die casting method to obtain two pieces of partially crosslinked hydrogel with a partial runner structure;
(2) and (3) attaching two pieces of partially crosslinked hydrogel, butting flow channel structures of the two pieces of partially crosslinked hydrogel, applying a secondary crosslinking condition, bonding the two pieces of hydrogel and completely crosslinking the two pieces of hydrogel to obtain the gel-based microfluidic chip based on secondary crosslinking.
When the mould pouring method is adopted, the structure of the mould can be designed in advance, and the design of the runner structure in the micro-control chip is realized through the design of the mould structure, for example, the mould structure with the bulge is designed, so that the partially-crosslinked hydrogel with the groove structure can be obtained. Preferably, the method for manufacturing the gel-based microfluidic chip based on secondary crosslinking comprises the following steps:
(I-1) casting the hydrogel precursor solution on two molds with set convex structures;
(I-2) applying a primary crosslinking condition to the poured hydrogel precursor solution to obtain a partially crosslinked hydrogel;
(I-3) releasing the hydrogel from the corresponding mold to obtain two pieces of partially crosslinked hydrogels each having a partial flow channel structure;
and (I-4) attaching two pieces of partially crosslinked hydrogel, butting flow channel structures of the two pieces of partially crosslinked hydrogel, applying a secondary crosslinking condition, bonding the two pieces of hydrogel, and completely crosslinking to obtain the gel-based microfluidic chip based on secondary crosslinking.
The manufacturing of the whole gel-based micro-fluidic chip is completed after the steps are completed, and the flow channel of the gel-based micro-fluidic chip prepared by the method has complicated and changeable shape and good biocompatibility.
When the three-dimensional printing method is adopted, two three-dimensional models of the partially cross-linked hydrogel with the partial flow channel structure can be designed in advance, and the manufacture of the two partially cross-linked hydrogels with the partial flow channel structure can be realized through three-dimensional printing, for example, the partially cross-linked hydrogel sheet with the groove structure is constructed through a three-dimensional printing mode. In the three-dimensional printing process, the setting of the curing conditions needs to be determined according to the composition of the hydrogel precursor solution. The three-dimensional printing mode can be an extrusion mode, an ink-jet mode, a photocuring mode and the like).
In the step (I-1), the two molds have the same structure, and the raised structures can correspond to the flow channel structures of the gel-based microfluidic chip which are processed according to actual needs and can be processed according to needs. The mold model can be constructed by adopting the existing mature technology, a three-dimensional model drawing can be obtained by Computer Aided Design (CAD) software, and the design can be realized by commercial CAD software such as CorelDraw, Solidworks and the like. The mold may be fabricated using known techniques such as three-dimensional printing, microfabrication, machining, and the like. In order to improve the precision of the flow channel and improve the precision of the mould, the mould is preferably manufactured by adopting photocuring three-dimensional printing, and the material is photosensitive resin. The depth of the flow channel protrusion can be set to be 150 and 500 micrometers according to requirements.
In the step (1) or the step (I-1), the hydrogel precursor solution may be prepared in advance.
In the present invention, the first crosslinking and the second crosslinking may be different only in time, for example, a single gel system with a relatively slow crosslinking speed may be selected in the present invention, and partial crosslinking in step (1) or step (I-2) may be achieved by controlling the crosslinking conditions, where the first crosslinking condition may be a specific time period, and the length of the time period is determined according to the properties of the actual hydrogel. In step (I-4), secondary crosslinking is achieved by extending the crosslinking time, completing the final crosslinking. Of course, the primary and secondary crosslinking may also be a difference in temperature. Or a different match of time and temperature, etc.
Of course, the primary crosslinking and the secondary crosslinking can be completely different crosslinking conditions, such as crosslinking by temperature control, ion diffusion or light curing, and the like. Preferably, the primary crosslinking conditions and the secondary crosslinking conditions are independently selected from temperature control, ion diffusion, or photo-curing.
As further preferred, the conditions for the primary crosslinking in step (1) or step (I-2) are two: condensation or ionic crosslinking. In the step (2) or the step (I-34), the conditions for the secondary crosslinking are two: ionic crosslinking and photocuring.
Preferably, the hydrogel precursor solution is prepared from hydrogels of two different crosslinking systems, and a composite hydrogel solution of two different crosslinking systems can be obtained; the primary crosslinking conditions correspond to the crosslinking conditions of the hydrogels of one crosslinking system, and the secondary crosslinking conditions correspond to the crosslinking conditions of the other hydrogels.
In the invention, the structure of the mould can be designed according to the actual use requirement, the composite hydrogel component can be selected according to the actual use requirement, the primary crosslinking and secondary crosslinking conditions are determined according to the crosslinking systems of the two components of the composite hydrogel, and the primary crosslinking and secondary crosslinking time is determined according to the size of the gel substrate chip.
Preferably, the hydrogel of the two different crosslinking systems is selected from any two of gelatin, sodium alginate, GelMA (methacrylated gelatin).
In the invention, the composite hydrogel material has three types: gelatin-sodium alginate, gelatin-GelMA, sodium alginate-GelMA.
Preferably, the corresponding crosslinking condition of the gelatin is standing at the temperature of 2-5 ℃; the corresponding crosslinking conditions of the sodium alginate are as follows: soaking in calcium salt solution with calcium ion concentration of 2-5%; the crosslinking conditions corresponding to GelMA are related to the initiator, and the initiator that can be used includes photoinitiator 2959(2-hydroxy-1(4- (hydroxyethoxy) phenyl) -2-methyl-1-propanone photoinitiator (Irgacure 2959)) or photoinitiator LAP (lithonium phenyl-2,4, 6-trimethylbenzoylphosphine), and when the photoinitiator LAP is used, the crosslinking conditions are as follows: the wavelength of the light is 400-410 nm, the light is irradiated for 5-20s, and the concentration of the LAP photoinitiator is 0.5-1%. When 2959 photoinitiator is adopted, the crosslinking conditions are as follows: the full-wave band light source illuminates for 30-60s, and the concentration of 2959 photoinitiator is 0.5% -1%.
Preferably, the method comprises the following steps:
when the hydrogel of the two different crosslinking systems is selected from gelatin and GelMA, the mass ratio of the two is 1 (0.5-2);
when the hydrogel of the two different crosslinking systems is selected from sodium alginate and GelMA, the mass ratio of the two is 1 (4-6);
when the hydrogel of the two different crosslinking systems is selected from the group consisting of gelatin and sodium alginate, the mass ratio of the two is (4-6): 1.
Preferably, the method comprises the following steps: the gel-based microfluidic chip is provided with a forked flow channel structure; and a forked convex structure corresponding to the forked flow passage structure is arranged in the mold cavity.
The preparation method of the invention can be used for manufacturing gel-based microfluidic chips in various flow channel forms.
The invention also provides a gel-based microfluidic chip prepared by the manufacturing method of the gel-based microfluidic chip based on secondary crosslinking in any technical scheme.
Preferably, the inner diameter of the flow channel is 150-800 micrometers.
The invention also provides a gel-based vascular chip, which comprises a gel-based chip substrate with a branched flow channel and endothelial cells inoculated on the inner wall of the flow channel.
Preferably, the gel-based blood vessel chip is a GelMA composite hydrogel material prepared from pigskin gelatin and pigskin gelatin.
Preferably, the inner diameter of the gel-based blood vessel chip flow channel is 500-600 microns.
Preferably, the gel-based vascular chip is seeded with endothelial cells at a density of 10M cells per ml.
Preferably, the gel-based blood vessel chip adopts 405nm blue light and 0.5 percent of LAP photoinitiator, and the illumination time is 10 seconds.
Compared with the prior art, the invention has the following advantages:
(1) the demolding and bonding of the hydrogel sheet completely depend on the crosslinking characteristics of the composite hydrogel material, and other substances are not required to be introduced;
(2) the secondary crosslinking principle can be applied to the combination of any two hydrogel materials with different crosslinking mechanisms;
(3) the two pieces of bonded hydrogel form a whole without any boundary;
(4) the construction of various different runner forms can be conveniently realized through the design of the die;
(5) can be applied to material combinations with good biocompatibility;
(6) complex experimental configuration is not needed, and the whole process is simple and convenient to operate.
The gel-based microfluidic chip has various structures, good biocompatibility, good strength and good perfusion performance.
Drawings
Fig. 1 is a schematic diagram of a microfluidic chip die according to the present invention.
FIG. 2 is a schematic representation of a partially crosslinked hydrogel sheet of the invention.
Fig. 3 is a schematic diagram of a gel-based microfluidic chip finally prepared in the present invention.
Fig. 4 is a diagram of a gel-based microfluidic chip with a complex internal flow channel manufactured by the method.
FIG. 5 is a drawing curve of a gel-based microfluidic chip according to the method of the present invention.
FIG. 6 is a compression curve of a gel-based microfluidic chip according to the method of the present invention.
FIG. 7 is a diagram of a cytooptic microscope of the gel-based vascular chip of the present invention.
FIG. 8 is a confocal image of live and dead staining fluorescence of the gel-based vascular chip of the present invention.
FIG. 9 is a confocal view of cytoskeleton staining fluorescence of the gel-based vascular chip of the present invention.
FIG. 10 is a cell electron microscope image of the cross section of the flow channel of the gel-based microfluidic chip according to the method of the present invention.
FIG. 11 is the electron microscope image of the gel-based vascular chip of the present invention.
FIG. 12 is an immunofluorescent-stained confocal image of a gel-based vascular chip of the present invention, wherein
(a) Focal adhesion immunofluorescent staining (b) CD31 immunofluorescent staining (c) VE-Cadherin immunofluorescent staining.
FIG. 13 is a bar graph showing cell viability of the gel-based vascular chip of the present invention.
FIG. 14 is a histogram of cell proliferation of the gel-based vascular chip of the present invention.
FIG. 15 shows the expression of ICAM mRNA under the inflammatory stimulation by the gel-based vascular chip of the present invention.
FIG. 16 shows the mRNA expression of VCAM under the inflammatory stimulation of the gel-based vascular chip of the present invention.
FIG. 17 is a fluorescence map of the diffusion of fluorescent particles in a gel-based microfluidic chip according to the method of the present invention.
FIG. 18 is a schematic view showing the diffusion of fluorescent particles in a gel-based microfluidic chip according to the method of the present invention.
FIG. 19 shows the diffusion rate of fluorescent particles in a gel-based microfluidic chip according to the method of the present invention.
In the figure, 1-photosensitive resin mold, 2-mold convex groove, 3-part cross-linked hydrogel sheet, 4-flow channel groove, 5-gel-based micro-fluidic chip, and 6-flow channel in the gel-based chip.
Detailed Description
Example 1:
the fabrication process of the present invention will be further described by taking the fabrication of gelatin-GelMA based microfluidic chip with a bifurcated flow channel and the construction of vascular chip as examples.
Fig. 1 shows a three-dimensional model of a designed bifurcated runner mold, starting the mold manufacturing process. The existing printing material can be selected, a corresponding three-dimensional printing mode can be adopted, for example, polylactic acid, photosensitive resin and the like can be selected, FDM three-dimensional printing, photocuring printing and the like can be adopted, an existing micro-manufacturing process or a traditional machining process can also be adopted, and the photosensitive resin and photocuring printing mode is selected in the embodiment. And after printing is finished, taking down the photosensitive resin mold 1 from the printer, and sterilizing the mold convex groove 2 for forming the forked groove structure in the inner cavity of the photosensitive resin mold 1 for later use.
Preparing a 1% w/v (g/ml) LAP (LAP blue light initiator) solution by using a basic culture medium and LAP powder, weighing a certain amount of GelMA material, dissolving GelMA by using the LAP solution through vortex oscillation to obtain a 10% w/v (g/ml) GelMA precursor solution, and filtering through a 220nm filter; weighing a certain amount of gelatin particles, and dissolving gelatin by deionized water through magnetic stirring at a constant temperature of 37 ℃ to obtain a 10% w/v (g/ml) gelatin precursor solution; mixing the two precursor solutions (wherein the volume ratio of the gelatin precursor solution to the GelMA precursor solution is 1:1), uniformly blowing to obtain a composite hydrogel precursor solution, and placing in a 37-degree water bath for later use.
And respectively pouring the prepared composite hydrogel precursor solution into the two printed part molds.
Placing the mixture in a refrigerator with the temperature of 4 ℃, standing for 10 minutes and then taking out; completing the primary crosslinking to obtain a partially crosslinked hydrogel sheet, as shown in FIG. 2;
disassembling the mold, and separating the partially crosslinked composite hydrogel from the mold to obtain a hydrogel sheet with a forked groove (formed by a forked bulge in the mold) on the surface;
the surfaces of two hydrogel sheets with grooves are tightly attached, the two grooves are aligned to form a complete six-channel structure, and a blue-light flashlight with the wavelength of 405nm illuminates the surfaces for 5 seconds from the upper surface and the lower surface respectively to obtain the gel-based microfluidic chip 5 with the forked flow channel 6, as shown in fig. 3.
And digesting the endothelial cells by pancreatin to prepare a cell suspension of 10M cells per milliliter, injecting the cell suspension into the prepared chip flow channel, turning the chip flow channel for 90 degrees every 15 minutes within 3 hours, and then statically culturing the chip flow channel in a constant-temperature incubator at 37 degrees to obtain the endothelialization flow channel with certain functions.
The gel-based microfluidic chip prepared by the method of the experiment is used for carrying out endothelial cell inoculation experiment. And forming a blood chip on the gel substrate chip through the structure of the flow channel. The supply of nutrients is achieved by the good osmotic properties of the hydrogel material.
FIG. 7 is a schematic diagram of a cytooptic microscope of the gel-based blood vessel chip prepared in this example; FIG. 8 is a confocal diagram of live and dead staining fluorescence of the gel-based vascular chip prepared in this example; FIG. 9 is a fluorescent confocal image of cytoskeleton staining of the gel-based vascular chip prepared in this example; FIG. 10 is a cross-sectional electron microscope of a flow channel of the gel-based microfluidic chip prepared in this example; FIG. 11 is a schematic diagram of a cell electron microscope showing the gel-based vascular chip prepared in this example; fig. 12 is an immunofluorescence staining fluorescence confocal diagram of the gel-based vascular chip prepared in this example, wherein: (a) immunofluorescent staining for focal adhesions (b) for CD31 immunofluorescent staining (c) for VE-Cadherin immunofluorescent staining: FIG. 12 illustrates the adhesion between cells and the material substrate, the expression of the vascular function protein, and the close relationship between cells; FIG. 13 is a histogram of the cell viability of the gum-based vascular chip prepared in this example; the cell survival rate is not greatly different from that of the control group, and the biocompatibility of the selected material combination is good; FIG. 14 is a histogram of cell proliferation of the gel-based vascular chip prepared in this example: FIG. 14 illustrates that continuous proliferation of cells on selected materials is achieved, illustrating that the biocompatibility of the selected material combination is good; FIG. 15 shows the expression of ICAM mRNA under the inflammatory stimulation by the gel-based vascular chip prepared in this example; FIG. 16 shows the mRNA expression of VCAM under the stimulation of inflammation in the gel-based vascular chip prepared in this example: figure 16 illustrates that the configured endothelialized flow channel can be used for the study of vascular inflammation stimulation.
A series of characteristics show that the gel-based microfluidic chip prepared by the method has good potential in constructing a vascular chip. In addition, the gel-based microfluidic chip prepared by the invention can realize the application of physical and chemical loads by constructing a specific flow channel form, and can realize the construction of applying specific stimulation and gradient to specific cells at specific positions. Fully shows that the microfluidic chip manufactured by the method has huge application prospect in the aspect of organ chips. FIG. 17, FIG. 18, and FIG. 19 show the permeation performance of the composite hydrogel material used in the present experiment. As can be seen from the confocal images of the diffusion of the fluorescent particles with different molecular weights of 10kDa and 40kDa, the composite hydrogel material adopted by the method has good permeability and is convenient for the diffusion of nutrition, the diffusion speed of the fluorescent particles with the molecular weights smaller than the molecular weights is slow, and the diffusion speed of the fluorescent particles in the flow channel with endothelial cell adhesion is slower than that in the blank flow channel without the cells, which indicates that the endothelialization flow channel has the barrier function.
Example 2
The manufacturing process of the present invention is further illustrated by taking the manufacturing of a gelatin-sodium alginate-based microfluidic chip as an example.
Weighing a certain amount of gelatin and sodium alginate particles, dissolving the gelatin and sodium alginate by deionized water through magnetic stirring at a constant temperature of 37 ℃ to obtain a composite hydrogel precursor solution of 2% of sodium alginate and 9% of w/v gelatin, and placing the composite hydrogel precursor solution in a water bath at 37 ℃ for later use.
And pouring the prepared composite hydrogel precursor solution into the assembled mould.
Placing the mixture in a refrigerator with the temperature of 4 ℃, standing for 10 minutes and then taking out;
disassembling the mold, and separating the partially crosslinked composite hydrogel from the mold to obtain a hydrogel sheet with a forked groove on the surface;
and (3) tightly attaching the surfaces of the two hydrogel sheets with the grooves, and soaking in a 4% calcium chloride solution for 10 minutes to obtain the chip with the forked flow channel structure.
Example 3
The manufacturing process of the present invention is further illustrated by taking the manufacturing of a sodium alginate-GelMA-based microfluidic chip as an example.
Preparing a 1% w/v (g/ml) LAP solution by using a basic culture medium and LAP powder, weighing a certain amount of GelMA material, dissolving GelMA by using the LAP solution through vortex oscillation to obtain a 10% w/v (g/ml) GelMA precursor solution, and filtering through a 220nm filter; weighing a certain amount of sodium alginate particles, dissolving the sodium alginate by GelMA precursor solution through magnetic stirring at a constant temperature of 37 ℃ to obtain 2% w/v (g/ml) of sodium alginate and 10% GelMAw/v (g/ml) of composite hydrogel precursor solution, and placing the composite hydrogel precursor solution in a water bath at 37 ℃ for later use.
And pouring the prepared composite hydrogel precursor solution into the assembled mould.
Soaking the mixture in 4% calcium chloride solution, standing for 10 min, and taking out;
disassembling the mold, and separating the partially crosslinked composite hydrogel from the mold to obtain a hydrogel sheet with a forked groove on the surface;
the surfaces of two hydrogel sheets with grooves are tightly attached, and a blue flashlight with the wavelength of 405nm is used for illuminating for 5 seconds from the upper surface and the lower surface respectively to obtain the chip with the forked flow channel structure.
By using the method of the invention, a gel-based microfluidic chip with a complex internal flow channel can be manufactured, and the physical diagram of the gel-based microfluidic chip is shown in FIG. 4.
Claims (4)
1. A manufacturing method of a gel-based micro-fluidic chip based on secondary crosslinking is characterized by comprising the following steps:
(1) matching the hydrogel precursor solution with a primary crosslinking condition by a three-dimensional printing method or a die casting method to obtain two pieces of partially crosslinked hydrogel with a partial runner structure;
(2) bonding two pieces of partially crosslinked hydrogel, butting flow channel structures of the two pieces of partially crosslinked hydrogel, applying a secondary crosslinking condition, bonding the two pieces of hydrogel and completely crosslinking the two pieces of hydrogel to obtain a gel-based microfluidic chip based on secondary crosslinking;
the hydrogel precursor solution is prepared from hydrogels of two different crosslinking systems, wherein the primary crosslinking condition corresponds to the crosslinking condition of the hydrogel of one crosslinking system, and the secondary crosslinking condition corresponds to the crosslinking condition of the other hydrogel; the hydrogel of the two different crosslinking systems is selected from any two of gelatin, sodium alginate and GelMA;
the gel-based microfluidic chip is provided with a forked flow channel structure;
the gelatin is subjected to a corresponding crosslinking condition of standing at a temperature of 2-5 ℃; the corresponding crosslinking conditions of the sodium alginate are as follows: soaking in calcium salt solution with calcium ion concentration of 2-5%; the crosslinking conditions corresponding to GelMA are as follows: illuminating for 5-20s at the wavelength of 400-410 nm, and initiating by using LAP (layered double hydroxide) photoinitiator, wherein the concentration of the LAP photoinitiator is 0.5-1%; or the full-wave band light source irradiates for 30-60s, 2959 photoinitiator is adopted, and the concentration of the 2959 photoinitiator is 0.5% -1%.
2. The method for manufacturing a gel-based microfluidic chip based on secondary crosslinking according to claim 1, wherein:
when the hydrogel of the two different crosslinking systems is selected from gelatin and GelMA, the mass ratio of the two is 1 (0.5-2);
when the hydrogel of the two different crosslinking systems is selected from sodium alginate and GelMA, the mass ratio of the two is 1 (4-6);
when the hydrogel of the two different crosslinking systems is selected from the group consisting of gelatin and sodium alginate, the mass ratio of the two is (4-6): 1.
3. The gel-based microfluidic chip prepared by the method for manufacturing the gel-based microfluidic chip based on secondary crosslinking according to any one of claims 1 to 2.
4. A gel-based vascular chip comprising a substrate comprising the gel-based vascular chip of claim 3, and endothelial cells seeded on the inner wall of the flow channel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811145368.1A CN109289947B (en) | 2018-09-29 | 2018-09-29 | Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811145368.1A CN109289947B (en) | 2018-09-29 | 2018-09-29 | Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109289947A CN109289947A (en) | 2019-02-01 |
| CN109289947B true CN109289947B (en) | 2020-09-25 |
Family
ID=65165018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811145368.1A Active CN109289947B (en) | 2018-09-29 | 2018-09-29 | Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109289947B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110479390A (en) * | 2019-07-31 | 2019-11-22 | 浙江大学 | A kind of sample production method and application based on microwire frame template |
| CN114308161B (en) * | 2021-12-31 | 2023-07-25 | 上海中航光电子有限公司 | Microfluidic chip and manufacturing method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1718728A2 (en) * | 2003-09-23 | 2006-11-08 | Massachusetts Institute of Technology | Fabrication and packaging of suspended microchannel detectors |
| CN107930542A (en) * | 2017-11-13 | 2018-04-20 | 王华楠 | One-step method continuously prepares the microflow control technique of calcium alginate microgel |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105381826B (en) * | 2015-11-25 | 2017-08-11 | 太原理工大学 | A kind of preparation method of micro-fluidic three dimensional gel chip model |
| CN107286359B (en) * | 2017-06-27 | 2020-05-22 | 上普博源（北京）生物科技有限公司 | Hydrogel with heterogeneous multilayer structure and preparation method thereof |
| CN108659440B (en) * | 2018-06-08 | 2020-06-16 | 天津工业大学 | Preparation method for obtaining high-strength hydrogel through secondary swelling and crosslinking |
-
2018
- 2018-09-29 CN CN201811145368.1A patent/CN109289947B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1718728A2 (en) * | 2003-09-23 | 2006-11-08 | Massachusetts Institute of Technology | Fabrication and packaging of suspended microchannel detectors |
| CN107930542A (en) * | 2017-11-13 | 2018-04-20 | 王华楠 | One-step method continuously prepares the microflow control technique of calcium alginate microgel |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109289947A (en) | 2019-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Skylar-Scott et al. | Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels | |
| Domachuk et al. | Bio‐microfluidics: biomaterials and biomimetic designs | |
| Gao et al. | Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery | |
| AU2018336901B2 (en) | Tissue construct, methods of producing and using the same | |
| Khalil et al. | Bioprinting endothelial cells with alginate for 3D tissue constructs | |
| Leng et al. | Mosaic hydrogels: one‐step formation of multiscale soft materials | |
| Choi et al. | Microfluidic scaffolds for tissue engineering | |
| Zhou | The recent development and applications of fluidic channels by 3D printing | |
| US10544036B2 (en) | Microfluidic devices and methods for the extrusion of tubular structures | |
| US20140306371A1 (en) | Devices and methods for producing planar polymeric materials using microfluidics | |
| CN109289947B (en) | Gel-based micro-fluidic chip based on secondary crosslinking and manufacturing method thereof | |
| CN106349487B (en) | Preparation method of hydrogel with microfluidic channel | |
| Brunel et al. | Engineered assistive materials for 3D bioprinting: support baths and sacrificial inks | |
| EP3464554A2 (en) | 3d printing of gel networks | |
| CN113667603A (en) | Liver organoid culture chip and preparation method and application thereof | |
| He et al. | Layer-by-layer micromolding of natural biopolymer scaffolds with intrinsic microfluidic networks | |
| CN113893387A (en) | Tissue engineering scaffold assembled by cell-loaded microgel and preparation method and application thereof | |
| He et al. | Sequential assembly of 3D perfusable microfluidic hydrogels | |
| Farshidfar et al. | The feasible application of microfluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs: a direction for future research | |
| CN110607271B (en) | Preparation method of in vitro vascularized 3D tissue based on micromachining technology | |
| CN107286359B (en) | Hydrogel with heterogeneous multilayer structure and preparation method thereof | |
| CN103120808B (en) | Preparation method of three-dimensional soft bracket | |
| Gao et al. | One‐Step Formation of Protein‐Based Tubular Structures for Functional Devices and Tissues | |
| CN110479390A (en) | A kind of sample production method and application based on microwire frame template | |
| Qi et al. | A 3D bioprinted hydrogel multilevel arc vascular channel combined with an isomaltol core sacrificial process |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |